TWI535844B - Production of n - Butanol Strain and Its Application - Google Patents
Production of n - Butanol Strain and Its Application Download PDFInfo
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本發明關於一種利用基因工程技術建構的菌株，且特別關於一種可生產正丁醇的菌株及其應用。The present invention relates to a strain constructed using genetic engineering techniques, and in particular to a strain capable of producing n-butanol and uses thereof.
石化產業雖與我們的日常生活息息相關，但石化燃料價格不斷上漲、存量日趨減少與對環境造成的傷害逐日嚴重已侷限這項產業的發展。因此，確實有必要開發更多再生且環保的燃料，利用生物過程製造化學製品便應蘊而生。一般而言，生物所製造的化學製品多為微生物的發酵產物。於微生物發酵過程中，發生了許多以NADH與NAD ＋為輔因子的氧化還原反應。當NAD ＋作為電子接受者時，醣類代謝會伴隨NADH產生。之後，於NADH存在下還原醣類代謝中間物而再次產生NAD ＋。於大腸桿菌內，此種還原反應通常會產生乙醇、乳酸、琥珀酸。故，維持NADH與NAD ＋間的氧化還原平衡乃是確保微生物發酵持續的關鍵。 Although the petrochemical industry is closely related to our daily lives, the rising prices of petrochemical fuels, the declining stocks and the damage to the environment have severely limited the development of this industry. Therefore, it is indeed necessary to develop more renewable and environmentally friendly fuels, and the use of biological processes to manufacture chemicals should come into being. In general, the chemicals produced by organisms are mostly fermentation products of microorganisms. During the microbial fermentation process, many redox reactions with NADH and NAD + as cofactors occurred. When NAD + acts as an electron acceptor, carbohydrate metabolism is accompanied by NADH production. Thereafter, the saccharide metabolism intermediate is reduced in the presence of NADH to regenerate NAD + . In E. coli, this reduction usually produces ethanol, lactic acid, and succinic acid. Therefore, maintaining the redox balance between NADH and NAD + is the key to ensuring the continued fermentation of microorganisms.
梭菌屬（ Clostridium）的正丁醇製造為微生物發酵的典型例（Jones and Woods 1986）。這種發酵製造分成二階段：產酸（acidogenesis）與產溶劑（solventogenesis）（Lee et al. 2008）。產酸時，梭菌屬會發酵葡萄糖而產生乙酸、丁酸。產溶劑時，梭菌屬於再吸收這些有機酸的同時製造丙酮、正丁醇與乙醇為最終產物。依這種合成路徑，自葡萄糖直接合成正丁醇會導致NADH/NAD ＋氧化還原失衡，主因在於此合成路徑所需的NADH較於醣解途徑所產生的NADH多。正丁醇為一可替代的燃料，且就能量密度、蒸氣壓、吸濕性而言，正丁醇優於乙醇（Mussatto et al. 2008）。以特定比例混合正丁醇與汽油後，此混合物可作為交通運輸的燃料，並直接使用已存在的輸油管運輸。上述性質讓微生物所製造的正丁醇極受業界注目。 Typical examples of (Jones and Woods 1986) for the microbial fermentation of Clostridium (Clostridium) in n-butanol manufactured. This fermentation process is divided into two phases: acidogenesis and solventogenesis (Lee et al. 2008). When producing acid, Clostridium ferments glucose to produce acetic acid and butyric acid. When producing a solvent, Clostridium belongs to reabsorbing these organic acids while producing acetone, n-butanol and ethanol as final products. According to this synthetic route, the direct synthesis of n-butanol from glucose leads to an NADH/NAD + redox imbalance, mainly due to the more NADH required for this synthetic pathway than the NADH produced by the glycolytic pathway. n-Butanol is an alternative fuel, and n-butanol is superior to ethanol in terms of energy density, vapor pressure, and hygroscopicity (Mussatto et al. 2008). After mixing n-butanol with gasoline in a specific ratio, the mixture can be used as a fuel for transportation and transported directly using existing pipelines. The above properties make n-butanol produced by microorganisms extremely attractive to the industry.
目前已有許多於不同菌種內製造正丁醇（Atsumi et al. 2008、Bhandiwad et al. 2014、Berezina et al. 2010、Lan and Liao 2011、Nielsen et al. 2009、Steen et al. 2008、Tong et al. 2014）的方案。這些方案雖然可行，但因其正丁醇的產量低而無法普及。大腸桿菌已廣泛作為生物科技的工具以用來製造高價值的化學製品和生物燃料。於引用梭菌屬的合成途徑至大腸桿菌後，利用大腸桿菌製造正丁醇將大有可為。即使如此，胞內氧化還原狀態仍舊為一難以解決的問題。之後，此問題透過增加大腸桿菌內的NADH量獲致解決（Lim at el. 2013、Shen et al. 2011）。然而，這些先前技術所用的TB培養基（成分為：1.2g/L胰化蛋白（tryptone）、24g/L酵母萃取物、2.31g/L磷酸二氫鉀（KH 2PO 4）、12.54g/L磷酸氫二鉀（K 2HPO 4）、4mL/L甘油）價格不斐，限制了它們的應用。 Many n-butanols have been produced in different strains (Atsumi et al. 2008, Bhandiwad et al. 2014, Berezina et al. 2010, Lan and Liao 2011, Nielsen et al. 2009, Steen et al. 2008, Tong Et al. 2014). Although feasible, these programs are not universal due to their low production of n-butanol. E. coli has been widely used as a tool for biotechnology to make high value chemicals and biofuels. After the synthetic route of Clostridium to Escherichia coli, it is highly probable that E. coli can be used to make n-butanol. Even so, the intracellular redox state is still an intractable problem. Later, this problem was solved by increasing the amount of NADH in E. coli (Lim at el. 2013, Shen et al. 2011). However, the TB medium used in these prior art (ingredients: 1.2 g/L tryptone, 24 g/L yeast extract, 2.31 g/L potassium dihydrogen phosphate (KH 2 PO 4 ), 12.54 g/L) Dipotassium hydrogen phosphate (K 2 HPO 4 ), 4 mL/L glycerol) is not expensive and limits their use.
調控一種特殊大腸桿菌內NADH含量可提升正丁醇的產生，且引用梭菌屬的CoA依賴合成途徑至此菌株內。這種調控為透過修飾菌株的中心代謝途徑完成的，包含有：（1）將丙酮酸導入乙醯輔酶A；（2）放大五碳糖磷酸途徑（pentose phosphate pathway，PP pathway）；（3）將醣解通量引導至五碳糖磷酸途徑；（4）自檸檬酸循環（tricarboxylic acid cycle，TCA cycle）轉向乙醯輔酶A。如此一來，此菌株可培養於外加葡萄糖的M9Y培養基（含有M9鹽類與酵母萃取物）並有效地製造正丁醇。Regulation of a specific E. coli content of NADH can increase the production of n-butanol, and the CoA-dependent synthetic pathway of Clostridium is used to the strain. This regulation is accomplished through the central metabolic pathway of the modified strain, including: (1) introduction of pyruvate into acetamyl coenzyme A; (2) amplification of the pentose phosphate pathway (PP pathway); (3) The glycolysis flux is directed to the five-carbon sugar phosphate pathway; (4) from the tricarboxylic acid cycle (TCA cycle) to the acetaminophen coenzyme A. In this way, the strain can be cultured in M9Y medium (containing M9 salt and yeast extract) supplemented with glucose and efficiently produced n-butanol.
依上述概念，本發明提出一種可生產正丁醇的菌株，其屬於大腸桿菌且含有：一丙酮丁醇梭桿菌（ Clostridium acetobutylicum）的 hbd基因、一丙酮丁醇梭桿菌的 crt基因、一丙酮丁醇梭桿菌的 adhE2基因、一鉤蟲貪銅菌（ Cupriavidus necator）的 phaA基因、一齒垢密螺旋體（ Terponema denticola）的 ter基因、一釀酒酵母（ Saccharomyces cerevisiae）的 fdh1基因、一啟動子PλP L、一操作子 lacO。啟動子PλP L為鑲嵌於菌株的染色體，以調控染色體上的 zwf基因、 pgl基因、 udhA基因、以及 aceEF基因操縱組-E354K點突變的 lpdA基因，操作子 lacO為鑲嵌於菌株的染色體，以調控色體上的 gltA基因，且菌株缺少 pgi基因、 ldhA基因、 pta基因、 adhE基因、 frdA基因。 According to the above concept, the present invention provides a strain capable of producing n-butanol, which belongs to Escherichia coli and contains: hbd gene of Clostridium acetobutylicum , crt gene of Fusobacterium acetobutylicum , and acetone. The adhE2 gene of Clostridium kawaii , the phaA gene of Cupriavidus necator , the ter gene of Terponema denticola , the fdh1 gene of Saccharomyces cerevisiae , a promoter PλP L , An operator lacO . LpdA gene promoter PλP L is embedded in the chromosome of the strain to the zwf gene regulation on the chromosome, gene PGL, udhA genes, genetic manipulation and aceEF group -E354K point mutations, operation sub-chromosomal lacO mosaic strains, to regulate The gltA gene on the chromosome, and the strain lacks the pgi gene, the ldhA gene, the pta gene, the adhE gene, and the frdA gene.
於一實施例中，菌株為衍生自大腸桿菌BL21。In one embodiment, the strain is derived from E. coli BL21.
於另一實施例中，啟動子PλP L為鑲嵌於 zwf基因、 pgl基因、 udhA基因、及 aceEF基因操縱組-E354K點突變之 lpdA基因的上游區域。 In another embodiment, the promoter PλP L is an upstream region of the lpdA gene embedded in the zwf gene, the pgl gene, the udhA gene, and the aceEF gene manipulation group-E354K point mutation.
於又一實施例中，操作子 lacO為鑲嵌於 gltA基因的上游區域。 In yet another embodiment, the operator lacO is embedded in the upstream region of the gltA gene.
於再一實施例中， hbd基因、 crt基因、 adhE2基因、 phaA基因、 ter基因、 fdh1基因為受另一啟動子PλP L調控。 In still another embodiment, the hbd gene, the crt gene, the adhE2 gene, the phaA gene, the ter gene, and the fdh1 gene are regulated by another promoter PλP L .
此外，於本發明揭露的範圍內，更提出一種生產正丁醇的方法，其包含：培養上述菌株於一含葡萄糖的M9Y培養基中。Further, within the scope of the present disclosure, a method for producing n-butanol is further provided, which comprises: cultivating the above strain in a glucose-containing M9Y medium.
於一實施例中，培養基含有6g/L磷酸氫二鈉（Na 2HPO 4）、3g/L磷酸二氫鉀、0.5g/L氯化鈉、1g/L氯化銨、1mM硫酸鎂、0.1mM氯化鈣、10mg/L維他命B1、5g/L酵母萃取物、20g/L葡萄糖。 In one embodiment, the medium contains 6 g/L disodium hydrogen phosphate (Na 2 HPO 4 ), 3 g/L potassium dihydrogen phosphate, 0.5 g/L sodium chloride, 1 g/L ammonium chloride, 1 mM magnesium sulfate, 0.1. mM calcium chloride, 10 mg/L vitamin B1, 5 g/L yeast extract, 20 g/L glucose.
