TWI756604B - Ornithine decarboxylase variants, methods for producing putrescine using the same, polynucleotide, microorganism, method for preparing polyamine, and composition for preparing polyamide - Google Patents

Abstract

本申請案是有關於一種鳥胺酸脫羧酶或蛋白質變異體、編 碼該鳥胺酸脫羧酶或蛋白質變異體的聚核苷酸、包括其的微生物、利用其生產丁二胺的方法、聚醯胺的製備方法以及聚醯胺製備用組成物。本發明達成如下效果：增大丁二胺的生產性、生產效率或生產選擇性，抑制副反應而減少精製丁二胺時的費用。 This application relates to an ornithine decarboxylase or protein variant, ed. A polynucleotide encoding the ornithine decarboxylase or protein variant, a microorganism including the same, a method for producing butanediamine using the same, a method for preparing polyamide, and a composition for preparing polyamide. The present invention achieves the following effects: increasing the productivity, production efficiency or production selectivity of butanediamine, suppressing side reactions and reducing the cost of purifying butanediamine.

Description

鳥胺酸脫羧酶變異體、使用其製造丁二胺之方 法、聚核苷酸、微生物、聚醯胺的製備方法以及聚醯胺製備用組成物 Ornithine decarboxylase variant and method for producing butanediamine using it Method, polynucleotide, microorganism, preparation method of polyamide and composition for preparation of polyamide

本申請案是有關於一種鳥胺酸脫羧酶變異體、編碼鳥胺酸脫羧酶變異體的基因、包括鳥胺酸脫羧酶變異體的微生物及利用其進行的丁二胺合成。 The present application relates to an ornithine decarboxylase variant, a gene encoding the ornithine decarboxylase variant, a microorganism including the ornithine decarboxylase variant, and the synthesis of butanediamine using the same.

本申請案主張基於在2018年12月28日申請的台灣專利申請案第107147749號的優先權的利益，該專利申請案的文獻中所揭示的所有內容包括作本說明書的一部分。 This application claims the benefit of priority based on Taiwan Patent Application No. 107147749 filed on December 28, 2018, all content disclosed in the documents of this patent application being incorporated as a part of this specification.

丁二胺(putrescine，1,4-diamino-butane)雖為導致腐爛的有機體產生惡臭的物質，但可用於合成4,6-尼龍，因此於工業上進行生產。目前，因石油資源而每年生產10,000噸以上的丁二胺，但因石油價格頻繁變動而存在原料供需不穩定的問題。另外，存在 因於生產過程中生成大量的毒性物質而會導致環境污染的問題。 Butanediamine (putrescine, 1,4-diamino-butane) is a substance that causes putrefaction of organisms to produce odor, but it can be used to synthesize 4,6-nylon, and thus is produced industrially. At present, more than 10,000 tons of butanediamine is produced annually due to petroleum resources, but there is a problem of unstable supply and demand of raw materials due to frequent fluctuations in petroleum prices. In addition, there is The problem of environmental pollution is caused by the production of a large amount of toxic substances in the production process.

為瞭解決此種問題，最近正在積極地研究來自生物的丁二胺的合成。例如，正在對自來自生物的鳥胺酸合成丁二胺或利用糖自微生物大量生產丁二胺的方法進行研究。 In order to solve such a problem, the synthesis of bio-derived butanediamine is being actively studied recently. For example, studies are being conducted on methods for synthesizing butanediamine from ornithine derived from organisms or for mass-producing butanediamine from microorganisms using sugars.

就自微生物生產丁二胺的方法而言，一直以來使用用以增加丁二胺的產量的各種生物學工程方法。上述方法可為如下者：例如，利用啟動子調節參與丁二胺的生物合成的酶的活性；過度表現反向轉運體以容易地向細胞外釋出丁二胺；或阻斷分解丁二胺的路徑。其中，已知調節微生物內的參與丁二胺的生物合成的酶的活性大幅有助於增加丁二胺的產量。 As a method for producing butanediamine from microorganisms, various biological engineering methods for increasing the production of butanediamine have been used. Such methods may be as follows: for example, using a promoter to modulate the activity of an enzyme involved in the biosynthesis of butanediamine; overexpressing an antiporter to readily release butanediamine extracellularly; or blocking the breakdown of butanediamine route of. Among them, it is known that regulation of the activity of an enzyme involved in the biosynthesis of butanediamine in microorganisms greatly contributes to an increase in the production of butanediamine.

鳥胺酸脫羧酶作為切割鳥胺酸的末端羧基而合成丁二胺的酶，其是於丁二胺的生物合成中執行重要作用的酶之一。然而，鳥胺酸脫羧酶不僅自鳥胺酸合成丁二胺，而且同時具有自離胺酸合成為戊二胺(1,5-diamino-pentane)的活性(副反應)，故而於提高其活性的情形時，會連同丁二胺一併生成戊二胺而降低丁二胺的產量。上述戊二胺於精製丁二胺時亦會導致許多問題。具體而言，於藉由蒸餾方法精製微生物培養液的過程中，由於丁二胺(H₂N(CH₂)₄NH₂)與戊二胺(H₂N(CH₂)₅NH₂)的結構非常相似，故而為了選擇性地精製上述微生物培養液而花費較多的費用及時間。 Ornithine decarboxylase, as an enzyme that cleaves the terminal carboxyl group of ornithine to synthesize butanediamine, is one of the enzymes that performs an important role in the biosynthesis of butanediamine. However, ornithine decarboxylase not only synthesizes butanediamine from ornithine, but also has the activity of synthesizing 1,5-diamino-pentane from lysine at the same time (a side reaction), so its activity is improved. In the case of butanediamine, pentamethylenediamine will be formed together with butanediamine and the yield of butanediamine will be reduced. The above-mentioned pentamethylene diamine also causes many problems when purifying butanediamine. Specifically, in the process of purifying the microbial culture liquid by the distillation method, due to the difference between butanediamine (H2N( _CH2 ) _4NH2 ) and pentamethylenediamine ₍ H2N _{( CH2 ) 5NH2 )} Since the structures are very similar, it takes a lot of time and money to selectively purify the above-mentioned microorganism culture liquid.

因此，於欲調節鳥胺酸脫羧酶的活性的情形時，非常重要的是保持自鳥胺酸合成丁二胺的活性，並且降低自離胺酸合成為戊二胺的活性(副反應)。 Therefore, when the activity of ornithine decarboxylase is to be adjusted, it is very important to maintain the activity of synthesizing butanediamine from ornithine and reduce the activity (side reaction) of synthesizing pentamethylenediamine from lysine.

因此，本發明者等人發掘出新穎的鳥胺酸脫羧酶，藉由確 認上述鳥胺酸脫羧酶中戊二胺的合成活性較低且丁二胺的合成活性較高而完成本申請案。 Therefore, the inventors of the present invention discovered a novel ornithine decarboxylase by identifying It is considered that the synthetic activity of pentamethylenediamine is low and the synthetic activity of butanediamine is high in the above-mentioned ornithine decarboxylase, and the present application is completed.

本申請案提供一種鳥胺酸脫羧酶或其變異體。 The present application provides an ornithine decarboxylase or a variant thereof.

另外，本申請案提供一種編碼上述鳥胺酸脫羧酶或其變異體的聚核苷酸。 In addition, the present application provides a polynucleotide encoding the above-mentioned ornithine decarboxylase or a variant thereof.

本申請案提供一種生產包括上述鳥胺酸脫羧酶或其變異體的丁二胺的微生物。 The present application provides a microorganism for producing butanediamine comprising the above-mentioned ornithine decarboxylase or a variant thereof.

本申請案的又一目的在於提供一種包括於培養基培養上述微生物的步驟的丁二胺生產方法。 Yet another object of the present application is to provide a method for producing butanediamine comprising the step of culturing the above-mentioned microorganism in a medium.

本申請案的又一目的在於提供一種包括於培養基培養上述微生物的步驟的增加丁二胺的純度的方法。 Yet another object of the present application is to provide a method for increasing the purity of butanediamine comprising the step of culturing the above-mentioned microorganism in a medium.

本申請案的又一目的在於提供一種包括於培養基培養上述微生物的步驟的增加丁二胺相對於戊二胺的比率的方法。 Yet another object of the present application is to provide a method of increasing the ratio of butanediamine to pentamethylenediamine comprising the step of culturing the above-mentioned microorganism in a medium.

而且，本申請案的又一目的在於提供一種上述丁二胺合成聚醯胺類聚合物的用途。 Furthermore, another object of the present application is to provide a use of the above-mentioned butanediamine to synthesize a polyamide-based polymer.

若具體地對其進行說明，則如下。另一方面，本申請案中所揭示的各者的說明及實施方式亦可適用於各者的其他說明及實施方式。即，本申請案中所揭示的各種要素的所有組合屬於本申請案的範疇。另外，本申請案的範疇並不限制於以下所記述的具體敍述。 Specifically, it is as follows. On the other hand, the descriptions and embodiments of each disclosed in this application can also be applied to other descriptions and embodiments of each. That is, all combinations of the various elements disclosed in this application belong to the scope of this application. In addition, the scope of the present application is not limited to the specific description described below.

本申請案的一實施方式提供一種鳥胺酸脫羧酶變異體，具有於SEQ ID NO：1的胺基酸序列中包括一個以上的胺基酸取代的丁二胺生產活性。 One embodiment of the present application provides an ornithine decarboxylase variant having butanediamine production activity including one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

具體而言，本申請案提供一種蛋白質的變異體，於SEQ ID NO：1的胺基酸序列中，i)作為第713個胺基酸的丙胺酸由其他胺基酸取代及/或ii)作為第698個胺基酸的麩胺酸由其他胺基酸取代。上述胺基酸取代可包括：i)作為第713個胺基酸的丙胺酸由選自白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸及麩醯胺酸中的胺基酸取代；及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代。 Specifically, the present application provides a protein variant in which, in the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is substituted with other amino acids and/or ii) Glutamic acid, which is the 698th amino acid, is substituted by other amino acids. The above amino acid substitutions may include: i) alanine as the 713th amino acid is selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, tryptophan and gluten amino acid substitution in the amino acid; and/or ii) glutamic acid as the 698th amino acid is substituted with aspartic acid.

於本申請案中，用語"丁二胺"作為藉由鳥胺酸的脫羧反應或精胺的水解而生成的物質，雖亦存在於腐爛物中，但作為正常的成分廣泛分佈於生物體。作為多元胺的一種，具有構成核糖體，促進細胞生長或促進核糖核酸(Ribonucleic Acid，RNA)合成的功能。特別是，於工業上為用以生產包括尼龍4、尼龍6的聚醯胺4、聚醯胺6的重要的原料物質，且為仍需進行量產研究的物質。 In the present application, the term "butanediamine" is used as a substance produced by the decarboxylation reaction of ornithine or the hydrolysis of spermine. Although it also exists in decaying matter, it is widely distributed in living organisms as a normal component. As a kind of polyamine, it has the function of forming ribosomes, promoting cell growth or promoting the synthesis of ribonucleic acid (RNA). In particular, it is an industrially important raw material for producing polyamide 4 and polyamide 6 including nylon 4 and nylon 6, and it is a substance that still needs to be studied for mass production.

可藉由使用鳥胺酸作為基質的方法生產丁二胺。另外，可於使用成為鳥胺酸的前驅物的物質作為基質合成鳥胺酸後，自該鳥胺酸生產丁二胺。只要鳥胺酸的合成為可由業者容易地選擇者，則可無限制地使用。 Butanediamine can be produced by a method using ornithine as a substrate. In addition, after synthesizing ornithine using a substance that becomes a precursor of ornithine as a substrate, butanediamine can be produced from the ornithine. As long as the synthesis of ornithine can be easily selected by the manufacturer, it can be used without limitation.

於本申請案中，用語"鳥胺酸"是於鳥胺酸循環中發揮重要作用的鹼性胺基酸，特別是L-鳥胺酸廣泛發現於植物、動物、微生物中。通常，於具有鳥胺酸循環的生物體內，與要素生產相關而於代謝上發揮重要的作用。另外，可於生物體內與精胺酸、麩胺 酸、脯胺酸相互轉化，傳遞酮酸、乙醛酸及胺基。作為藉由鳥胺酸脫羧酶生成胺(丁二胺)的基質，由其合成為多元胺。於本發明中，特別是可為可用作鳥胺酸脫羧酶的基質的L-鳥胺酸。 In this application, the term "ornithine" is a basic amino acid that plays an important role in the ornithine cycle, especially L-ornithine is widely found in plants, animals, and microorganisms. Generally, in organisms having an ornithine cycle, it plays an important role in metabolism in relation to element production. In addition, it can interact with arginine, glutamine in vivo The acid and proline are converted into each other, and the keto acid, glyoxylic acid and amine group are transferred. A polyamine is synthesized from it as a substrate to generate an amine (butanediamine) by ornithine decarboxylase. In the present invention, in particular, L-ornithine can be used as a substrate for ornithine decarboxylase.

於本申請案中，用語"鳥胺酸脫羧酶(ornithine decarboxylase，ODC)"是促進作為合成多元胺時的最初步驟及丁二胺(putrescine)生產路徑中的最後步驟的下述反應式的酶。於本申請案中，鳥胺酸脫羧酶可與鳥胺酸去羧酶(ornithine decarboxylase)混用。ODC以L-鳥胺酸為基質生產丁二胺，磷酸吡哆醛(Pyridoxal phosphate，PLP)作為輔助因子(co-factor)發揮作用。 In this application, the term "ornithine decarboxylase (ODC)" is an enzyme that promotes the following reaction formula as the first step in the synthesis of polyamines and the last step in the production path of butanediamine (putrescine) . In the present application, ornithine decarboxylase may be used in combination with ornithine decarboxylase. ODC uses L-ornithine as a substrate to produce butanediamine, and pyridoxal phosphate (PLP) acts as a co-factor.

[反應式]L-鳥胺酸<=>丁二胺+CO₂ [Reaction formula] L-ornithine <=> butanediamine+CO ₂

圖1表示利用鳥胺酸脫羧酶以鳥胺酸為基質合成丁二胺的過程的化學反應式。另外，表示需抑制的鳥胺酸脫羧酶的副反應即戊二胺(cadaverine)合成路徑。 Fig. 1 shows the chemical reaction formula of the process of synthesizing butanediamine with ornithine as a substrate by ornithine decarboxylase. In addition, the side reaction of ornithine decarboxylase to be inhibited, that is, a cadaverine synthesis pathway is shown.

於本申請案中，獲得ODC(ornithine decarboxylase)的方法可應用本領域中熟知的各種方法。作為該方法的示例，可基於包括以可於通常廣泛用於酶表現的微生物中以高效率獲得酶的方式進行密碼子最佳化的基因合成技術及微生物的大量基因組資訊，利用生物資訊學方法而藉由對有用的酶資源進行篩選的方法獲得，且不限制於此。 In the present application, the method of obtaining ODC (ornithine decarboxylase) can apply various methods well known in the art. As an example of this method, bioinformatics methods can be utilized based on gene synthesis techniques including codon-optimized gene synthesis techniques and a large amount of genomic information of microorganisms in such a way that enzymes can be obtained with high efficiency in microorganisms generally widely used for enzyme expression. However, it is obtained by a method of screening useful enzyme resources, but is not limited thereto.

於本申請案中，SEQ ID NO：1是指具有丁二胺生產活性的鳥胺酸脫羧酶的胺基酸序列。上述SEQ ID NO：1的胺基酸序列可自作為公知的資料庫的美國國家生物技術資訊中心(National Center for Biotechnology Information，NCBI)的基因庫(GenBank)其序列。作為一例，鳥胺酸脫羧酶可來自乳桿菌屬(Lactobacillus sp.)、酵母屬(Saccharomyces sp.)或大腸桿菌(Escherichia coli，E.coli)，具體而言，可來自雞源乳桿菌(Lactobacillus saerimneri)，但並不限制於此，只要為具有與包括上述胺基酸序列的蛋白質相同的活性的蛋白質的胺基酸序列，則可無限制地包括。另外，作為本申請案中的具有丁二胺生產活性的鳥胺酸脫羧酶，記載了包括SEQ ID NO：1的胺基酸序列的蛋白質，但並不排除於SEQ ID NO：1的胺基酸序列前後無意義地追加序列或可自然發生的突變、或者其潛伏性突變(silent mutation)，只要為具有與包括SEQ ID NO：1的胺基酸序列的蛋白質彼此相同或相應的活性的情形，則業者明白相當於具有本申請案的丁二胺的活性的蛋白質。具體而言，例如本申請案的具有丁二胺生產活性的蛋白質可為由SEQ ID NO：1的胺基酸序列、或與其具有80%、85%、90%、95%、96%、97%、98%或99%以上的同源性或一致性的胺基酸序列構成的蛋白質。另外，只要為具有此種同源性或一致性且表現出與上述蛋白質相應的功效的胺基酸序列，則應明白具有一部分序列缺失、變形、經取代或加成的胺基酸序列的蛋白質亦包括於成為本申請案的變異對象的蛋白質的範圍內。 In this application, SEQ ID NO: 1 refers to the amino acid sequence of ornithine decarboxylase having butanediamine production activity. The amino acid sequence of the above-mentioned SEQ ID NO: 1 can be obtained from the National Center for Biotechnology Information (National Center for Biotechnology Information), which is a well-known database. Center for Biotechnology Information, NCBI) of the GenBank (GenBank) its sequence. As an example, ornithine decarboxylase can be derived from Lactobacillus sp., Saccharomyces sp. or Escherichia coli (E. coli), specifically, Lactobacillus saerimneri), but not limited thereto, and can be included without limitation as long as it is an amino acid sequence of a protein having the same activity as the protein including the above-mentioned amino acid sequence. In addition, as the ornithine decarboxylase having butanediamine production activity in the present application, a protein including the amino acid sequence of SEQ ID NO: 1 is described, but it is not excluded from the amino acid sequence of SEQ ID NO: 1 Insignificant addition sequences before and after the acid sequence, naturally occurring mutations, or silent mutations thereof, as long as they have the same or corresponding activities as the proteins including the amino acid sequence of SEQ ID NO: 1. , the industry understands that it corresponds to a protein having the activity of butanediamine of the present application. Specifically, for example, the protein with butanediamine production activity of the present application may be composed of the amino acid sequence of SEQ ID NO: 1, or have 80%, 85%, 90%, 95%, 96%, 97% therewith %, 98%, or more than 99% homology or identity of amino acid sequences composed of proteins. In addition, as long as it is an amino acid sequence having such homology or identity and showing the function corresponding to the above-mentioned protein, it should be understood that a protein having a partial sequence deletion, modification, substitution or addition of the amino acid sequence should be understood. It is also included in the scope of the protein that is the subject of mutation in the present application.

即，於本申請案中，即便記載有"具有以特定SEQ ID NO記載的胺基酸序列的蛋白質或多肽"、"包括以特定SEQ ID NO記載的胺基酸序列的蛋白質或多肽"，只要為具有與包括相應的SEQ ID NO的胺基酸序列的多肽相同或相應的活性的情形，則應明白具有一部分序列缺失、變形、經取代或加成的胺基酸序列的蛋白質 亦可使用於本申請案。例如，只要為"包括SEQ ID NO：1的胺基酸序列的多肽"為與SEQ ID NO：1相應的序列、或具有與其相同或相應的活性的序列的情形，則應明白可屬於"包括SEQ ID NO：1的胺基酸序列的多肽"。例如，只要為具有與上述變異體蛋白質相同或相應的活性的情形，則不排除於上述胺基酸序列前後追加不變更蛋白質的功能的序列、可自然發生的突變、其潛伏性突變(silent mutation)或保留性取代，且應明白於具有此種序列追加或突變的情形時，亦屬於本申請案的範圍內。 That is, in the present application, even if "a protein or polypeptide having the amino acid sequence described in a specific SEQ ID NO" or "a protein or polypeptide including the amino acid sequence described in a specific SEQ ID NO" is described, as long as In the case of having the same or corresponding activity as a polypeptide comprising the amino acid sequence of the corresponding SEQ ID NO, it should be understood that a protein having a partial sequence deletion, modification, substitution or addition of the amino acid sequence can also be used in this application. For example, as long as the "polypeptide including the amino acid sequence of SEQ ID NO: 1" is a sequence corresponding to SEQ ID NO: 1, or a sequence having the same or corresponding activity, it should be understood that "including Polypeptide of the amino acid sequence of SEQ ID NO: 1". For example, as long as it has the same or corresponding activity as the above-mentioned variant protein, it is not excluded to add a sequence that does not change the function of the protein before and after the above-mentioned amino acid sequence, a naturally occurring mutation, and its latent mutation (silent mutation). ) or retention substitutions, and it should be understood that in the case of such sequence additions or mutations, it is also within the scope of this application.

