TWI512105B - Bacterial strain for producing butyrate, bacterial strain for producing n-butanol, and method of production of n-butanol from butyrate - Google Patents
Bacterial strain for producing butyrate, bacterial strain for producing n-butanol, and method of production of n-butanol from butyrate Download PDFInfo
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本發明關於一種利用代謝工程建構的菌株，且特別攸關一種利用此技術建構而成並可生產丁酸的菌株、與生產正丁醇的菌株。The present invention relates to a strain constructed using metabolic engineering, and particularly relates to a strain constructed by this technique and capable of producing butyric acid, and a strain producing n-butanol.
丁酸為一種短鏈脂肪酸並可用於多種不同用途。舉例而言，丁酸的衍生物丁酸酯(butyrate ester)可為飲料、食品或化妝品的調味劑(參閱Armstronga and Yamazakib 1986、Dwidar et al.2012)。另舉例而言，丁酸與纖維素組成的聚合物可用來製造塑膠或紡織纖維(參閱Cao et al.2011、El-Shafee et al.2001)。更舉例而言，丁酸具備抗癌療效(參閱Rephaeli et al.2000)。此外，由於丁酸為正丁醇等生物燃料的前驅物，故其商業價值相當可觀。已有文獻提出觸媒轉化丁酸成正丁醇的方法，如微生物的生物轉化、或化學催化劑涉及的氫化反應(參閱Dwidar et al.2012、Kim et al.2011)。Butyric acid is a short chain fatty acid and can be used in a variety of different applications. For example, the butyrate ester of butyric acid can be a flavoring agent for beverages, foods or cosmetics (see Armstronga and Yamazakib 1986, Dwidar et al. 2012). As another example, polymers of butyric acid and cellulose can be used to make plastic or textile fibers (see Cao et al. 2011, El-Shafee et al. 2001). More specifically, butyric acid has anticancer effects (see Rephaeli et al. 2000). In addition, since butyric acid is a precursor of biofuels such as n-butanol, its commercial value is considerable. Catalysts for the conversion of butyric acid to n-butanol have been proposed in the literature, such as biotransformation of microorganisms or hydrogenation reactions involving chemical catalysts (see Dwidar et al. 2012, Kim et al. 2011).
丁酸目前於商業上的製造為採用原油為原料的化學合成(參閱Cascone 2008)。然而，鑒於對全球暖化的關切與自然產物的需求已迫使產業重視丁酸的發酵製造。梭菌屬(Clostridium )為一格蘭氏陽性並產孢子的絕對厭氧菌。由於梭菌屬具高丁酸產率與產效，長久以來一直研究此菌 的丁酸生成(參閱Zhang et al.2009)。不過，梭菌屬的發酵相當繁瑣，故須相當留意影響丁酸生成的變因(參閱Dwidar et al.2012)。梭菌屬的典型發酵依序有產酸(acidogenesis)階段與產溶劑(solventogenesis)階段。於第一階段，丁酸、乙酸及氫為主要產物。接著，所產之酸於第二階段中的再吸收將生成丙酮、正丁醇與乙醇，這即為所謂的「ABE發酵(ABE fermentation)」(參閱Jones and Woods 1986)。另外，梭菌屬之基因工具的缺乏與對其生理資訊之瞭解的不足亦阻卻利用此菌生產丁酸的發展。Butyric acid is currently commercially produced as a chemical synthesis using crude oil as a raw material (see Cascone 2008). However, given the concerns about global warming and the demand for natural products, the industry has been forced to pay attention to the fermentation of butyric acid. Clostridium (Clostridium) is a Gram-positive and anaerobic spore-absolute. Due to the high butyric acid yield and productivity of Clostridium, the production of butyric acid has been studied for a long time (see Zhang et al. 2009). However, the fermentation of Clostridium is quite cumbersome, so it is necessary to pay attention to the factors that affect the formation of butyric acid (see Dwidar et al. 2012). The typical fermentation of Clostridium has an acidogenesis phase and a solventogenesis phase. In the first stage, butyric acid, acetic acid and hydrogen are the main products. Next, the re-absorption of the acid produced in the second stage will produce acetone, n-butanol and ethanol, which is called "ABE fermentation" (see Jones and Woods 1986). In addition, the lack of genetic tools of Clostridium and the lack of understanding of its physiological information also hindered the development of the production of butyric acid.
近年來，正丁醇已逐漸受到國內、外大廠的重視，此主因在於正丁醇具有高能源密度與接近汽油的性質，且正丁醇還可產生較乙醇多的能量並具有低腐蝕性。於是，無須更換既有的汽油輸送管線及儲存設備，便能將汽油替換為正丁醇，且正丁醇的運送亦無安全疑慮。另外，正丁醇本身即可作為液態燃料。舉例而言，85%正丁醇與汽油的混合物可直接用於既有的汽油引擎，且燃燒後，僅排放二氧化碳，不會產生SOX 、NOX 與一氧化碳等有毒氣體。倘若正丁醇的來源為生物質，則其燃燒便可完成自然界的二氧化碳循環，因此正丁醇亦可視為環境友好的綠色能源(參閱Duerre 2007)。正丁醇的傳統製造為微生物的發酵。在西元1916年，人類便知道利用丙酮丁醇梭菌(Clostridium acetobutylicum )生產丙酮、正丁醇及乙醇，此為ABE發酵(參閱Lee et al.2008)。在發酵過程中，先生產丁酸、丙酸、乙酸與乳酸，當發酵液的pH值下降時，則會走向所謂蝴蝶(butterfly)」代謝途徑，以產生丙酮、正丁醇及乙醇(參閱Jones and Woods 1986)。由此可知，各界均重視如何從生物資來製得正丁醇。可想而知的是：生物質的利用將能大幅降低石化燃料造成的溫室效應。In recent years, n-butanol has gradually attracted the attention of domestic and foreign manufacturers. The main reason is that n-butanol has high energy density and close to gasoline, and n-butanol can produce more energy than ethanol and has low corrosivity. . As a result, gasoline can be replaced with n-butanol without replacing the existing gasoline pipeline and storage equipment, and there is no safety concern with the delivery of n-butanol. In addition, n-butanol itself can be used as a liquid fuel. For example, a mixture of gasoline with 85% n-butanol may be used directly in existing petrol engines, and combustion, only carbon dioxide emissions, no toxic gases SO X, NO X and carbon monoxide. If the source of n-butanol is biomass, it burns to complete the natural carbon dioxide cycle, so n-butanol can also be considered an environmentally friendly green energy source (see Duerre 2007). The traditional manufacture of n-butanol is the fermentation of microorganisms. In 1916, humans knew that acetone, n-butanol and ethanol were produced using Clostridium acetobutylicum , which is ABE fermentation (see Lee et al. 2008). In the fermentation process, butyric acid, propionic acid, acetic acid and lactic acid are produced first. When the pH of the fermentation broth drops, it will go to the so-called "butterfly" metabolic pathway to produce acetone, n-butanol and ethanol (see Jones). And Woods 1986). It can be seen from this that all sectors pay attention to how to produce n-butanol from biological resources. It is conceivable that the use of biomass will significantly reduce the greenhouse effect caused by fossil fuels.
大腸桿菌(Escherichia coli ，E.coli )易培養，且易進行基因操作，因而已廣泛用為生物材料。另一方面，大腸桿菌的生理資訊與發酵技術均已詳載於許多工具書。許多研究亦已提出利用大腸桿菌製造生質燃料與高價值的化學物質(參閱Clomburg and Gonzalez 2010、Yu et al.2011)。為達成有效生產丁酸與正丁醇的目標，大腸桿菌的代謝工程顯然為一可行的策略。E. coli (Escherichia coli, E.coli) easy to culture, and easy genetic manipulation, and thus has been widely used as biomaterials. On the other hand, the physiological information and fermentation technology of E. coli are detailed in many reference books. Many studies have also proposed the use of E. coli to produce biofuels and high-value chemicals (see Clomburg and Gonzalez 2010, Yu et al. 2011). In order to achieve the goal of efficiently producing butyric acid and n-butanol, the metabolic engineering of E. coli is clearly a viable strategy.