為讓本發明更明顯易懂，文中使用的基因全名於下： aceEF-lpdA，丙酮酸去氫酶複合體（pyruvate dehydrogenase complex）； adhE，丁醛/丁醇去氫酶（butyraldehyde/butanol dehydrogenase）； adhE2，丁醛/丁酸去氫酶（butyraldehyde/butanol dehydrogenase）； crt，巴豆酸酶（crotonase）； fdh1，甲酸去氫酶（formate dehydrogenase）； frdA，富馬酸還原酶（fumarate reductase）； gltA，檸檬酸合成酶（citrate synthase）； hbd，3-羥基丁基輔酶A去氫酶（3-hydroxybutyryl-CoA dehydorgenase）； ldhA，乳酸去氫酶（lactate dehydrogenase）； pgi，磷酸葡糖異構酶（phosphoglucose isomerase）； pgl，内酯酶（ lactonase）； 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）； udhA，吡啶核苷酸轉氫酶（pyridine nucleotide transhydrogenase）； zwf，葡萄糖-6-磷酸去氫酶（glucose-6-phosphate dehydrogenase）。 To make the invention more apparent, the full name of the gene used herein is as follows: aceEF-lpdA , pyruvate dehydrogenase complex; adhE , butyraldehyde/butanol dehydrogenase adhE2 , butyraldehyde/butanol dehydrogenase; crt , crotonase; fdh1 , formate dehydrogenase; frdA , fumarate reductase ; gltA , citrate synthase; hbd , 3-hydroxybutyryl-CoA dehydorgenase; ldhA , lactate dehydrogenase; pgi , phosphoglucose Phosphoglucose isomerase; pgl , lactonase ; phaA , β-ketothiolase; pta , phosphate acetyltransferase; ter , trans olefinic acid Kiev A reductase (trans-enoyl-coA reductase) ; udhA, pyridine nucleotide transhydrogenase (pyridine nucleotide transhydrogenase); zwf Glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase).
茲以下述實施例，詳細說明本發明：The invention will be described in detail by the following examples:
製備例1：菌株的培養Preparation Example 1: Cultivation of strains
接種液為透過將大腸桿菌生長於含2g/L葡萄糖的Luria-Bertani培養液中至隔夜而取得。細胞密度為於光線波長550nm測得。接著，將接種液培養於50mL的M9Y培養液中，其含有：6g/L磷酸氫二鈉、3g/L磷酸二氫鉀）、0.5g/L氯化鈉、1g/L氯化銨、1mM硫酸鎂、0.1mM氯化鈣、10mg/L維他命B1、5g/L酵母萃取物、20g/L葡萄糖。於光線波長550nm下測得的細胞密度達0.1時，提供有限供氧條件給菌株。The inoculum was obtained by growing Escherichia coli in Luria-Bertani medium containing 2 g/L of glucose until overnight. Cell density was measured at a light wavelength of 550 nm. Next, the inoculum was cultured in 50 mL of M9Y medium containing: 6 g/L disodium hydrogen phosphate, 3 g/L potassium dihydrogen phosphate), 0.5 g/L sodium chloride, 1 g/L ammonium chloride, 1 mM. Magnesium sulfate, 0.1 mM calcium chloride, 10 mg/L vitamin B1, 5 g/L yeast extract, 20 g/L glucose. When the cell density measured at a light wavelength of 550 nm is 0.1, limited oxygen supply conditions are provided to the strain.
製備例2：菌株的建構Preparation 2: Construction of the strain
本實施例的菌株與引子列示於表1。 表1 <TABLE border="1" borderColor="#000000" width="_0004"><TBODY><tr><td> </td><td> 主要特徵 </td><td> 參考文獻 </td></tr><tr><td><u>菌株</u></td><td> </td><td> </td></tr><tr><td> BuT-8 </td><td> Δ<i>frdA</i> φ80<i>attB</i>::PλP<sub>L</sub>-<i>ter</i> λ<i>attB</i>::PλP<sub>L</sub>-<i>crt</i> Δ<i>adhE</i>::φ80<i>attB</i>::PλP<sub>L</sub>-<i>pha</i>-<i>hbd</i> Δ<i>ldhA</i>::λ<i>attB</i>::PλP<sub>L</sub>-<i>adhE2</i></td><td> Saini et al. 2015 </td></tr><tr><td> BuT-8-Fdh1 </td><td> 同BuT-8 P21<i>attB</i>::Ptac-<i>fdh1</i></td><td> － </td></tr><tr><td> BuT-9 </td><td> 同BuT-8-Fdh1 Δ<i>lpdA</i> λ<i>attB</i>::PλP<sub>L</sub>-<i>lpdA</i>* PλP<sub>L</sub>-<i>aceEF</i></td><td> － </td></tr><tr><td> BuT-10 </td><td> 同BuT-9 PλP<sub>L</sub>-<i>zwf</i> PλP<sub>L</sub>-<i>UdhA</i></td><td> － </td></tr><tr><td> BuT-12 </td><td> 同BuT-10 Δ<i>atoD</i>::PλP<sub>L</sub>-<i>pgl</i></td><td> － </td></tr><tr><td> BuT-13 </td><td> 同BuT-12 Δ<i>pgi</i></td><td> － </td></tr><tr><td> BuT-14 </td><td> 同BuT-13 <i>lacO</i>-<i>gltA</i></td><td> － </td></tr><tr><td><u>引子</u></td><td> </td><td> </td></tr><tr><td> RC10178 </td><td> 5’-ataaggatccatatctaacaccgtgcgtg-3’ </td><td> － </td></tr><tr><td> RC11210 </td><td> 5’-cacaccatatgttagaattcattaccttcg-3’ </td><td> － </td></tr><tr><td> RC11403 </td><td> 5’-tttgcggtaccaagccctttgcaaattgc-3’ </td><td> － </td></tr><tr><td> RC11404 </td><td> 5’-cagcagagctcgaatggatcgcgttatc-3’ </td><td> － </td></tr><tr><td> RC11405 </td><td> 5’-agaatcatatggcggtaacgcaaacag-3’ </td><td> － </td></tr><tr><td> RC11406 </td><td> 5’-cttaaggatcctaacccggtacttaagccag-3’ </td><td> － </td></tr><tr><td> RC11407 </td><td> 5’-cgtaaggtacctgacgcatgcgcgtttg-3’ </td><td> － </td></tr><tr><td> RC11408 </td><td> 5’-acttagagctctaaatgcggcttccaccag-3’ </td><td> － </td></tr><tr><td> RC11409 </td><td> 5’-gccctcatatgccacattcctacgattac-3’ </td><td> － </td></tr><tr><td> RC11410 </td><td> 5’-tgttcggatccataaaagcaacagaatggtaac-3’ </td><td> － </td></tr><tr><td> RC11417 </td><td> 5’-ccaagccctttgcaaattgc-3’ </td><td> － </td></tr><tr><td> RC11418 </td><td> 5’-ctcgaatggatcgcgttatc-3’ </td><td> － </td></tr><tr><td> RC11419 </td><td> 5’-cctgacgcatgcgcgtttg-3’ </td><td> － </td></tr><tr><td> RC11420 </td><td> 5’-ctaaatgcggcttccaccag-3’ </td><td> － </td></tr><tr><td> RC12058 </td><td> 5’-aataacatatgtcagaacgtttcccaaatg-3’ </td><td> － </td></tr><tr><td> RC12059 </td><td> 5’-ctatctctagacgttgagttttctggaacc-3’ </td><td> － </td></tr><tr><td> RC12060 </td><td> 5’-ccagttcgaggtcttttttcg-3’ </td><td> － </td></tr><tr><td> RC12085 </td><td> 5’-tatggggtaccagttcgaggtcttttttcg-3’ </td><td> － </td></tr><tr><td> RC12086 </td><td> 5’-caatggagctctgcttcatctgctaagg-3’ </td><td> － </td></tr><tr><td> RC12154 </td><td> 5’-gcgatatcgtcggtcaacc-3’ </td><td> － </td></tr><tr><td> RC12155 </td><td> 5’-tgagaagcttcagtccgcatcaccagag-3’ </td><td> － </td></tr><tr><td> RC12171 </td><td> 5’-gcaagcttatttcttctgtccataagc-3’ </td><td> － </td></tr><tr><td> RC12215 </td><td> 5’-gtccatcgcctataccaaaccagaagttgcatg-3’ </td><td> － </td></tr><tr><td> RC12216 </td><td> 5’-catgcaacttctggtttggtataggcgatggac-3’ </td><td> － </td></tr><tr><td> RC12288 </td><td> 5’-aactgctcgagttacttcttcttcgctttcg-3’ </td><td> － </td></tr><tr><td> RC12289 </td><td> 5’-aagtggatccatacccgtcgtctttcagg-3’ </td><td> － </td></tr><tr><td> RC12290 </td><td> 5’-ccatgagctcggcttttttctggtaatctc-3’ </td><td> － </td></tr><tr><td> RC12314 </td><td> 5’-tctggggatccttctgaaatgagctgttgac-3’ </td><td> － </td></tr><tr><td> RC12331 </td><td> 5’-actctcgaattctggtcgtcctatcgcttc-3’ </td><td> － </td></tr><tr><td> RC13001 </td><td> 5’-ttgaattccgcctttaaagatcgccatg-3’ </td><td> － </td></tr><tr><td> RC13034 </td><td> 5’-catctcaccagatatcatgc-3’ </td><td> － </td></tr><tr><td> RC13035 </td><td> 5’-aatcggagctcgaaagtgaactgtttgg-3’ </td><td> － </td></tr><tr><td> RC13195 </td><td> 5’-atcttcccgggcggaattcattaccgttc-3’ </td><td> － </td></tr><tr><td> RC13196 </td><td> 5’-gaaattgttatccgctcacaattccgggtacccaattc-3’ </td><td> － </td></tr><tr><td> RC13197 </td><td> 5’-cagcaaaataccttcatcacc-3’ </td><td> － </td></tr><tr><td> RC13198 </td><td> 5’-ttcaggggaagagaggctg-3’ </td><td> － </td></tr><tr><td> RC13199 </td><td> 5’-tcaatgggcccacactgttacataagttaatc-3’ </td><td> － </td></tr><tr><td> RC13200 </td><td> 5’-ttaatgtcgacgattgctaagtacttgattcg-3’ </td><td> － </td></tr><tr><td> RC13201 </td><td> 5’-ggtacccagaagccacag-3’ </td><td> － </td></tr><tr><td> RC13292 </td><td> 5’-atcccgggaagcaaacagtttatatcgc-3’ </td><td> － </td></tr><tr><td> RC13293 </td><td> 5’-atctcgagttagtgtgcgttaaccaccac-3’ </td><td> － </td></tr><tr><td> RC14025 </td><td> 5’-gaggaattctgtaggctggagctgcttc-3’ </td><td> － </td></tr><tr><td> RC14026 </td><td> 5’-aacggtcgacatgggaattagccatgg-3’ </td><td> － </td></tr></TBODY></TABLE>The strains and primers of this example are shown in Table 1. Table 1 <TABLE border="1" borderColor="#000000" width="_0004"><TBODY><tr><td> </td><td> Key Features</td><td> References</td> </tr><tr><td><u>Strain</u></td><td> </td><td> </td></tr><tr><td> BuT-8 < /td><td> Δ<i>frdA</i> φ80<i>attB</i>::PλP<sub>L</sub>-<i>ter</i> λ<i>attB< /i>::PλP<sub>L</sub>-<i>crt</i> Δ<i>adhE</i>::φ80<i>attB</i>::PλP<sub>L </sub>-<i>pha</i>-<i>hbd</i> Δ<i>ldhA</i>::λ<i>attB</i>::PλP<sub>L< /sub>-<i>adhE2</i></td><td> Saini et al. 2015 </td></tr><tr><td> BuT-8-Fdh1 </td><td> Same as BuT-8 P21<i>attB</i>::Ptac-<i>fdh1</i></td><td> - </td></tr><tr><td> BuT-9 </td><td> with BuT-8-Fdh1 Δ<i>lpdA</i> λ<i>attB</i>::PλP<sub>L</sub>-<i>lpdA</i> >* PλP<sub>L</sub>-<i>aceEF</i></td><td> - </td></tr><tr><td> BuT-10 </td>< Td> with BuT-9 PλP<sub>L</sub>-<i>zwf</i> PλP<sub>L</sub>-<i>UdhA</i></td><td> - </td></tr><tr><td> BuT-12 </td><td> with BuT-10 Δ<i>atoD</i>::PλP<sub>L</sub>-< i>pgl</i></td><td> - </td></tr><tr><td> BuT-13 </td><td> with BuT-12 Δ<i>pgi</ i></td><td > - </td></tr><tr><td> BuT-14 </td><td> with BuT-13 <i>lacO</i>-<i>gltA</i></td ><td> - </td></tr><tr><td><u>Introduction</u></td><td> </td><td> </td></tr>< Tr><td> RC10178 </td><td> 5'-ataaggatccatatctaacaccgtgcgtg-3' </td><td> - </td></tr><tr><td> RC11210 </td><td> 5'-cacaccatatgttagaattcattaccttcg-3' </td><td> - </td></tr><tr><td> RC11403 </td><td> 5'-tttgcggtaccaagccctttgcaaattgc-3' </td><td > - </td></tr><tr><td> RC11404 </td><td> 5'-cagcagagctcgaatggatcgcgttatc-3' </td><td> - </td></tr><tr> <td> RC11405 </td><td> 5'-agaatcatatggcggtaacgcaaacag-3' </td><td> - </td></tr><tr><td> RC11406 </td><td> 5' -cttaaggatcctaacccggtacttaagccag-3' </td><td> - </td></tr><tr><td> RC11407 </td><td> 5'-cgtaaggtacctgacgcatgcgcgtttg-3' </td><td> - </td></tr><tr><td> RC11408 </td><td> 5'-acttagagctctaaatgcggcttccaccag-3' </td><td> - </td></tr><tr><td > RC11409 </td><td> 5'-gccctcatatgccacattcctacgattac-3' </td><td> - </td></tr><tr><td> RC11410 </td><td > 5'-tgttcggatccataaaagcaacagaatggtaac-3' </td><td> - </td></tr><tr><td> RC11417 </td><td> 5'-ccaagccctttgcaaattgc-3' </td>< Td> - </td></tr><tr><td> RC11418 </td><td> 5'-ctcgaatggatcgcgttatc-3' </td><td> - </td></tr><tr ><td> RC11419 </td><td> 5'-cctgacgcatgcgcgtttg-3' </td><td> - </td></tr><tr><td> RC11420 </td><td> 5 '-ctaaatgcggcttccaccag-3' </td><td> - </td></tr><tr><td> RC12058 </td><td> 5'-aataacatatgtcagaacgtttcccaaatg-3' </td><td> - </td></tr><tr><td> RC12059 </td><td> 5'-ctatctctagacgttgagttttctggaacc-3' </td><td> - </td></tr><tr>< Td> RC12060 </td><td> 5'-ccagttcgaggtcttttttcg-3' </td><td> - </td></tr><tr><td> RC12085 </td><td> 5'- Tatggggtaccagttcgagctcttttttcg-3' </td><td> - </td></tr><tr><td> RC12086 </td><td> 5'-caatggagctctgcttcatctgctaagg-3' </td><td> - < /td></tr><tr><td> RC12154 </td><td> 5'-gcgatatcgtcggtcaacc-3' </td><td> - </td></tr><tr><td> RC12155 </td><td> 5'-tgagaagcttcagtccgcatcaccagag-3' </td><t d> - </td></tr><tr><td> RC12171 </td><td> 5'-gcaagcttatttcttctcccataagc-3' </td><td> - </td></tr><tr ><td> RC12215 </td><td> 5'-gtccatcgcctataccaaaccagaagttgcatg-3' </td><td> - </td></tr><tr><td> RC12216 </td><td> 5 '-catgcaacttctggtttggtataggcgatggac-3' </td><td> - </td></tr><tr><td> RC12288 </td><td> 5'-aactgctcgagttacttcttcttcgctttcg-3' </td><td> - </td></tr><tr><td> RC12289 </td><td> 5'-aagtggatccatacccgtcgtctttcagg-3' </td><td> - </td></tr><tr>< Td> RC12290 </td><td> 5'-ccatgagctcggcttttttctggtaatctc-3' </td><td> - </td></tr><tr><td> RC12314 </td><td> 5'- Tctggggatccttctgaaatgagctgttgac-3' </td><td> - </td></tr><tr><td> RC12331 </td><td> 5'-actctcgaattctggtcgtcctatcgcttc-3' </td><td> - < /td></tr><tr><td> RC13001 </td><td> 5'-ttgaattccgcctttaaagatcgccatg-3' </td><td> - </td></tr><tr><td> RC13034 </td><td> 5'-catctcaccagatatcatgc-3' </td><td> - </td></tr><tr><td> RC13035 </td><td> 5'-aatcggagctcgaaagtgaactgtttgg- 3' </td> <td> - </td></tr><tr><td> RC13195 </td><td> 5'-atcttcccgggcggaattcattaccgttc-3' </td><td> - </td></tr>< Tr><td> RC13196 </td><td> 5'-gaaattgttatccgctcacaattccgggtacccaattc-3' </td><td> - </td></tr><tr><td> RC13197 </td><td> 5'-cagcaaaataccttcatcacc-3' </td><td> - </td></tr><tr><td> RC13198 </td><td> 5'-ttcaggggaagagaggctg-3' </td><td > - </td></tr><tr><td> RC13199 </td><td> 5'-tcaatgggcccacactgttacataagttaatc-3' </td><td> - </td></tr><tr> <td> RC13200 </td><td> 5'-ttaatgtcgacgattgctaagtacttgattcg-3' </td><td> - </td></tr><tr><td> RC13201 </td><td> 5' -ggtacccagaagccacag-3' </td><td> - </td></tr><tr><td> RC13292 </td><td> 5'-atcccgggaagcaaacagtttatatcgc-3' </td><td> - </td></tr><tr><td> RC13293 </td><td> 5'-atctcgagttagtgtgcgttaaccaccac-3' </td><td> - </td></tr><tr><td > RC14025 </td><td> 5'-gaggaattctgtaggctggagctgcttc-3' </td><td> - </td></tr><tr><td> RC14026 </td><td> 5'-aacggtcgacatgggaattagccatgg -3' </td><td> - </td></t r></TBODY></TABLE>
用RC12171引子（SEQ ID NO：22）與RC12314引子（SEQ ID NO：28）對質體pTrc-Fdh1（Chiang et al. 2012）進行PCR以增幅出一受 trc啟動子（Ptrc）調控的釀酒酵母 fdh1。以 BamHI限制酶處理增幅產物後，將產物連接至 BamHI與 NruI等限制酶處理過的質體pP21-Km來取得質體pP21-Fdh1。接著，用質體pP21-Fdh1將含Ptrc- fdh1的DNA片段***至大腸桿菌內，且移除***至菌株內的卡納黴素抗性基因（Chiang et al. 2008）。 PCR was performed on the plastid pTrc-Fdh1 (Chiang et al. 2012) using the RC12171 primer (SEQ ID NO: 22) and the RC12314 primer (SEQ ID NO: 28) to increase the S. cerevisiae fdh1 regulated by the trc promoter (Ptrc). . After the amplification product was treated with Bam HI restriction enzyme, the product was ligated to the restriction enzyme-treated plastid pP21-Km such as Bam HI and Nru I to obtain plastid pP21-Fdh1. Next, the DNA fragment containing Ptrc- fdh1 was inserted into E. coli with the plastid pP21-Fdh1, and the kanamycin resistance gene inserted into the strain was removed (Chiang et al. 2008).
用RC12154引子（SEQ ID NO：20）與RC12155引子（SEQ ID NO：21）對菌株BL21進行PCR以增幅出一 lpdA片段，並將增幅產物***至 NdeI與 XhoI等限制酶處理過的質體pMCS-5來取得質體pMSC-lpdA。以RC12215引子（SEQ ID NO：23）與RC12216引子（SEQ ID NO：24）對質體pMSC-lpdA中的 lpdA進行E354K點突變（亦即， lpdA的蛋白質產物中第354位置由谷胺酸變成賴胺酸）。於 NdeI與 XhoI等限制酶處理經點突變後的質體，取得一E354K點突變的 lpdA片段（以 lpdA*表示）。然後，將 lpdA*轉質至夾有LE*- kan-RE*-PλP L盒匣的質體pLoxKm-PR（Saini et al. 2014），得到的質體pLoxKm-lpdA*含有受LE*- kan-RE*-PλP L調控的 lpdA*（LE*- kan-RE*-PλP L- lpdA*）。另以RC12289引子（SEQ ID NO：26）與RC12290引子（SEQ ID NO：27）增幅出 lpdA的上游區域，並接合至 BamHI與 SacI等限制酶處理過的質體pBluescript以得到質體pBlue-ac。以 BamHI與 XhoI等限制酶處理質體pLoxKm-lpdA*，並將得到的LE*- kan-RE*-PλP L- lpdA*片段***至質體pBlue-ac以獲得質體pBlue-ac/lpdA*。此外，以RC11210引子（SEQ ID NO：2）與RC12331引子（SEQ ID NO：29）對質體pBlue-ac/lpdA*進行PCR。先以 EcoRI切割PCR產物，後自接成質體pBlue-Ac-lpdA，其具有受LE*- kan-RE*阻斷的 lpdA。為剔除 lpdA，以RC12288引子（SEQ ID NO：25）與RC12290引子（SEQ ID NO：27）對質體pBlue-Ac-lpdA進行PCR以取得一截斷（truncated）的lpdA，並電穿孔至大腸桿菌菌株內。最後，以RC10178引子（SEQ ID NO：1）與RC12288引子（SEQ ID NO：25）對質體pBlue-ac/lpdA*進行PCR來取得一含PλP L- lpdA*的DNA片段，並以 BamHI限制酶處理此片段。透過接合此片段與經 BamHI與 EcoRV等限制酶處理的質體pLam-Crt（Saini et al. 2014）來取得質體pLam-LpdA*。最後，將含有PλP L- lpdA*的DNA片段***至大腸桿菌內後，移除***的標記（Chiang et al. 2012）。 The strain BL21 was subjected to PCR using the RC12154 primer (SEQ ID NO: 20) and the RC12155 primer (SEQ ID NO: 21) to increase an lpdA fragment, and the amplified product was inserted into a restriction enzyme-treated substance such as Nde I and Xho I. The body pMCS-5 was used to obtain the plastid pMSC-lpdA. E354K point mutation was performed on lpdA in plastid pMSC-lpdA with RC12215 primer (SEQ ID NO: 23) and RC12216 primer (SEQ ID NO: 24) (ie, position 354 of lpdA protein product was changed from glutamic acid to Lai Amino acid). The point-mutated plastids were treated with restriction enzymes such as Nde I and Xho I to obtain an E354K point mutation lpdA fragment (expressed as lpdA *). Then, to the lpdA * transplastomic interposed LE * - L cassette of plasmid kan -RE * -PλP pLoxKm-PR (. Saini et al 2014), the resulting plasmid comprising receiving pLoxKm-lpdA * LE * - kan -RE*-PλP L regulated lpdA *(LE*- kan -RE*-PλP L - lpdA *). Further, an RC12289 primer (SEQ ID NO: 26) and an RC12290 primer (SEQ ID NO: 27) were used to increase the upstream region of lpdA , and ligated to a restriction enzyme-treated plastid pBluescript such as Bam HI and Sac I to obtain a plastid pBlue. -ac. In other Xho I and Bam HI restriction enzyme plasmid pLoxKm-lpdA *, and the resultant LE * - kan -RE * -PλP L - lpdA * fragment was inserted into plasmid pBlue-ac to obtain plasmid pBlue-ac / lpdA*. In addition, PCR was performed on the plastid pBlue-ac/lpdA* with the RC11210 primer (SEQ ID NO: 2) and the RC12331 primer (SEQ ID NO: 29). First with Eco RI PCR product was cut, since then into plasmid pBlue-Ac-lpdA, by having a LE * - lpdA kan -RE * blocked. To eliminate lpdA , PCR was performed on plastid pBlue-Ac-lpdA with RC12288 primer (SEQ ID NO: 25) and RC12290 primer (SEQ ID NO: 27) to obtain a truncated lpdA and electroporated to E. coli strain Inside. Finally, PCR was performed on the plastid pBlue-ac/lpdA* with the RC10178 primer (SEQ ID NO: 1) and the RC12288 primer (SEQ ID NO: 25) to obtain a DNA fragment containing PλP L - lpdA *, which was restricted by Bam HI. This fragment was treated with an enzyme. The plastid pLam-LpdA* was obtained by conjugating this fragment to the plastid pLam-Crt (Saini et al. 2014) treated with restriction enzymes such as Bam HI and Eco RV. Finally, after inserting the DNA fragment containing PλP L - lpdA * into E. coli, the inserted marker was removed (Chiang et al. 2012).