於本申請案中，用語"保留性取代(conservative substitution)"是指使一個胺基酸由具有相似的結構及/或化學性質的其他胺基酸取代。上述變體可仍保留一個以上的生物學活性，並且具有例如一個以上的保留性取代。此種胺基酸取代通常可基於殘基的極性、電荷、溶解度、疏水性、親水性及/或兩親媒性(amphipathic nature)中的相似性而發生。例如，具有帶電荷的側鏈(electrically charged amino acid)的胺基酸中帶正電荷的(鹼性)胺基酸包括精胺酸、離胺酸及組胺酸，帶負電荷的(酸性)胺基酸包括麩胺酸及天冬胺酸，具有不帶電荷的側鏈(uncharged amino acid)的胺基酸中的非極性胺基酸(nonpolar amino acid)包括甘胺酸、丙胺酸、纈胺酸、白胺酸、異白胺酸、甲硫胺酸、***酸、色胺酸及脯胺酸，極性(polar)或親水性(hydrophilic)胺基酸包括絲胺酸、蘇胺酸、半胱胺酸、酪胺酸、天冬醯胺及麩醯胺酸，上述非極性胺基酸中的芳香族胺基酸包括***酸、色胺酸及酪胺酸。 In this application, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may still retain more than one biological activity, and have, for example, one or more retaining substitutions. Such amino acid substitutions can typically occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids in amino acids with electrically charged side chains include arginine, lysine, and histidine, and negatively charged (acidic) Amino acids include glutamic acid and aspartic acid, non-polar amino acids in amino acids with uncharged side chains (uncharged amino acid) include glycine, alanine, valine Amino acid, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, polar or hydrophilic amino acids including serine, threonine, Cysteine, tyrosine, asparagine and glutamic acid, the aromatic amino acids in the above non-polar amino acids include phenylalanine, tryptophan and tyrosine.

於本申請案中，用語"變異體(variant)"是指一個以上的 胺基酸於保留性取代(conservative substitution)及/或變形(modification)中與所列舉的上述序列(the recited sequence)不同，但保持上述蛋白質的功能(functions)或特性(properties)的蛋白質。變異體因數個胺基酸取代、缺失或加成而與識別的序列(identified sequence)不同。通常，可將上述蛋白質的胺基酸序列中的一個以上胺基酸變形，評估變形的上述蛋白質的特性而識別此種變異體。即，與天然蛋白質(native protein)相比，變異體的能力會增加、不變或降低。另外，一部分變體可包括去除如N-末端前導序列或跨膜域(transmembrane domain)的一個以上的部分而成的變異體。其他變異體可包括自成熟蛋白質(mature protein)的N-及/或C-末端去除一部分而成的變異體。上述用語"變異體"可使用變體、變形、變異的蛋白質、變異體多肽、變異等用語(英文表示為modification、modified protein、modified polypeptide、mutant、mutein、divergent、variant等)，只要為以變異的含義使用的用語，則並不限制於此。就本申請案的目的而言，上述變異體可為變異的蛋白質的活性較天然的野生型或非變形蛋白質增加者，但並不限制於此。 In this application, the term "variant" refers to more than one A protein whose amino acid differs from the recited sequence in conservative substitution and/or modification, but retains the functions or properties of the protein. Variants differ from an identified sequence by several amino acid substitutions, deletions, or additions. Generally, one or more amino acids in the amino acid sequence of the protein described above can be mutated, and the variant can be identified by evaluating the properties of the mutated protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. In addition, a portion of the variants may include variants in which one or more portions such as the N-terminal leader sequence or the transmembrane domain have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of a mature protein. The above term "variant" may use terms such as variant, variant, variant protein, variant polypeptide, variant (expressed in English as modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), as long as it is based on variant The terms used in the meaning of , are not limited to this. For the purposes of this application, the aforementioned variants may be, but are not limited to, those with increased activity of the mutated protein over the native wild-type or non-mutated protein.

另外，變異體可包括對多肽的特性與二級結構影響最小的胺基酸的缺失或加成。例如，多肽可與在轉譯-同時(co-translationally)或轉譯-後(post-translationally)參與蛋白質的轉移(transfer)的蛋白質N-末端的訊號(或前導)序列共軛。另外，上述多肽可與其他序列或連接子共軛以可對多肽進行確認、精製或合成。 In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a polypeptide can be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in the transfer of the protein either co-translationally or post-translationally. In addition, the above-mentioned polypeptides can be conjugated with other sequences or linkers so that the polypeptides can be identified, purified or synthesized.

本申請案的蛋白質變異體可為鳥胺酸脫羧酶變異體。於 本申請案中，用語"鳥胺酸脫羧酶變異體"可與"變異體ODC蛋白質、ODC變異體、變異體鳥胺酸脫羧酶、變異體鳥胺酸去羧酶、變異體ODC蛋白質、ODC變異體、變異體ODC酶蛋白質、變異體ODC酶"等混用。 The protein variants of the present application may be ornithine decarboxylase variants. At In this application, the term "ornithine decarboxylase variant" may be used in conjunction with "variant ODC protein, ODC variant, variant ornithine decarboxylase, variant ornithine decarboxylase, variant ODC protein, ODC Variant, variant ODC enzyme protein, variant ODC enzyme" etc. are mixed.

上述變異體可為SEQ ID NO：1的胺基酸序列中第713個及第698個胺基酸中的任一個以上的胺基酸由與取代前的胺基酸不同的胺基酸取代而成者。 The above variant can be any one or more amino acids in the 713th and 698th amino acids in the amino acid sequence of SEQ ID NO: 1 are replaced by amino acids different from the amino acids before substitution. adult.

只要為與取代前的胺基酸不同的胺基酸，則上述"由其他胺基酸取代"並無限制。例如，可包括作為SEQ ID NO：1的胺基酸序列的第713個胺基酸的丙胺酸由除丙胺酸以外的疏水性胺基酸、鹼性胺基酸、酸性胺基酸、中性胺基酸或芳香族性胺基酸取代。即，只要為作為SEQ ID NO：1的胺基酸序列的第713個胺基酸的丙胺酸由除丙胺酸以外的其他胺基酸殘基取代、或作為第698個胺基酸的麩胺酸由除麩胺酸以外的其他胺基酸殘基取代，則並無限制。另一方面，於本申請案中，於表述為"已取代特定胺基酸"的情形時，即便未另外表明由其他胺基酸取代，亦應明白為由與取代前的胺基酸不同的胺基酸取代者。 The above-mentioned "substituted with another amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. For example, alanine, which can be included as the 713th amino acid of the amino acid sequence of SEQ ID NO: 1, is composed of hydrophobic amino acids, basic amino acids, acidic amino acids, neutral Amino acid or aromatic amino acid substitution. That is, as long as alanine which is the 713th amino acid of the amino acid sequence of SEQ ID NO: 1 is substituted with other amino acid residues other than alanine, or glutamine which is the 698th amino acid The acid is substituted with amino acid residues other than glutamic acid, without limitation. On the other hand, in the present application, when it is expressed as "a specific amino acid that has been substituted", even if it is not indicated to be substituted by other amino acids, it should be understood that it is caused by a different amino acid from the amino acid before the substitution. Amino Acid Substitutes.

具體而言，上述變異體可為於SEQ ID NO：1的胺基酸序列中，i)作為第713個胺基酸的丙胺酸由其他胺基酸取代、及/或ii)作為第698個胺基酸的麩胺酸由其他胺基酸取代的變異體。上述由其他胺基酸取代可為如下者：i)作為第713個胺基酸的丙胺酸由選自白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸及麩醯胺酸中的胺基酸取代；及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代。更具體而言，上述變異體可為i)作為第713 個胺基酸的丙胺酸由選自白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸及麩醯胺酸中的胺基酸取代、及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代的變異體。 Specifically, the above variant may be in the amino acid sequence of SEQ ID NO: 1, i) the alanine as the 713th amino acid is substituted with another amino acid, and/or ii) as the 698th amino acid Variants of amino acids in which glutamic acid is substituted by other amino acids. The above-mentioned substitution by other amino acids may be as follows: i) Alanine as the 713th amino acid is selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, tryptamine amino acid substitution in acid and glutamic acid; and/or ii) glutamic acid as the 698th amino acid is substituted with aspartic acid. More specifically, the above variant may be i) as the 713th The alanine of the amino acid is substituted with an amino acid selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, tryptophan and glutamic acid, and/or ii ) as a variant in which the 698th amino acid has glutamic acid replaced by aspartic acid.

於SEQ ID NO：1的胺基酸序列中，i)作為第713個胺基酸的丙胺酸由選自白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸及麩醯胺酸中的胺基酸取代、及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代的變異體可包括選自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列，具體而言，可必須由SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列構成(consisting essentially of)，更具體而言，可包括SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列，但並不限制於此。 In the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is selected from the group consisting of leucine, isoleucine, valine, arginine, aspartic acid, color Amino acid substitutions in amino acid and glutamic acid, and/or ii) Variants in which glutamic acid as the 698th amino acid is substituted with aspartic acid may include those selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 4, SEQ ID NO: 4, SEQ ID NO: 4 Any amino acid sequence of ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23, specifically, may necessarily consist of SEQ ID NO: 4, SEQ ID NO: 8, Any amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23 constitutes (consisting essentially of), more specifically, may include SEQ ID NO: 4, SEQ ID NO: 8 , SEQ ID NO: 9, any amino acid sequence of SEQ ID NO: 19 to SEQ ID NO: 23, but not limited thereto.

上述變異體可為如下者：於與SEQ ID NO：1的第713個及/或第698個位置相應的位置包括其他胺基酸的取代，與SEQ ID NO：1的胺基酸序列具有至少80%、90%、95%、96%、97%、98%或99%以上且未滿100%的序列同源性，且具有丁二胺生產活性。 The above variant may be as follows: the substitution of other amino acids at positions corresponding to the 713th and/or the 698th position of SEQ ID NO: 1 and having at least one amino acid sequence with the amino acid sequence of SEQ ID NO: 1 is included. 80%, 90%, 95%, 96%, 97%, 98% or 99% or more and less than 100% sequence homology, and have butanediamine production activity.

另外，上述變異體可包括SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9及SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列、或與上述胺基酸序列具有80%以上的同源性或一致性的胺基酸序列，但並不限制於此。具體而言，本申請案的變異體可包括與SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9及SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列具有至少80%、90%、95%、96%、97%、98%或99%同源性或一致性的多肽。另外，只要為具 有此種同源性或一致性且表現出與上述蛋白質相應的功效的胺基酸序列，則具有除第713個或第698個胺基酸位置以外，一部分序列缺失、變形、經取代或加成的胺基酸序列的蛋白質亦包括於本申請案的範圍內。 In addition, the above-mentioned variant may include any amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 19 to SEQ ID NO: 23, or the amino acid sequence with the above-mentioned amino acid. The amino acid sequence has more than 80% homology or identity to the amino acid sequence, but is not limited thereto. Specifically, the variants of the present application may include amino acid sequences related to any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 19 to SEQ ID NO: 23 Polypeptides having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity. In addition, as long as the Amino acid sequences that have such homology or identity and exhibit the corresponding efficacy to the above-mentioned proteins have, except for the 713th or 698th amino acid position, a part of the sequence deleted, deformed, substituted or added. Proteins of the resulting amino acid sequence are also included within the scope of this application.

於本申請案中，用語"同源性(homology)"或"一致性(identity)"是指與兩個給出的胺基酸序列或鹼基序列相關的程度，能夠以百分比表示。用語同源性及一致性時常可互換使用。 In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

藉由標準排列演算法確定保留的(conserved)聚核苷酸或多肽的序列同源性或一致性，可一併利用藉由使用的程式確立的預設空隙處罰。實質上，具有同源性(homologous)或相同(identical)的序列通常可沿著整個序列或整體-長度的至少約50%、60%、70%、80%或90%於中等或高度嚴格的條件(stringent conditions)下雜交。雜交亦考慮於聚核苷酸中含有簡並密碼子來代替密碼子的聚核苷酸。 Conserved polynucleotide or polypeptide sequence homology or identity is determined by standard alignment algorithms, together with preset gap penalties established by the program used. Substantially, homologous or identical sequences generally can be moderately or highly stringent along at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or entire-length. Hybridization under stringent conditions. Hybridization also contemplates polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

例如，可利用如Pearson et al(1988)[美國科學學院學報(Proc.Natl.Acad.Sci.USA)85]：2444中的預設參數而藉由如"FASTA"程式的公知的電腦演算法確定任意兩個聚核苷酸或多肽序列是否具有同源性、相似性或一致性。或者，可使用如於EMBOSS包的尼德曼程式(EMBOSS：歐洲分子生物學開放軟體包(The European Molecular Biology Open Software Suite)，Rice et al.,2000，遺傳學趨勢(Trends Genet.)16：276-277)(5.0.0版本或更高版本)中執行的尼德曼-翁施(Needleman-Wunsch)演算法(Needleman and Wunsch,1970，分子生物學雜誌(J.Mol.Biol.)48：443-453)確定(包括GCG程式包(Devereux,J.,et al，核酸研 究(Nucleic Acids Research)12：387(1984))、BLASTP、BLASTN、FASTA(Atschul,[S.][F.,][ET AL,J MOLEC BIOL 215]：403(1990)；Guide to Huge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego,1994，及[CARILLO ETA/.](1988)工業和應用數學學會(SIAM J Applied Math)48：1073)。例如，可利用國立生物工程學資訊資料庫中心的BLAST或ClustalW確定同源性、相似性或一致性。 For example, well-known computer algorithms such as the "FASTA" program can be performed using preset parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 Determine whether any two polynucleotide or polypeptide sequences share homology, similarity or identity. Alternatively, the Needlemann program as in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite), Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.) 48 : 443-453) determined (including the GCG package (Devereux, J., et al, Nucleic Acids Research) Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers , Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Bioengineering Information.

例如，如Smith and Waterman，應用數學進展(Adv.Appl.Math)(1981)2：482中所公告，例如利用如Needleman et al.(1970)，分子生物學雜誌(J Mol Biol.)48：443的GAP電腦程式對序列資訊進行比較，藉此可確定聚核苷酸或多肽的同源性、相似性或一致性。簡要而言，GAP程式定義為將相似的排列的符號(即，核苷酸或胺基酸)的數量除以兩個序列中更短者的符號的總數所得的值。用於GAP程式的預設參數可包括：(1)二進制比較矩陣(為了一致性而含有1的值，並且為了非-一致性而含有0的值)、及如Schwartz and Dayhoff,eds.,蛋白質序列和結構圖集(Atlas Of Protein Sequence And Structure)，國家政務醫學研究基金會(National Biomedical Research Foundation)，pp.353-358(1979)所揭示，Gribskov et al(1986)核酸研究(Nucl.Acids Res.)14：6745的加權比較矩陣(或EDNAFULL(NCBI NUC4.4的EMBOSS版本)取代矩陣)；(2)用於各空隙的3.0處罰及各空隙中用於各符號的追加的0.10處罰(或空隙開放處罰10、空隙擴展處罰0.5)；及(3)用於末端空隙的無處罰。因此，作為本申請案中所使用者，用語"同源性"或"一致性"表示序列間的相關性(relevance)。 For example, as published in Smith and Waterman, Adv. Appl. Math (1981) 2:482, using, for example, Needleman et al. (1970), J Mol Biol. 48: 443's GAP computer program compares sequence information, whereby homology, similarity or identity of polynucleotides or polypeptides can be determined. Briefly, the GAP formula is defined as the value obtained by dividing the number of similarly arranged symbols (ie, nucleotides or amino acids) by the total number of symbols of the shorter of the two sequences. Preset parameters for the GAP program may include: (1) a binary comparison matrix (with values of 1 for consistency and 0 for non-coincidence), and as in Schwartz and Dayhoff, eds., protein Atlas Of Protein Sequence And Structure, revealed by National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res.) 14: A weighted comparison matrix of 6745 (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each slot and an additional 0.10 penalty for each symbol in each slot ( or void opening penalty of 10, void expansion penalty of 0.5); and (3) no penalty for end voids. Thus, as used in this application, the terms "homology" or "identity" refer to the relationship between sequences.

另外，可藉由在定義的嚴格條件下藉由南方雜交實驗比較序列來確認任意兩個聚核苷酸或多肽序列是否具有同源性、相似性或一致性，定義的適當的雜交條件為相應技術範圍內，可藉由業者所熟知的方法(例如，J.Sambrook et al.,Molecular Cloning,A Laboratory Manual,2nd Edition,Cold Spring Harbor Laboratory press,Cold Spring Harbor,New York,1989；F.M.Ausubel et al.,Current Protocols in Molecular Biology,John Wiley & Sons,Inc.,New York)確定。 In addition, homology, similarity or identity of any two polynucleotide or polypeptide sequences can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, where appropriate hybridization conditions are corresponding Within the technical scope, it can be carried out by methods well known to the artisan (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FMAusubel et al. al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

於本申請案中，用語"鳥胺酸脫羧酶變異體"可與具有丁二胺生產能力的鳥胺酸脫羧酶的變異體多肽、鳥胺酸脫羧酶蛋白質的變異體、鳥胺酸脫羧酶蛋白質的變異體多肽、鳥胺酸脫羧酶變異體多肽、鳥胺酸脫羧酶的變體、鳥胺酸脫羧酶蛋白質的變體、變異體鳥胺酸脫羧酶、變異體鳥胺酸脫羧酶蛋白質等混用。另外，上述鳥胺酸脫羧酶可來自乳桿菌屬(Lactobacillus sp.)、酵母屬(Saccharomyces sp.)或大腸桿菌(Escherichia coli，E.coli)，但並不限制於此。 In the present application, the term "ornithine decarboxylase variant" may be used in combination with variant polypeptides of ornithine decarboxylase, ornithine decarboxylase protein variants, ornithine decarboxylase, which have the ability to produce butanediamine. protein variant polypeptide, ornithine decarboxylase variant polypeptide, ornithine decarboxylase variant, ornithine decarboxylase protein variant, variant ornithine decarboxylase, variant ornithine decarboxylase protein etc. mixed. In addition, the above-mentioned ornithine decarboxylase may be derived from Lactobacillus sp., Saccharomyces sp. or Escherichia coli (E. coli), but is not limited thereto.

上述鳥胺酸脫羧酶變異體可於SEQ ID NO：1的胺基酸序列中第713個及/或第698個位置包括變異，即便為於SEQ ID NO：1加成、缺失胺基酸的胺基酸序列，只要為與自SEQ ID NO：1的N-末端起713號及/或698號胺基酸相應的位置的胺基酸經取代的變異體，則包括於本申請案的範圍。 The above-mentioned ornithine decarboxylase variants may include variations at positions 713 and/or 698 in the amino acid sequence of SEQ ID NO: 1, even if the amino acid is added or deleted in SEQ ID NO: 1. Amino acid sequences, as long as they are substituted variants of amino acids at positions corresponding to amino acids 713 and/or 698 from the N-terminus of SEQ ID NO: 1, are included in the scope of this application .

關於胺基酸殘基位置，本申請案中所揭示的用語"相應的(correspondingto)"是指蛋白質或肽中列舉的位置的胺基酸殘基，或者與蛋白質或肽中列舉的殘基相似、相同或同源的胺基酸殘基。 本申請案中所使用的"相應區域"通常是指相關蛋白質或參考蛋白質中的相似的位置。 With regard to amino acid residue positions, the term "corresponding to" as disclosed in this application refers to the amino acid residue at the recited position in the protein or peptide, or is similar to the recited residue in the protein or peptide , identical or homologous amino acid residues. A "corresponding region" as used in this application generally refers to a similar position in a related or reference protein.