迄今僅有少數之利用大腸桿菌製造丁酸的研究。一研究使用菌株脂肪酸逆向β氧化反應(reverse β-oxidation)，此未涉及任何外源基因，且於48小時發酵後，菌株可將30g/L葡萄糖反應成1.3g/L丁酸(參閱Seregina et al.2010)。另一研究藉由梭菌屬途徑來導入丁酸合成途徑至大腸桿菌內，從而還原乙醯乙醯輔酶A(acetoacetyl-CoA)成丁醯輔酶A(butyryl-CoA)，並導入內源tesB 基因，從而轉化丁醯輔酶A成丁酸(參閱Lim et al.2013)，此研究結果顯示丁酸/乙酸重量比值(B/A比值)為41。為改進Lim等人提出的丁酸製備，近期的研究採取合成支架的方法，以結合途徑中的hbd 基因、crt 基因與ter 基因等外源基因，而其菌株於葡萄糖饋料批式供應48小時下，可將19g/L葡萄糖轉化為7.2g/L丁酸(參閱Back et al.2013)。儘管Back等人的技術可達到高丁酸產量，但同時伴隨生產4g/L乙酸(亦即，B/A比值約為1.8)。一般而言，B/A比值(或稱「丁酸選擇率(butyrate selectivity)」)越低，後續須耗費更多金錢、時間或人力等成本來純化丁酸，故B/A比值為一衡量梭菌屬發酵表現的指標(參閱Zhang et al.2009)。即使不活化乙酸合成途徑，酪丁酸梭菌(C.tyrobutyricum )依然具有為介於5-7的B/A比值(參閱Liu et al.2006)。最新的研究指出，重新建構困難梭菌(C.difficile )的丁酸 合成途徑於大腸桿菌內，所建構之菌株的丁酸產率為0.27g/L，且同時僅會產生相當微量的乙酸(參閱Aboulnaga et al.2013)。To date, only a few studies have been conducted on the production of butyric acid using E. coli. One study used reverse β-oxidation of the fatty acid of the strain, which did not involve any exogenous genes, and after 48 hours of fermentation, the strain could react 30 g/L glucose to 1.3 g/L butyric acid (see Sergina et Al.2010). Another study introduced the butyric acid synthesis pathway into Escherichia coli by the Clostridium pathway, thereby reducing acetoacetyl-CoA into butyryl-CoA and introducing the endogenous tesB gene. Thus, the conversion of Ding Coenzyme A to butyric acid (see Lim et al. 2013) showed that the butyric acid/acetic acid weight ratio (B/A ratio) was 41. In order to improve the preparation of butyric acid proposed by Lim et al., a recent study adopts a synthetic scaffold method to bind the exogenous genes such as hbd gene, crt gene and ter gene in the pathway, and the strain is supplied in a batch of glucose feed for 48 hours. Next, 19 g/L glucose can be converted to 7.2 g/L butyric acid (see Back et al. 2013). Although Back et al.'s technology can achieve high butyric acid production, it is accompanied by the production of 4 g/L acetic acid (ie, a B/A ratio of about 1.8). In general, the lower the B/A ratio (or "butyrate selectivity"), the later it will cost more money, time or labor to purify butyric acid, so the B/A ratio is a measure. An indicator of the performance of Clostridium fermentation (see Zhang et al. 2009). Even without activating the acetic acid synthesis pathway, C. tyrobutyricum still has a B/A ratio between 5 and 7 (see Liu et al. 2006). The latest research indicates that the re-construction of C. difficile 's butyric acid synthesis pathway in E. coli has a yield of butyric acid of 0.27 g/L and produces only a relatively small amount of acetic acid (at the same time). See Aboulnaga et al. 2013).
本發明之第一方案提出一種可生產丁酸的菌株，其為一大腸桿菌，且包含一λ噬菌體PL 啟動子、一齒垢密螺旋體(Treponema denticola )的ter 基因、一丙酮丁醇梭菌的crt 基因、一鉤蟲貪銅菌(Cupriavidus necator )的phaA 基因、及一丙酮丁醇梭菌的hbd 基因。λ噬菌體PL 啟動子為鑲嵌於菌株的染色體，以調控染色體上atoDA 基因操縱組的表現，而ter 基因、crt 基因、phaA 基因、及hbd 基因為鑲嵌於菌株的染色體，且菌株缺少adhE 基因、frdA 基因、及ldhA 基因。The first aspect of the present invention provides a strain capable of producing butyric acid, which is an Escherichia coli, and comprises a lambda phage P L promoter, a ter gene of Treponema denticola , and Clostridium acetobutylicum hbd gene crt gene, a C. necator (Cupriavidus necator) of the phaA gene, a Clostridium acetone and butanol. The λ phage P L promoter is embedded in the chromosome of the strain to regulate the expression of the atoDA gene manipulation group on the chromosome, and the ter gene, crt gene, phaA gene, and hbd gene are embedded in the chromosome of the strain, and the strain lacks the adhE gene, frdA gene, and ldhA gene.
於本方案的一實施方式，λ噬菌體PL 啟動子為鑲嵌於染色體上atoDA 基因操縱組的上游區域，而較佳地為取代染色體上atoDA 基因操縱組的啟動子。In one embodiment of the present invention, the lambda phage P L promoter is inserted in the upstream region of the atoDA gene manipulation group on the chromosome, and is preferably a promoter of the atoDA gene manipulation group on the substitution chromosome.
於本方案的一實施方式，ter 基因、crt 基因、phaA 基因、及hbd 基因的表現為受另一λ噬菌體PL 啟動子調控。In one embodiment of the present invention, the ter gene, the crt gene, the phaA gene, and the hbd gene are expressed by another lambda phage P L promoter.
於本方案的一實施方式，菌株為大腸桿菌BL21的衍生品系，而較佳地為BL-A1。In one embodiment of the present invention, the strain is a derivative of E. coli BL21, and is preferably BL-A1.
本發明之第二方案提出一種可生產丁酸的菌株，其為一大腸桿菌並培養於一外加乙酸的培養液中，以利用乙酸來生成丁酸，且包含一λ噬菌體PL 啟動子、一齒垢密螺旋體的ter 基因、一丙酮丁醇梭菌的crt 基因、一鉤蟲貪銅菌的phaA 基因、及一丙酮丁醇梭菌的hbd 基因。λ噬菌體PL 啟動子為鑲嵌於菌株的染色體，以調控染色體上atoDA 基因操縱組的表現，而ter 基因、crt 基因、phaA 基因、及hbd 基因為鑲嵌於菌株的染色體，且菌株缺少adhE 基因、frdA 基因、ldhA 基因、及pta 基因。The second aspect of the present invention provides a strain capable of producing butyric acid, which is an Escherichia coli and cultured in a culture medium of acetic acid to produce butyric acid using acetic acid, and comprises a lambda phage P L promoter, hbd gene ter gene of Treponema denticola, a CRT acetone C. acetobutylicum genes, phaA gene Cupriavidus a bacteria, a C. acetobutylicum and acetone. The λ phage P L promoter is embedded in the chromosome of the strain to regulate the expression of the atoDA gene manipulation group on the chromosome, and the ter gene, crt gene, phaA gene, and hbd gene are embedded in the chromosome of the strain, and the strain lacks the adhE gene, The frdA gene, the ldhA gene, and the pta gene.