為提昇內源基因的表現，將啟動子PλP L置於特定基因的前方以取代原本的啟動子，詳細操作於下。首先，以RC11403引子（SEQ ID NO：3）與RC11404引子（SEQ ID NO：4）對菌株BL21進行PCR以增幅出一含 zwf上游區域與5’-端區域的片段；以RC11407引子（SEQ ID NO：7）與RC11408引子（SEQ ID NO：8）對菌株BL21進行PCR以取得一含 udh上游區域與5’-端區域的片段；以RC12085引子（SEQ ID NO：18）與RC12086引子（SEQ ID NO：19）對菌株BL21進行PCR以取得一含 aceE上游區域與5’-端區域的片段。每一片段經 KpnI與 SacI等限制酶處理後，***至質體pBluescript以分別取得質體pBlue-zwf、pBlue-udhA與pBlue-aceE。之後，以RC11405引子（SEQ ID NO：5）與RC11406引子（SEQ ID NO：6）對質體pBlue-zwf進行PCR以建立 NdeI與 BamHI等限制酶的切點於質體上；以RC11409引子（SEQ ID NO：9）與RC11410引子（SEQ ID NO：10）對質體pBlue-udhA進行PCR以建立 NdeI與 BamHI等限制酶的切點於質體上；以RC12058引子（SEQ ID NO：15）與RC12059引子（SEQ ID NO：16）對質體pBlue-aceE進行PCR以建立 NdeI與 XbaI等限制酶的切點於質體上。以 NdeI與 BamHI（或 NdeI與 XbaI）等限制酶處理質體pLoxKm-PR以取得LE*- kan-RE*-PλP L盒匣，並***至質體pBlue-zwf、pBlue-udhA與pBlue-aceE來各別取得質體pPR-zwf、pPR-udhA、pPR-aceE。最終，以RC11417引子（SEQ ID NO：11）與RC11418引子（SEQ ID NO：12）對質體pPR-zwf進行PCR以增幅出一客座DNA（passenger DNA）；以RC11419引子（SEQ ID NO：13）與RC11420引子（SEQ ID NO：14）對質體pPR-udhA進行PCR以增幅出另一客座DNA；以RC12060引子（SEQ ID NO：17）與RC12086引子（SEQ ID NO：19）對質體pPR-aceE進行PCR以增幅出又一客座DNA。以電穿孔法將此三客座DNA***至菌株，並移除標記基因（Chiang et al. 2012）。 In order to enhance the expression of the endogenous gene, the promoter PλP L is placed in front of the specific gene to replace the original promoter, and the details are as follows. First, PCR was performed on strain BL21 with RC11403 primer (SEQ ID NO: 3) and RC11404 primer (SEQ ID NO: 4) to increase a fragment containing the upstream region and the 5'-end region of zwf ; RC11407 primer (SEQ ID) NO: 7) and RC11408 primer (SEQ ID NO: 8) PCR was performed on strain BL21 to obtain a fragment containing the udh upstream region and the 5'-end region; RC12085 primer (SEQ ID NO: 18) and RC12086 primer (SEQ ID NO: 19) PCR was carried out on strain BL21 to obtain a fragment containing the upstream region and the 5'-end region of aceE . Each fragment was treated with restriction enzymes such as Kpn I and Sac I, and inserted into plastid pBluescript to obtain plastids pBlue-zwf, pBlue-udhA and pBlue-aceE, respectively. Thereafter, PCR was performed on the plastid pBlue-zwf with the RC11405 primer (SEQ ID NO: 5) and the RC11406 primer (SEQ ID NO: 6) to establish a cleavage point of the restriction enzymes such as Nde I and Bam HI on the plastid; the RC11409 primer ( SEQ ID NO: 9) and RC11410 primer (SEQ ID NO: 10) were subjected to PCR on plastid pBlue-udhA to establish a cleavage point of restriction enzymes such as Nde I and Bam HI on plastids; RC12058 primer (SEQ ID NO: 15) The plastid pBlue-aceE was subjected to PCR with the RC12059 primer (SEQ ID NO: 16) to establish a cleavage point of the restriction enzymes such as Nde I and Xba I on the plastid. In Nde I and Bam HI (or Nde I and Xba I) and the like plasmid restriction enzyme treatment to obtain pLoxKm-PR LE * - L cassette kan -RE * -PλP, and inserted into the plasmid pBlue-zwf, pBlue-udhA The plastids pPR-zwf, pPR-udhA, and pPR-aceE were obtained separately from pBlue-aceE. Finally, PCR was performed on plastid pPR-zwf with RC11417 primer (SEQ ID NO: 11) and RC11418 primer (SEQ ID NO: 12) to increase a guest DNA (passenger DNA); RC11419 primer (SEQ ID NO: 13) PCR of plastid pPR-udhA with RC11420 primer (SEQ ID NO: 14) to increase the other guest DNA; plastid pPR-aceE with RC12060 primer (SEQ ID NO: 17) and RC12086 primer (SEQ ID NO: 19) PCR was performed to increase the additional guest DNA. This three-site DNA was inserted into the strain by electroporation and the marker gene was removed (Chiang et al. 2012).
為取得 pgl，用RC13292引子（SEQ ID NO：40）與RC13293引子（SEQ ID NO：41）對菌株MG1655進行PCR。於PCR產物經 EcoRV與 SacI等限制酶處理後，與質體pBluescript接合成一質體pBlue-pgl。接著，先以 SmaI與 XhoI等限制酶處理質體pBlue-pgl，再將處理後的產物與質體pLoxKm-PL接合。如此，可取得一含與LE*- kan-RE*-PλP L融合之 pgl的質體pSPL-pgl。然後，利用RC13001引子（SEQ ID NO：30）與RC13293引子（SEQ ID NO：41）對質體pSPL-pgl進行PCR增幅出LE*- kan-RE*-PλP L- pgl片段。之後，將此增幅片段接合至經 EcoRI與 NruI等限制酶處理過的質體pSPL-ato（Saini et al. 2014）。同樣，以RC13034引子（SEQ ID NO：31）與RC13035引子（SEQ ID NO：32）對接合後的質體pSPL-ato進行PCR以得到一客座DNA，並電穿孔至菌株內。最後，移除***的標記基因。 To obtain pgl , PCR was carried out on strain MG1655 using RC13292 primer (SEQ ID NO: 40) and RC13293 primer (SEQ ID NO: 41). After the PCR product was treated with a restriction enzyme such as Eco RV and Sac I, it was ligated with the plastid pBluescript to form a plastid pBlue-pgl. Next, the plastid pBlue-pgl is first treated with a restriction enzyme such as Sma I and Xho I, and the treated product is ligated to the plastid pLoxKm-PL. Thus, and can be obtained having a LE * - pSPL-pgl pgl plasmid fused to the kan -RE * -PλP L. Kan -RE * -PλP L - - pgl increase confrontation PCR fragment thereof pSPL-pgl a LE *: RC13293 and primers (41 SEQ ID NO): Then, using primers RC13001 (30 SEQ ID NO). Thereafter, this amplified fragment was ligated to the plastid pSPL-ato (Saini et al. 2014) treated with restriction enzymes such as Eco RI and Nru I. Similarly, the ligated pSPL-ato was ligated with the RC13034 primer (SEQ ID NO: 31) and the RC13035 primer (SEQ ID NO: 32) to obtain a guest DNA, and electroporated into the strain. Finally, the inserted marker gene is removed.
為調控 gltA的表現，以操作子 lacO取代啟動子P2，取代過程如下。首先，以RC13195引子（SEQ ID NO：33）與RC13196引子（SEQ ID NO：34）對質體pLoxKm-PR進行PCR以增幅出一含操作子 lacO的片段。於此片段經 SmaI限制酶處理後，自黏成質體pLoxCm-LacO，此質體融合有LE*- kan-RE*- lacO。其次，以RC13197引子（SEQ ID NO：35）與RC13198引子（SEQ ID NO：36）對菌株BL21進行PCR以增幅出一含 gltA上游區域與5’-端區域的片段。將增幅得到的片段***至經 KpnI與 SmaI等限制酶處理過的質體pBluescript來得到一質體pBlue-GltA。再者，以RC13199引子（SEQ ID NO：37）與RC13200引子（SEQ ID NO：38）對質體pBlue-GltA進行PCR以建立 ApaI與 SalI等限制酶的切點於質體上。以 ApaI與 SalI等限制酶處理質體pLoxCm-LacO以取得LE*- kan-RE*- lacO盒匣，並接合至建立有 ApaI與 SalI等限制酶之切點的質體pBlue-GltA來取得一質體pBlue-GltO。最後，以RC14025引子（SEQ ID NO：42）與RC14026引子（SEQ ID NO：43）對質體pKD3進行PCR以得到FRT- Cm-FRT盒匣。將PCR產物***至經 EcoRI與 SalI等限制酶處理過的質體pBlue-GltO，使FRT- Cm-FRT盒匣取代LE*- kan-RE*盒匣並得到一質體pB-gltO-Cm。同樣，以RC13197引子（SEQ ID NO：35）與RC13201引子（SEQ ID NO：39）對質體pB-gltO-Cm進行PCR以得到一客座DNA，並電穿孔至菌株內和移除***的標記基因。 To regulate the performance of gltA , the promoter P2 was replaced with the operator lacO , and the substitution procedure was as follows. First, PCR was performed on the plastid pLoxKm-PR with the RC13195 primer (SEQ ID NO: 33) and the RC13196 primer (SEQ ID NO: 34) to increase a fragment containing the operator lacO . After this fragment was treated with Sma I restriction enzyme, it was self-adhesive into plastid pLoxCm-LacO, which was fused with LE*- kan -RE*- lacO . Next, PCR of strain BL21 was carried out with RC13197 primer (SEQ ID NO: 35) and RC13198 primer (SEQ ID NO: 36) to increase a fragment containing the upstream region and the 5'-end region of gltA . The amplified fragment was inserted into a plastid pBluescript treated with a restriction enzyme such as Kpn I and Sma I to obtain a plastid pBlue-GltA. Furthermore, plastid pBlue-GltA was subjected to PCR using RC13199 primer (SEQ ID NO: 37) and RC13200 primer (SEQ ID NO: 38) to establish a cleavage point of restriction enzymes such as Apa I and Sal I on the plastid. In Apa I and Sal I and other restriction enzyme plasmid pLoxCm-LacO to obtain LE * - kan -RE * - lacO cassette, and engaged to establish Apa I and Sal I and other restriction endonuclease point of the plasmid pBlue-GltA To obtain a plastid pBlue-GltO. Finally, plastid pKD3 was subjected to PCR with RC14025 primer (SEQ ID NO: 42) and RC14026 primer (SEQ ID NO: 43) to obtain FRT- Cm- FRT cassette. The PCR product was inserted into the Eco RI and Sal I and other restriction enzyme treated plasmid pBlue-GltO, so FRT- Cm -FRT cassette substituted LE * - kan -RE * cassette and get a plasmid pB-gltO- Cm. Similarly, PCR was performed on the plastid pB-gltO-Cm with the RC13197 primer (SEQ ID NO: 35) and the RC13201 primer (SEQ ID NO: 39) to obtain a guest DNA, and electroporation into the strain and removal of the inserted marker gene. .