上述鳥胺酸脫羧酶蛋白質的變異體為SEQ ID NO：1的胺基酸序列中第713個及/或第698個胺基酸由其他胺基酸取代者，可為包括SEQ ID NO：1的胺基酸序列或具有較來自野生型微生物的變異前鳥胺酸脫羧酶強化的活性的變異體鳥胺酸脫羧酶蛋白質。如上所述的鳥胺酸脫羧酶蛋白質的變異體是指與以上所說明的SEQ ID NO：1的胺基酸序列具有至少80%、85%、90%、95%、96%、97%、98%或99%以上的同源性或一致性的胺基酸中與SEQ ID NO：1的第713個或第698個相應的位置的胺基酸變異者。 The variant of the above-mentioned ornithine decarboxylase protein is that the 713th and/or the 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted by other amino acids, which may include SEQ ID NO: 1 The amino acid sequence of or a variant ornithine decarboxylase protein having enhanced activity compared to a pre-mutated ornithine decarboxylase from a wild-type microorganism. The variant of the ornithine decarboxylase protein as described above is meant to have at least 80%, 85%, 90%, 95%, 96%, 97%, 80%, 85%, 90%, 95%, 96%, 97%, The amino acid variation at the position corresponding to the 713th or 698th position of SEQ ID NO: 1 among the amino acids with 98% or more homology or identity.

上述第713個及/或第698個胺基酸變異可為i)作為第713個胺基酸的丙胺酸由白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸或麩醯胺酸取代、及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代者。 The above-mentioned 713th and/or 698th amino acid variation can be i) alanine as the 713th amino acid is composed of leucine, isoleucine, valine, arginine, aspartic acid , tryptophan or glutamic acid substitution, and/or ii) glutamic acid as the 698th amino acid is substituted by aspartic acid.

具體而言，上述鳥胺酸脫羧酶變異體可為於SEQ ID NO：1的胺基酸序列中，i)作為第713個胺基酸的丙胺酸由白胺酸、異白胺酸、纈胺酸、精胺酸、天冬胺酸、色胺酸或麩醯胺酸取代、及/或ii)作為第698個胺基酸的麩胺酸由天冬胺酸取代者，且可為具有較包括上述SEQ ID NO：1的胺基酸序列的蛋白質或來自野生型微生物的變異前鳥胺酸脫羧酶蛋白質強化的活性者。 Specifically, the ornithine decarboxylase variant can be in the amino acid sequence of SEQ ID NO: 1, i) alanine as the 713th amino acid is composed of leucine, isoleucine, valeric acid amino acid, arginine, aspartic acid, tryptophan or glutamic acid, and/or ii) glutamic acid as the 698th amino acid is substituted by aspartic acid, and may have Those with enhanced activity compared to a protein comprising the amino acid sequence of SEQ ID NO: 1 above or a premutated ornithine decarboxylase protein from a wild-type microorganism.

就本申請案的目的而言，於包括上述鳥胺酸脫羧酶蛋白質的變異體的微生物的情形時，特徵在於增加丁二胺的產量、增加丁二胺的純度或增加丁二胺的生產選擇性。本申請案的蛋白質變異體的特徵在於：具有基因調節活性以較天然的野生型或非變異 鳥胺酸脫羧酶增加丁二胺的生產能力、丁二胺的純度或丁二胺的生產選擇性。特別是，於可藉由導入有本申請案的蛋白質變異體的微生物阻礙作為鳥胺酸脫羧酶的副反應之一的戊二胺(cadaverine)的合成且增加丁二胺產量的方面存在意義。 For the purposes of this application, in the case of microorganisms comprising variants of the above-described ornithine decarboxylase proteins, characterized by increased production of butanediamine, increased purity of butanediamine, or increased selection of butanediamine production sex. The protein variants of the present application are characterized in that they have gene regulatory activity to compare with the natural wild-type or non-variant Ornithine decarboxylase increases butanediamine production capacity, butanediamine purity, or butanediamine production selectivity. In particular, it is significant in that the synthesis of cadaverine, which is one of the side reactions of ornithine decarboxylase, can be inhibited by microorganisms into which the protein variant of the present application is introduced, and the production of butanediamine can be increased.

本申請案的又一實施方式提供一種編碼上述鳥胺酸脫羧酶蛋白質的變異體的聚核苷酸。 Yet another embodiment of the present application provides a polynucleotide encoding a variant of the above-described ornithine decarboxylase protein.

包括SEQ ID NO：1的胺基酸序列的鳥胺酸脫羧酶蛋白質及其變異體如上所述。 Ornithine decarboxylase proteins comprising the amino acid sequence of SEQ ID NO: 1 and variants thereof are described above.

於本申請案中，用語"聚核苷酸"作為核苷酸單體(monomer)藉由共價鍵較長地連成鏈狀的核苷酸的聚合物(polymer)，其是固定長度以上的(Deoxyribonucleic Acid，DNA)或RNA鏈，更具體而言，是指編碼上述變體的聚核苷酸片段。 In the present application, the term "polynucleotide" is used as a polymer of nucleotides of nucleotide monomers (monomers) long linked by covalent bonds to form a chain of nucleotides, which is a fixed length or more. Deoxyribonucleic Acid (DNA) or RNA strands, more specifically, refers to polynucleotide fragments encoding the above variants.

本申請案的編碼鳥胺酸脫羧酶變異體的聚核苷酸只要為編碼本申請案的具有丁二胺的生產活性的變異體多肽的聚核苷酸序列，則可無限制地包括。於本申請案中，編碼鳥胺酸脫羧酶蛋白質的胺基酸序列的基因例如可為speC、odc、spe1或speF基因，上述基因可來自乳桿菌屬、酵母屬或大腸桿菌(Escherichia coli，E.coli)，但並不受此限制。另外，上述基因可為編碼SEQ ID NO：1、SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：19至SEQ ID NO：23中的任一胺基酸序列的鹼基序列，更具體而言，可為包括SEQ ID NO：10、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：18及SEQ ID NO：24至SEQ ID NO：28中的任一鹼基序列的序列，但並不限制於此。 The polynucleotide encoding the ornithine decarboxylase variant of the present application can be included without limitation as long as it is a polynucleotide sequence encoding the variant polypeptide having the production activity of butanediamine of the present application. In the present application, the gene encoding the amino acid sequence of the ornithine decarboxylase protein can be, for example, a speC, odc, spel or speF gene, which can be derived from Lactobacillus, Saccharomyces or Escherichia coli (Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli .coli), but is not subject to this restriction. In addition, the above-mentioned gene can be any amino acid sequence encoding SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 23 The base sequence of , more specifically, may include SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 24 to SEQ ID NO: 28 The sequence of any base sequence, but not limited thereto.

具體而言，本申請案的聚核苷酸因密碼子的簡並性 (degeneracy)或考慮到欲表現上述多肽的生物所喜好的密碼子而可於不改變多肽的胺基酸序列的範圍內對編碼區域實現各種變形。具體而言，只要為編碼SEQ ID NO：1的胺基酸序列中第713個及/或第698個胺基酸由其他胺基酸取代的鳥胺酸脫羧酶蛋白質的變異體的聚核苷酸序列，則可無限制地包括。 Specifically, the polynucleotides of the present application are due to the degeneracy of codons Various modifications can be made to the coding region within a range that does not alter the amino acid sequence of the polypeptide in consideration of the codons preferred by the organism in which the polypeptide is to be expressed (degeneracy). Specifically, as long as it is a polynucleoside encoding a variant of ornithine decarboxylase protein in which the 713th and/or 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted by other amino acids acid sequences can be included without limitation.

另外，只要為編碼於嚴格的條件下與可自公知的基因序列製備的探針、例如上述鹼基序列整體或一部分的互補序列雜交而SEQ ID NO：1的胺基酸序列中第713個及/或第698個胺基酸由其他胺基酸取代的具有丁二胺生產活性的鳥胺酸脫羧酶蛋白質的序列，則可無限制地包括。上述"嚴格的條件(stringent condition)"是指可使聚核苷酸間實現特異性雜交的條件。於文獻(例如，J.Sambrook et al.,同源)中有具體地記載有此種條件。例如，可列舉如下條件：同源性或一致性較高的基因間雜交、具有40%以上、具體而言為90%以上、更具體而言為95%以上、進而具體而言為97%以上、特別具體而言為99%以上的同源性或一致性的基因間雜交，同源性或一致性低於其的基因間不雜交的條件；或普通的南方雜交(southern hybridization)的清洗條件、即於相當於60℃、1×SSC、0.1%十二烷基磺酸鈉(Sodium Dodecyl Sulfonate，SDS)、具體而言為60℃、0.1×SSC、0.1% SDS、更具體而言為68℃、0.1×SSC、0.1% SDS的鹽濃度及溫度下清洗1次、具體而言為2次至3次的條件。 In addition, the 713th and 713th amino acid sequences of SEQ ID NO: 1 and /or the sequence of the ornithine decarboxylase protein having butanediamine producing activity in which the 698th amino acid is substituted by other amino acids may be included without limitation. The above-mentioned "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically documented in the literature (eg, J. Sambrook et al., homologs). For example, the following conditions can be mentioned: intergenic hybridization with high homology or identity, 40% or more, specifically 90% or more, more specifically 95% or more, and more specifically 97% or more , In particular, intergenic hybridization with more than 99% homology or identity, and the conditions of non-hybridization between genes with homology or identity lower than it; or ordinary southern hybridization (southern hybridization) cleaning conditions , that is, equivalent to 60°C, 1×SSC, 0.1% Sodium Dodecyl Sulfonate (SDS), specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68 ℃, 0.1×SSC, and 0.1% SDS salt concentration and temperature under the conditions of one wash, specifically two to three times.

雜交即便根據雜交的嚴格度而會發生鹼基間的失配(mismatch)，但要求兩個核酸具有互補序列。用語"互補"用於記述可彼此雜交的核苷酸鹼基間的關係。例如，關於DNA，腺苷與胸 嘧啶互補，胞嘧啶與鳥嘌呤互補。因此，本申請案不僅可包括實質上相似的核酸序列，而且亦包括與整個序列互補的單離的核酸片段。 Hybridization requires that the two nucleic acids have complementary sequences even though a mismatch between bases may occur depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine and thymus Pyrimidine is complementary, cytosine is complementary to guanine. Thus, the present application can include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

具體而言，具有同源性或一致性的聚核苷酸可於55℃的Tm值下使用包括雜交步驟的雜交條件，並使用上述條件而進行探測。另外，上述Tm值可為60℃、63℃或65℃，但並不限制於此，可根據其目的由業者適當地調節。 Specifically, polynucleotides having homology or identity can be probed using hybridization conditions including a hybridization step at a Tm value of 55°C using the conditions described above. In addition, although the said Tm value may be 60 degreeC, 63 degreeC, or 65 degreeC, it is not limited to this, It can adjust suitably by a manufacturer according to the objective.

雜交聚核苷酸的適當的嚴格度依存於聚核苷酸的長度及互補性程度，參數於本技術領域內熟知(參照Sambrook et al.,supra,9.50-9.51,11.7-11.8)。 Appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, parameters well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

本申請案的又一實施方式提供一種包括編碼鳥胺酸脫羧酶變異體的聚核苷酸的載體。 Yet another embodiment of the present application provides a vector comprising a polynucleotide encoding a variant of ornithine decarboxylase.

包括SEQ ID NO：1的胺基酸序列的鳥胺酸脫羧酶、其變異體及上述聚核苷酸如上所述。 The ornithine decarboxylase comprising the amino acid sequence of SEQ ID NO: 1, its variants, and the above-mentioned polynucleotides are as described above.

本申請案中所使用的用語"載體(vector)"是指含有編碼以可於適當的宿主內表現目標多肽的方式可進行動作地連接於適當的調節序列的上述目標多肽的聚核苷酸的鹼基序列的DNA製備物。上述調節序列可包括可開始進行轉錄的啟動子、用以調節此種轉錄的任意的操縱序列、編碼適當的信使核糖核酸(Messenger RNA，mRNA)核糖體結合位點的序列、及調節轉錄及解碼的終止的序列。載體轉形至適當的宿主細胞內後，可與宿主基因組無關地複製或發揮功能，可整合至基因組本身。 The term "vector" as used in this application refers to a polynucleotide comprising a polynucleotide encoding the above-mentioned polypeptide of interest operably linked to appropriate regulatory sequences in such a manner that the polypeptide of interest can be expressed in a suitable host. DNA preparations of base sequences. Such regulatory sequences may include a promoter that can initiate transcription, any operator sequence to regulate such transcription, a sequence encoding an appropriate messenger RNA (mRNA, mRNA) ribosome binding site, and regulation of transcription and decoding terminating sequence. After the vector is transformed into an appropriate host cell, it can replicate or function independently of the host genome, and can be integrated into the genome itself.

本申請案中使用的載體並無特別限定，可利用業界內已知的任意的載體。作為通常使用的載體的示例，可列舉：天然狀態 或重組狀態的質體、黏質體、病毒及細菌噬菌體。例如，作為噬菌體載體或黏質體載體，可使用pWE15、M13、MBL3、MBL4、IXII、ASHII、APII、t10、t11、Charon4A及Charon21A等，作為質體載體，可使用pBR類、pUC類、pBluescriptII類、pGEM類、pTZ類、pCL類及pET類等。具體而言，可使用pDZ、pACYC177、pACYC184、pCL、pECCG117、pUC19、pBR322、pMW118、pCC1BAC載體等。 The carrier used in this application is not particularly limited, and any carrier known in the industry can be used. Examples of commonly used carriers include: natural state or recombinant plastids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR, pUC, pBluescriptII, etc. can be used as plastid vectors. class, pGEM class, pTZ class, pCL class and pET class, etc. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

作為一例，可藉由細胞內染色體***用載體將於染色體內編碼目標多肽的聚核苷酸***至染色體內。可藉由業界內已知的任意方法、例如同源重組(homologous recombination)將上述聚核苷酸***至染色體內，但並不限定於此。可更包括用以確認上述染色體***與否的篩選標誌(selection marker)。篩選標誌用以篩選以載體轉形的細胞、即確認目標核酸分子的***與否，可使用賦予如耐藥物性、營養需求性、對細胞毒性劑的耐性或表面多肽的表現的可選擇的表型的標誌。於經選擇劑(selective agent)處理的環境中，僅存活表現篩選標誌的細胞或表現出不同的表型性狀，因此可篩選轉形的細胞。 As an example, the intrachromosomal polynucleotide encoding the polypeptide of interest can be inserted into the chromosome by a vector for intracellular chromosome insertion. The above-mentioned polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. It may further include a selection marker for confirming the above-mentioned chromosomal insertion. Screening markers are used to screen cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule, using selectable tables that confer, for example, drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides. type logo. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit different phenotypic traits, and thus transformed cells can be screened.

作為本申請案的又一實施方式，本申請案提供一種包括上述鳥胺酸脫羧酶或其變異體，或者包括編碼上述酶的聚核苷酸而生產丁二胺的微生物。 As a further embodiment of the present application, the present application provides a microorganism comprising the above-mentioned ornithine decarboxylase or a variant thereof, or a polynucleotide encoding the above-mentioned enzyme to produce butanediamine.

於本申請案中，用語"包括變異體多肽的微生物"或"包括鳥胺酸脫羧酶變異體的微生物"只要為包括本申請案的蛋白質變異體而可生產丁二胺的微生物，則均可，但並不限制於此。例如，包括本申請案的蛋白質變異體的微生物可為於天然的野生型微生 物或生產丁二胺的微生物中表現本申請案的蛋白質變異體，從而丁二胺的生產能力、丁二胺的生產純度或丁二胺的生產選擇性增加的重組微生物。上述重組微生物可為丁二胺的生產能力、生產純度或丁二胺的生產選擇性較天然的野生型微生物或非變形微生物增加的微生物，但並不限制於此。 In the present application, the terms "microorganisms including variant polypeptides" or "microorganisms including ornithine decarboxylase variants" can be used as long as they are microorganisms that can produce butanediamine including the protein variants of the present application. , but not limited to this. For example, microorganisms comprising protein variants of the present application may be native wild-type microorganisms A recombinant microorganism that expresses the protein variant of the present application in an organism or a butanediamine-producing microorganism, thereby increasing the production capacity of butanediamine, the production purity of butanediamine, or the production selectivity of butanediamine. The above-mentioned recombinant microorganism may be a microorganism whose production capacity, production purity, or production selectivity of butanediamine is increased compared to a natural wild-type microorganism or a non-transformed microorganism, but is not limited thereto.

具體而言，上述微生物作為表現包括SEQ ID NO：1的胺基酸序列內的一個以上胺基酸變異的鳥胺酸脫羧酶變異體的微生物，上述胺基酸變異可包括自N-末端起第713個及/或第698個胺基酸由其他胺基酸取代。另外，上述微生物可為於SEQ ID NO：1的胺基酸序列中第713個或第698個胺基酸由其他胺基酸取代且具有丁二胺的生產活性的表現變異體多肽的微生物，但並不限制於此。 Specifically, the above-mentioned microorganisms are microorganisms that express ornithine decarboxylase variants including one or more amino acid variations within the amino acid sequence of SEQ ID NO: 1, and the above-mentioned amino acid variations may include starting from the N-terminus. The 713th and/or the 698th amino acid is substituted with other amino acids. In addition, the above-mentioned microorganism may be a microorganism expressing a variant polypeptide in which the 713th or 698th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted by another amino acid and has the production activity of butanediamine, But not limited to this.

上述丁二胺、包括SEQ ID NO：1的胺基酸序列的鳥胺酸脫羧酶蛋白質及其變異體如上所述。 The above-mentioned butanediamine, ornithine decarboxylase protein comprising the amino acid sequence of SEQ ID NO: 1, and variants thereof are as described above.

於本申請案中，用語"以表現/表現"蛋白質是指靶蛋白導入至微生物內、或以於微生物內表現的方式變形的形態。於上述靶蛋白為存在於微生物內的蛋白質的情形時，指活性較內在或變形前強化的狀態。就本申請案的目的而言，"靶蛋白"可為上述具有丁二胺生產能力的鳥胺酸脫羧酶蛋白質的變異體。 In the present application, the term "expressed/expressed" protein refers to a form in which a target protein is introduced into a microorganism or deformed so as to be expressed in a microorganism. When the above-mentioned target protein is a protein existing in a microorganism, it refers to a state in which the activity is relatively intensified or before deformation. For the purposes of this application, a "target protein" may be a variant of the above-described ornithine decarboxylase protein having the ability to produce butanediamine.

具體而言，"蛋白質的導入"是指表現出微生物原本未具有的特定蛋白質的活性，或表現出較上述蛋白質的內在活性或修飾前活性提高的活性。例如，可為編碼特定蛋白質的聚核苷酸導入至微生物內的染色體，或包括編碼特定蛋白質的聚核苷酸的載體導入至微生物內而表現出其活性。另外，"活性的強化"是指活性較微 生物所具有的特定蛋白質的內在活性或修飾前活性提高。上述"內在活性"是指於因自然或人為因素引起的遺傳性變異而微生物轉形的情形時，轉形前的母菌株原本所具有的特定蛋白質的活性。 Specifically, "introduction of a protein" refers to expressing the activity of a specific protein not originally possessed by a microorganism, or expressing an activity that is improved over the intrinsic activity or the activity before modification of the above-mentioned protein. For example, a polynucleotide encoding a specific protein can be introduced into a chromosome of a microorganism, or a vector including a polynucleotide encoding a specific protein can be introduced into a microorganism to exhibit its activity. In addition, "enhanced activity" means that the activity is less The intrinsic activity or pre-modification activity of a specific protein possessed by an organism is increased. The above-mentioned "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

具體而言，本申請案的活性強化可藉由選自由如下方法所組成的族群中的任一種以上的方法實現，但並不限制於此：增加編碼本申請案的蛋白質變異體的基因的細胞內的複製數，將變異導入至編碼上述蛋白質變異體的基因的表現調節序列的方法；將編碼鳥胺酸脫羧酶蛋白質變異體的基因表現調節序列更換為活性強烈的序列的方法；以編碼上述蛋白質變異體的基因代替染色體上的編碼鳥胺酸脫羧酶的野生型蛋白質的基因的方法；以強化上述蛋白質變異體的活性的方式亦將變異導入至編碼上述鳥胺酸脫羧酶蛋白質的基因的方法；及將蛋白質變異體導入至微生物的方法。 Specifically, the potentiation of the activity of the present application can be achieved by any one or more methods selected from the group consisting of, but not limited to: cells that increase the gene encoding the protein variant of the present application A method for introducing variation into the expression regulatory sequence of the gene encoding the above-mentioned protein variant; the method for replacing the expression regulatory sequence of the gene encoding the ornithine decarboxylase protein variant with a highly active sequence; to encode the above-mentioned protein variant A method in which the gene of the protein variant replaces the gene encoding the wild-type protein of ornithine decarboxylase on the chromosome; the mutation is also introduced into the gene encoding the above-mentioned ornithine decarboxylase protein in a manner to enhance the activity of the protein variant. methods; and methods of introducing protein variants into microorganisms.