於本方案的一實施方式，λ噬菌體PL 啟動子為鑲嵌於染色體上atoDA 基因操縱組的上游區域，而較佳地為取代染色體上atoDA 基因操縱組的啟動子。In one embodiment of the present invention, the lambda phage P L promoter is inserted in the upstream region of the atoDA gene manipulation group on the chromosome, and is preferably a promoter of the atoDA gene manipulation group on the substitution chromosome.
於本方案的一實施方式，ter 基因、crt 基因、phaA 基因、及hbd 基因的表現為受另一λ噬菌體PL 啟動子調控。In one embodiment of the present invention, the ter gene, the crt gene, the phaA gene, and the hbd gene are expressed by another lambda phage P L promoter.
於本方案的一實施方式，菌株為大腸桿菌BL21的衍生品系，而較佳地為BL-A1。In one embodiment of the present invention, the strain is a derivative of E. coli BL21, and is preferably BL-A1.
本發明之第三方案提出一種可生產正丁醇的菌株，其為一大腸桿菌，且包含一λ噬菌體PL 啟動子、及一丙酮丁醇梭菌的adhE2 基因。λ噬菌體PL 啟動子為鑲嵌於菌株的染色體，以調控染色體上atoDA 基因操縱組的表現，而adhE2 基因為鑲嵌於菌株的染色體，且菌株缺少adhE 基因、frdA 基因、ldhA 基因、及pta 基因。A third aspect of the present invention provides a strain capable of producing n-butanol, which is an Escherichia coli, and comprises a lambda phage PL promoter, and an adhE2 gene of Clostridium acetobutylicum. The λ phage P L promoter is a chromosome embedded in the strain to regulate the expression of the atoDA gene manipulation group on the chromosome, and the adhE2 gene is a chromosome embedded in the strain, and the strain lacks the adhE gene, the frdA gene, the ldhA gene, and the pta gene.
於本方案的一實施方式，λ噬菌體PL 啟動子為鑲嵌於染色體上atoDA 基因操縱組的上游區域，而較佳地為取代染色體上atoDA 基因操縱組的啟動子。In one embodiment of the present invention, the lambda phage P L promoter is inserted in the upstream region of the atoDA gene manipulation group on the chromosome, and is preferably a promoter of the atoDA gene manipulation group on the substitution chromosome.
於本方案的一實施方式，adhE2 基因的表現為受另一λ噬菌體PL 啟動子調控。In one embodiment of the present scheme, the adhE2 gene is expressed by another lambda phage P L promoter.
於本方案的一實施方式，菌株為大腸桿菌BL21的衍生品系，而較佳地為BL-A1。In one embodiment of the present invention, the strain is a derivative of E. coli BL21, and is preferably BL-A1.
本發明之第四方案提出一種自丁酸生成正丁醇的方法，此方 法包含以下步驟：共同培養如第二方案的菌株與如第三方案的菌株於一含乙酸及葡萄糖的培養液中，藉此前者的菌株利用葡萄糖及乙酸來生成丁酸，而後者的菌株再利用丁酸及葡萄糖來生成正丁醇及乙酸。A fourth aspect of the present invention provides a method for producing n-butanol from butyric acid, which is The method comprises the steps of co-cultivating a strain according to the second embodiment and a strain according to the third embodiment in a culture solution containing acetic acid and glucose, whereby the former strain utilizes glucose and acetic acid to form butyric acid, and the latter strain Butyric acid and glucose are used to form n-butanol and acetic acid.
於本方案的一實施方式，培養液中的乙酸濃度為2g/L。In one embodiment of the present embodiment, the concentration of acetic acid in the culture solution is 2 g/L.
於本方案的一實施方式，後者之菌株相對於前者之菌株的起始細胞濃度比為1：3至2：1，而較佳地為1：2。In one embodiment of the present invention, the initial cell concentration ratio of the latter strain relative to the former strain is from 1:3 to 2:1, and preferably 1:2.
第1圖為菌株BuT-8LA的丁酸合成途徑。Figure 1 shows the butyric acid synthesis pathway of the strain BuT-8LA.
第2圖為菌株BuT-8L-ato的丁酸合成途徑。Figure 2 shows the butyric acid synthesis pathway of the strain BuT-8L-ato.
第3圖說明著菌株BuT-8L、BuT-8LA、及BuT-8L-ato於含葡萄糖的培養液中發酵24小時後產生的丁酸量及葡萄糖的剩餘量。Fig. 3 is a graph showing the amount of butyric acid and the amount of glucose remaining after fermentation of the strains BuT-8L, BuT-8LA, and BuT-8L-ato in a glucose-containing culture solution for 24 hours.
第4圖說明著菌株BuT-8L-ato於含不同濃度乙酸鈉之培養液中發酵24小時後產生的丁酸量及葡萄糖的剩餘量。Figure 4 is a graph showing the amount of butyric acid and the remaining amount of glucose produced by the strain BuT-8L-ato after 24 hours of fermentation in a medium containing different concentrations of sodium acetate.
第5圖說明著菌株BuT-8L-ato先培養於含12g/L葡萄糖與8g/L乙酸鈉的培養液中24小時，再添加6g/L葡萄糖至培養液中持續培養後產生的丁酸量及葡萄糖的剩餘量。Figure 5 shows the amount of butyric acid produced by the strain BuT-8L-ato cultured in a medium containing 12 g/L glucose and 8 g/L sodium acetate for 24 hours, and then adding 6 g/L glucose to the culture medium for continuous culture. And the remaining amount of glucose.
第6圖說明著菌株BuT-8L-ato先培養於含12g/L葡萄糖與8g/L乙酸鈉的培養液中24小時，再添加8g/L葡萄糖至培養液中持續培養後產生的丁酸量及葡萄糖的剩餘量。Figure 6 shows the amount of butyric acid produced by the strain BuT-8L-ato grown in a medium containing 12g/L glucose and 8g/L sodium acetate for 24 hours, and then adding 8g/L glucose to the culture medium for continuous culture. And the remaining amount of glucose.
第7圖說明著菌株BuT-8L-ato先培養於含12g/L葡萄糖與8g/L乙酸鈉的培養液中24小時，再添加10g/L葡萄糖至培養液中持續培養後產生的丁酸量及葡 萄糖的剩餘量。Figure 7 shows the amount of butyric acid produced by the strain BuT-8L-ato cultured in a medium containing 12 g/L glucose and 8 g/L sodium acetate for 24 hours, and then adding 10 g/L glucose to the culture medium for continuous culture. And Portugal The remaining amount of sugar.
第8圖為菌株BuT-3BE的正丁醇合成途徑。Figure 8 is the n-butanol synthesis pathway of the strain BuT-3BE.
第9圖說明著菌株BuT-3BE培養於外加5g/L酵母萃取物、20g/L葡萄糖及不同濃度丁酸的M9 mineral培養液中24小時後產生的正丁醇量與乙酸量。Fig. 9 is a graph showing the amount of n-butanol and the amount of acetic acid produced by the strain BuT-3BE cultured in an M9 mineral medium supplemented with 5 g/L of yeast extract, 20 g/L of glucose and different concentrations of butyric acid for 24 hours.
第10圖為於共同培養菌株BuT-8L-ato與菌株BuT-3BE下的正丁醇合成途徑。Figure 10 shows the n-butanol synthesis pathway in the co-cultured strain BuT-8L-ato and the strain BuT-3BE.
第11圖說明著以不同菌株BuT-8L-ato與菌株BuT-3BE的起始細胞濃度比共同培養此二菌株於外加2g/L乙酸的培養液中24小時後產生的正丁醇量與丁酸量。Figure 11 shows the amount of n-butanol produced by the co-cultivation of the two strains in the culture medium supplemented with 2 g/L acetic acid after 24 hours of the initial cell concentration of the different strains of BuT-8L-ato and the strain BuT-3BE. The amount of acid.