製備例3：分析方法Preparation Example 3: Analytical method
此處分析方法主要參照Saini et al, 2015。使用配有Reflective Index RID-10A的高效液相層析儀（High Performance Liquid Chromatography，HPLC；購自Shimadzu，日本）測量葡萄糖與甘油。使用氣相層析儀Trace 130測量正丁醇（購自Thermo Scientific，美國）。The analysis method here mainly refers to Saini et al, 2015. Glucose and glycerol were measured using a High Performance Liquid Chromatography (HPLC; available from Shimadzu, Japan) equipped with a Reflective Index RID-10A. n-Butanol (purchased from Thermo Scientific, USA) was measured using a gas chromatograph Trace 130.
胞內NADH含量為用螢光NAD ＋/NADH檢測套組（購自Cell Technology，美國）測得的。此項測量流程可參考使用者說明書。簡言之，先對細菌培養液進行離心，並將離心得到的細胞團回溶於200μL分解緩衝液與200μLNADH萃取液。將所得的混合液置於60℃下約20分鐘。於對混合液離心後，取出離心所得的上層物並與反應試劑混合。將得到的混合物置於暗處、室溫下1小時以進行反應。最後，以波長530至570nm的激發光與波長590至600nm的放射光測量反應產物以取得NADH含量。 Intracellular NADH levels were determined using a fluorescent NAD + /NADH assay kit (purchased from Cell Technology, USA). Refer to the user manual for this measurement procedure. Briefly, the bacterial culture was first centrifuged, and the pellet obtained by centrifugation was dissolved back into 200 μL of the decomposition buffer and 200 μL of the NADH extract. The resulting mixture was placed at 60 ° C for about 20 minutes. After centrifuging the mixture, the supernatant obtained by centrifugation was taken out and mixed with the reaction reagent. The resulting mixture was placed in the dark at room temperature for 1 hour to carry out the reaction. Finally, the reaction product was measured with excitation light having a wavelength of 530 to 570 nm and emitted light having a wavelength of 590 to 600 nm to obtain a NADH content.
製備例4：酵素活性分析Preparation Example 4: Enzyme activity analysis
細胞培養液離心後，將離心得到的細胞團回溶於1mL溶解緩衝液。先以超音波震盪破壞細胞，再離心。收集離心取得的上層物並稱之為「無細胞萃取物（cell-free extract）」。無細胞萃取物中的總蛋白質濃度為採用Bio-Rad蛋白質分析套組測得的。丙酮酸去氫酶的活性如Bond-Watts et al. 2011所述於室溫下用波長340nm監測NAD ＋的還原，所用的反應溶液含有50mM磷酸鉀（pH7.9）、5mM丙酮酸鈉、1.3mM輔酶A、0.5mM硫胺素焦磷酸（thiamine pyrophosphate）、與5mM氯化鎂。為開啟還原反應，取100μL無細胞萃取物至1mL反應溶液。葡萄糖-6-磷酸去氫酶的活性如Snoep et al. 1996所述用波長340nm監測NADP ＋的還原，所用反應溶液含2mM葡萄糖-6-磷酸、0.67mMNADP ＋、10mM氯化鎂、與50mMTris-氯化氫（pH7.5）。為開啟還原反應，於30℃下取100μL無細胞萃取物至1mL反應溶液中。6-磷酸葡萄糖酸内酯酶之活性的測量如葡萄糖-6-磷酸去氫酶的活性測量（Sinha and Maitra 1992），除了所用的反應溶液有50μM葡萄糖-6-磷酸酶、0.5mMNADP ＋、50mMTris-氯化氫、10mM氯化鎂、與50mMTris-氯化氫（pH7.5）。此外，檸檬酸合成酶之活性的測量如Walsh and Koshland Jr 1985所述，所使用的反應溶液含有0.1mM乙醯輔酶A、0.5mM草醋酸、0.2mM5,5’-二硫代雙（2-硝基苯甲酸）（5,5'-Dithiobis(2-nitrobenzoic Acid)）、與50mMTris-氯化氫（pH7.5）。 After the cell culture solution was centrifuged, the pellet obtained by centrifugation was dissolved in 1 mL of the lysis buffer. First destroy the cells with ultrasonic shock and centrifuge. The supernatant obtained by centrifugation was collected and referred to as "cell-free extract". The total protein concentration in the cell free extract was measured using the Bio-Rad Protein Assay Kit. Pyruvate dehydrogenase activity The NAD + reduction was monitored at room temperature using a wavelength of 340 nm as described in Bond-Watts et al. 2011. The reaction solution used contained 50 mM potassium phosphate (pH 7.9), 5 mM sodium pyruvate, 1.3. mM CoA, 0.5 mM thiamine pyrophosphate, and 5 mM magnesium chloride. To initiate the reduction reaction, 100 μL of cell-free extract was taken to 1 mL of the reaction solution. Glucose-6-phosphate dehydrogenase activity The NADP + reduction was monitored by a wavelength of 340 nm as described by Snoep et al. 1996. The reaction solution used contained 2 mM glucose-6-phosphate, 0.67 mM NADP + , 10 mM magnesium chloride, and 50 mM Tris-hydrogen chloride ( pH 7.5). To initiate the reduction reaction, 100 μL of cell-free extract was taken at 30 ° C into 1 mL of the reaction solution. Measurement of the activity of 6-phosphogluconolactonease, such as glucose-6-phosphate dehydrogenase activity (Sinha and Maitra 1992), except that the reaction solution used was 50 μM glucose-6-phosphatase, 0.5 mM NADP + , 50 mM Tris. - Hydrogen chloride, 10 mM magnesium chloride, and 50 mM Tris-hydrogen chloride (pH 7.5). In addition, the activity of citrate synthase was measured as described by Walsh and Koshland Jr 1985, and the reaction solution used contained 0.1 mM acetamidine CoA, 0.5 mM oxalic acid, 0.2 mM 5,5'-dithiobis (2- Nitrobenzoic acid (5,5'-Dithiobis (2-nitrobenzoic acid)), and 50 mM Tris-hydrogen chloride (pH 7.5).
分析例1：丙酮酸氧化途徑的放大Analysis Example 1: Magnification of pyruvate oxidation pathway
本實施例的起始菌株為大腸桿菌菌株BuT-8（Saini et al. 2015），其具備了生產正丁醇的輔酶A依賴途徑，並含有丙酮丁醇梭桿菌的 hbd基因、 crt基因、與 adhE2基因、鉤蟲貪銅菌的 phaA基因、及齒垢密螺旋體的 ter基因。此外，為減少碳源耗損與保存NADH，自此菌株中移除涉入不必要途徑的內源基因，如 adhE基因、 ldhA基因、 pta基因、 frdA基因。如圖1所示，自一個葡萄糖還原合成成一個正丁醇需要的NADH量比醣解所提供者來的多。依此，可期待透過補充NADH來促進正丁醇的生產。於本實施例中，連結醣解的丙酮酸節點（node）與檸檬酸循環顯然為一可行的標的。於大腸桿菌內，丙酮酸在有氧環境下透過丙酮酸去氫酶氧化成乙醯輔酶A，而在發酵條件下透過丙酮酸甲酸裂解酶（pyruvate-formate lyase）氧化成乙醯輔酶A（White 2007）。甲酸為丙酮酸甲酸裂解酶反應的還原產物。其他微生物的甲酸去氫酶，如博伊丁假絲酵母（ Candida boidinii） fdh、釀酒酵母 fdh1，能氧化甲酸成二氧化碳與NADH（Berrios-Rivera et al. 2002）。此二基因早已用於大腸桿菌內來提升胞內NADH，藉此增加正丁醇的生產（Chiang et al. 2012、Shen et al. 2011）。於是，受 trc啟動子調控的釀酒酵母 fdh1***至菌株BuT-8內。如圖2所示，所得到的菌株BuT-8-Fdh1於有限供氧條件下生長24小時可產生3.1g/L正丁醇，且其正丁醇濃度較菌株BuT-8的正丁醇濃度約增加25％。 The starting strain of this example is Escherichia coli strain BuT-8 (Saini et al. 2015), which has a coenzyme A-dependent pathway for producing n-butanol, and contains the hbd gene, crt gene, and The adhE2 gene, the phaA gene of C. necator , and the ter gene of Treponema pallidum. In addition, in order to reduce carbon source depletion and preserve NADH, endogenous genes involved in unnecessary pathways such as adhE gene, ldhA gene, pta gene, and frdA gene were removed from this strain. As shown in Figure 1, the amount of NADH required to synthesize a n-butanol from a glucose reduction is greater than that provided by the glycolysis. Accordingly, it is expected to promote the production of n-butanol by supplementing NADH. In this example, the pyrolysis of the pyruvate node and the citric acid cycle is clearly a viable target. In Escherichia coli, pyruvic acid is oxidized to acetaminophen coenzyme A by pyruvate dehydrogenase in an aerobic environment, and oxidized to acetaminophen coenzyme A by pyrylation of pyruvate-formate lyase under fermentation conditions. 2007). Formic acid is the reduction product of the pyruvate formate lyase reaction. Formate dehydrogenase of other microorganisms, such as Candida boidinii (Candida boidinii) fdh, Saccharomyces cerevisiae fdhl, formic acid can be oxidized to carbon dioxide and NADH (Berrios-Rivera et al. 2002). This two genes have long been used in E. coli to enhance intracellular NADH, thereby increasing the production of n-butanol (Chiang et al. 2012, Shen et al. 2011). Thus, S. cerevisiae fdh1 regulated by the trc promoter was inserted into the strain BuT-8. As shown in Figure 2, the obtained strain BuT-8-Fdh1 was grown for 24 hours under limited oxygen supply to produce 3.1 g/L n-butanol, and its n-butanol concentration was higher than that of the strain BuT-8. About 25% increase.