於上述內容中，增加基因的複製數並無特別限制，但能夠以可進行動作地連接於載體的形態執行、或藉由***至宿主細胞內的染色體內而執行。具體而言，可將如下載體導入至宿主細胞內：以可進行動作的方式連接有編碼本申請案的蛋白質的聚核苷酸且可與宿主無關地複製並發揮功能。或者，可將如下載體導入至宿主細胞的染色體內：以可進行動作的方式連接有上述聚核苷酸且可將上述聚核苷酸***至宿主細胞內的染色體內。可藉由業界內已知的任意方法、例如同源重組將上述聚核苷酸***至染色體內。 In the above, increasing the number of copies of a gene is not particularly limited, but can be performed in a form operably linked to a vector, or by insertion into a chromosome in a host cell. Specifically, a vector in which a polynucleotide encoding the protein of the present application is operably linked and capable of replicating and functioning independently of the host can be introduced into a host cell. Alternatively, a vector in which the above-described polynucleotide is operably linked and inserted into the chromosome of the host cell can be introduced into the chromosome of the host cell. The above-described polynucleotides can be inserted into chromosomes by any method known in the art, such as homologous recombination.

以增加聚核苷酸的表現的方式變形表現調節序列並無特別限制，但可藉由缺失、***、非保留性或保留性取代或其組合對 核酸序列誘導序列上的變異以進一步強化上述表現調節序列的活性來執行，或藉由更換為具有更強的活性的核酸序列來執行。上述表現調節序列並不特別限制於此，可包括啟動子、操縱序列、編碼核糖體結合位點的序列、調節轉錄及解碼的終止的序列等。 Morphing the expression regulatory sequence in a manner that increases the expression of the polynucleotide is not particularly limited, but can be modified by deletions, insertions, non-retained or retained substitutions, or a combination thereof. Nucleic acid sequence-induced sequence variation is performed to further enhance the activity of the above-described expressive regulatory sequence, or by replacement with a nucleic acid sequence with greater activity. The above-mentioned expression regulation sequences are not particularly limited thereto, and may include promoters, operator sequences, sequences encoding ribosome binding sites, sequences regulating termination of transcription and decoding, and the like.

可於上述聚核苷酸表現單位的上部連接強大的啟動子來代替原本的啟動子，且並不限定於此。於公知的強大的啟動子的示例中，有cj1至cj7啟動子(韓國註冊專利第10-0620092號)、lac啟動子、trp啟動子、trc啟動子、tac啟動子、λ噬菌體PR啟動子、PL啟動子、tet啟動子、gapA啟動子、SPL7啟動子、SPL13(sm3)啟動子(韓國註冊專利第10-1783170號)、O2啟動子(韓國註冊專利第10-1632642號)、tkt啟動子及yccA啟動子等，但並不限定於此。 A strong promoter may be connected to the upper part of the above-mentioned polynucleotide expression unit instead of the original promoter, and it is not limited to this. Examples of known powerful promoters include cj1 to cj7 promoters (Korea Registered Patent No. 10-0620092), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Korea Registered Patent No. 10-1783170), O2 promoter (Korea Registered Patent No. 10-1632642), tkt promoter and yccA promoter, etc., but not limited to this.

染色體上的聚核苷酸序列的變形並無特別限制，可藉由缺失、***、非保留性或保留性取代或其組合對核酸序列誘導表現調節序列上的變異以進一步強化上述聚核苷酸序列的活性來執行，或藉由更換為以具有更強的活性的方式改良的聚核苷酸序列來執行。 The modification of the polynucleotide sequence on the chromosome is not particularly limited, and the above-mentioned polynucleotide can be further strengthened by inducing the variation in the expression regulatory sequence of the nucleic acid sequence by deletion, insertion, non-retention or retention substitution or a combination thereof. activity of the sequence, or by replacing it with a polynucleotide sequence modified in a way that has greater activity.

如上所述的蛋白質活性的導入及強化可為相應的蛋白質的活性或濃度以野生型或非變形微生物菌株中的蛋白質的活性或濃度為基準而通常增加至最小1%、10%、25%、50%、75%、100%、150%、200%、300%、400%或500%，最大1000%或2000%，但並不限制於此。 The introduction and enhancement of the protein activity as described above can be performed by increasing the activity or concentration of the corresponding protein, typically to a minimum of 1%, 10%, 25%, 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, maximum 1000% or 2000%, but not limited thereto.

於本申請案中，包括上述鳥胺酸脫羧酶變異體或包括編碼上述鳥胺酸脫羧酶變異體的聚核苷酸的微生物可為藉由以包括 上述聚核苷酸的載體轉形而製備的重組微生物，但並不限制於此。 In the present application, a microorganism comprising the above-mentioned ornithine decarboxylase variant or a polynucleotide encoding the above-mentioned ornithine decarboxylase variant can be obtained by including The recombinant microorganism prepared by transforming the above-mentioned polynucleotide vector is not limited to this.

於本申請案中，用語"轉形"是指將包括編碼靶蛋白的聚核苷酸的載體導入至宿主細胞內而可使上述聚核苷酸編碼的蛋白質可於宿主細胞內表現。轉形的聚核苷酸只要可於宿主細胞內表現，則可包括***至宿主細胞的染色體內定位或位於染色體外兩者。另外，上述聚核苷酸包括編碼靶蛋白的DNA及RNA。上述聚核苷酸只要為可導入至宿主細胞內而表現者，則能夠以任一形態導入。例如，上述聚核苷酸能夠以作為包括自行表現所需的所有要素的基因結構體的表現卡匣(expression cassette)的形態導入至宿主細胞。上述表現卡匣通常可包括以可進行動作的方式連接於上述聚核苷酸的啟動子(promoter)、轉錄終止訊號、核糖體結合位點及轉譯終止訊號。上述表現卡匣可為可實現自複製的表現載體形態。另外，上述聚核苷酸亦可為以其本身的形態導入至宿主細胞而於宿主細胞以可進行動作的方式與表現所需的序列連接者，且並不限定於此。 In this application, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include both intrachromosomal location or extrachromosomal location into the host cell as long as it can be expressed in the host cell. In addition, the above-mentioned polynucleotide includes DNA and RNA encoding the target protein. The above-mentioned polynucleotide can be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the above-mentioned polynucleotide can be introduced into a host cell in the form of an expression cassette as a gene structure including all elements required for self-expression. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The above-mentioned presentation cassette can be in the form of a presentation carrier that can realize self-replication. In addition, the above-mentioned polynucleotide may be introduced into a host cell in its own form and linked to a sequence required for expression in an operable manner in the host cell, but is not limited to this.

另外，於上述內容中，用語"以可進行動作的方式連接"是指使編碼本申請案的目標多肽的聚核苷酸開始轉錄及進行媒介的啟動子序列與上述基因序列功能性地連接。 In addition, in the above content, the term "operably linked" means that a promoter sequence that initiates transcription and mediates the polynucleotide encoding the polypeptide of interest of the present application is functionally linked to the above-mentioned gene sequence.

本申請案的用語"非變形微生物"是指天然型菌株本身、不包括本申請案的蛋白質變異體的微生物、或未以包括編碼本申請案的蛋白質變異體的聚核苷酸的載體轉形的微生物。 The term "non-transformed microorganism" as used herein refers to the native strain itself, a microorganism that does not include the protein variant of the present application, or is not transformed with a vector that includes a polynucleotide encoding the protein variant of the present application of microorganisms.

本申請案的"微生物"只要為可生產丁二胺的微生物，則可包括原核微生物及真核微生物中的任一者。 The "microorganism" in the present application may include any of a prokaryotic microorganism and a eukaryotic microorganism as long as it is a microorganism capable of producing butanediamine.

於本申請案中，用語"生產丁二胺的微生物"是指天然具有 丁二胺生產能力的野生型微生物、或藉由將野生型或變異體導入至無丁二胺生產能力或生產能力明顯較低的母菌株而具有丁二胺生產能力的微生物。具體而言，包括自然或人為地引發遺傳性變形的所有微生物，作為因***外部基因、強化內在基因的活性、或失活等原因而特定機制弱化或強化的微生物，可以是為了生產目標丁二胺而發生遺傳性變異、或強化活性的微生物。就本申請案的目的而言，本申請案的微生物包括本申請案的蛋白質變異體，因此可增加目標丁二胺的生產能力、生產純度或丁二胺的生產選擇性。具體而言，本申請案的微生物可為如下微生物：丁二胺生物合成路徑內的基因的一部分強化或弱化，或者丁二胺分解路徑內的基因的一部分強化或弱化。就本申請案的目的而言，生產丁二胺的微生物可指特徵如下的微生物：包括上述鳥胺酸脫羧酶變異體，因此欲自培養基中的碳源生成的丁二胺的產量、丁二胺的純度或丁二胺的生產選擇性較野生型或非變形微生物增加增加。於本申請案中，上述"生產丁二胺的微生物"可與"具有丁二胺的生產能力的微生物"或"生產丁二胺的微生物"混用。 In this application, the term "microorganism producing butanediamine" refers to a A butanediamine-producing wild-type microorganism, or a microorganism having butanediamine-producing ability by introducing a wild-type or a variant into a parent strain having no butanediamine-producing ability or a significantly lower production ability. Specifically, it includes all microorganisms that cause genetic deformation naturally or artificially, as microorganisms whose specific mechanisms are weakened or enhanced due to insertion of external genes, enhancement of the activity of internal genes, or inactivation, etc. Microorganisms with genetic variation or enhanced activity due to amines. For the purposes of the present application, the microorganisms of the present application include the protein variants of the present application, and thus may increase the production capacity, production purity, or production selectivity of butanediamine of interest. Specifically, the microorganism of the present application may be a microorganism in which a part of genes in the butanediamine biosynthesis pathway is enhanced or weakened, or a part of genes in the butanediamine decomposition pathway is enhanced or weakened. For the purposes of this application, a butanediamine-producing microorganism may refer to a microorganism characterized by including the ornithine decarboxylase variants described above, and thus the production of butanediamine to be generated from the carbon source in the culture medium, the production of butanediamine The purity of the amine or the selectivity for the production of butanediamine is increased compared to wild-type or non-transformed microorganisms. In the present application, the above-mentioned "microorganisms producing butanediamine" may be used in combination with "microorganisms having the ability to produce butanediamine" or "microorganisms producing butanediamine".

上述生產丁二胺的微生物可為重組微生物。上述重組微生物如上所述。 The above-mentioned butanediamine-producing microorganism may be a recombinant microorganism. The above-mentioned recombinant microorganisms are as described above.

生產上述丁二胺的微生物只要可生產丁二胺，則其種類並無特別限制，具體而言，可為屬於棒狀桿菌(Corynebacterium)屬、埃希氏菌(Escherichia)屬、腸桿菌(Enterbacter)屬、歐文氏桿菌(Erwinia)屬、沙雷氏菌(Serratia)屬、普羅威登菌(Providencia)屬及短桿菌(Brevibacterium)屬的微生物，更具體而言，可為屬於棒狀桿菌(Corynebacterium)屬或埃希氏菌 (Escherichia)屬的微生物。 The type of the microorganism that produces the above-mentioned butanediamine is not particularly limited as long as it can produce butanediamine. ), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providencia) and Brevibacterium (Brevibacterium) genera microorganisms, more specifically, may belong to Corynebacterium ( Corynebacterium) or Escherichia (Escherichia) microorganisms.

更進一步具體而言，埃希氏菌屬(Escherichia)微生物可為大腸桿菌(Escherichia coli)，棒狀桿菌(Corynebacterium)屬微生物可為谷胺酸棒狀桿菌(Corynebacterium glutamicum)、產胺棒狀桿菌(Corynebacterium ammoniagenes)、克氏棒狀桿菌(Corynebacterium crudilactis)、荒漠棒狀桿菌(Corynebacterium deserti)、高效棒狀桿菌(Corynebacterium efficiens)、帚石南棒狀桿菌(Corynebacterium callunae)、停滯棒狀桿菌(Corynebacterium stationis)、奇棒狀桿菌(Corynebacterium singulare)、耐鹽棒狀桿菌(Corynebacterium halotolerans)、紋狀體棒狀桿菌(Corynebacterium striatum)、污染棒狀桿菌(Corynebacterium pollutisoli)、亞胺棒狀桿菌(Corynebacterium imitans)、睾丸棒狀桿菌(Corynebacterium testudinoris)或微黃棒狀桿菌(Corynebacterium flavescens)等，且可為谷胺酸棒狀桿菌(Corynebacterium glutamicum)，但可無限制地包括屬於可導入或強化鳥胺酸脫羧酶蛋白質而增加丁二胺的產量、丁二胺的純度、丁二胺的生產選擇性的棒狀桿菌屬或埃希氏菌屬的微生物。 More specifically, the microorganism of the genus Escherichia can be Escherichia coli, and the microorganism of the genus Corynebacterium can be Corynebacterium glutamicum, Corynebacterium amine-producing bacteria. (Corynebacterium ammoniagenes), Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stasis stationis), Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans ), Corynebacterium testudinoris (Corynebacterium testudinoris) or Corynebacterium flavescens (Corynebacterium flavescens), etc., and can be Corynebacterium glutamicum (Corynebacterium glutamicum), but can include without limitation those that can introduce or fortify ornithine Microorganisms of the genus Corynebacterium or Escherichia that increase the yield of butanediamine, the purity of butanediamine, and the production selectivity of butanediamine by decarboxylase protein.

於本申請案中，生產以表現鳥胺酸脫羧酶蛋白質或上述蛋白質的變異體的方式變形的丁二胺的微生物的母菌株只要為生產丁二胺的微生物，則並無特別限制。 In the present application, the parent strain of a butanediamine-producing microorganism modified to express an ornithine decarboxylase protein or a variant of the protein is not particularly limited as long as it is a butanediamine-producing microorganism.

棒狀桿菌屬微生物雖無丁二胺的生物合成路徑，但若自外部導入鳥胺酸脫羧酶(ornithine decarboxylase，ODC)，則可合成丁二胺。 Microorganisms belonging to the genus Corynebacterium do not have a biosynthetic pathway for butanediamine, but they can synthesize butanediamine when ornithine decarboxylase (ODC) is introduced from outside.

另外，生產丁二胺的上述微生物並無特別限制，進而可為 如下者：鳥胺酸中參與精胺酸合成的鳥胺酸胺基甲醯轉移酶(ornithine carbamoyltransfrase，ArgF)、參與麩胺酸的排出的蛋白質(NCgl1221)失活者。 In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited, and may be The following: in ornithine, the ornithine carbamoyltransfrase (ArgF) involved in the synthesis of arginine and the protein involved in the excretion of glutamic acid (NCgl1221) are inactivated.

另外，生產丁二胺的上述微生物並無特別限制，例如可為如下者：為了強化自麩胺酸至鳥胺酸的生物合成路徑，將麩胺酸轉化為乙醯谷胺酸(N-acetylglutamate)的乙醯谷胺酸合酶、或將乙醯鳥胺酸轉化為鳥胺酸的鳥胺酸乙醯轉移酶(ArgJ)、將乙醯谷胺酸轉化為乙醯谷胺醯磷酸(N-acetylglutamyl phosphate)的乙醯谷胺酸激酶(ArgB)、將乙醯谷胺醯磷酸轉化為乙醯谷胺酸半醛(N-acetylglutamate semialdehyde)的乙醯谷胺醯-磷酸還原酶(ArgC)、將乙醯谷胺酸半醛轉化為乙醯鳥胺酸(N-acetylornithine)的乙醯鳥胺酸轉胺酶(ArgD)的活性較內在活性強化，從而用作丁二胺的生物合成原料的鳥胺酸的生產性提高。 In addition, the above-mentioned microorganisms that produce butanediamine are not particularly limited, and may be, for example, the following: in order to strengthen the biosynthetic pathway from glutamic acid to ornithine, glutamic acid is converted into acetylglutamate (N-acetylglutamate) ) of acetoglutamate synthase, or ornithine acetyltransferase (ArgJ), which converts acetonitrile to ornithine, and acetonitrile to acetonitrile phosphate (N -acetylglutamyl phosphate), acetylglutamate kinase (ArgB), and acetylglutamate-phosphate reductase (ArgC), which converts acetylglutamyl phosphate to N-acetylglutamate semialdehyde (N-acetylglutamate semialdehyde) , The activity of acetonitrile transaminase (ArgD), which converts acetonitrile semialdehyde into acetylornithine (N-acetylornithine), is stronger than its intrinsic activity, so it can be used as a raw material for the biosynthesis of butanediamine. The productivity of ornithine increases.

另外，生產丁二胺的上述微生物並無特別限制，可進而為丁二胺乙醯轉移酶的活性弱化且具有丁二胺的生產能力的棒狀桿菌屬微生物。而且，生產上述丁二胺的微生物並無特別限制，可為丁二胺排出蛋白質的活性強化者，但並不限制於此。 In addition, the above-mentioned microorganism for producing butanediamine is not particularly limited, and may further be a microorganism of the genus Corynebacterium that has a butanediamine-producing ability with weakened butanediamine acetyltransferase activity. Furthermore, the microorganisms producing the above-mentioned butanediamine are not particularly limited, and may be an activity enhancer of butanediamine-excreted proteins, but are not limited thereto.

於本申請案中，用語"強化/增加"為包括活性較內在活性增加的所有概念。 In this application, the term "enhancing/increasing" is intended to include all concepts of increased activity compared to intrinsic activity.

可藉由應用本領域內所熟知的各種方法而達成此種基因活性的強化或增加。作為上述方法的示例，可包括選自由如下方法所組成的族群中的任一種以上的方法，亦可藉由其組合達成，但並不特別限制於上述示例：增加基因的細胞內複製數；將變異導入至基因的表現調節序列的方法；將基因表現調節序列更換為活性強 烈的序列的方法；進而將變異導入至相應基因以強化基因的活性的方法；及將外來基因導入至微生物的方法。 Such enhancement or increase in gene activity can be achieved by applying various methods well known in the art. Examples of the above-mentioned methods include any one or more methods selected from the group consisting of the following methods, and can also be achieved by a combination thereof, but are not particularly limited to the above-mentioned examples: increasing the intracellular copy number of a gene; A method for introducing mutation into the expression regulatory sequence of a gene; replacing the gene expression regulatory sequence with a highly active one A method of introducing a strong sequence; a method of introducing a mutation into a corresponding gene to enhance the activity of the gene; and a method of introducing a foreign gene into a microorganism.

於本申請案中，用語"失活"為包括活性較內在活性弱化或無活性的所有概念。 In this application, the term "inactivation" includes all concepts that are less active than intrinsically active or inactive.