第12圖說明著先以菌株BuT-8L-ato與菌株BuT-3BE的起始細胞濃度比為1：3共同培養此二菌株於外加2g/L乙酸的培養液中16小時，再另加入含菌株BuT-3BE的菌液至培養液中使培養液總體OD550 值為2.0並培養8小時後產生的正丁醇量與乙酸量。Figure 12 shows that the two strains were co-cultured with the initial cell concentration ratio of the strain BuT-8L-ato and the strain BuT-3BE for 1:16, and then added with 2 g/L acetic acid in the culture solution for 16 hours. BuT-3BE strain of bacteria to the culture broth generally manipulation was OD 550 of 2.0 and incubated for an amount of n-butanol and the amount of acetic acid produced after 8 hours.
首先，介紹本發明提到之基因的全名，如下：adhE ，乙醛輔酶A/乙醇去氫酶(acetaldehyde-CoA/alcohol dehydrogenase)；adhE2 ，丁醛/丁酸去氫酶(butyraldehyde/butanol dehydrogenase)；atoDA ，乙醯乙醯輔酶A轉移酶(acetoacetyl-CoA transferase)；crt ，巴豆酸酶(crotonase)；frdA ，富馬酸還原酶(fumarate reductase)；hbd ，3-羥基丁基輔酶A去氫酶(3-hydroxybutyryl-CoA dehydorgenase)；ldhA ，乳酸去氫酶(lactate dehydrogenase)；phaA ，β-酮硫解酶(β-ketothiolase)；pta ，磷酸乙醯轉移酶(phosphate acetyltransferase)；ter ，反烯醯基輔酶A還原酶(trans-enoyl-coA reductase)。First, the full name of the gene mentioned in the present invention is introduced as follows: adhE , acetaldehyde-CoA/alcohol dehydrogenase; adhE2 , butyraldehyde/butanol dehydrogenase (butyraldehyde/butanol dehydrogenase) atoDA , acetoacetyl-CoA transferase; crt , crotonase; frdA , fumarate reductase; hbd , 3-hydroxybutyrate A Hydrogenase (3-hydroxybutyryl-CoA dehydorgenase); ldhA , lactate dehydrogenase; phaA , β-ketothiolase; pta , phosphate acetyltransferase; ter , Trans-enoyl-coA reductase.
其次，為讓本發明上述及/或其他目的、功效及特徵能更明顯易懂，下文特舉具體實施例做詳細說明：In the following, in order to make the above and/or other objects, functions and features of the present invention more obvious and obvious, the following detailed description is given in detail.
以下實施例使用的實驗方法與材料，茲如下所述：The experimental methods and materials used in the following examples are as follows:
I、基因的剔除與***I, gene culling and insertion
使用的菌株、質體與引子列示於表1。DNA操作為透過大腸桿菌DH5α(pir)為中介菌株來完成，發酵則為利用大腸桿菌BL21的衍生品系BL-A1來進行。The strains, plastids and primers used are shown in Table 1. The DNA operation was carried out by using Escherichia coli DH5α (pir) as an intermediate strain, and the fermentation was carried out using the derivative line BL-A1 of Escherichia coli BL21.
大腸桿菌之frdA 基因的剔除為參考Chiang et al.2008、Chiang et al.2011、Chiang et al.2013等文獻提出的方法來操作。詳言之，先利用FrdA1引子(SEQ ID NO.：1)與FrdA2引子(SEQ ID NO.：2)對大腸桿菌CGSC10964的染色體進行增幅，以得到一含***至FRT-kan -FRT盒匣(cassette)之frdA 基因的DNA片段。接著，電穿孔轉形此片段至一有質體pKD46的菌株內(參閱Datsenko and Wanner 2000)。經菌株染色體與此片段 進行同源重組後，篩選出染色體帶有此片段的菌株。最後，參考Datsenko and Wanner 2000以協助質體pCP20移除此片段的標記基因。同理，利用LdhA1引子(SEQ ID NO.：3)與LdhA2引子(SEQ ID NO.：4)增幅大腸桿菌CGSC9216的染色體，以得到一含有***至FRT-kan -FRT盒匣之ldhA 基因的DNA片段，再利用此片段完成大腸桿菌之ldhA 基因的剔除；利用Pta1引子(SEQ ID NO.：5)與Pta2引子(SEQ ID NO.：6)對質體pMC-ptaKm進行增幅，以得到一含***至FRT-kan -FRT盒匣之pta 基因的DNA片段，而利用此片段完成大腸桿菌之pta 基因的剔除。The knockdown of the frdA gene of Escherichia coli was performed by referring to the methods proposed in the literatures of Chiang et al. 2008, Chiang et al. 2011, Chiang et al. 2013, and the like. In particular, the chromosome of E. coli CGSC10964 was first amplified using the FrdA1 primer (SEQ ID NO.: 1) and the FrdA2 primer (SEQ ID NO.: 2) to obtain an insertion into the FRT- kan- FRT cassette ( A DNA fragment of the frdA gene of cassette. This fragment was then electroporated into a strain with plastid pKD46 (see Datsenko and Wanner 2000). After the homologous recombination of the chromosome of the strain with this fragment, the strain carrying the fragment on the chromosome is screened. Finally, reference is made to Datsenko and Wanner 2000 to assist the plastid pCP20 in removing the marker gene of this fragment. Similarly, the chromosome of E. coli CGSC9216 was amplified by LdhA1 primer (SEQ ID NO.: 3) and LdhA2 primer (SEQ ID NO.: 4) to obtain a DNA containing the ldhA gene inserted into the FRT- kan- FRT cassette. Fragment, and then using this fragment to complete the knockdown of the ldhA gene of Escherichia coli; using the Pta1 primer (SEQ ID NO.: 5) and the Pta2 primer (SEQ ID NO.: 6) to increase the plastid pMC-ptaKm to obtain an insertion The DNA fragment of the pta gene of the FRT- kan- FRT cassette was used, and the fragment of the pta gene of Escherichia coli was used to complete the deletion.