相對丙酮酸甲酸裂解酶，丙酮酸去氫酶的反應生成了NADH作為還原產物。因此，可期望透過控制丙酮酸去氫酶的表現來改變胞內的NADH。這可於菌株BuT-8-Fdh1中透過PλP L啟動子結合至 aceEF基因操縱組達成。為使丙酮酸去氫酶對NADH的抑制不靈敏，去除內源 lpdA基因並額外建構一E354K點突變的 lpdA（亦即 lpdA*）於菌株BuT-8-Fdh1內，且 lpdA*受PλP L啟動子控制。此時，得到的菌株稱為菌株BuT-9。如圖3所示，於同樣條件下，菌株BuT-9可生產4.3g/L正丁醇。又如表2所示，菌株BuT-9中的丙酮酸去氫酶活性約為菌株BuT-8的1.3倍，且前者菌株中的NADH量較後者菌株多45％。 表2 <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> 菌株 </td><td> 正丁醇濃度（g/L） </td><td> 正丁醇生產速率（g/L/h） </td><td> 正丁醇葡萄糖轉換率（g/g） </td><td> NADH（μmol/細胞重量g） </td><td> 酵素活性（U/每mg的蛋白質） </td></tr><tr><td> PDH </td><td> Zwf </td><td> Pgl </td><td> GltA </td></tr><tr><td> BuT-8 </td><td> 2.7 </td><td> 0.11 </td><td> 0.14 </td><td> 42.2 </td><td> 0.7 </td><td> - </td><td> - </td><td> - </td></tr><tr><td> BuT-9 </td><td> 4.3 </td><td> 0.18 </td><td> 0.22 </td><td> 60.9 </td><td> 1.6 </td><td> 6.1 </td><td> - </td><td> - </td></tr><tr><td> BuT-10 </td><td> 4.9 </td><td> 0.20 </td><td> 0.25 </td><td> 75.8 </td><td> - </td><td> 18.3 </td><td> 0.5 </td><td> - </td></tr><tr><td> BuT-12 </td><td> 5.4 </td><td> 0.23 </td><td> 0.27 </td><td> 82.6 </td><td> - </td><td> </td><td> 6.2 </td><td> 2.2 </td></tr><tr><td> BuT-14 </td><td> 6.1 </td><td> 0.21 </td><td> 0.31 </td><td> 96.1 </td><td> - </td><td> - </td><td> - </td><td> 1.5 </td></tr></TBODY></TABLE>The reaction with pyruvate formate lyase and pyruvate dehydrogenase produces NADH as a reduction product. Therefore, it can be expected to change the intracellular NADH by controlling the performance of pyruvate dehydrogenase. This was achieved in strain BuT-8-Fdh1 by binding to the aceEF gene manipulation group via the PλP L promoter. In order to make pyruvate dehydrogenase insensitive to NADH inhibition, the endogenous lpdA gene was removed and an E354K point mutation lpdA (ie lpdA* ) was constructed in strain BuT-8-Fdh1, and lpdA* was initiated by PλP L Sub-control. At this time, the obtained strain was referred to as strain BuT-9. As shown in Figure 3, under the same conditions, the strain BuT-9 produced 4.3 g/L of n-butanol. As shown in Table 2, the pyruvate dehydrogenase activity in the strain BuT-9 was about 1.3 times that of the strain BuT-8, and the amount of NADH in the former strain was 45% more than that of the latter strain. Table 2 <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td>strain</td><td> n-butanol concentration (g/L) </ Td><td> n-butanol production rate (g/L/h) </td><td> n-butanol glucose conversion rate (g/g) </td><td> NADH (μmol/cell weight g) </td><td> Enzyme activity (U/mg of protein) </td></tr><tr><td> PDH </td><td> Zwf </td><td> Pgl </ Td><td> GltA </td></tr><tr><td> BuT-8 </td><td> 2.7 </td><td> 0.11 </td><td> 0.14 </td ><td> 42.2 </td><td> 0.7 </td><td> - </td><td> - </td><td> - </td></tr><tr><td > BuT-9 </td><td> 4.3 </td><td> 0.18 </td><td> 0.22 </td><td> 60.9 </td><td> 1.6 </td><td > 6.1 </td><td> - </td><td> - </td></tr><tr><td> BuT-10 </td><td> 4.9 </td><td> 0.20 </td><td> 0.25 </td><td> 75.8 </td><td> - </td><td> 18.3 </td><td> 0.5 </td><td> - </td></tr><tr><td> BuT-12 </td><td> 5.4 </td><td> 0.23 </td><td> 0.27 </td><td> 82.6 </ Td><td> - </td><td></td><td> 6.2 </td><td> 2.2 </td></tr><tr><td> BuT-14 </td><td> 6.1 </td><td> 0.21 </td><td> 0.31 </td><td> 96.1 </td><td> - </td><td> - </td><td> - </td><td> 1.5 </td></tr></TBODY></TABLE>
同樣方式早已應用於具備輔酶A依賴合成途徑之大腸桿菌的正丁醇生成（Shen et al. 2012、Bond-Watt et al. 2011、Lim et al. 2013）。然而，習用的菌株為培養於價格昂貴的TB培養液中；反之，本實施例的菌株為培養於相對便宜的M9Y培養液。The same approach has long been applied to the production of n-butanol in Escherichia coli with a coenzyme A-dependent synthetic pathway (Shen et al. 2012, Bond-Watt et al. 2011, Lim et al. 2013). However, the conventional strain is cultured in an expensive TB culture solution; otherwise, the strain of the present example is cultured in a relatively inexpensive M9Y culture solution.
分析例2：五碳糖磷酸途徑的放大Analysis Example 2: Magnification of the five-carbon sugar phosphate pathway
醣解途徑於葡萄糖-6-磷酸結點分支。以葡萄糖-6-磷酸為起始代謝物，五碳糖磷酸途徑產生供核酸與芳香族胺基酸合成的前驅物，並供應大量供還原性生物合成的NADPH（White 2007）。於是，可透過調節葡萄糖-6-磷酸結點來提升NADH的取得。葡萄糖-6-磷酸去氫酶可於五碳糖磷酸途徑中催化第一步，故菌株BuT-9的 zwf基因融合有PλP L啟動子。於大腸桿菌內，吡啶核苷酸轉氫酶的功能為NADH與NADPH的互相轉變（Canocaco et al. 2001），故菌株BuT-9的 udhA基因融合有PλP L啟動子。如圖4所示，所得到的菌株BuT-10可生產4.9g/L正丁醇。如表2所示，相較於菌株BuT-9，菌株BuT-10中的葡萄糖-6-磷酸去氫酶活性約增加2倍，且生產的正丁醇量約增加10％。 The glycolytic pathway is branched into the glucose-6-phosphate node. Glucose-6-phosphate is the starting metabolite, and the five-carbon sugar phosphate pathway produces a precursor for the synthesis of nucleic acids and aromatic amino acids, and supplies a large amount of NADPH for reducing biosynthesis (White 2007). Thus, the acquisition of NADH can be enhanced by adjusting the glucose-6-phosphate node. Glucose-6-phosphate dehydrogenase catalyzes the first step in the five-carbon sugar phosphate pathway, so the zwf gene of the strain BuT-9 is fused with the PλP L promoter. In E. coli, the function of pyridine nucleotide transhydrogenase is the mutual transformation of NADH and NADPH (Canocaco et al. 2001), so the udhA gene of strain BuT-9 is fused with the PλP L promoter. As shown in Figure 4, the resulting strain BuT-10 produced 4.9 g/L of n-butanol. As shown in Table 2, the glucose-6-phosphate dehydrogenase activity in the strain BuT-10 was approximately 2-fold increased compared to the strain BuT-9, and the amount of n-butanol produced was increased by about 10%.
由於菌株BuT-8衍生自大腸桿菌BL21，故其缺乏 pgl基因（Meier et al. 2012）。内酯酶主要於五碳糖磷酸途徑中負責葡萄糖-6-磷酸去氫酶之後的步驟。因此，由提升表現之葡萄糖-6-磷酸去氫酶引導至五碳糖磷酸途徑中的碳流量可能會於内酯酶調控的步驟受限。為解決此問題，自大腸桿菌K-12取得受PλP L啟動子調控的 pgl基因並導入至菌株BuT-10。最後，如圖5所示，所得到的菌株BuT-12可生產5.4g/L正丁醇。如表2所示，相較於菌株BuT-10，菌株BuT-12中的内酯酶活性約增加10倍，生成的NADH量約增加36％，且其正丁醇量約增加25.6％。 Since the strain BuT-8 is derived from E. coli BL21, it lacks the pgl gene (Meier et al. 2012). The lactonase is primarily responsible for the step following the glucose-6-phosphate dehydrogenase in the five carbon sugar phosphate pathway. Therefore, the carbon flux directed to the five-carbon sugar phosphate pathway by the elevated glucose-6-phosphate dehydrogenase may be limited in the steps of lactosterase regulation. To solve this problem, the pgl gene regulated by the PλP L promoter was obtained from E. coli K-12 and introduced into the strain BuT-10. Finally, as shown in Figure 5, the resulting strain BuT-12 produced 5.4 g/L of n-butanol. As shown in Table 2, the lactonase activity in the strain BuT-12 was increased by about 10 times, the amount of NADH produced was increased by about 36%, and the amount of n-butanol was increased by 25.6% as compared with the strain BuT-10.
由上可知，於醣解與五碳糖磷酸途徑中碳流量的重新分布可影響胞內NADH的含量。須注意的是，一個葡萄糖進入五碳糖磷酸途徑會產生二個還原性等效物並浪費一個二氧化碳。儘管如此，相較於菌株BuT-8，菌株BuT-12生成的NADH量約增加96％，且生產的正丁醇量約增加為2倍。It can be seen from the above that the redistribution of carbon flux in the glycolytic and five-carbon sugar phosphate pathways can affect the intracellular NADH content. It should be noted that a glucose entry into the five-carbon sugar phosphate pathway produces two reducing equivalents and wastes a carbon dioxide. Nevertheless, compared to the strain BuT-8, the amount of NADH produced by the strain BuT-12 increased by about 96%, and the amount of n-butanol produced was increased by about 2 times.
分析例3：經五碳糖磷酸途徑的葡萄糖代謝Analysis Example 3: Glucose metabolism via the five-carbon sugar phosphate pathway
依大腸桿菌的中央代謝，每單位葡萄糖經五碳糖磷酸途徑較經醣解多生成85％的還原效能。故，透過將碳流量自醣解轉移至五碳糖磷酸途徑可有效提升胞內NADH量。磷酸葡糖異構酶負責於葡萄糖-6-磷酸的異構化，且失活可讓五碳糖磷酸途徑為葡萄糖代謝的主要途徑（Hua et al. 2003）。故，藉由剔除菌株BuT-12中的 pgi基因來得到菌株BuT-13。如圖6所示，相較於菌株BuT-12，菌株BuT-13於生物質量與葡萄糖利用約減少32％與30％。於發酵30小時後，菌株BuT-13並無法完全消耗葡萄糖且產生較少的正丁醇（4.6g/L）。 According to the central metabolism of E. coli, each unit of glucose produces 85% of the reducing efficiency through the five-carbon sugar phosphate pathway. Therefore, the amount of intracellular NADH can be effectively increased by transferring the carbon flux from the glycolysis to the five-carbon sugar phosphate pathway. Phosphoglucose isomerase is responsible for the isomerization of glucose-6-phosphate, and inactivation allows the five-carbon sugar phosphate pathway to be the major pathway for glucose metabolism (Hua et al. 2003). Therefore, the strain BuT-13 was obtained by knocking out the pgi gene in the strain BuT-12. As shown in Figure 6, strain BuT-13 was reduced by about 32% and 30% in biomass and glucose utilization compared to strain BuT-12. After 30 hours of fermentation, the strain BuT-13 did not completely consume glucose and produced less n-butanol (4.6 g/L).
透過缺乏 pgi基因的菌株可實現改善NADPH的取得，但此菌株卻於比生長率（specific growth rate）減少47％（Chemler et al. 2010）。這種缺乏 pgi基因所致的生長缺失乃因過剩的NADPH會傷害細胞的生理狀態（Canonaco et al. 2001）。有趣的是，葡萄糖-6-磷酸去氫酶與吡啶核苷酸轉氫酶的高活性可促進缺乏 pgi基因的菌株生長（Canonaco et al. 2001、Flores et al. 2004）。然而，菌株BuT-13雖有高葡萄糖-6-磷酸去氫酶與吡啶核苷酸轉氫酶的活性、及正丁醇合成途徑，但仍受到生長缺失所影響。此結果意謂著此菌株存在失衡的還原狀態。 Improved NADPH can be achieved by strains lacking the pgi gene, but this strain is reduced by a specific growth rate of 47% (Chemler et al. 2010). This lack of growth due to the lack of the pgi gene is due to the fact that excess NADPH can damage the physiological state of the cell (Canonaco et al. 2001). Interestingly, the high activity of glucose-6-phosphate dehydrogenase and pyridine nucleotide transhydrogenase promotes the growth of strains lacking the pgi gene (Canonaco et al. 2001, Flores et al. 2004). However, although the strain BuT-13 has high glucose-6-phosphate dehydrogenase and pyridine nucleotide transhydrogenase activity, and n-butanol synthesis pathway, it is still affected by growth loss. This result means that the strain has an unbalanced reduced state.