可藉由應用本領域內所熟知的各種方法而達成此種基因活性的失活。作為上述方法的示例，有如下等方法，亦可藉由其組合而達成，但並不特別限制於上述示例：包括去除上述基因的活性的情形在內而使染色體上的基因整體或一部分缺失的方法；將染色體上的編碼上述蛋白質的基因替代為以減少相應蛋白質的活性的方式突變的基因的方法；將變異導入至編碼上述蛋白質的染色體上的基因的表現調節序列的方法；將編碼上述蛋白質的基因的表現調節序列更換為活性較弱或無活性的序列的方法(例如，將上述基因的啟動子更換為弱於內在啟動子的啟動子的方法)；使編碼上述蛋白質的染色體上的基因整體或一部分缺失的方法；導入互補結合至上述染色體上的基因的轉錄體而阻礙自上述mRNA轉譯為蛋白質的反義寡核苷酸(例如，反義RNA)的方法；於編碼上述蛋白質的基因的SD序列前端人為地附加與SD序列互補的序列而形成二級結構物來使核糖體(ribosome)無法附著的方法；及以逆轉錄至相應序列的開放閱讀框(open reading frame，ORF)的3'末端的方式附加啟動子的逆轉錄工程(Reverse transcription engineering，RTE)方法等。於本申請案中，用語"內在活性"是指於因自然或人為因素引起的遺傳性變異而微生物轉形的情形時，轉形前的母菌株原本所具有的特定蛋白質的活性。 Inactivation of such gene activity can be achieved by applying various methods well known in the art. Examples of the above-mentioned methods include the following methods, which can also be achieved by a combination thereof, but are not particularly limited to the above-mentioned examples: deletion of the entire or a part of the gene on the chromosome, including the case of removing the activity of the above-mentioned gene method; a method for replacing a gene encoding the above-mentioned protein on a chromosome with a gene mutated in a manner that reduces the activity of the corresponding protein; a method for introducing mutation into the expression regulatory sequence of the gene on the chromosome encoding the above-mentioned protein; the method for encoding the above-mentioned protein A method of replacing the expression regulatory sequence of the gene with a weaker or inactive sequence (for example, a method of replacing the promoter of the above-mentioned gene with a promoter that is weaker than the intrinsic promoter); making the gene on the chromosome encoding the above-mentioned protein A method for deletion in whole or in part; a method for introducing an antisense oligonucleotide (for example, antisense RNA) that is complementary to the above-mentioned mRNA and translating into a protein by introducing a transcript of the gene that is complementary to the above-mentioned mRNA; in a gene encoding the above-mentioned protein A method of artificially attaching a sequence complementary to the SD sequence to the front of the SD sequence to form a secondary structure to prevent ribosomes from attaching; Reverse transcription engineering (RTE) method of adding a promoter in the form of a 3' end. In the present application, the term "intrinsic activity" refers to the activity of a specific protein originally possessed by the parent strain before transformation when the microorganism is transformed due to genetic variation caused by natural or human factors.

作為本申請案的又一實施方式，提供一種包括於培養基 培養生產丁二胺的上述微生物的步驟的丁二胺生產方法。 As another embodiment of the present application, there is provided a A butanediamine production method of the step of culturing the above-mentioned microorganisms producing butanediamine.

上述丁二胺、包括SEQ ID NO：1的胺基酸序列的鳥胺酸脫羧酶、其變異體、蛋白質的表現及微生物如上所述。 The above-mentioned butanediamine, ornithine decarboxylase comprising the amino acid sequence of SEQ ID NO: 1, variants thereof, protein expressions and microorganisms are as described above.

於本申請案中，用語"培養"是指使上述微生物於適當調節的環境條件下生長。本申請案的培養過程可藉由業界內已知的適當的培養基與培養條件實現。業者可根據所選擇的菌株容易地進行調整而使用此種培養過程。具體而言，上述培養可為分批式、連續式及流加式，但並不限制於此。 In this application, the term "culturing" refers to growing the above-mentioned microorganisms under appropriately regulated environmental conditions. The culturing process of the present application can be realized by appropriate medium and culturing conditions known in the industry. This cultivation process can be easily adapted by the practitioner depending on the strain of choice. Specifically, the above-mentioned culturing can be batch type, continuous type and fed-batch type, but is not limited thereto.

於本申請案中，用語"培養基"是指將培養上述微生物所需的營養物質作為主成分混合的物質，包括存活及發育中不可或缺的水在內而供給營養物質及發育因子等。具體而言，用於培養本申請案的微生物的培養基及其他培養條件只要為用於培養普通微生物的培養基，則無特別限制地使用任一培養基，但可於含有適當的碳源、氮源、磷、無機化合物、胺基酸及/或維生素等的普通培養基中，於好氧條件下調節溫度、pH值等而培養本申請案的微生物。 In the present application, the term "medium" refers to a substance in which nutrients necessary for culturing the above-mentioned microorganisms are mixed as main components, and supplies nutrients, development factors, etc., including water indispensable for survival and development. Specifically, as long as the medium and other culture conditions for culturing the microorganisms of the present application are those used for culturing ordinary microorganisms, any medium can be used without particular limitation, and any medium may be used that contains an appropriate carbon source, nitrogen source, The microorganism of the present application is cultivated in a common medium containing phosphorus, inorganic compounds, amino acids, and/or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, and the like.

於上述方法中，培養上述微生物的步驟並無特別限制，可藉由公知的分批式培養方法、連續式培養方法、流加式培養方法等而執行。此時，培養條件並無特別限制，可使用鹼性化合物(例如，氫氧化鈉、氫氧化鉀或胺)或酸性化合物(例如，磷酸或硫酸)來調節適當大pH值(例如，pH5至pH9，具體而言為pH6至pH8，最具體而言為pH6.8)，可將氧氣或含氧氣體混合物導入至培養物而保持好氧條件。培養溫度可保持20℃至45℃，具體而言為25℃至40℃，可培養約10小時至160小時，但並不限制於此。藉由上述培養生產的丁二胺可分泌至培養基中或殘留於細胞內。 In the above-mentioned method, the step of culturing the above-mentioned microorganism is not particularly limited, and can be performed by a known batch culture method, continuous culture method, fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited, and a basic compound (for example, sodium hydroxide, potassium hydroxide, or amine) or an acidic compound (for example, phosphoric acid or sulfuric acid) can be used to adjust an appropriately large pH value (for example, pH5 to pH9) , specifically pH 6 to pH 8, most specifically pH 6.8), oxygen or oxygen-containing gas mixtures can be introduced into the culture to maintain aerobic conditions. The culturing temperature may be maintained at 20°C to 45°C, specifically 25°C to 40°C, and may be cultured for about 10 hours to 160 hours, but is not limited thereto. The butanediamine produced by the above-mentioned culture can be secreted into the medium or remain in the cells.

而且，使用的培養用培養基可單獨使用或混合使用糖及碳水化合物(例如，葡萄糖、蔗糖、乳糖、果糖、麥芽糖、糖蜜、澱粉及纖維素)、油脂及脂肪(例如，大豆油、葵花籽油、花生油及椰子油)、脂肪酸(例如，棕櫚酸、硬脂酸及亞麻油酸)、醇(例如，甘油及乙醇)及有機酸(例如，乙酸)等作為碳供給源，但並不限制於此。可單獨使用或混合使用含氮有機化合物(例如，蛋白腖、酵母萃取液、肉汁、麥芽萃取液、玉米漿、大豆粕粉及尿素)、或無機化合物(例如，硫酸銨、氯化銨、磷酸銨、碳酸銨及硝酸銨)等作為氮供給源，但並不限制於此。可單獨使用或混合使用磷酸二氫鉀、磷酸氫二鉀、與其相應的含鈉鹽等作為磷供給源，但並不限制於此。另外，培養基可包括其他金屬鹽(例如，硫酸鎂或硫酸鐵)、胺基酸及如維生素的必需生長促進物質。 Furthermore, the culture medium to be used can be used alone or in combination with sugars and carbohydrates (for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (for example, soybean oil, sunflower oil) , peanut oil and coconut oil), fatty acids (for example, palmitic acid, stearic acid, and linoleic acid), alcohols (for example, glycerol and ethanol), and organic acids (for example, acetic acid), etc. as carbon supply sources, but not limited to this. Nitrogen-containing organic compounds (eg, egg whites, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, and urea), or inorganic compounds (eg, ammonium sulfate, ammonium chloride, phosphoric acid), can be used alone or in combination ammonium, ammonium carbonate, and ammonium nitrate) etc. are used as nitrogen supply sources, but are not limited thereto. As the phosphorus supply source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salts corresponding thereto, and the like may be used alone or in combination, but are not limited thereto. Additionally, the medium may include other metal salts (eg, magnesium sulfate or ferric sulfate), amino acids, and essential growth promoting substances such as vitamins.

回收本申請案的上述培養步驟中生產的丁二胺的方法可根據培養方法利用本領域內公知的適當的方法自培養液收集目標胺基酸。例如，可使用離心分離、過濾、陰離子交換層析法、結晶化及高效液相層析法(High Performance Liquid Chromatography，HPLC)等，可利用本領域內公知的適當的方法自培養基或微生物回收目標丁二胺。回收上述丁二胺的方法可更包括精製步驟。 In the method for recovering the butanediamine produced in the above-mentioned culturing step of the present application, the target amino acid can be collected from the culture solution by an appropriate method known in the art according to the culturing method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and high performance liquid chromatography (HPLC), etc. can be used, and the target can be recovered from the culture medium or microorganisms using appropriate methods known in the art Butanediamine. The method for recovering the above-mentioned butanediamine may further include a refining step.

作為本申請案的又一實施方式，提供一種包括培養生產上述丁二胺的微生物的步驟的提高丁二胺的純度的方法。另外，提供一種包括培養生產上述丁二胺的微生物的步驟的增加丁二胺相對於戊二胺的比率的方法。上述丁二胺及微生物如上所述。 As yet another embodiment of the present application, there is provided a method for improving the purity of butanediamine including the step of culturing the above-mentioned butanediamine-producing microorganism. In addition, there is provided a method of increasing the ratio of butanediamine to pentamethylenediamine comprising the step of culturing the above-mentioned butanediamine-producing microorganism. The above-mentioned butanediamine and microorganisms are as described above.

作為本申請案的又一實施方式，提供一種培養生產上述丁二胺的微生物而製備的丁二胺的用以製備聚醯胺的用途。另外， 提供一種包括生產上述丁二胺的微生物的聚醯胺製備用組成物。上述丁二胺及微生物如上所述。 As yet another embodiment of the present application, there is provided a use of butanediamine prepared by culturing a microorganism producing the above-mentioned butanediamine for preparing polyamide. in addition, Provided is a polyamide preparation composition comprising a microorganism producing the above-mentioned butanediamine. The above-mentioned butanediamine and microorganisms are as described above.

生產上述丁二胺的微生物包括包含如下的鳥胺酸脫羧酶的微生物：包括SEQ ID NO：1的多肽、或於SEQ ID NO：1的a)第713個、b)第698個或c)與第713個及第698個相應的位置包括胺基酸取代，於SEQ ID NO：1的多肽包括具有至少80%以上且未滿100%的序列同源性的多肽。另外，培養上述生產丁二胺的微生物的步驟包括培養包括如下的鳥胺酸脫羧酶的微生物的步驟：包括SEQ ID NO：1的多肽、或於SEQ ID NO：1的a)第713個、b)第698個或c)與第713個及第698個相應的位置包括胺基酸取代，於SEQ ID NO：1的多肽包括具有至少80%以上且未滿100%的序列同源性的多肽。 The microorganisms that produce the above-mentioned butanediamine include microorganisms comprising the following ornithine decarboxylase: the polypeptide comprising SEQ ID NO: 1, or a) No. 713, b) No. 698 or c) of SEQ ID NO: 1 The positions corresponding to the 713th and 698th positions include amino acid substitutions, and the polypeptide in SEQ ID NO: 1 includes a polypeptide having sequence homology of at least 80% or more and less than 100%. In addition, the step of culturing the above-mentioned butanediamine-producing microorganism includes the step of culturing a microorganism comprising ornithine decarboxylase comprising the polypeptide of SEQ ID NO: 1, or the 713th item in a) of SEQ ID NO: 1, b) the 698th or c) positions corresponding to the 713th and 698th include amino acid substitutions, and the polypeptide in SEQ ID NO: 1 includes at least 80% or more and less than 100% sequence homology peptide.

上述聚醯胺作為活用於各種素材的物質，因醯胺鍵間的氫鍵而耐熱性、耐藥品性等優異，因此開發為各種素材的材料。例如，上述聚醯胺可為纖維原料，具體而言可為尼龍的原料。聚醯胺纖維於高強度、耐磨耗性、柔軟性、光澤特性、染色鮮明性等方面具有優異的特徵，因此可用於絲襪等腿部穿戴物(leg wear)、內衣(inner wear)、運動裝(sports wear)等服飾類製品。另外，上述聚醯胺可為醫藥品、界面活性劑、膜、塑膠等的原料。例如，於利用聚醯胺製備膜的情形時，可實現優異的光學物性及機械物性，同時亦具備柔軟性，因此可用作各種成形品的材料，上述聚醯胺膜可應用於顯示用基板、顯示用保護膜、觸控面板、摺疊式設備的窗蓋等。 The above-mentioned polyamides are used for various materials because they are excellent in heat resistance, chemical resistance, etc. due to hydrogen bonds between amide bonds, and thus have been developed as materials for various materials. For example, the above-mentioned polyamide may be a fiber raw material, specifically, a nylon raw material. Polyamide fibers have excellent characteristics in terms of high strength, abrasion resistance, softness, gloss properties, and dyeing clarity, so they can be used for leg wear such as stockings, inner wear, sportswear, etc. Apparel products such as sports wear. In addition, the above-mentioned polyamide can be used as a raw material for pharmaceuticals, surfactants, films, plastics, and the like. For example, when a film is prepared from polyamide, it can achieve excellent optical properties and mechanical properties, and also has flexibility, so it can be used as a material for various molded products, and the above-mentioned polyamide film can be applied to display substrates , Display protective film, touch panel, window cover of folding equipment, etc.

本申請案的鳥胺酸脫羧酶具有增大丁二胺的生產性或生產效率且抑制副反應的效果。特別是，本申請案具有阻礙作為鳥胺酸脫羧酶的副反應之一的戊二胺合成的效果，因此達成簡化丁二胺精製/分離製程且減少生產費用的效果。 The ornithine decarboxylase of the present application has the effect of increasing the productivity or production efficiency of butanediamine and suppressing side reactions. In particular, the present application has the effect of inhibiting the synthesis of pentamethylenediamine, which is one of the side reactions of ornithine decarboxylase, thus achieving the effect of simplifying the purification/separation process of butanediamine and reducing production costs.

另外，本申請案可藉由量產丁二胺而實現聚合物前驅物、醫藥品、化學添加劑等的各種活用。 In addition, the present application can realize various utilizations of polymer precursors, pharmaceuticals, chemical additives, and the like by mass-producing butanediamine.

圖1是表示於本申請案中以鳥胺酸為基質而利用鳥胺酸脫羧酶的丁二胺合成的示意圖。另外，表示作為需抑制的鳥胺酸脫羧酶的副反應的戊二胺合成路徑。 FIG. 1 is a schematic diagram showing the synthesis of butanediamine using ornithine decarboxylase using ornithine as a substrate in the present application. In addition, the synthetic route of pentamethylenediamine, which is a side reaction of ornithine decarboxylase to be inhibited, is shown.

圖2是確認各種來源的鳥胺酸脫羧酶活性者，表示將鳥胺酸用作基質時的反應性及將離胺酸用作基質時的反應性(副反應)的相對活性度。ODC_Lb來自雞源乳桿菌(誘導型(inducible))，ODC_Sc來自釀酒酵母(誘導型)，ODC_Ec來自大腸桿菌(組成型(constitutive))，ODC_Ef來自大腸桿菌(誘導型)。 Fig. 2 shows the activity of ornithine decarboxylase from various sources confirmed, and shows the relative activity of the reactivity when ornithine is used as a substrate and the reactivity (side reaction) when lysine is used as a substrate. ODC_Lb is from Lactobacillus hen (inducible), ODC_Sc is from Saccharomyces cerevisiae (inducible), ODC_Ec is from Escherichia coli (constitutive), and ODC_Ef is from Escherichia coli (inducible).

圖3是表示利用來自精製的乳桿菌的野生型鳥胺酸脫羧酶及如下變異體分別對(a)鳥胺酸比活度與(b)離胺酸比活度進行定量後進行比較的圖：野生型鳥胺酸脫羧酶的第696個丙胺酸由麩胺酸取代(A696E)；野生型鳥胺酸脫羧酶的第702個纈胺酸由甘胺酸取代(V702G)；野生型鳥胺酸脫羧酶的第713個丙胺酸由白胺酸取代(A713L)；野生型鳥胺酸脫羧酶的第696個丙胺酸與第713個丙胺酸由麩胺酸與白胺酸取代(A696E/A713L)；野生型鳥 胺酸脫羧酶的第702個纈胺酸與第713個丙胺酸由甘胺酸與白胺酸取代(V702G/A713L)；野生型鳥胺酸脫羧酶的第696個丙胺酸、第702個纈胺酸及第713個丙胺酸由麩胺酸、甘胺酸及白胺酸取代(A696E/V702G/A713L)；野生型鳥胺酸脫羧酶的第698個麩胺酸由天冬胺酸取代(E698D)；野生型鳥胺酸脫羧酶的第698個麩胺酸與第713個丙胺酸由天冬胺酸與白胺酸取代(E698D/A713L)。 Fig. 3 is a graph showing the comparison of the quantification of (a) ornithine specific activity and (b) lysine specific activity by wild-type ornithine decarboxylase derived from purified Lactobacillus and the following variants, respectively : The 696th alanine of wild-type ornithine decarboxylase is replaced by glutamic acid (A696E); the 702nd valine of wild-type ornithine decarboxylase is replaced by glycine (V702G); wild-type ornithine The 713th alanine of acid decarboxylase is replaced by leucine (A713L); the 696th and 713th alanine of wild-type ornithine decarboxylase are replaced by glutamic acid and leucine (A696E/A713L) ); wild type bird The 702nd valine and 713th alanine of amino acid decarboxylase are replaced by glycine and leucine (V702G/A713L); the 696th alanine and 702nd valine of wild-type ornithine decarboxylase amino acid and the 713th alanine were replaced by glutamic acid, glycine and leucine (A696E/V702G/A713L); the 698th glutamic acid of wild-type ornithine decarboxylase was replaced by aspartic acid ( E698D); the 698th glutamic acid and 713th alanine of wild-type ornithine decarboxylase were replaced by aspartic acid and leucine (E698D/A713L).

圖4是利用來自精製的乳桿菌的野生型鳥胺酸脫羧酶及如下變異體對離胺酸與鳥胺酸的動力學係數進行比較的圖：野生型鳥胺酸脫羧酶的第698個麩胺酸由天冬胺酸取代(E698D)；野生型去羧酶的第713個丙胺酸由白胺酸取代(A713L)；野生型鳥胺酸脫羧酶的第698個麩胺酸與第713個丙胺酸由天冬胺酸與白胺酸取代(E698D/A713L)。 Figure 4 is a graph comparing the kinetic coefficients of lysine and ornithine using wild-type ornithine decarboxylase from purified Lactobacillus and the following variants: 698th gluten of wild-type ornithine decarboxylase Amino acid is replaced by aspartic acid (E698D); 713 alanine of wild-type decarboxylase is replaced by leucine (A713L); 698 glutamic acid and 713 of wild-type ornithine decarboxylase Alanine is replaced by aspartic acid and leucine (E698D/A713L).

圖5是對各種條件下的生物轉化反應進行比較的圖。(a)是以鳥胺酸基質定量丁二胺的合成，使來自精製的乳桿菌的野生型鳥胺酸脫羧酶於0.37M濃度緩衝液中反應時(參照●)、使野生型鳥胺酸脫羧酶的第713個丙胺酸由白胺酸取代的變異體於0.37M濃度緩衝液中反應時(參照○)、使野生型鳥胺酸脫羧酶於0.1M濃度緩衝液中反應時(參照◆)、使野生型去羧酶的第713個丙胺酸由白胺酸取代的變異體於0.1M濃度緩衝液中反應時(參照◇)的圖。(b)是以離胺酸為基質定量戊二胺合成，使來自精製的乳桿菌的野生型鳥胺酸脫羧酶於0.37M濃度緩衝液中反應時(參照●)、使野生型鳥胺酸脫羧酶的第713個丙胺酸由白胺酸取代的變異體於0.37M濃度緩衝液中反應時(參照○)、使野生型鳥胺酸脫羧酶於0.1M濃度緩衝液中反應時(參照◆)、使野生型鳥胺酸脫羧酶 的第713個丙胺酸由白胺酸取代的變異體於0.1M濃度緩衝液中反應時(參照◇)的圖。 Figure 5 is a graph comparing biotransformation reactions under various conditions. (a) When the synthesis of butanediamine was quantified based on ornithine substrate, wild-type ornithine decarboxylase derived from purified Lactobacillus was reacted in a buffer solution with a concentration of 0.37 M (refer to ●), wild-type ornithine was reacted with When the variant in which the 713th alanine of decarboxylase was substituted by leucine was reacted in a buffer of 0.37M concentration (refer to ○), and when wild-type ornithine decarboxylase was reacted in a buffer of 0.1M concentration (refer to ◆ ), and when the variant in which the 713th alanine of the wild-type decarboxylase was substituted with leucine was reacted in a 0.1 M concentration buffer (refer to ◇). (b) When quantifying pentamethylenediamine synthesis using lysine as a substrate, and reacting wild-type ornithine decarboxylase derived from purified Lactobacillus in 0.37 M buffer solution (refer to ●), wild-type ornithine decarboxylase was reacted with When the variant in which the 713th alanine of decarboxylase was substituted by leucine was reacted in a buffer of 0.37M concentration (refer to ○), and when wild-type ornithine decarboxylase was reacted in a buffer of 0.1M concentration (refer to ◆ ), making wild-type ornithine decarboxylase The 713th variant of alanine substituted by leucine was reacted in 0.1M buffer (refer to ◇).