外源基因***至大腸桿菌染色體的操作係參考Chiang et al.2012報導的方法來完成的。詳言之，先提供一含有可表現φ80整合酶(φ80 integrase)之質體pAH123的大腸桿菌。接著，轉形一有齒垢密螺旋體DSM14222之ter 基因的質體pPhi-Ter至菌株內(參閱Haldimann and Wanner 2001)。於菌株染色體與此片段進行同源重組後，篩選出染色體基因帶有ter 基因之片段於染色體之φ80attB 位的菌株。最後，參考Chiang et al.2012以協助質體pTH19-CreCs移除此片段的標記基因。同樣地，***丙酮丁醇梭菌DSM792之crt 基因於菌株之染色體的λattB 位亦可實現。詳言之，先提供一含有可表現λ整合酶之質體pINT-ts的大腸桿菌；後轉形一有crt 基因的質體pLam-Crt至菌株內(參閱Haldimann and Wanner 2001)。此外，得到之菌株的染色體還有一額外的φ80attB 位位於菌株染色體的adhE 基因。詳細地說，先對一有夾於φ80attB -LE*-gen -RE*盒匣之二adhE 基因衍生區域的DNA片段增幅，此些衍生區域為利用AdE1引子(SEQ ID NO.：7)與AdE2引子(SEQ ID NO.：8)、及AdE3引子(SEQ ID NO.：9)與AdE4引子(SEQ ID NO.：10) 對質體pBlue-P80Gn進行重疊PCR(overlapping PCR)取得的(參閱Chiang et al.2013)。於增幅之DNA片段電穿孔送進一有質體pKD46的菌株後，篩選出染色體之φ80attB 位有adhE 基因的菌株。接著，利用質體pPhi-PhaAHbd來將鉤蟲貪銅菌之phaA 基因與丙酮丁醇梭菌DSM792之hbd 基因***至菌株染色體的φ80attB 位，並剔除其adhE 基因。最後，以質體pLam-AdhE***丙酮丁醇梭菌DSM792之adhE2 基因於菌株染色體的λattB 位。The manipulation of the insertion of the foreign gene into the E. coli chromosome is done by reference to the method reported by Chiang et al. 2012. In particular, an Escherichia coli containing the plastid pAH123 which can express φ80 integrase (φ80 integrase) is first provided. Next, the plastid pPhi-Ter of the ter gene of Treponema pallidum DSM14222 was transformed into the strain (see Haldimann and Wanner 2001). After the homologous recombination of the chromosome of the strain with the fragment, a strain having a chromosomal gene with a fragment of the ter gene at the φ80 attB position of the chromosome was screened. Finally, refer to Chiang et al. 2012 to assist the plastid pTH19-CreCs to remove the marker gene of this fragment. Similarly, the insertion of the crt gene of Clostridium acetobutylicum DSM792 can also be achieved at the λ attB position of the chromosome of the strain. Specifically, an Escherichia coli containing a plastid pINT-ts expressing λ integrase was first provided; and a plastid pLam-Crt having a crt gene was transformed into the strain (see Haldimann and Wanner 2001). In addition, the chromosome of the obtained strain has an additional φ80 attB located in the adhE gene of the strain chromosome. In detail, firstly there is sandwiched a φ80 attB -LE * - DNA fragment derived growth two adhE gene region gen -RE * cassette, the use of such derivatives AdE1 region primers (SEQ ID NO.:7) and AdE2 primer (SEQ ID NO.: 8), and AdE3 primer (SEQ ID NO.: 9) and AdE4 primer (SEQ ID NO.: 10) were obtained by overlapping PCR of plastid pBlue-P80Gn (see Chiang Et al. 2013). After the amplified DNA fragment was electroporated into a strain with plastid pKD46, the strain with the adhE gene at the φ80 attB of the chromosome was screened. Next, the phA gene of C. necator and the hbd gene of Clostridium acetobutylicum DSM792 were inserted into the φ80 attB position of the chromosome of the strain using the plastid pPhi-PhaAHbd, and the adhE gene was deleted. Finally, the adhE2 gene of Clostridium acetobutylicum DSM792 was inserted into the λ attB position of the chromosome of the strain with plastid pLam-AdhE.
II、內源基因表現的加強II. Enhancement of endogenous gene expression
使用質體pPL-Gn將λ噬菌體PL 啟動子嵌入至質體pBlue-LamGn。詳言之，先以LPL1引子(SEQ ID NO.：11)與LPL2引子(SEQ ID NO.：12)對質體pBlue-LamGn增幅，後以LPL1引子與LPL3引子(SEQ ID NO.：13)對得到之產物增幅。第二次增幅的產物與質體pPL-Gn經限制酶Eco RI剪切後，接合剪切產物使質體pPL-Gn具有融合LE*-gen -RE*盒匣的λ噬菌體PL 啟動子。另外，以RC13034引子(SEQ ID NO.：14)與RC13035引子(SEQ ID NO.：15)增幅大腸桿菌BL21的染色體來得到一含atoD 基因上游區域及其5’端區域的DNA片段。於DNA片段與質體pBluescript經限制酶Eco RV與Sac I剪切後，接合剪切產物以取得質體pBlue-atoD。利用RC13036引子(SEQ ID NO.：16)與RC13037引子(SEQ ID NO.：17)建立出限制酶Nde I與Bam HI辨識位於質體pBlue-atoD。此外，從質體pPL-Gn以限制酶Nde I與Bam HI剪切來獲得一具有融合LE*-gen -RE*盒匣之λ噬菌體PL 啟動子的片段，並將此片段接合到質體pBlue-atoD，以取得質體pSPL-atoD，其中質體pSPL-atoD有一依序為atoD 基因上游區域、LE*-gen -RE*盒匣、λ噬菌體PL 啟動子與atoD 基因5’端區域的DNA片段。最後，於RC13034引子與RC13035 引子增幅DNA片段後，將增幅片段電穿孔送入一含質體pKD46的菌株，讓λ噬菌體PL 啟動子嵌入至菌株的染色體，以調控染色體上的atoDA 基因操縱組。The lambda phage P L promoter was inserted into the plastid pBlue-LamGn using the plastid pPL-Gn. In particular, the plastid pBlue-LamGn was first amplified with the LPL1 primer (SEQ ID NO.: 11) and the LPL2 primer (SEQ ID NO.: 12), followed by the LPL1 primer and the LPL3 primer (SEQ ID NO.: 13). The product obtained is increased. The second product to increase the plasmid pPL-Gn after restriction enzyme Eco RI cut, so that the engagement cleavage product having a plasmid pPL-Gn fusion LE * - gen -RE * cassette of phage λ P L promoter. In addition, the chromosome of E. coli BL21 was amplified with RC13034 primer (SEQ ID NO.: 14) and RC13035 primer (SEQ ID NO.: 15) to obtain a DNA fragment containing the upstream region of the atoD gene and its 5' end region. After the DNA fragment and the plastid pBluescript were cleaved by the restriction enzymes Eco RV and Sac I, the cleavage product was ligated to obtain the plastid pBlue-atoD. The restriction enzymes Nde I and Bam HI were identified using the RC13036 primer (SEQ ID NO.: 16) and the RC13037 primer (SEQ ID NO.: 17) to be located in the plastid pBlue-atoD. Further, from the plasmid pPL-Gn restriction enzymes Nde I and Bam HI to obtain a cut having a fusion LE * - gen -RE * cassette fragment of λ phage P L promoter, and the fragment bonded to the plastid pBlue-atoD, to obtain plasmid pSPL-atoD, wherein pSPL-atoD plasmid has a gene sequence of the upstream region atoD, LE * - gen -RE * cassette, λ phage P L promoter atoD gene 5 'end region DNA fragment. Finally, after the RC13034 primer and RC13035 primer were used to amplify the DNA fragment, the amplified fragment was electroporated into a strain containing plastid pKD46, and the λ phage P L promoter was inserted into the chromosome of the strain to regulate the atoDA gene manipulation group on the chromosome. .
III、菌株的培養III. Cultivation of strains
將大腸桿菌置於於37℃的LB培養液內隔夜培養，菌體濃度則是根據菌液的OD550 值決定。為產生丁酸，將隔夜培養過的菌株接植於容積125mL的錐形瓶內，瓶內有50mL修飾TB培養液(含12g/L胰蛋白(tryptone)、24g/L酵母萃取物(yeast extract)、2.13g/L磷酸二氫鉀(KH2 PO4 )、12.54g/L磷酸氫二鉀(K2 HPO4 ))外加12g/L葡萄糖，且菌液的起始OD550 值為0.1。為製備正丁醇，菌株是生長於M9 mineral培養液(參閱Miller 1972)加上20g/L葡萄糖及5g/L酵母萃取物，且菌液的起始OD550 值為0.2。Escherichia coli was cultured overnight in an LB medium at 37 ° C, and the cell concentration was determined based on the OD 550 value of the bacterial solution. In order to produce butyric acid, the overnight cultured strain was transplanted into a 125 mL conical flask containing 50 mL of modified TB medium (containing 12 g/L tryptone, 24 g/L yeast extract (yeast extract) ), 2.13g / L potassium dihydrogen phosphate (KH 2 PO 4), 12.54g / L potassium phosphate dibasic (K 2 HPO 4)) plus 12g / L glucose, bacteria and starting OD 550 was 0.1. For the preparation of n-butanol, the strain is grown in medium M9 mineral (see Miller 1972) plus 20g / L glucose and 5g / L yeast extract, and the broth of the initial OD 550 value of 0.2.