分析例4：自檸檬酸循環之碳流量的轉移Analysis Example 4: Transfer of carbon flux from the citric acid cycle
乙醯輔酶A是合成正丁醇的前驅物且亦可於檸檬酸循環中氧化。為適應氧氣張力，檸檬酸循環可進行氧化途徑或還原途徑以產生不同程度的還原等效物（White 2007）。檸檬酸合成酶可催化檸檬酸循環的第一步。可以期待透過降低檸檬酸合成酶活性來從檸檬酸循環將碳流量轉移，從而保留乙醯輔酶A與調控還原等效物的產生。此可透過於菌株BuT-13中以操作子 lacO取代檸檬酸合成酶原來的啟動子P2。接著，培養所得的菌株BuT-14並分析其發酵能力。因此，幾乎回復菌株BuT-14生長且其生物質量與菌株BuT-12相當。如圖7所示，菌株BuT-14的葡萄糖利用稍為緩慢並於29小時後產生6.1g/L正丁醇。如表2所示，相較於菌株BuT-12，菌株BuT-14生成的NADH量約增加16％，且其檸檬酸合成酶活性約降低32％。為進一步瞭解菌株BuT-14對於更低檸檬酸合成酶活性的反應，將 lacI Q 導入至此菌株內。發現相較於菌株BuT-12，所得之菌株BuT-14-A的檸檬酸合成酶活性約降低50％。而且，此菌株亦展現不佳的生長、僅耗損40％葡萄糖且於30小時產生1.8g/L正丁醇（結果未示）。過去咸知，降低檸檬酸合成酶活性90％並未影響大腸桿菌於葡萄糖中的生長（Walsh and Koshland Jr 1985）。相對地，缺乏 pgi基因的菌株於葡萄糖的生長則與檸檬酸合成酶的活性相關。透過調控檸檬酸合成酶的活性，可讓菌株自缺乏 pgi基因導致的缺陷復原。這可能是檸檬酸合成酶的調控破壞胞內的還原狀態。顯然地，此菌株展現了對胞內還原狀態的高感受性，且適度調節檸檬酸合成酶活性對於確保菌株優異的表現是必要的。 Ethylene coenzyme A is a precursor for the synthesis of n-butanol and can also be oxidized in the citric acid cycle. To accommodate oxygen tension, the citric acid cycle can be subjected to an oxidation pathway or a reduction pathway to produce varying degrees of reduction equivalents (White 2007). Citric acid synthase can catalyze the first step in the citric acid cycle. It is expected that the carbon flux can be transferred from the citric acid cycle by reducing the citrate synthase activity, thereby preserving the production of acetamyl coenzyme A and the regulatory reduction equivalent. This is permeable to the original promoter P2 of the citrate synthase by the operator lacO in the strain BuT-13. Next, the obtained strain BuT-14 was cultured and analyzed for its fermentation ability. Therefore, almost the recovery strain BuT-14 was grown and its biological quality was comparable to that of the strain BuT-12. As shown in Figure 7, the glucose utilization of strain BuT-14 was slightly slow and produced 6.1 g/L n-butanol after 29 hours. As shown in Table 2, the amount of NADH produced by the strain BuT-14 was increased by about 16% compared to the strain BuT-12, and its citrate synthase activity was decreased by about 32%. To further understand the response of the strain BuT-14 to lower citrate synthase activity, lacI Q was introduced into this strain. The citrate synthase activity of the resulting strain BuT-14-A was found to be reduced by about 50% compared to the strain BuT-12. Moreover, this strain also exhibited poor growth, only depleted 40% glucose and produced 1.8 g/L n-butanol at 30 hours (results not shown). In the past, it was known that reducing citrate synthase activity by 90% did not affect the growth of E. coli in glucose (Walsh and Koshland Jr 1985). In contrast, the growth of glucose in strains lacking the pgi gene is related to the activity of citrate synthase. By modulating the activity of citrate synthase, the strain can be restored from defects caused by the lack of the pgi gene. This may be due to the regulation of citrate synthase disrupting the intracellular reduction state. Obviously, this strain exhibits high sensitivity to the intracellular reduction state, and moderate regulation of citrate synthase activity is necessary to ensure excellent performance of the strain.
甲酸去氫酶的使用及丙酮酸去氫酶表現的提升為大腸桿菌內用來提升NADH取得的常見方法。透過建構乙醯輔酶A與NADH的驅動，Shen et al. 2011已使用甲酸去氫酶來達到0.2g/L/h正丁醇產生速率與88％轉換率。另外，Bond-Watts et al. 2011已透過丙酮酸去氫酶來達到0.065g/L/h正丁醇產生速率。再者，Lim et al. 2013已透過甲酸去氫酶與丙酮酸去氫酶來達0.26g/L/h正丁醇產生速率與0.27g/g正丁醇葡萄糖轉換率。須注意的是，這些前案使用的培養基為昂貴的TB培養基，且培養基中非葡萄糖的組份可貢獻15％正丁醇生產（Shen et al. 2011）。於本實施例，採用較便宜的M9Y培養基來系統性地調控胞內NADH。首先，於菌株BuT-8中提升甲酸去氫酶及丙酮酸去氫酶的活性，此菌株的正丁醇生產速率為0.18g/L/h、正丁醇葡萄糖轉換率為0.22g/g。其次，菌株BuT-12可引導碳流量至五碳糖循環來增加NADPH並透過吡啶核苷酸轉氫酶將NADPH轉變為NADH，其正丁醇生產速率與正丁醇葡萄糖轉換率各為0.23g/L/h、0.27g/g。最後，於菌株BuT-14中透過引導碳流量至五碳糖循環並自檸檬酸循環轉移碳流量來改善還原效力。菌株BuT-14的NADH量與正丁醇濃度均為起始菌株BuT-8的1.3倍，且其正丁醇生產速率為0.21g/L/h、正丁醇葡萄糖轉換率為0.31g/g。理論上，於經五碳糖磷酸循環的葡萄糖代謝中每莫耳葡萄糖會產生0.85莫耳（非1莫耳）正丁醇，這歸咎於二氧化碳的產生。如此一來，正丁醇葡萄糖之轉換率的理論值為0.35g/g。經計算後，菌株BuT-14的正丁醇葡萄糖轉換率約為此理論值的89％。The use of formate dehydrogenase and the improvement in pyruvate dehydrogenase performance are common methods used to enhance NADH in E. coli. Through the construction of acetaminophen A and NADH, Shen et al. 2011 have used formate dehydrogenase to achieve a 0.2 g/L/h n-butanol production rate and an 88% conversion rate. In addition, Bond-Watts et al. 2011 has achieved a rate of 0.065 g/L/h n-butanol production by pyruvate dehydrogenase. Furthermore, Lim et al. 2013 have passed the formate dehydrogenase and pyruvate dehydrogenase to achieve a 0.26 g/L/h n-butanol production rate and a 0.27 g/g n-butanol glucose conversion rate. It should be noted that the medium used in these previous cases was an expensive TB medium, and the non-glucose component of the medium contributed 15% n-butanol production (Shen et al. 2011). In this example, less expensive M9Y medium was used to systematically modulate intracellular NADH. First, the activity of formate dehydrogenase and pyruvate dehydrogenase was increased in strain BuT-8. The n-butanol production rate of this strain was 0.18 g/L/h, and the n-butanol glucose conversion rate was 0.22 g/g. Secondly, the strain BuT-12 can direct carbon flux to the five-carbon sugar cycle to increase NADPH and convert NADPH to NADH by pyridine nucleotide transhydrogenase. The n-butanol production rate and the n-butanol glucose conversion rate are each 0.23g. /L/h, 0.27g/g. Finally, the reduction efficiency was improved in strain BuT-14 by directing carbon flux to the five carbon sugar cycle and transferring carbon flux from the citric acid cycle. The amount of NADH and n-butanol in the strain BuT-14 were 1.3 times that of the original strain BuT-8, and the n-butanol production rate was 0.21 g/L/h, and the n-butanol glucose conversion rate was 0.31 g/g. . Theoretically, 0.85 moles (not 1 mole) of n-butanol is produced per mole of glucose in the glucose metabolism of the five carbon sugar phosphate cycle, which is attributed to the production of carbon dioxide. As a result, the theoretical conversion of n-butanol glucose is 0.35 g/g. After calculation, the n-butanol glucose conversion rate of strain BuT-14 was about 89% of this theoretical value.
綜上所述，說明著本發明所構築的菌株可有效地生產正丁醇，且正丁醇的生產可於相對便宜的培養基中實現。In summary, it is illustrated that the strain constructed by the present invention can efficiently produce n-butanol, and the production of n-butanol can be achieved in a relatively inexpensive medium.
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.
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圖1為大腸桿菌菌株BuT-14的正丁醇生產途徑；圖中符號○＋ 為強化基因的表現，○－ 為減弱或抑制基因的表現。 圖2為大腸桿菌菌株BuT-8-Fdh1於培養液中培養24小時後的各種物質濃度。 圖3為大腸桿菌菌株BuT-9於培養液中培養24小時後的各種物質濃度。 圖4為大腸桿菌菌株BuT-10於培養液中培養24小時後的各種物質濃度。 圖5為大腸桿菌菌株BuT-12於培養液中培養24小時後的各種物質濃度。 圖6為大腸桿菌菌株BuT-13於培養液中培養不同時間後的各種物質濃度。 圖7為大腸桿菌菌株BuT-14於培養液中培養不同時間後的各種物質濃度。Figure 1 shows the n-butanol production pathway of E. coli strain BuT-14; the symbol ○+ in the figure is the expression of the enhanced gene, and ○- is the expression of the weakened or suppressed gene. Fig. 2 shows the concentrations of various substances after the Escherichia coli strain BuT-8-Fdh1 was cultured for 24 hours in the culture solution. Fig. 3 shows the concentrations of various substances after the Escherichia coli strain BuT-9 was cultured for 24 hours in the culture solution. Fig. 4 shows the concentrations of various substances after the Escherichia coli strain BuT-10 was cultured for 24 hours in the culture solution. Fig. 5 shows the concentrations of various substances after the Escherichia coli strain BuT-12 was cultured for 24 hours in the culture solution. Figure 6 shows the concentrations of various substances after the E. coli strain BuT-13 was cultured in the culture medium for various times. Figure 7 shows the concentrations of various substances after the E. coli strain BuT-14 was cultured in the culture medium for various times.