圖6是表示來自四種菌株的重組鳥胺酸脫羧酶基因的表現量。ODC_e.coli_SpeC來自大腸桿菌(組成型)，ODC_e.coli_SpeF來自大腸桿菌(誘導型)，ODC_乳桿菌來自雞源乳桿菌(誘導型)，ODC_釀酒酵母來自釀酒酵母(誘導型)。 Figure 6 shows the expression levels of recombinant ornithine decarboxylase genes from four strains. ODC_e.coli_SpeC is from Escherichia coli (constitutive), ODC_e.coli_SpeF is from Escherichia coli (inducible), ODC_Lactobacillus is from Lactobacillus chicken origin (inducible), and ODC_Saccharomyces cerevisiae is from Saccharomyces cerevisiae (inducible).

以下，藉由實施例而更詳細地對本申請案進行說明。然而，上述實施例用以例示性地對本申請案進行說明，本申請案的範圍並不限制於上述實施例，對於本申請案所屬的技術領域內具有常識者而言是顯而易見的。 Hereinafter, the present application will be described in more detail by way of examples. However, the above-mentioned embodiments are used to illustrate the present application, and the scope of the present application is not limited to the above-mentioned embodiments, which are obvious to those skilled in the technical field to which the present application belongs.

實施例1.各種來源的鳥胺酸脫羧酶的活性比較Example 1. Activity comparison of ornithine decarboxylase from various sources

對來自四種微生物的鳥胺酸脫羧酶的基質的反應性進行比較。以來自雞源乳桿菌(Lactobacillus saerimneri(誘導型))、釀酒酵母(saccharomyces cerevisiae(誘導型))、大腸桿菌(E.coli(組成型))、大腸桿菌(E.coli(誘導型))的野生型鳥胺酸脫羧酶為對象，將其分別標記為ODC_Lb、ODC_Sc、ODC_Ec、ODC_Ef。將與上述酶相應的基因***至pET24ma載體後，利用大腸桿菌BL21(DE3)於0.1mM的異丙基硫代半乳糖苷(Isopropyl Thiogalactoside，IPTG)及18℃的條件下表現蛋白質。此後，利用10%細胞萃取物於45℃下比較初始反應速度。分別對使用4m的鳥胺酸作為基質的情形與使用4mM的離胺酸作為基質的情形進行比較。 The reactivity of ornithine decarboxylase substrates from four microorganisms was compared. With Lactobacillus saerimneri (inducible), Saccharomyces cerevisiae (inducible), Escherichia coli (E.coli (constitutive)), Escherichia coli (E.coli (inducible)) The wild-type ornithine decarboxylase was used as the target, which were marked as ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef, respectively. After inserting the gene corresponding to the above enzyme into the pET24ma vector, the protein was expressed using Escherichia coli BL21 (DE3) under the conditions of 0.1 mM Isopropyl Thiogalactoside (IPTG) and 18°C. Thereafter, initial reaction rates were compared using 10% cell extracts at 45°C. The case of using 4 m of ornithine as the substrate was compared with the case of using 4 mM of lysine as the substrate, respectively.

圖2表示來自上述四種微生物的ODC酶的活性。具體而言，表示將鳥胺酸用作基質而生產丁二胺的鳥胺酸脫羧酶活性、及將離胺酸用作基質而生產戊二胺的離胺酸脫羧酶活性。離胺酸脫羧酶活性全部相似，但來自乳桿菌的鳥胺酸脫羧酶(ODC_Lb)的鳥胺酸脫羧酶活性最優異。即，於ODC_Lb中，離胺酸脫羧酶活性相對於鳥胺酸脫羧酶活性、即副反應的生成比率最低，因此基於如上所述的內容製作變異體。 Figure 2 shows the activity of ODC enzymes from the above four microorganisms. Specifically, it shows the ornithine decarboxylase activity of producing butanediamine using ornithine as a substrate, and the lysine decarboxylase activity of producing pentamethylenediamine using lysine as a substrate. The lysine decarboxylase activities were all similar, but the ornithine decarboxylase activity of Lactobacillus-derived ornithine decarboxylase (ODC_Lb) was the best. That is, in ODC_Lb, the lysine decarboxylase activity relative to the ornithine decarboxylase activity, that is, the generation ratio of the side reaction was the lowest, so a variant was prepared based on the above.

實施例2.鳥胺酸脫羧酶的變異位置的選擇Example 2. Selection of variant positions of ornithine decarboxylase

已知乳桿菌鳥胺酸脫羧酶的結晶結構，可藉由分析結構預測基質進出酶的隧道。為了選擇預測的隧道部分中執行飽和變異的功能性殘基(functional residues)，執行利用生物資訊學的序列資訊的多序列排列(multiple sequence alignment)，自本發明中利用的鳥胺酸脫羧酶胺基酸序列的N末端選擇A696、V702、A713、E698的位置作為變異位置。 Knowing the crystal structure of Lactobacillus ornithine decarboxylase, the tunneling of substrates into and out of the enzyme can be predicted by analyzing the structure. To select the functional residues in the predicted tunnel moiety that perform saturation variation, multiple sequence alignments using sequence information from bioinformatics were performed, from the ornithine decarboxylase amine utilized in the present invention. The positions of A696, V702, A713, and E698 were selected as the variation positions at the N-terminus of the amino acid sequence.

於蛋白質結構內保存有特定位置的胺基酸殘基的殘基於上述蛋白質中在結構與功能方面發揮非常重要的作用，特別是於觸媒過程中發揮直接作用的可能性較高，故而排除其為變異殘基。 Residues containing amino acid residues at specific positions in the protein structure play a very important role in structure and function in the above-mentioned proteins, especially in the catalytic process. for the variant residues.

實施例3.對鳥胺酸脫羧酶的功能性殘基執行飽和變異及探索變異體Example 3. Saturation mutation of functional residues of ornithine decarboxylase and exploration of variants

飽和變異(saturation mutagenesis)是指於基因的指定位置導入各種鹼基序列的變化。飽和變異是指於鍵結至模板鏈的互補序列的引子(primer)上***NNK密碼子(codon)來代替欲使變異的序列來藉由聚合酶鏈反應(Polymerase Chain Reaction，PCR)***變異。此時，於NNK密碼子中，N是指核苷酸的A、T、G、 C，K是指T、G。 Saturation mutation (saturation mutagenesis) refers to changes in various nucleotide sequences introduced into a given position of a gene. Saturation mutation refers to inserting a NNK codon (codon) into a primer bound to the complementary sequence of the template strand instead of the sequence to be mutated to insert mutation by polymerase chain reaction (PCR). In this case, in the NNK codon, N refers to A, T, G, C, K refers to T, G.

使用NNK密碼子對所選擇的功能性殘基執行飽和變異後，對變異體庫執行篩選。所有庫藉由全細胞反應進行一次及二次篩選。上述全細胞反應是指如下反應：對包括特定酶的細胞進行粉碎而利用細胞內容物，或不對酶進行分離精製而利用完整的細胞整體。藉由鳥胺酸全細胞反應進行一次篩選，選擇與野生型進行比較時表現出相似或更快的活性的變異體作為吸光度的變化。藉由離胺酸全細胞反應進行二次篩選，對於在一次篩選的篩選出的變異體，若對離胺酸的反應性低於野生型，則選擇。 After performing saturation mutations on selected functional residues using NNK codons, screening of the variant library was performed. All libraries were subjected to primary and secondary screening by whole cell reactions. The whole-cell reaction described above refers to a reaction in which cells containing a specific enzyme are pulverized and cell contents are used, or whole cells are used without separating and purifying the enzyme. A single screen was performed by ornithine whole cell reaction, and variants showing similar or faster activity when compared to wild type were selected as changes in absorbance. Secondary screening was performed by whole cell reaction with lysine, and variants screened in the primary screening were selected if their reactivity to lysine was lower than that of the wild type.

對於篩選出的變異體酶，測定使用鳥胺酸或離胺酸作為基質時的比活度。比活度(specific activity)表示藉由酶精製去除雜質及其他蛋白質的純蛋白質的單位活性，通常以1分鐘促進1μmol的基質變化的酶量為單位而表示為每1mg的單位數。具體而言，將來自乳桿菌的野生型及變異體的鳥胺酸脫羧酶轉形至大腸桿菌BL21(DE3)而利用作為誘導體的IPTG以50mL的培養體積表現後，利用Ni-NTA管柱僅精製純蛋白質。首先，於蛋白質表現後，藉由音波粉碎機對細胞進行粉碎，於離心分離後獲得細胞萃取液。將細胞萃取液放入至藉由添加有300mM的氯化鈉的50mM磷酸鹽(phosphate)緩衝液(pH8.0)平衡化的管柱而於0℃下與鎳樹脂(resin)結合1小時。此後，將未能與樹脂結合的蛋白質流棄，藉由包括50mM咪唑的Tris緩衝液去除非特異性地結合的其他蛋白質。最後，利用包括250mM咪唑的Tris緩衝液僅溶出所期望的蛋白質。為了自溶出的蛋白質去除咪唑，執行利用過濾管柱的脫鹽過程而最終僅獲得有活性的蛋白質。此後，使用布拉德 福(Bradford)蛋白質定量套組測定蛋白質量，使用相同量的蛋白質測定比活度。 For the screened variant enzymes, the specific activity was determined using ornithine or lysine as substrates. Specific activity refers to the unit activity of pure protein from which impurities and other proteins have been removed by enzymatic purification, and is usually expressed as a unit per 1 mg in terms of the amount of enzyme that promotes 1 μmol of substrate change in 1 minute. Specifically, the Lactobacillus-derived wild-type and mutant ornithine decarboxylase was transformed into Escherichia coli BL21 (DE3) and expressed in a culture volume of 50 mL using IPTG as an inducer, and then using a Ni-NTA column. Only pure protein is refined. First, after protein expression, the cells are pulverized by a sonic pulverizer, and a cell extract is obtained after centrifugation. The cell extract was placed into a column equilibrated with 50 mM phosphate buffer (pH 8.0) supplemented with 300 mM sodium chloride and bound to nickel resin for 1 hour at 0°C. Thereafter, proteins that failed to bind to the resin were discarded and other proteins bound nonspecifically were removed by a Tris buffer including 50 mM imidazole. Finally, only the desired protein was eluted using Tris buffer including 250 mM imidazole. In order to remove imidazole from the eluted protein, a desalting process using a filter column is performed to finally obtain only the active protein. After this, use brad The Bradford Protein Quantitation Kit determines protein amount and uses the same amount of protein to determine specific activity.

藉由HPLC(High-performance liquid chromatography)分析法測定將鳥胺酸或離胺酸用作基質時的來自乳桿菌的野生型及變異體的鳥胺酸脫羧酶的比活度。於50℃下執行30分鐘至300分鐘的反應而求出三次實驗的平均值。測定表現出10%至25%的轉化產率時的初始反應速度。使用陽離子交換管柱，流動相包括0.6g/L檸檬酸、4g/L酒石酸、1.4g/L乙二胺、5%甲醇及95%水。所使用的pH緩衝液於pH5.0的情形時，使用檸檬酸鹽緩衝液(citric-sodium citrate buffer)。野生型及變異體的鳥胺酸脫羧酶的比活度如圖3所示。 The specific activity of Lactobacillus-derived wild-type and mutant ornithine decarboxylase was measured by HPLC (High-performance liquid chromatography) analysis method using ornithine or lysine as a substrate. The reaction was performed at 50°C for 30 minutes to 300 minutes and the average value of three experiments was obtained. The initial reaction rate was determined at which conversion yields from 10% to 25% were exhibited. A cation exchange column was used, and the mobile phase included 0.6 g/L citric acid, 4 g/L tartaric acid, 1.4 g/L ethylenediamine, 5% methanol and 95% water. When the pH buffer used was pH 5.0, a citrate-sodium citrate buffer was used. The specific activities of ornithine decarboxylase of the wild type and the variant are shown in FIG. 3 .

如圖3(a)及圖3(b)所示，確認野生型及變異體(A696E、V702G、A713L、A696E/A713L、V702G/A713L、A696E/V702G/A713L、E698D及E698D/A713L)酶的比活度，結果確認到上述功能性殘基(A696、V702、A713、E698)均位於活性部位(active site)或基質接近隧道。 As shown in Figures 3(a) and 3(b), wild-type and variant (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D and E698D/A713L) enzymes were confirmed As a result, it was confirmed that the above-mentioned functional residues (A696, V702, A713, E698) were all located in the active site (active site) or the substrate near the tunnel.

具體而言，於使用鳥胺酸作為基質時，確認到變異體(A696E、V702G、A713L、A696E/A713L、V702G/A713L、A696E/V702G/A713L、E698D、E698D/A713L)的比活度相對於野生型的比活度分別為19.9%、4.3%、89.4%、12.8%、4.9%、0.1%、75.6%、74.4%(圖3(a))。 Specifically, when ornithine was used as a substrate, it was confirmed that the specific activities of the variants (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) relative to The specific activities of the wild type were 19.9%, 4.3%, 89.4%, 12.8%, 4.9%, 0.1%, 75.6%, and 74.4%, respectively (Fig. 3(a)).

於使用離胺酸作為基質時，變異體(A696E、V702G、A713L、A696E/A713L、V702G/A713L、A696E/V702G/A713L、E698D、E698D/A713L)酶的比活度相對於野生型酶的比活度分別 為16.9%、0.6%、42.4%、4.4%、0.9%、0.7%、50.8%、29.2%而確認到副反應得到抑制(圖3(b))。 Ratio of specific activity of variant (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) enzymes to wild-type enzymes when using lysine as substrate activity respectively Suppression of side reactions was confirmed at 16.9%, 0.6%, 42.4%, 4.4%, 0.9%, 0.7%, 50.8%, and 29.2% ( FIG. 3( b )).

實施例4.確認鳥胺酸脫羧酶的功能性殘基的動力學係數Example 4. Confirmation of kinetic coefficients of functional residues of ornithine decarboxylase

更細緻地確認上述實施例3中所使用的鳥胺酸脫羧酶變異體中具有70%以上的比活度的變異體A713L、E698D及E698D/A713L的特性。為了比較上述變異體及野生型的動力學係數(Kinetic parameter)，利用各種濃度條件的離胺酸。動力學係數利用具有不同的濃度的基質溶液來表示酶的基質親和度及基質轉化能力數值。 The properties of variants A713L, E698D, and E698D/A713L having a specific activity of 70% or more among the ornithine decarboxylase variants used in Example 3 were confirmed in more detail. In order to compare the kinetic parameters (Kinetic parameters) of the variant and the wild type, lysine at various concentrations was used. Kinetic coefficients use substrate solutions with different concentrations to express the substrate affinity and substrate conversion capacity values of enzymes.

具體而言，為了確認完成蛋白質精製的野生型及變異體的鳥胺酸脫羧酶的離胺酸的動力學係數，使用0.45mM至140mM的離胺酸濃度。pH緩衝液使用pH5.0檸檬酸鹽緩衝液(citric-sodium citrate buffer)，於200μl的反應體積下進行。藉由所明示的上述HPLC分析方法進行分析，求出三次實驗的平均值。野生型及變異體的離胺酸去羧酶的動力學係數如圖4所示。 Specifically, in order to confirm the kinetic coefficient of lysine of the wild-type and variant ornithine decarboxylases that have completed protein purification, lysine concentrations of 0.45 mM to 140 mM were used. The pH buffer was carried out in a reaction volume of 200 μl using a pH 5.0 citrate-sodium citrate buffer. The analysis was carried out by the above-mentioned HPLC analysis method as indicated, and the average value of the three experiments was obtained. Kinetic coefficients of lysine decarboxylase of wild type and variants are shown in FIG. 4 .

如圖4所示，確認到變異體(A713L)的離胺酸的k_cat值較與野生型下降2.16倍。確認到因k_cat值的減小而變異體(A713L)的離胺酸的k_cat/K_M值較野生型減少1.93倍。其結果，確認到變異體(A713L)可降低離胺酸脫羧酶活性。確認到變異體E698D及E698D/A713L的離胺酸的k_cat值亦分別降低2.08倍與2.59倍，且確認到離胺酸的k_cat/K_M值減小1.28倍及1.67倍。即，確認到上述變異體可減少副反應。 As shown in FIG. 4 , the k _cat value of lysine of the variant (A713L) was confirmed to be 2.16 times lower than that of the wild type. It was confirmed that the k _cat /KM value of lysine in the variant ( _A713L ) was 1.93-fold lower than that of the wild type due to the decrease in the k _cat value. As a result, it was confirmed that the variant (A713L) reduced lysine decarboxylase activity. The k _cat values of lysine of the variants E698D and E698D/ _A713L were also confirmed to be reduced by 2.08 times and 2.59 times, respectively, and the k _cat /KM values of lysine were confirmed to be reduced by 1.28 times and 1.67 times. That is, it was confirmed that the above variant reduces side reactions.

實施例5.變異體鳥胺酸脫羧酶的特性分析Example 5. Characterization of variant ornithine decarboxylase

欲研究變異體中作為具有最高的鳥胺酸比活度的變異體的鳥 胺酸脫羧酶(A713L)對丁二胺或戊二胺的生成產生的影響。分別進行將高濃度51.5g/L(0.39M)的鳥胺酸用作基質的情形、及將濃度為2.57g/L(17.6mM)的離胺酸用作基質的情形。為了於在兩種基質條件下進行反應時設定適宜的反應條件，於兩種條件(0.1M或0.37M)下進行使pH適當的緩衝液濃度。 Among the variants to be studied, the bird with the highest ornithine specific activity The effect of amino acid decarboxylase (A713L) on the production of butanediamine or pentamethylenediamine. The case where ornithine at a high concentration of 51.5 g/L (0.39 M) was used as the substrate and the case where lysine at a concentration of 2.57 g/L (17.6 mM) was used as the substrate were respectively performed. In order to set the appropriate reaction conditions when performing the reaction under both substrate conditions, pH-appropriate buffer concentrations were performed under both conditions (0.1 M or 0.37 M).

具體而言，於反應中使用0.1mg的完成蛋白質精製的野生型及變異體酶。使用0.39M的鳥胺酸或17.6mM的離胺酸作為基質，使用0.1M或0.37M的檸檬酸鹽緩衝液(citric-sodium citrate buffer，pH5.0)作為緩衝液。使用0.1mM的PLP輔酶，於50℃下進行反應，以2mL的反應體積進行。將其結果示於圖5。 Specifically, 0.1 mg of protein-purified wild-type and mutant enzymes were used in the reaction. 0.39M ornithine or 17.6mM lysine was used as matrix, and 0.1M or 0.37M citrate buffer (pH 5.0) was used as buffer. Reactions were performed at 50°C using 0.1 mM PLP coenzyme in a 2 mL reaction volume. The results are shown in FIG. 5 .

圖5(a)是將51.5g/L(0.39M)的鳥胺酸用作基質的情形。於將鳥胺酸轉化為丁二胺的情形時，在使用0.37M的緩衝液時(參照圖5(a)的●及○)，4小時後自野生型及變異體(A713L)分別生成33.0g/L及31.6g/L的丁二胺。另外，於使用低濃度的緩衝液(0.1M)時(參照圖5(a)的◆及◇)，7小時後野生型及變異體(A713L)分別生成20.2g/L及20.7g/L的丁二胺。確認到野生型及變異體(A713L)的丁二胺生產能力相似，且確認到使用高濃度緩衝液(0.37M)有益於反應性。 Figure 5(a) is the case where 51.5 g/L (0.39 M) ornithine was used as a substrate. In the case of converting ornithine to butanediamine, when a 0.37M buffer was used (refer to ● and ○ in Fig. 5(a) ), 33.0% were generated from the wild type and the mutant (A713L) after 4 hours, respectively. g/L and 31.6 g/L butanediamine. In addition, when a low-concentration buffer (0.1M) was used (see ◆ and ◇ in Fig. 5(a)), the wild-type and the variant (A713L) produced 20.2 g/L and 20.7 g/L, respectively, after 7 hours. Butanediamine. The wild-type and variant (A713L) were confirmed to have similar butanediamine productivity, and the use of high concentration buffer (0.37M) was confirmed to be beneficial for reactivity.