IV、成分或酵素活性的測定Determination of IV, composition or enzyme activity
葡萄糖、有機酸(如丁酸)及正丁醇的濃度主要是採用高效液相層析(high-performance liquid chromatography，HPLC)與氣相層析(gas chromatography，GC)來測定的(參閱Chiang et al.2012)。為測量酵素AtoDA的活性，菌株於培養24小時後，離心收集於67mM Tris-HCl(pH 8.0)中。接著，超音波震盪以打破菌株後，將震盪後的溶液離心並取出離心得到的上清液(亦稱作「無細胞萃取液(cell-free extract，CFX)」)。混合10μL CFX與90μL反應液(含20mM丁醯輔酶A、0.4mM乙酸鈉(sodium acetate)、67mM Tris-HCl(pH 8.0))後，進行酵素反應。反應約進行20分鐘，並加熱混合液至100℃，持溫10分鐘來終止反應。利用HPLC定量混合液中的丁酸濃度， 而酵素活性(U/mg)表示為單位重量之CFX於每分鐘內所產生的丁酸莫耳數。The concentrations of glucose, organic acids (such as butyric acid) and n-butanol are mainly determined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) (see Chiang et al). Al.2012). To measure the activity of the enzyme AtoDA, the strain was collected by centrifugation in 67 mM Tris-HCl (pH 8.0) after 24 hours of culture. Next, after the ultrasonic wave is shaken to break the strain, the oscillating solution is centrifuged and the supernatant obtained by centrifugation (also referred to as "cell-free extract (CFX)") is taken out. After 10 μL of CFX and 90 μL of a reaction solution (containing 20 mM of butazone coenzyme A, 0.4 mM sodium acetate, and 67 mM Tris-HCl (pH 8.0)), an enzyme reaction was carried out. The reaction was carried out for about 20 minutes, and the mixture was heated to 100 ° C, and the reaction was terminated by holding the temperature for 10 minutes. Quantify the concentration of butyric acid in the mixture by HPLC, The enzyme activity (U/mg) is expressed as the number of moles of butyric acid produced per minute per unit weight of CFX.
實施例1：菌株之丁酸合成途徑的建構Example 1: Construction of a butyric acid synthesis pathway of a strain
丁酸合成途徑是建構於大腸桿菌BL-A1中。根據文獻Chiang et al.2013報導，此菌株缺少poxB 基因且其原先的傳送葡萄糖系統已置換為運動發酵單胞菌(Zymomonas mobilis )的glf 基因。首先，剔除菌株的adhE 基因、ldhA 基因、frdA 基因，以減少NADH的消耗並減少副產物的生成。接著，受λ噬菌體PL 啟動子調控的外源phaA 基因、hbd 基因、crt 基因與ter 基因***至菌株的染色體，此時的菌株稱作「BuT-8L」。據前文所述之「實驗方法與材料」得知，於***phaA 基因及hbd 基因的同時，亦剔除菌株的adhE 基因。如此一來，丁酸合成途徑中的碳通量自乙醯輔酶A(acetyl-CoA)轉移至丁醯輔酶A。最後，透過融合λ噬菌體PL 啟動子至菌株BuT-8L染色體的atoDAEB 基因操縱組來強化內源atoDA 基因操縱組的表現，此融合菌株特別命名為「BuT-8LA」(見第1圖)。依照前文「酵素AtoDA的活性測試」，菌株BuT-8LA具有較菌株BuT-8L大於8倍的酵素活性(菌株BuT-8LA：0.56U/mg，菌株BuT-8L：0.07U/mg)。The butyric acid synthesis pathway was constructed in E. coli BL-A1. Chiang et al.2013 according to literature reports, this strain lacks poxB gene and its original glucose transport system has been replaced with a gene glf Z. mobilis (Zymomonas mobilis) a. First, the adhE gene, ldhA gene, and frdA gene of the strain were eliminated to reduce the consumption of NADH and reduce the production of by-products. Next, the exogenous phaA gene, hbd gene, crt gene and ter gene regulated by the lambda phage P L promoter are inserted into the chromosome of the strain, and the strain at this time is called "BuT-8L". According to the "Experimental Methods and Materials" described above, the adhE gene of the strain was also removed while inserting the phaA gene and the hbd gene. As a result, the carbon flux in the butyric acid synthesis pathway is transferred from aceto-coenzyme A (acetyl-CoA) to butyl sulfonamide A. Finally, to atoDAEB genetic manipulation group strain BuT-8L chromosomes to strengthen the performance of the endogenous atoDA genetic manipulation group through fusion λ phage P L promoter, the fused strain typically named "BuT-8LA" (see FIG. 1). According to the above "Activity AtoDA Activity Test", the strain BuT-8LA has an enzyme activity greater than 8 times that of the strain BuT-8L (strain BuT-8LA: 0.56 U/mg, strain BuT-8L: 0.07 U/mg).
實施例2：菌株的丁酸生成Example 2: Butyric acid production of strains
為製造丁酸，將菌株BuT-8LA與BuT-8L各別搖晃培養於含葡萄糖之培養液的錐形瓶中。於發酵24小時後，菌株BuT-8LA可產生3.4g/L丁酸；反觀，菌株BuT-8L僅可產生1.0g/L丁酸(見第3圖)。由此可證實，菌株BuT-8LA具有較高之酵素AtoDA的活性，因此其能促進催化乙酸與丁醯輔酶A成丁酸及乙醯輔酶A。於菌株BuT-8LA中，乙酸轉化成丁酸以及丁醯輔酶 A轉化成乙醯輔酶A等此二反應均以乙醯輔酶A為反應前驅物。顯然地，此二轉化反應的反應速率為不相等的，因而侷限了丁酸的生成。此現象從外加乙酸至菌株BuT-8LA可提升丁酸生成來獲得證實(見第3圖)。不過，菌株BuT-8LA建構的合成途徑仍可增進丁酸的製造。To produce butyric acid, the strains BuT-8LA and BuT-8L were separately shaken and cultured in an Erlenmeyer flask containing the culture solution containing glucose. After 24 hours of fermentation, the strain BuT-8LA produced 3.4 g/L of butyric acid; in contrast, the strain BuT-8L produced only 1.0 g/L of butyric acid (see Figure 3). It can be confirmed that the strain BuT-8LA has a higher activity of the enzyme AtoDA, so it can promote the catalysis of acetic acid and butyl hydrazine coenzyme A into butyric acid and acetaminophen coenzyme A. In the strain BuT-8LA, acetic acid is converted into butyric acid and butyl hydrazine coenzyme The conversion of A to acetamyl coenzyme A and the like were carried out using acetaminophen coenzyme A as a reaction precursor. Obviously, the reaction rates of the two conversion reactions are unequal, thus limiting the formation of butyric acid. This phenomenon was confirmed by the addition of acetic acid to the strain BuT-8LA to increase butyric acid production (see Figure 3). However, the synthetic pathway constructed by the strain BuT-8LA can still improve the production of butyric acid.