＜110＞逢甲大學 ＜120＞可生產正丁醇的菌株及其應用 ＜160＞43 ＜210＞1 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC10178引子 ＜400＞1 ataaggatcc atatctaaca ccgtgcgtg 29 ＜210＞2 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11210引子 ＜400＞2 cacaccatat gttagaattc attaccttcg 30 ＜210＞3 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11403引子 ＜400＞3 tttgcggtac caagcccttt gcaaattgc 29 ＜210＞4 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11404引子 ＜400＞4 cagcagagct cgaatggatc gcgttatc 28 ＜210＞5 ＜211＞27 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11405引子 ＜400＞5 agaatcatat ggcggtaacg caaacag 27 ＜210＞6 ＜211＞31 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11406引子 ＜400＞6 cttaaggatc ctaacccggt acttaagcca g 31 ＜210＞7 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11407引子 ＜400＞7 cgtaaggtac ctgacgcatg cgcgtttg 28 ＜210＞8 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11408引子 ＜400＞8 acttagagct ctaaatgcgg cttccaccag 30 ＜210＞9 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11409引子 ＜400＞9 gccctcatat gccacattcc tacgattac 29 ＜210＞10 ＜211＞33 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11410引子 ＜400＞10 tgttcggatc cataaaagca acagaatggt aac 33 ＜210＞11 ＜211＞20 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11417引子 ＜400＞11 ccaagccctt tgcaaattgc 20 ＜210＞12 ＜211＞20 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11418引子 ＜400＞12 ctcgaatgga tcgcgttatc 20 ＜210＞13 ＜211＞19 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11419引子 ＜400＞13 cctgacgcat gcgcgtttg 19 ＜210＞14 ＜211＞20 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC11420引子 ＜400＞14 ctaaatgcgg cttccaccag 20 ＜210＞15 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12058引子 ＜400＞15 aataacatat gtcagaacgt ttcccaaatg 30 ＜210＞16 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12059引子 ＜400＞16 ctatctctag acgttgagtt ttctggaacc 30 ＜210＞17 ＜211＞21 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12060引子 ＜400＞17 ccagttcgag gtcttttttc g 21 ＜210＞18 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12085引子 ＜400＞18 tatggggtac cagttcgagg tcttttttcg 30 ＜210＞19 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12086引子 ＜400＞19 caatggagct ctgcttcatc tgctaagg 28 ＜210＞20 ＜211＞19 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12154引子 ＜400＞20 gcgatatcgt cggtcaacc 19 ＜210＞21 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12155引子 ＜400＞21 tgagaagctt cagtccgcat caccagag 28 ＜210＞22 ＜211＞27 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12171引子 ＜400＞22 gcaagcttat ttcttctgtc cataagc 27 ＜210＞23 ＜211＞33 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12215引子 ＜400＞23 gtccatcgcc tataccaaac cagaagttgc atg 33 ＜210＞24 ＜211＞33 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12216引子 ＜400＞24 catgcaactt ctggtttggt ataggcgatg gac 33 ＜210＞25 ＜211＞31 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12288引子 ＜400＞25 aactgctcga gttacttctt cttcgctttc g 31 ＜210＞26 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12289引子 ＜400＞26 aagtggatcc atacccgtcg tctttcagg 29 ＜210＞27 ＜211＞ ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12290引子 ＜400＞27 ccatgagctc ggcttttttc tggtaatctc 30 ＜210＞28 ＜211＞31 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12314引子 ＜400＞28 tctggggatc cttctgaaat gagctgttga c 31 ＜210＞29 ＜211＞30 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC12331引子 ＜400＞29 actctcgaat tctggtcgtc ctatcgcttc 30 ＜210＞30 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13001引子 ＜400＞30 ttgaattccg cctttaaaga tcgccatg 28 ＜210＞31 ＜211＞20 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13034引子 ＜400＞31 catctcacca gatatcatgc 20 ＜210＞32 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13035引子 ＜400＞32 aatcggagct cgaaagtgaa ctgtttgg 28 ＜210＞33 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13195引子 ＜400＞33 atcttcccgg gcggaattca ttaccgttc 29 ＜210＞34 ＜211＞38 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13196引子 ＜400＞34 gaaattgtta tccgctcaca attccgggta cccaattc 38 ＜210＞35 ＜211＞21 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13197引子 ＜400＞35 cagcaaaata ccttcatcac c 21 ＜210＞36 ＜211＞19 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13198引子 ＜400＞36 ttcaggggaa gagaggctg 19 ＜210＞37 ＜211＞32 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13199引子 ＜400＞37 tcaatgggcc cacactgtta cataagttaa tc 32 ＜210＞38 ＜211＞32 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13200引子 ＜400＞38 ttaatgtcga cgattgctaa gtacttgatt cg 32 ＜210＞39 ＜211＞18 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13201引子 ＜400＞39 ggtacccaga agccacag 18 ＜210＞40 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13292引子 ＜400＞40 atcccgggaa gcaaacagtt tatatcgc 28 ＜210＞41 ＜211＞29 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC13293引子 ＜400＞41 atctcgagtt agtgtgcgtt aaccaccac 29 ＜210＞42 ＜211＞28 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC14025引子 ＜400＞42 gaggaattct gtaggctgga gctgcttc 28 ＜210＞43 ＜211＞27 ＜212＞DNA ＜213＞人工序列 ＜220＞ ＜223＞RC14026引子 ＜400＞43 aacggtcgac atgggaatta gccatgg 27<110>Fengjia University <120> can produce n-butanol strain and its application <160>43 <210>1 <211>29 <212>DNA <213> artificial sequence <220> <223>RC10178 primer <400 >1 ataaggatcc atatctaaca ccgtgcgtg 29 <210>2 <211>30 <212>DNA <213>Artificial sequence<220> <223>RC11210 Intro <400>2 cacaccatat gttagaattc attaccttcg 30 <210>3 <211>29 <212> DNA <213>Artificial sequence<220> <223>RC11403 primer<400>3 tttgcggtac caagcccttt gcaaattgc 29 <210>4 <211>28 <212>DNA <213>Artificial sequence<220> <223>RC11404 primer<400> 4 cagcagagct cgaatggatc gcgttatc 28 <210>5 <211>27 <212>DNA <213>Artificial sequence<220> <223>RC11405 Intro <400>5 agaatcatat ggcggtaacg caaacag 27 <210>6 <211>31 <212>DNA <213>Artificial sequence <220> <223>RC11406 primer <400>6 cttaaggatc ctaacccggt acttaagcca g 31 <210>7 <211>28 <212>DNA <213>Artificial sequence<220> <223>RC11407 primer< 400>7 cgtaaggtac ctgacgcatg cgcgtttg 28 <210>8 <211>30 <212>DNA <213>Artificial sequence<220> <223>RC11408 Intro <400>8 acttagagct ctaaatgcgg cttccaccag 30 <210>9 <211>29 <212 >DNA <213>Artificial sequence<220> <223>RC11409 primer<400>9 gccctcatat gccacattcc tacgattac 29 <210>10 <211>33 <212>DNA <213>Artificial sequence<220> <223>RC11410 primer<400 >10 tgttcggatc cataaaagca acagaatggt aac 33 <210>11 <211>20 <212>DNA <213>Artificial sequence<220> <223>RC11417 primer<400>11 ccaagccctt tgcaaattgc 20 <210>12 <211>20 <212> DNA <213>Artificial sequence<220> <223>RC11418 Initiative <400>12 ctcgaatgga tcgcgttatc 20 <210>13 <211>19 <212>DNA <213>Artificial sequence<220> <223>RC11419Introduction <400>13 Cctgacgcat gcgcgtttg 19 <210>14 <211>20 <212>DNA <213>Artificial sequence<220> <223>RC11420 Intro <400>14 ctaaatgcgg cttccaccag 20 <210>15 <211>30 <212>DNA <213>Artificial sequence<220> <223>RC12058 Initiator <400>15 aataacatat gtcagaacgt ttcccaaatg 30 <210>16 <211>30 <212>DNA <213>Artificial sequence<220> <223>RC12059 Introduction <400>16 ctatctctag acgttgagtt ttctggaacc 30 <210>17 <211>21 <212>DNA <213>Artificial sequence<220> <223>RC12060 Intro <400>17 ccagttcgag gtcttttttc g 21 <210>18 <211>30 < 212>DNA <213>Artificial sequence<220> <223>RC12085 Intron <400>18 tatggggtac cagttcgagg tcttttttcg 30 <210>19 <211>28 <212>DNA <213>Artificial sequence<220> <223>RC12086 Initiator< 400>19 caatggagct ctgcttcatc tgctaagg 28 <210>20 <211>19 <212>DNA <213>Artificial sequence<220> <223>RC12154 primer<400>20 gcgatatcgt cggtcaacc 19 <210>21 <211>28 <212> DNA <213>Artificial sequence<220> <223>RC12155 primer<400>21 tgagaagctt cagtccgcat caccagag 28 <210>22 <211>27 <212>DNA <213>Artificial sequence<220> <223>RC12171 primer <400>22 gcaagcttat ttcttctgtc cataagc 27 <210>23 <211>33 <212>DNA <213>Artificial sequence<220> <223>RC12215 primer<400>23 gtccatcgcc tataccaaac cagaagttgc atg 33 <210> 24 <211>33 <212>DNA <213>Artificial sequence<220> <223>RC12216 primer<400>24 catgcaactt ctggtttggt ataggcgatg gac 33 <210>25 <211>31 <212>DNA <213>Artificial sequence<220 > <223>RC12288 primer <400>25 aactgctcga gttacttctt cttcgctttc g 31 <210>26 <211>29 <212>DNA <213>Artificial sequence<220> <223>RC12289Introduction <400>26 aagtggatcc atacccgtcg tctttcagg 29 <210 >27 <211> <212>DNA <213>Artificial sequence<220> <223>RC12290 Intro <400>27 ccatgagctc ggcttttttc tggtaatctc 30 <210>28 <211>31 <212>DNA <213>Artificial sequence<220> <223>RC12314 primer <400>28 tctggggatc cttctgaaat gagctgttga c 31 <210>29 <211>30 <212>DNA <213>Artificial sequence<220> <223>RC123 31 primer <400>29 actctcgaat tctggtcgtc ctatcgcttc 30 <210>30 <211>28 <212>DNA <213> artificial sequence <220> <223>RC13001 primer <400>30 ttgaattccg cctttaaaga tcgccatg 28 <210>31 <211> 20 <212>DNA <213>Artificial sequence<220> <223>RC13034 primer <400>31 catctcacca gatatcatgc 20 <210>32 <211>28 <212>DNA <213>Artificial sequence<220> <223>RC13035 primer <400>32 aatcggagct cgaaagtgaa ctgtttgg 28 <210>33 <211>29 <212>DNA <213>Artificial sequence<220> <223>RC13195 Intro <400>33 atcttcccgg gcggaattca ttaccgttc 29 <210>34 <211>38 < 212>DNA <213>Artificial sequence<220> <223>RC13196 primer <400>34 gaaattgtta tccgctcaca attccgggta cccaattc 38 <210>35 <211>21 <212>DNA <213>Artificial sequence<220> <223>RC13197 primer <400>35 cagcaaaata ccttcatcac c 21 <210>36 <211>19 <212>DNA <213>Artificial sequence<220> <223>RC13198 primer <400>36 ttcaggggaa gaga Ggctg 19 <210>37 <211>32 <212>DNA <213>Artificial sequence<220> <223>RC13199 primer<400>37 tcaatgggcc cacactgtta cataagttaa tc 32 <210>38 <211>32 <212>DNA <213 >Artificial sequence<220> <223>RC13200 primer <400>38 ttaatgtcga cgattgctaa gtacttgatt cg 32 <210>39 <211>18 <212>DNA <213>Artificial sequence<220> <223>RC13201Introduction <400>39 ggtacccaga Agccacag 18 <210>40 <211>28 <212>DNA <213>Artificial sequence<220> <223>RC13292 primer<400>40 atcccgggaa gcaaacagtt tatatcgc 28 <210>41 <211>29 <212>DNA <213> Artificial sequence <220> <223> RC13293 primer <400>41 atctcgagtt agtgtgcgtt aaccaccac 29 <210>42 <211>28 <212>DNA <213> artificial sequence <220> <223>RC14025 primer <400>42 gaggaattct gtaggctgga gctgcttc 28 <210>43 <211>27 <212>DNA <213>Artificial sequence<220> <223>RC14026Introduction <400>43 aacggtcgac atgggaatta gccatgg 27
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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 |
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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 |
| US10633677B2 (en) | 2016-12-27 | 2020-04-28 | National Tsing Hua University | Butanol expression cassette, recombinant plasmid and butanol production related gene expression method |
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