圖5(b)是將2.57g/L(17.6mM)的離胺酸用作基質的情形。於將離胺酸轉化成戊二胺的情形時，在使用0.37M的緩衝液時(參照圖5(b)的●及○)，4小時後自野生型及變異體(A713L)分別生成0.03g/L及0.007g/L的戊二胺。另外，於使用0.1M的緩衝液時(參照圖5(b)的◆及◇)，生成戊二胺的副反應增加，於7小時後自野生型及變異體(A713L)分別生成0.59g/L及0.38 g/L的戊二胺作為副反應。藉此，確認到自變異體(A713L)生成戊二胺的副反應得到抑制，且確認到於使用高濃度的緩衝液(0.37M)時，上述副反應抑制效果更突出。 Figure 5(b) is the case where 2.57 g/L (17.6 mM) of lysine was used as a matrix. When lysine was converted into pentamethylenediamine, when 0.37M buffer was used (refer to ● and ○ in Fig. 5(b)), 0.03 was generated from the wild type and the mutant (A713L) after 4 hours, respectively. g/L and 0.007g/L of pentamethylenediamine. In addition, when a 0.1 M buffer was used (see ◆ and ◇ in Fig. 5(b)), the side reaction to generate pentamethylene diamine increased, and 0.59 g/mol of pentamethylene diamine was generated from the wild type and the mutant (A713L) after 7 hours, respectively. L and 0.38 g/L pentamethylene diamine as a side reaction. Thereby, it was confirmed that the side reaction of pentamethylenediamine from the variant (A713L) was suppressed, and it was confirmed that the effect of suppressing the side reaction was more prominent when a high-concentration buffer solution (0.37M) was used.

實施例6.重組ODC基因於棒狀菌株中的表現量的比較測定Example 6. Comparative determination of expression levels of recombinant ODC genes in coryneform strains

用以表現實施例1中所提及的來自四種微生物的鳥胺酸脫羧酶ODC_Lb、ODC_Sc、ODC_Ec、ODC_Ef的重組基因的製作方法如下。 The production methods for expressing the recombinant genes of ornithine decarboxylase ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef from the four microorganisms mentioned in Example 1 are as follows.

具體而言，利用雞源乳桿菌(ACCESSION no.P43099)、釀酒酵母(ACCESSION no.J02777.1)及大腸桿菌str.K-12(ACCESSION no.BAA35349)遺傳體資訊以表1中明示的基因序列藉由PCR對鳥胺酸脫羧酶基因進行基因編碼區域擴增後，對PCR生成物處理限制酶而***至質體。 Specifically, the genes shown in Table 1 were determined using the genetic information of Lactobacillus gallis (ACCESSION no. P43099), Saccharomyces cerevisiae (ACCESSION no. J02777.1), and Escherichia coli str. After amplifying the gene coding region of the ornithine decarboxylase gene by PCR, the PCR product was treated with restriction enzymes and inserted into the plastid.

以可於蛋白質C-末端另外轉譯His-tag的方式製作重組 的基因。將大腸桿菌DH5α用作用以操作DNA的宿主菌株，將大腸桿菌BL21(DE3)用作用以表現C-末端His6-tag ODC基因的宿主菌株。於含有50μg/mL的康黴素的50mL LB培養基中在37℃下生長重組大腸桿菌BL21。於培養液於OD₆₀₀條件下達到0.8時，將0.2mM IPTG添加至培養液。於18℃至30℃下誘導蛋白質表現後，收穫細胞。將細胞再懸浮至溶解緩衝液，進行超音波處理而破壞細胞。將所獲得的重組ODC於4℃下精製成Quiagen(Hilden，Germany)的Ni-NTA瓊脂糖樹脂。連同100kDa的分子質量切斷(cut off)一併使用Centriplus YM-30(Millipore，Bedford，MA)獲得重組蛋白質。表現結果如圖6。 Recombinant genes are made in such a way that a His-tag can be additionally translated at the C-terminus of the protein. Escherichia coli DH5α was used as a host strain for DNA manipulation, and Escherichia coli BL21(DE3) was used as a host strain for expressing the C-terminal His6-tag ODC gene. Recombinant E. coli BL21 was grown at 37°C in 50 mL of LB medium containing 50 μg/mL of kanamycin. When the broth reached 0.8 at _OD600 , 0.2 mM IPTG was added to the broth. After induction of protein expression at 18°C to 30°C, cells were harvested. The cells were resuspended in lysis buffer and sonicated to disrupt the cells. The obtained recombinant ODC was refined into Ni-NTA agarose resin from Quiagen (Hilden, Germany) at 4°C. Recombinant proteins were obtained using Centriplus YM-30 (Millipore, Bedford, MA) along with a 100 kDa molecular mass cut off. The performance results are shown in Figure 6.

若於十二烷基磺酸鈉聚丙烯醯胺凝膠電泳(Sodium Dodecyl Sulfonate Polyacrylamide Gel Electrophoresis，SDS-PAGE)凝膠(gel)上分析30℃表現誘導條件的結果，則可確認到重組ODC_Lb與ODC_Ec的表現量高於ODC_Sc、ODC_Ef。然而，於ODC_Ec的情形時，確認到中溫條件表現時可溶性蛋白質量明顯減少。進而，於進行37℃表現時，ODC_Ec的可溶性蛋白質量進一步減少。 When the results of the induction conditions at 30°C were analyzed on a Sodium Dodecyl Sulfonate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel (gel), it was confirmed that the recombinant ODC_Lb interacted with The performance of ODC_Ec is higher than that of ODC_Sc and ODC_Ef. However, in the case of ODC_Ec, it was confirmed that the amount of soluble protein was significantly reduced when the mesophilic condition was exhibited. Furthermore, the amount of soluble protein in ODC_Ec was further reduced when expressed at 37°C.

於棒狀菌株中，表現ODC_Lb與ODC_Ec基因而對表現為可溶性蛋白質的量進行比較評估。於ODC_Lb基因及ODC_Ec基因的起始密碼子前導入CJ7啟動子(KCCM10617，韓國註冊專利第10-0620092號)。首先，為了獲得包括CJ7啟動子序列的基因，以谷胺酸棒狀桿菌ATCC13032的基因組DNA為模板執行利用表2中明示的引子對的PCR。PCR反應是反覆進行30次如下過程而實施：於95℃下改質30秒、於55℃下退火30秒及於72℃下 伸長30秒。 In the coryneform strain, the ODC_Lb and ODC_Ec genes were expressed and the amount expressed as a soluble protein was evaluated comparatively. The CJ7 promoter (KCCM10617, Korean Registered Patent No. 10-0620092) was introduced before the start codons of the ODC_Lb gene and the ODC_Ec gene. First, in order to obtain a gene including the CJ7 promoter sequence, PCR using the primer pairs indicated in Table 2 was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The PCR reaction was carried out by repeating the following procedures 30 times: modification at 95°C for 30 seconds, annealing at 55°C for 30 seconds and at 72°C Elongate for 30 seconds.

於利用1.5%瓊脂糖凝膠進行電泳後，確認具有400鹼基對(bp)的尺寸的PCR核酸產物。利用PCR prep kit(GeneAll，首爾)對所獲得的PCR產物進行精製。將BamHI與XbaI放入至精製的PCR產物與pSCEC載體溶液樣品，藉由於37℃下反應4小時而進行限制酶處理，於利用1.5%瓊脂糖凝膠進行電泳後，切斷具有400bp的尺寸的PCR核酸產物條帶與載體尺寸的條帶，之後利用Gel prep kit(GeneAll，Seoul)獲得核酸片段。於利用T4連接酶(ligase)將精製的各1mg的CJ7啟動子片段與載體連接後，對大腸桿菌DH5α菌株進行電致孔(electrophoration)。以2500V施加電致孔。將回收的菌株塗抹至包括50μg/L觀黴素(spectinomycin)的LB平板培養基而於37℃下培養1日後，篩選表現出耐性的菌株。於篩選18個菌株而以SEQ ID NO：9及SEQ ID NO：10進行菌落PCR後，可確認具有400bp尺寸的PCR產物。根據菌落PCR結果確認具有CJ7啟動子的pSCEC_cj7的製作。 After electrophoresis using a 1.5% agarose gel, a PCR nucleic acid product with a size of 400 base pairs (bp) was confirmed. The obtained PCR product was purified using PCR prep kit (GeneAll, Seoul). BamHI and XbaI were put into the purified PCR product and pSCEC carrier solution sample, and subjected to restriction enzyme treatment by reacting at 37°C for 4 hours. The PCR nucleic acid product band and the vector-sized band were then used to obtain nucleic acid fragments using Gel prep kit (GeneAll, Seoul). After 1 mg of each purified CJ7 promoter fragment was ligated to the vector using T4 ligase, electrophoration was performed on Escherichia coli DH5α strain. Electroporation was applied at 2500V. The recovered strains were spread on LB plate medium containing 50 μg/L spectinomycin and cultured at 37° C. for 1 day, and then strains showing resistance were screened. After screening 18 strains and performing colony PCR with SEQ ID NO: 9 and SEQ ID NO: 10, a PCR product having a size of 400 bp was confirmed. The production of pSCEC_cj7 having the CJ7 promoter was confirmed from the results of colony PCR.

基於所獲得的pSCEC_cj7載體而利用表3中所明示的引子來藉由PCR對2個基因ODC_Lb與ODC_Ec進行擴增。 Two genes, ODC_Lb and ODC_Ec, were amplified by PCR using the primers shown in Table 3 based on the obtained pSCEC_cj7 vector.

藉由限制酶XbaI與SalI對所獲得的上述PCR產物及pSCEC_cj7載體進行處理。對經限制酶處理的核酸進行凝膠預備而將ODC_Lb、ODC_Ec及pSCEC_cj7核酸片段連接，***至大腸桿菌DH5α菌株。自確認到***的篩選出的菌株分別獲得pSCEC_cj7_ODC_Lb及pSCEC_cj7_ODC_Ec，對生產丁二胺的棒狀桿菌屬微生物KCCM11240P分別以2500確V進行電致孔(electrophoration)。 The obtained PCR product and pSCEC_cj7 vector were treated with restriction enzymes XbaI and SalI. The restriction enzyme-treated nucleic acid was subjected to gel preparation to ligate the ODC_Lb, ODC_Ec, and pSCEC_cj7 nucleic acid fragments, and inserted into E. coli DH5α strain. From the screened strains confirmed to be inserted, pSCEC_cj7_ODC_Lb and pSCEC_cj7_ODC_Ec were obtained, respectively, and electrophoration was performed on the butanediamine-producing Corynebacterium microorganism KCCM11240P at 2500 confirm V, respectively.

於包括50μg/L觀黴素(spectinomycin)的BHIS平板培養基(腦心浸液(Braine heart infusion)37g/l，山梨醇91g/l，瓊脂2%)塗抹上述菌株進行培養，藉此形成菌落。確認到篩選出的上述菌株可於包括50μg/L觀黴素(spectinomycin)的CM培養基(葡萄糖(glucose)10g/L，多聚蛋白腖(polypeptone)10g/L，酵母萃(yeast extract)5g/L，牛肉萃(beef extract)5g/L，NaCl 2.5 g/L，尿素(Urea)2g/L，pH6.8)進行振盪培養。於將所製作的2種谷胺酸棒狀桿菌變異株培養3mL後，進行離心分離而獲得菌體。藉由超音波處理方法對所獲得的菌體進行細胞粉碎後，進行離心分離而獲得包括可溶性蛋白質的溶液。 The above strains were smeared and cultured on BHIS plate medium (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) including 50 μg/L spectinomycin, whereby colonies were formed. It was confirmed that the selected strains can be grown in CM medium containing 50μg/L spectinomycin (glucose 10g/L, polypeptone 10g/L, yeast extract 5g/L) , beef extract (beef extract) 5g/L, NaCl 2.5 g/L, urea (Urea) 2g/L, pH 6.8) for shaking culture. After culturing 3 mL of the prepared two types of Corynebacterium glutamicum mutant strains, centrifugation was performed to obtain bacterial cells. The obtained bacterial cells were cell-pulverized by ultrasonication, and then centrifuged to obtain a solution containing soluble proteins.

利用Ni-NTA離心柱(Spin Columns)(Hilden，Germany)分別對包括His-tag的ODC_Lb、ODC_Ec蛋白質進行精製。利用Nano drop獲得而對蛋白質的濃度進行測定。基於測定值計算的重組蛋白質濃度分別為ODC_Lb：1.282g/L、ODC_Ec：0.039g/L，確認到獲得棒狀菌株中ODC_Lb較ODC_Ec為30倍以上的可溶性蛋 白質。 ODC_Lb and ODC_Ec proteins including His-tag were purified by Ni-NTA spin columns (Spin Columns) (Hilden, Germany). The protein concentration was measured by Nano drop acquisition. The recombinant protein concentrations calculated based on the measured values were ODC_Lb: 1.282 g/L, ODC_Ec: 0.039 g/L, respectively, and it was confirmed that soluble proteins with ODC_Lb more than 30 times higher than ODC_Ec in the coryneform strain were obtained. white matter.

可確認到於在中溫條件下將大腸桿菌與棒狀菌株用作宿主而表現ODC_Lb時，因高表現量與正常蛋白質皺褶而可生產可溶性較高的蛋白質。 It was confirmed that when ODC_Lb was expressed by using Escherichia coli and a coryneform strain as hosts under mesophilic conditions, a highly soluble protein could be produced due to the high expression level and normal protein folds.

實施例7.導入有變異體鳥胺酸脫羧酶的棒狀丁二胺生產菌株的製備及丁二胺生產能力的測定Example 7. Preparation of rod-shaped butanediamine-producing strains introduced with variant ornithine decarboxylase and measurement of butanediamine production capacity

為了研究本申請案的鳥胺酸脫羧酶變異體對丁二胺生產產生的影響，製作將上述鳥胺酸脫羧酶變異體導入至丁二胺生產能力提高的棒狀桿菌屬微生物的菌株。 In order to study the effect of the ornithine decarboxylase variant of the present application on the production of butanediamine, a strain of the microorganism belonging to the genus Corynebacterium having improved butanediamine production ability was prepared by introducing the ornithine decarboxylase variant.

具體而言，作為上述丁二胺生產能力提高的棒狀桿菌屬微生物，使用專利申請案(韓國公開專利第2013-0082478號)中所揭示的具有丁二胺生產能力的棒狀桿菌屬微生物(KCCM11240P)。具有丁二胺生產能力的上述棒狀桿菌屬微生物(KCCM11240P)為於由谷胺酸棒狀桿菌ATCC13032製備的微生物(ATCC 13032 △argF △NCgl1221 P(CJ7)-argCJBD bioAD：：P(CJ7)-speC(Ec)：KCCM11138P(韓國公開專利第2012-0064046號))內缺失NCgl1469的微生物。 Specifically, as the microorganism of the genus Corynebacterium having the improved butanediamine-producing ability, the microorganism of the genus Corynebacterium having the ability to produce butanediamine disclosed in the patent application (Korean Laid-Open Patent Publication No. 2013-0082478 ) was used ( KCCM11240P). The above-mentioned Corynebacterium microorganism (KCCM11240P) having the ability to produce butanediamine is a microorganism prepared from Corynebacterium glutamicum ATCC13032 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)- speC(Ec): A microorganism in which NCgl1469 is deleted in KCCM11138P (Korean Laid-Open Patent Publication No. 2012-0064046).

製作用以於上述丁二胺生產微生物內以來自上述乳桿菌的鳥胺酸脫羧酶變異體取代鳥胺酸脫羧酶的載體。更具體而言，利用下述表4中揭示的ODC_Lb_start(EcoRV)_5、ODC_Lb_stop(MfeI)_3引子對上述實施例1及實施例3中製作的來自乳桿菌的鳥胺酸脫羧酶變異體的DNA進行擴增。具體而言，於所製作的上述pET24ma載體***野生型及變異體(E698D、A713L)的乳桿菌鳥胺酸脫羧酶而分別將其作為模板，使用下述表4中揭示的兩個 引子L-odc_start(EcoRV)_5、L-odc_stop(MfeI)_3執行PCR。 A vector for replacing ornithine decarboxylase with the Lactobacillus-derived ornithine decarboxylase variant in the above-mentioned butanediamine-producing microorganism was prepared. More specifically, utilize the ODC_Lb_start(EcoRV)_5, ODC_Lb_stop(MfeI)_3 primers disclosed in the following table 4 to the DNA of the ornithine decarboxylase variant from Lactobacillus prepared in the above-mentioned Example 1 and Example 3. Amplify. Specifically, Lactobacillus ornithine decarboxylase of wild-type and variant (E698D, A713L) was inserted into the prepared pET24ma vector and used as templates, respectively, and two of the following Table 4 were used. Primers L-odc_start(EcoRV)_5, L-odc_stop(MfeI)_3 perform PCR.

利用EcoRV與MfeI限制酶對藉由PCR擴增獲得的基因片段進行處理(37℃、3小時)，將來自乳桿菌的野生型及變異體(E698D、A713L)鳥胺酸脫羧酶的基因片段***至利用與韓國公開專利第2012-0064046號中揭示的方法相同的方法製作的pDZ-bioAD-P(CJ7)載體。於上述方法中，使用EcoRV與MfeI限制酶。藉由序列分析對藉由上述方法製作的染色體***用重組載體(pDZ-ODC_Lb、pDZ-ODC_Lb_E698D、pDZ-ODC_Lb_A713L)進行確認。 The gene fragment obtained by PCR amplification was treated with EcoRV and MfeI restriction enzymes (37°C, 3 hours), and the gene fragment of Lactobacillus-derived wild-type and variant (E698D, A713L) ornithine decarboxylase was inserted To the pDZ-bioAD-P(CJ7) vector produced by the same method as that disclosed in Korean Laid-Open Patent No. 2012-0064046. In the above method, EcoRV and MfeI restriction enzymes were used. The recombinant vectors for chromosomal insertion (pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, pDZ-ODC_Lb_A713L) produced by the above method were confirmed by sequence analysis.

為了獲得於染色體內***有來自乳桿菌的野生型及變異體鳥胺酸脫羧酶的棒狀菌株，利用電穿孔法將以上所製作的pDZ-ODC_Lb、pDZ-ODC_Lb_E698D、pDZ-ODC_Lb_A713L重組載體分別轉染至KCCM11240P菌株後，塗抹至BHIS平板培養基(腦心浸液37g/l，山梨醇91g/l，瓊脂(agar)2%，1L基準+康黴素(kanamycin)25ug/ml)。 In order to obtain a coryneform strain with wild-type and variant ornithine decarboxylase from Lactobacillus inserted into the chromosome, the recombinant vectors of pDZ-ODC_Lb, pDZ-ODC_Lb_E698D and pDZ-ODC_Lb_A713L prepared above were respectively transformed by electroporation. After staining to KCCM11240P strain, spread to BHIS plate medium (brain heart infusion 37g/l, sorbitol 91g/l, agar 2%, 1L standard + kanamycin 25ug/ml).

藉由是否在包括X-gal(5-溴-4-氯-3-吲哚基-β-D-半乳糖苷)的固體培養基中顯示出藍色來判別載體成功***至染色體內。於營養培養基中振盪培養(30℃，8小時)***有一次染色體的菌株後，分別進行連續稀釋而塗抹於包括X-gal的固體培養基。大部分菌落呈藍色，相反地，能夠以低比率篩選白色的菌落，篩選出的 菌落可藉由二次交叉(cross over)獲得於染色體導入有最終乳桿菌鳥胺酸脫羧酶變異體的菌株。最後，藉由變異體的序列分析確認菌株。將確認到的菌株命名為KCCM11240P：：ODC_Lb、KCCM11240P：：ODC_Lb_E698D、KCCM11240P：：ODC_Lb_A713L。 Successful insertion of the vector into the chromosome was judged by whether it showed blue color in the solid medium including X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). After shaking cultured in a nutrient medium (30° C., 8 hours), the primary chromosome-inserted strains were serially diluted and spread on a solid medium including X-gal. Most of the colonies are blue, conversely, white colonies can be screened at a low rate, and the screened Colonies can be obtained by secondary cross over to the strains into which the final Lactobacillus ornithine decarboxylase variant has been chromosomally introduced. Finally, strains were confirmed by sequence analysis of the variants. The confirmed strains were named KCCM11240P::ODC_Lb, KCCM11240P::ODC_Lb_E698D, KCCM11240P::ODC_Lb_A713L.