酵素AtoAD催化丁醯輔酶A成丁酸的反應需要乙酸。這代表著可進一步瞭解乙酸於菌株BuT-8LA中對丁酸生成的影響。首先，於外加2g/L乙酸至培養液中，菌株BuT-8LA的丁酸產量可提高至4.1g/L(見第3圖)。須注意的是，pta 基因與ackA 基因均涉及菌株BuT-8LA的乙酸生成。於是，將菌株BuT-8LA的pta 基因剔除，以獲致菌株BuT-8L-ato(見第2圖)，菌株BuT-8L-ato僅可生產2.5g/L丁酸。綜上，證實了乙酸為影響建構之丁酸合成途徑中的關鍵。The enzyme AtoAD catalyzes the reaction of butyric acid coenzyme A into butyric acid, which requires acetic acid. This represents a further understanding of the effect of acetic acid on the formation of butyric acid in the strain BuT-8LA. First, the addition of 2 g/L acetic acid to the culture solution increased the butyric acid yield of the strain BuT-8LA to 4.1 g/L (see Figure 3). It should be noted that both the pta gene and the ackA gene are involved in the production of acetic acid from the strain BuT-8LA. Thus, the pta gene of the strain BuT-8LA was knocked out to obtain the strain BuT-8L-ato (see Fig. 2), and the strain BuT-8L-ato produced only 2.5 g/L of butyric acid. In summary, it was confirmed that acetic acid is the key to the formation of the butyric acid synthesis pathway.
實施例3：乙酸對丁酸生成的影響Example 3: Effect of acetic acid on the formation of butyric acid
如第1圖，酵素Pta、AckA與酵素AtoDA的協同可於胞內循環製造乙酸。然而，乙酸的胞內循環製造對於菌株BuT-8LA的丁酸製造並無顯著的功效(亦即，較宜地外加乙酸至菌株)。這意謂著乙酸的取得可限制丁酸的生成。因此，透過外加乙酸至菌株的方式將可提升丁酸的生成。為此，使用缺乏pta 基因的菌株BuT-8L-ato來驗證上述推論。如第4圖所示，菌株BuT-8L-ato於24小時發酵後，可完全消耗掉葡萄糖，且菌株可依外加之乙酸鈉含量的增加而提升丁酸產量。於外加乙酸鈉含量為8g/L的狀態下，菌株BuT-8L-ato的丁酸產量可達到6.8g/L。此外，此菌株亦可提升葡萄糖的消耗。由於葡萄糖能提供菌株之丁酸生成所需的乙醯輔酶A，故丁醯輔酶A與乙酸的反應將驅使葡萄糖的消耗。綜上，可推論出乙酸對於丁酸生成途徑具有 正面貢獻。As shown in Fig. 1, the synergy of the enzymes Pta, AckA and the enzyme AtoDA can produce acetic acid in the intracellular circulation. However, the intracellular circulation of acetic acid produced no significant effect on the production of butyric acid of the strain BuT-8LA (i.e., it is preferred to add acetic acid to the strain). This means that the acquisition of acetic acid can limit the formation of butyric acid. Therefore, the formation of butyric acid can be enhanced by the addition of acetic acid to the strain. To this end, the above inference was verified using the strain BuT-8L-ato lacking the pta gene. As shown in Fig. 4, the strain BuT-8L-ato can completely consume glucose after fermentation for 24 hours, and the strain can increase the yield of butyric acid according to the increase of the added sodium acetate content. Under the condition of adding sodium acetate content of 8g/L, the yield of butyric acid of strain BuT-8L-ato can reach 6.8g/L. In addition, this strain can also increase glucose consumption. Since glucose can provide the acetaminophen coenzyme A required for the production of butyric acid in the strain, the reaction of acetoin C with acetic acid will drive the consumption of glucose. In summary, it can be inferred that acetic acid has a positive contribution to the formation of butyric acid.
實施例4：葡萄糖供料對丁酸生成的影響Example 4: Effect of glucose feed on butyric acid production
可發現到菌株BuT-8L-ato於發酵結束後，仍有一半的外加乙酸未反應掉。因此，推測可透過外加葡萄糖的方式來消耗掉剩餘的乙酸。首先，將菌株BuT-8L-ato培養於含12g/L葡萄糖與8g/L乙酸鈉的培養液中。於培養24小時後，添加不同量的葡萄糖至培養液中並持續培養菌株。如第5至7圖所示，隨著培養時間愈久，菌株的丁酸產量愈多。而且，如第6、7圖所示，於外加葡萄糖之濃度為8g/L及10g/L的條件下，菌株於發酵48小時後，可產生超過10g/L丁酸。然而，此菌株的培養液僅剩餘相當微量的乙酸(小於0.07g/L)。It was found that after the fermentation of the strain BuT-8L-ato, half of the added acetic acid was not reacted. Therefore, it is speculated that the remaining acetic acid can be consumed by the addition of glucose. First, the strain BuT-8L-ato was cultured in a culture solution containing 12 g/L of glucose and 8 g/L of sodium acetate. After 24 hours of culture, different amounts of glucose were added to the culture medium and the strain was continuously cultured. As shown in Figures 5 to 7, the longer the incubation time, the more the butyric acid yield of the strain. Further, as shown in Figs. 6 and 7, the strain can produce more than 10 g/L of butyric acid after 48 hours of fermentation under the conditions of the concentration of added glucose of 8 g/L and 10 g/L. However, only a relatively small amount of acetic acid (less than 0.07 g/L) remained in the culture solution of this strain.
綜合本實施例，菌株BuT-8L-ato培養於共20g/L葡萄糖與8g/L乙酸下，可於48小時內產生至少10g/L丁酸以及相當微量的乙酸。經計算得到：菌株的B/A比值約為143，丁酸產率約為0.22g/L/h。如此，證明了本發明提出之可生產丁酸的菌株具備高丁酸產量與產率、以及極高的B/A比值，此意謂著後續僅須耗費少數的金錢、時間或人力等成本來純化丁酸。In general, the strain BuT-8L-ato was cultured in a total of 20 g/L glucose and 8 g/L acetic acid to produce at least 10 g/L butyric acid and a relatively small amount of acetic acid in 48 hours. It was calculated that the strain had a B/A ratio of about 143 and a butyric acid yield of about 0.22 g/L/h. Thus, it was proved that the strain for producing butyric acid proposed by the present invention has high butyric acid yield and yield, and a very high B/A ratio, which means that only a small amount of money, time or labor costs are required for subsequent follow-up. Purified butyric acid.
實施例5：從丁酸的正丁醇生成Example 5: Formation from n-butanol of butyric acid
如第8圖所示，正丁醇生成菌株的建構包含以下步驟。首先，剔除菌株的adhE 基因、ldhA 基因、frdA 基因、及pta 基因，以減少NADH的耗費並減少副產物的生成。接著，菌株染色體之內源atoDA 基因操縱組的表現透過融合λ噬菌體PL 啟動子至atoDAEB 基因操縱組來提高。最後，受λ噬菌體PL 啟動子調控的外源adhE2 基因***至菌株染色體內，此建構的菌株特別命名為「BuT-3BE」。As shown in Fig. 8, the construction of the n-butanol-producing strain comprises the following steps. First, the adhE gene, ldhA gene, frdA gene, and pta gene of the strain are eliminated to reduce the cost of NADH and reduce the production of by-products. Next, the expression of the endogenous atoDA gene manipulation group of the strain chromosome was enhanced by fusing the lambda phage P L promoter to the atoDAEB gene manipulation group. Finally, the exogenous adhE2 gene regulated by the lambda phage P L promoter was inserted into the chromosome of the strain, and the constructed strain was specifically named "BuT-3BE".