為了確認導入來自乳桿菌的野生型及變異體鳥胺酸脫羧酶對丁二胺生產菌株的丁二胺生產能力產生的影響，對丁二胺生產能力進行評估。 In order to confirm the influence of the introduction of wild-type and variant ornithine decarboxylase derived from Lactobacillus on the butanediamine-producing ability of the butanediamine-producing strain, the butanediamine-producing ability was evaluated.

具體而言，將以上所製作的菌株於包括1mM精胺酸的CM平板培養基(葡萄糖1%，多聚蛋白腖1%，酵母萃取物0.5%，牛肉萃0.5%，NaCl 0.25%，尿素0.2%，50%NaOH 100ul，瓊脂2%，pH6.8，1L基準)中以30℃培養16小時後，將約一種鉑接種至具有下述表5的組成的25ml效價培養基，之後將其於30℃下以200rpm振盪培養24小時。於醱酵時在培養基添加1mM精胺酸來培養所製作的所有菌株。 Specifically, the above strains were prepared in CM plate medium containing 1mM arginine (glucose 1%, polyprotein 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, After culturing at 30°C for 16 hours in 50% NaOH 100ul, agar 2%, pH 6.8, 1L reference), about one platinum was inoculated into 25 ml of titer medium having the composition of Table 5 below, which was then incubated at 30°C. Shaking at 200 rpm for 24 hours. All the produced strains were cultured by adding 1 mM arginine to the medium during fermentation.

其結果，如表6所示，於培養12小時後，在導入來自乳桿菌的變異體(A713L)鳥胺酸脫羧酶的菌株中，與具有大腸桿菌 鳥胺酸脫羧酶的菌株(KCCM11240P)相比，丁二胺產量增加約115%P。另外，於導入A713L鳥胺酸脫羧酶的菌株中，表現出較具有來自乳桿菌的野生型鳥胺酸脫羧酶的菌株(KCCM11240P：：ODC_Lb)增加約40%P的狀態。 As a result, as shown in Table 6, after culturing for 12 hours, the Lactobacillus-derived variant (A713L) ornithine decarboxylase-introduced strains were compared with Escherichia coli. Compared with the ornithine decarboxylase strain (KCCM11240P), the production of butanediamine increased by about 115%P. In addition, the A713L ornithine decarboxylase-introduced strain exhibited an increase of about 40% P compared to the strain (KCCM11240P::ODC_Lb) having the wild-type Lactobacillus-derived ornithine decarboxylase.

另外，可知與KCCM11240P相比，導入有A713L鳥胺酸脫羧酶的菌株(KCCM11240P：：ODC_Lb_A713L)因生產丁二胺時的副反應產生的戊二胺減少約48%P，且根據殘留於培養液內的葡萄糖濃度推測，相同時間內的糖消耗量增加，生產性一併增加。 In addition, compared with KCCM11240P, it was found that the strain (KCCM11240P::ODC_Lb_A713L) into which the A713L ornithine decarboxylase was introduced was reduced by about 48%P due to the side reaction during the production of butanediamine, and the amount remaining in the culture solution was reduced by about 48%P. It is presumed that the sugar consumption in the same time increases, and the productivity also increases.

如上所述的結果表明，因導入來自乳桿菌的變異體鳥胺酸脫羧酶而不僅可自丁二胺生產菌株生產相對糖消耗而高於現有的濃度的丁二胺，而且具有減少戊二胺及提高生產性的效果。 The results as described above show that, by introducing the variant ornithine decarboxylase from Lactobacillus, it is possible not only to produce butanediamine at a concentration higher than the existing concentration relative to sugar consumption from butanediamine-producing strains, but also to reduce pentamethylenediamine. and improve productivity.

實施例8.乳桿菌鳥胺酸脫羧酶的第713個胺基酸殘基的飽和變異影響的預測Example 8. Prediction of the effect of saturation variation of amino acid residue 713 of Lactobacillus ornithine decarboxylase

於所選擇的變異中，針對乳桿菌鳥胺酸脫羧酶的第713個丙胺酸由白胺酸取代的功能性殘基(A713L)以除丙胺酸及白胺酸以外的其他胺基酸取代，之後導入至丁二胺生產菌株KCCM11240P而研究對丁二胺生產造成的影響。 In the selected variant, the functional residue (A713L) for the 713th alanine substituted by leucine for Lactobacillus ornithine decarboxylase was substituted with other amino acids than alanine and leucine, Then, it was introduced into butanediamine-producing strain KCCM11240P to study the effect on the production of butanediamine.

具體而言，製作如下的變異菌株：代替丁二胺生產能力提高的棒狀桿菌屬微生物(KCCM11240P)的染色體內的來自大腸桿 菌的野生型鳥胺酸脫羧酶取代於SEQ ID NO：1的胺基酸序列中以包括疏水性胺基酸的其他胺基酸取代自N-末端起第713個胺基酸的形態的變異體。更具體而言，為了分別製作以疏水性胺基酸之一的纈胺酸、鹼性胺基酸之一的精胺酸、酸性胺基酸之一的天冬胺酸、中性胺基酸之一的麩醯胺酸、芳香族性胺基酸之一的色胺酸取代的載體，以實施例7中製作的pDZ-ODC_Lb載體為模板而使用上述表4與下述表7中揭示的引子來執行PCR。以欲引發變異的部位為中心分別對前側部分(5')與後側部分(3')進行一次PCR，之後執行使兩個PCR片段吻合的二次PCR。例如，於將第713個胺基酸自丙胺酸取代為纈胺酸的變異(A713V)的情形時，前側部分使用ODC_Lb_start(EcoRV)_5與ODC_Lb_A713V_3引子而藉由PCR進行擴增，後側部分使用ODC_Lb_A713V_5與ODC_Lb_stop(MfeI)_3引子而藉由PCR進行擴增。將藉由一次PCR獲得的兩個PCR片段用作二次PCR的模板，使用ODC_Lb_start(EcoRV)_5與ODC_Lb_stop(MfeI)_3引子進行PCR。最終獲得的乳桿菌鳥胺酸脫羧酶變異體A713V基因片段藉由與實施例7相同的方法***至pDZ-bioAD-P(CJ7)載體。除此之外的剩餘變異體A713R、A713D、A713W、A713Q亦使用表7中揭示的引子而藉由與上述相同的方法進行PCR來***至pDZ-bioAD-P(CJ7)載體。藉由序列分析對所製作的染色體***用重組載體(pDZ-ODC_Lb_A713V、pDZ-ODC_Lb_A713R、pDZ-ODC_Lb_A713D、pDZ-ODC_Lb_A713W、pDZ-ODC_Lb_A713Q)進行確認。 Specifically, the following mutant strains were prepared in place of E. coli derived in the chromosome of the microorganism of the genus Corynebacterium (KCCM11240P) having improved butanediamine productivity. The wild-type ornithine decarboxylase of Bacteria is substituted in the amino acid sequence of SEQ ID NO: 1 with other amino acids including hydrophobic amino acids. Variation in the form of the 713th amino acid from the N-terminus body. More specifically, in order to prepare valine, which is one of hydrophobic amino acids, arginine, which is one of basic amino acids, aspartic acid, which is one of acidic amino acids, and neutral amino acid, respectively. One of the glutamic acid, the tryptophan-substituted carrier of one of the aromatic amino acids, using the pDZ-ODC-Lb carrier prepared in Example 7 as a template and using the above-mentioned table 4 and the following table 7. Revealed primers to perform PCR. The first PCR is performed on the front part (5') and the rear part (3') with the site to be mutated as the center, and then the second PCR is performed to match the two PCR fragments. For example, in the case of the variation (A713V) in which the 713th amino acid is substituted from alanine to valine, the front part is amplified by PCR using primers ODC_Lb_start(EcoRV)_5 and ODC_Lb_A713V_3, and the rear part is ODC_Lb_A713V_5 and ODC_Lb_stop(MfeI)_3 primers were amplified by PCR. The two PCR fragments obtained by the primary PCR were used as templates for the secondary PCR, and PCR was performed using ODC_Lb_start(EcoRV)_5 and ODC_Lb_stop(MfeI)_3 primers. The finally obtained Lactobacillus ornithine decarboxylase variant A713V gene fragment was inserted into the pDZ-bioAD-P(CJ7) vector by the same method as in Example 7. The remaining variants A713R, A713D, A713W, A713Q other than that were also inserted into the pDZ-bioAD-P(CJ7) vector by PCR using the primers disclosed in Table 7 by the same method as above. The produced recombinant vectors for chromosomal insertion (pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, pDZ-ODC_Lb_A713Q) were confirmed by sequence analysis.

[表7]

[Table 7]

為了獲得於染色體內***有乳桿菌鳥胺酸脫羧酶的第713個丙胺酸由包括疏水性胺基酸的其他胺基酸取代的形態的變異體的菌株，可藉由如實施例7的方法將以上所製作的pDZ-ODC_Lb_A713V、pDZ-ODC_Lb_A713R、pDZ-ODC_Lb_A713D、pDZ-ODC_Lb_A713W、pDZ-ODC_Lb_A713Q重組載體分別轉染至KCCM11240P菌株並進行篩選而最終獲得於染色體導入有最終乳桿菌鳥胺酸脫羧酶變異體的菌株。最後，藉由變異體的序列分析而確認菌株。將確認的菌株命名為KCCM11240P：：ODC_Lb_A713V、KCCM11240P：：ODC_Lb_A713R、KCCM11240P：：ODC_Lb_A713D、KCCM11240P：：ODC_Lb_A713Q、KCCM11240P：：ODC_Lb_A713W。 In order to obtain a strain in which the 713th alanine of Lactobacillus ornithine decarboxylase is chromosomally substituted by other amino acids including hydrophobic amino acids, the method of Example 7 can be used. The pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, pDZ-ODC_Lb_A713Q recombinant vectors produced above were respectively transfected into the KCCM11240P strain and screened to obtain the final Lactobacillus ornithine decarboxylase introduced into the chromosome. mutant strains. Finally, the strains were confirmed by sequence analysis of the variants. The confirmed strains were named KCCM11240P::ODC_Lb_A713V, KCCM11240P::ODC_Lb_A713R, KCCM11240P::ODC_Lb_A713D, KCCM11240P::ODC_Lb_A713Q, KCCM11240P::ODC_Lb_A713W.

為了確認導入乳桿菌鳥胺酸脫羧酶的第713個丙胺酸由包括疏水性胺基酸的其他胺基酸取代的形態的變異體對丁二胺生產菌株的丁二胺生產能力產生的影響，藉由如上述實施例7的方 法對丁二胺生產能力進行評估。 In order to confirm the effect of the variant in which the 713th alanine introduced into Lactobacillus ornithine decarboxylase was substituted with other amino acids including hydrophobic amino acids, on the butanediamine-producing ability of the butanediamine-producing strain, By the method as described above in Example 7 method to evaluate the production capacity of butanediamine.

其結果，如表8所示，導入有以包括疏水性的其他胺基酸取代的形態的乳桿菌鳥胺酸脫羧酶變異體的菌株的丁二胺產量亦較培養12小時後具有來自大腸桿菌的野生型鳥胺酸脫羧酶的菌株(KCCM11240P)平均增加約86%P。另外，表現出較具有來自乳桿菌的野生型鳥胺酸脫羧酶的菌株平均增加約21%P的狀態。 As a result, as shown in Table 8, the production of butanediamine of the strain in which the Lactobacillus ornithine decarboxylase variant having been substituted with other amino acids including hydrophobicity was introduced was higher than that from Escherichia coli after culturing for 12 hours. The wild-type ornithine decarboxylase strain (KCCM11240P) had an average increase of about 86% P. In addition, the state exhibited an average increase of about 21% P compared to the strain having the wild-type ornithine decarboxylase from Lactobacillus.

另外，可知因丁二胺生產時的副反應而戊二胺生產減少約41%P，並且根據殘留於培養液內的葡萄糖濃度推算，相同時間內的糖消耗量增加，生產性一併增加。 In addition, it was found that the production of pentamethylenediamine decreased by about 41%P due to a side reaction in the production of butanediamine, and the sugar consumption increased in the same time period, and the productivity was also increased, estimated from the glucose concentration remaining in the culture solution.

如上所述的結果表示，由於除乳桿菌鳥胺酸脫羧酶的第713個丙胺酸被白胺酸取代的變異體以外，被其他疏水性胺基酸中之一的纈胺酸、鹼性胺基酸中之一的精胺酸、酸性胺基酸中之一的天冬胺酸、中性胺基酸中之一的麩醯胺酸、芳香族性胺基酸中之一的色胺酸取代，因此，不僅可生產相對於丁二胺生產菌株中的糖消耗高於現有的濃度的丁二胺，而且具有減少戊二胺及提高生產性的效果。 The above results show that, except for the variant in which the 713th alanine of Lactobacillus ornithine decarboxylase is replaced by leucine, valine and basic amine, which are one of the other hydrophobic amino acids, are Arginine, one of the base acids, aspartic acid, one of the acidic amino acids, glutamic acid, one of the neutral amino acids, tryptophan, one of the aromatic amino acids Therefore, it is possible not only to produce butanediamine at a higher concentration than the conventional sugar consumption in the butanediamine-producing strain, but also to reduce pentamethylenediamine and improve productivity.

根據以上說明，本申請案所屬的技術領域內的業者應可 理解，本申請案可不變更其技術思想或必要特徵而以其他具體的形態實施。與此相關，應理解，以上所記述的實施例於所有方面均為例示，並無限制性含義。本申請案的範圍應解釋為根據下文敍述的申請專利範圍的含義、範圍及其等效概念導出的所有變更或變形的形態包括於本申請案的範圍，而並非上述詳細說明。 According to the above description, those in the technical field to which this application belongs should be able to It is understood that the present application can be implemented in other specific forms without changing its technical idea or essential features. In this connection, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning, scope and equivalent concepts of the scope of the application described below, rather than the above detailed description.

<110> CJ第一製糖股份有限公司(CJ CheilJedang Corporation) <110> CJ CheilJedang Corporation

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Claims (16)

一種鳥胺酸脫羧酶變異體，其在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置、或c)第713個位置及第698個位置處包括胺基酸取代。 An ornithine decarboxylase variant comprising an amino acid corresponding to a) the 713th position, b) the 698th position, or c) the 713th position and the 698th position of SEQ ID NO: 1 replace. 如申請專利範圍第1項所述的鳥胺酸脫羧酶變異體，其中所述第713個位置的胺基酸取代是由除丙胺酸以外的疏水性胺基酸、鹼性胺基酸、酸性胺基酸、中性胺基酸或芳香族性胺基酸取代。 The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is a hydrophobic amino acid other than alanine, a basic amino acid, an acidic Amino acid, neutral amino acid or aromatic amino acid substitution. 如申請專利範圍第1項所述的鳥胺酸脫羧酶變異體，其中所述第713個位置的胺基酸取代為A713L、A713I、A713V、A713R、A713D、A713W或A713Q。 The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W or A713Q. 如申請專利範圍第1項所述的鳥胺酸脫羧酶變異體，其中所述第698個位置的胺基酸取代為E698D。 The ornithine decarboxylase variant according to claim 1, wherein the amino acid substitution at the 698th position is E698D. 如申請專利範圍第1項所述的鳥胺酸脫羧酶變異體，其中所述第713個位置的胺基酸取代為A713L、A713I、A713V、A713R、A713D、A713W或A713Q，所述第698個位置的胺基酸取代為E698D。 The ornithine decarboxylase variant of claim 1, wherein the amino acid substitution at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W or A713Q, and the 698th position The amino acid substitution at position is E698D. 如申請專利範圍第1項所述的鳥胺酸脫羧酶變異體，其中所述鳥胺酸脫羧酶變異體包括選自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：9及SEQ ID NO：19至SEQ ID NO：23中的多肽。 The ornithine decarboxylase variant according to claim 1, wherein the ornithine decarboxylase variant comprises a variant selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 9 Polypeptides in ID NO: 19 to SEQ ID NO: 23. 一種聚核苷酸，其編碼如申請專利範圍第1項至第6項中任一項所述的鳥胺酸脫羧酶變異體。 A polynucleotide encoding the ornithine decarboxylase variant described in any one of items 1 to 6 of the application scope. 一種微生物，其包括如下的鳥胺酸脫羧酶：包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第713個位置及第698個位置處包括胺基酸取代的多肽。 A microorganism comprising the following ornithine decarboxylase: comprising the polypeptide of SEQ ID NO:1, or at a) the 713th position, b) the 698th position or c) the 713th position corresponding to SEQ ID NO:1 The amino acid substituted polypeptides are included at the 698th position and at the 698th position. 如申請專利範圍第8項所述的微生物，其中所述微生物為埃希氏菌屬或棒狀桿菌屬。 The microorganism according to claim 8, wherein the microorganism is Escherichia or Corynebacterium. 一種丁二胺的生產方法，其包括培養包含鳥胺酸脫羧酶的微生物，其中所述鳥胺酸脫羧酶包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第713個位置及第698個位置處包括胺基酸取代的多肽。 A method for producing butanediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1 or in a) corresponding to SEQ ID NO: 1 Polypeptides comprising amino acid substitutions at positions 713, b) 698 or c) 713 and 698. 如申請專利範圍第10項所述的丁二胺的生產方法，其包括於培養基內累積丁二胺。 The method for producing butanediamine according to item 10 of the application scope, comprising accumulating butanediamine in a culture medium. 如申請專利範圍第10項所述的丁二胺的生產方法，其包括自培養的所述微生物或培養基回收丁二胺。 The method for producing butanediamine as described in item 10 of the patent application scope, comprising recovering butanediamine from the cultured microorganism or culture medium. 一種增加丁二胺的純度的方法，其包括培養包含鳥胺酸脫羧酶的微生物，其中所述鳥胺酸脫羧酶包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第713個位置及第698個位置位置處包括胺基酸取代的多肽。 A method for increasing the purity of butanediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1, or in a corresponding to SEQ ID NO: 1 ) at position 713, b) at position 698, or c) at positions 713 and 698 comprising amino acid substitutions. 一種增加丁二胺相對於戊二胺的比率的方法，其包括培養包含鳥胺酸脫羧酶的微生物，其中所述鳥胺酸脫羧酶包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第 713個位置及第698個位置處包括胺基酸取代的多肽。 A method of increasing the ratio of butanediamine to pentamethylenediamine, comprising culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises the polypeptide of SEQ ID NO: 1, or in a manner corresponding to SEQ ID NO: 1 of a) 713th position, b) 698th position or c) th Amino acid substituted polypeptides are included at positions 713 and 698. 一種聚醯胺的製備方法，其是利用丁二胺製備聚醯胺的方法，所述丁二胺是藉由培養包括鳥胺酸脫羧酶的微生物而製備，所述鳥胺酸脫羧酶包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第713個位置及第698個位置處包括胺基酸取代的多肽。 A method for preparing polyamide, which is a method for preparing polyamide using butanediamine, the butanediamine is prepared by culturing a microorganism comprising ornithine decarboxylase, the ornithine decarboxylase comprising SEQ The polypeptide of ID NO: 1, or a polypeptide comprising amino acid substitutions at positions corresponding to a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 . 一種聚醯胺製備用組成物，其包括包含鳥胺酸脫羧酶的微生物，所述鳥胺酸脫羧酶包括SEQ ID NO：1的多肽、或在對應於SEQ ID NO：1的a)第713個位置、b)第698個位置或c)第713個位置及第698個位置處包括胺基酸取代的多肽。 A composition for the preparation of polyamide, comprising a microorganism comprising an ornithine decarboxylase, the ornithine decarboxylase comprising the polypeptide of SEQ ID NO: 1, or in a) section 713 corresponding to SEQ ID NO: 1 A polypeptide comprising amino acid substitutions at position 1, b) position 698, or c) position 713 and position 698.

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