將菌株BuT-3BE培養於外加酵母萃取物(5g/L)、葡萄糖(20g/L)及丁酸(不同濃度)的M9 mineral培養液中。於24小時發酵後，菌株可產生正丁醇，且正丁醇的產量隨著外加的丁酸濃度增加而提升。請參照第9圖，於外加6g/L丁酸下，菌株之正丁醇的最大產量為4.3g/L，乙酸的產量為3.6g/L，且正丁醇相對於丁酸之最大莫耳轉化率約為85%。然而，於外加7g/L丁酸下，菌株之正丁醇的產量則有減少的趨勢，此現象可能為過高濃度的丁酸對菌株造成細胞毒性所致。總括上述，本實施例說明著本發明之可生產正丁醇的菌株可轉化丁酸成正丁醇。The strain BuT-3BE was cultured in an M9 mineral culture solution supplemented with yeast extract (5 g/L), glucose (20 g/L), and butyric acid (different concentrations). After 24 hours of fermentation, the strain produced n-butanol and the yield of n-butanol increased with increasing concentration of butyric acid. Please refer to Figure 9. Under the addition of 6g/L butyric acid, the maximum yield of n-butanol in the strain is 4.3g/L, the yield of acetic acid is 3.6g/L, and the maximum molar mass of n-butanol relative to butyric acid. The conversion rate is about 85%. However, under the addition of 7g/L butyric acid, the yield of n-butanol in the strain decreased, which may be caused by excessive toxicity of butyric acid to the strain. In summary, the present embodiment demonstrates that the strain producing n-butanol of the present invention can convert butyric acid to n-butanol.
實施例6：採用二不同大腸桿菌的共培養來生成正丁醇Example 6: Co-culture of two different E. coli to produce n-butanol
由實施例4確知，菌株BuT-8L-ato需要乙酸來製造丁酸。另由實施例5確知，菌株BuT-3BE利用丁酸來產生正丁醇，並伴隨產生乙酸。因此，透過組合此二菌株自葡萄糖直接製造正丁醇應為可行的(見第10圖)。It was confirmed from Example 4 that the strain BuT-8L-ato required acetic acid to produce butyric acid. It was also confirmed from Example 5 that the strain BuT-3BE utilizes butyric acid to produce n-butanol with concomitant production of acetic acid. Therefore, it should be feasible to directly produce n-butanol from glucose by combining the two strains (see Figure 10).
本實施例是採用共同培養菌株BuT-8L-ato及菌株BuT-3BE來達到上述構思。二菌株是培養於M9 mineral培養液中。培養液除了外加5g/L酵母萃取物與20g/L葡萄萄外，還外加有2g/L乙酸來啟動丁酸的製造。如第11圖所示，當菌株BuT-3BE及菌株BuT-8L-ato的起始細胞濃度比為1：2時，於培養24小時後，可得到約4.1g/L正丁醇。且經估算後，正丁醇的產生速率約為0.171g/L/h。In the present example, the above concept was achieved by cocultivating the strain BuT-8L-ato and the strain BuT-3BE. The two strains were cultured in M9 mineral medium. In addition to the addition of 5 g/L yeast extract and 20 g/L grape, the culture medium was further supplemented with 2 g/L acetic acid to start the production of butyric acid. As shown in Fig. 11, when the initial cell concentration ratio of the strain BuT-3BE and the strain BuT-8L-ato was 1:2, about 4.1 g/L of n-butanol was obtained after 24 hours of culture. And after estimation, the production rate of n-butanol is about 0.171 g/L/h.
另外，當菌株BuT-3BE及菌株BuT-8L-ato的起始細胞濃度比為1：3時，於培養16小時後，另加入含菌株BuT-3BE的菌液至培養液中使得培養液的整體OD550 值為2.0，接著再另培養8小時。於總共24小時發酵後，可得到約5.5g/L正丁酸。且經估算後，正丁醇的產生速率約為0.23g/L/h。In addition, when the initial cell concentration ratio of the strain BuT-3BE and the strain BuT-8L-ato is 1:3, after 16 hours of culture, a bacterial solution containing the strain BuT-3BE is added to the culture solution to make the culture solution The overall OD 550 value was 2.0, followed by another 8 hours of incubation. After a total of 24 hours of fermentation, about 5.5 g/L of n-butyric acid was obtained. And after estimation, the production rate of n-butanol is about 0.23 g/L/h.
依據本實施例，證實著本發明提出之自丁酸生成正丁醇的方法為有效且為有潛力的。According to this example, it was confirmed that the method of producing n-butanol from butyric acid proposed by the present invention is effective and has potential.
惟以上所述者，僅為本發明之較佳實施例，但不能以此限定本發明實施之範圍；故，凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效改變與修飾，皆仍屬本發明專利涵蓋之範圍內。The above is only the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto; therefore, the simple equivalent changes and modifications made by the scope of the present invention and the contents of the description of the invention, All remain within the scope of the invention patent.
<110> 逢甲大學<110> Feng Chia University
<120> 可生產丁酸的菌株、可生產正丁醇的菌株可及自丁酸生成正丁醇的方法<120> A strain capable of producing butyric acid, a strain capable of producing n-butanol, and a method for producing n-butanol from butyric acid
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Claims (15)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW103101762A TWI512105B (en) | 2014-01-17 | 2014-01-17 | Bacterial strain for producing butyrate, bacterial strain for producing n-butanol, and method of production of n-butanol from butyrate |
| CN201410050883.7A CN104789487B (en) | 2014-01-17 | 2014-02-14 | Bacterial strain capable of respectively producing butyric acid and n-butanol and method for producing n-butanol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| TWI622648B (en) * | 2016-12-27 | 2018-05-01 | 國立清華大學 | Butanol expression cassette, recombinant plasmid and butanol production related gene expression method |
| WO2021127648A1 (en) * | 2019-12-19 | 2021-06-24 | Auburn University | Microbial ester production |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438056B (en) * | 2019-08-12 | 2021-05-28 | 江南大学 | Construction and application of escherichia coli engineering bacteria for producing n-butyric acid |
| CN114774338B (en) * | 2022-03-29 | 2023-08-08 | 北京航空航天大学 | Probiotics for producing butyric acid, construction method and application thereof |
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| CN101668852A (en) * | 2006-12-15 | 2010-03-10 | 生物普尔肯株式会社 | Enhanced butanol producing microorganisms and method for preparing butanol using the same |
| KR101277711B1 (en) * | 2009-09-22 | 2013-06-24 | 한국과학기술원 | Enhanced Butanol Producing Recombinant Microorganisms and Method for Preparing Butanol Using the Same |
| CN104220601A (en) * | 2011-12-16 | 2014-12-17 | 英威达技术有限责任公司 | Methods of producing 6-carbon chemicals via coa-dependent carbon chain elongation associated with carbon storage |
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Non-Patent Citations (3)
| Title |
|---|
| Baek JM et al. Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol Bioeng. 2013 Oct;110(10), p 2790-2794. Epub 2013 Apr 22. * |
| Christine A. Rabinovitch-Deere et al. Synthetic Biology and Metabolic Engineering Approaches To Produce Biofuels. Chem. Rev., 2013, 113 (7), p 4611-4632. http://www.genome.jp/dbget-bin/www_bget?ec:2.8.3.9 (2001/02/01) http://www.genome.jp/dbget-bin/www_bget?ec:2.8.3.8 (2008/06/27) * |
| Claire R. Shen et al. Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli. Appl. Environ. Microbiol. May 2011, 77(9), p 2905-2915 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI622648B (en) * | 2016-12-27 | 2018-05-01 | 國立清華大學 | Butanol expression cassette, recombinant plasmid and butanol production related gene expression method |
| US10633677B2 (en) | 2016-12-27 | 2020-04-28 | National Tsing Hua University | Butanol expression cassette, recombinant plasmid and butanol production related gene expression method |
| WO2021127648A1 (en) * | 2019-12-19 | 2021-06-24 | Auburn University | Microbial ester production |
Also Published As
| Publication number | Publication date |
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| TW201529846A (en) | 2015-08-01 |
| CN104789487A (en) | 2015-07-22 |
| CN104789487B (en) | 2018-11-06 |
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