弱化呼吸链水平对代谢工程大肠杆菌聚羟基丁酸乳酸酯合成的影响
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摘要
聚3-羟基丁酸乳酸酯[Poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA)],属于聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHA)家族的一员,是一种具有良好生物相容性和可降解性的天然高分子生物材料。文中通过在大肠杆菌中引入来源于富养罗尔斯通氏菌Ralstonia eutropha的β-酮硫解酶、乙酰乙酰Co A还原酶、来源于丙酸梭菌Clostridium propionicum的丙酰Co A转移酶突变体以及荧光假单胞菌Pseudomonas fluorescens strain 2P24来源的PHA合成酶突变体等异源酶,成功实现了一步法利用葡萄糖合成P(3HB-co-LA),其中乳酸组分的摩尔百分比达到1.6%,聚合物含量为83.9wt%。在此基础上,通过敲除辅酶Q8合成所需的黄素异戊烯基转移酶基因(ubi X)来弱化呼吸链水平,从而增强乳酸积累,并进一步缺失乳酸脱氢酶基因(dld)以减少乳酸在发酵后期转化成丙酮酸,最终将P(3HB-co-LA)中乳酸组分的摩尔百分比提高至14.1%,而聚合物含量为81.7wt%。上述实验结果表明,采用弱化呼吸链水平策略可有效提高聚合物中乳酸组分的摩尔百分比,从而提供了一种改变生物合成聚合物中单体组分含量的新思路。
Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates(PHA) family andpossesses promising properties including biocompatibility and biodegradability.In this study,we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha,the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli.The polymer content was 83.9%(W/W),and the molar percentage of lactate reached 1.6%.On this basis,in order to accumulate lactate,we reduced the activity of respiratory chain by deleting the ubiX gene,which is involved in the synthesis of coenzyme Q8.Moreover,we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation.With these manipulations,the molar percentage of lactate in the polymer was improved to 14.1%,with an 81.7%(W/W) of polymer content.The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer,and it contributed a new approach to change the content of monomer components in the polymer.
Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates(PHA) family andpossesses promising properties including biocompatibility and biodegradability.In this study,we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha,the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli.The polymer content was 83.9%(W/W),and the molar percentage of lactate reached 1.6%.On this basis,in order to accumulate lactate,we reduced the activity of respiratory chain by deleting the ubiX gene,which is involved in the synthesis of coenzyme Q8.Moreover,we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation.With these manipulations,the molar percentage of lactate in the polymer was improved to 14.1%,with an 81.7%(W/W) of polymer content.The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer,and it contributed a new approach to change the content of monomer components in the polymer.
引文
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[3]Tsuji H.Poly(lactide)stereocomplexes:formation,structure,properties,degradation,and applications.Macromol Biosci,2005,5(7):569-597.
[4]Chen GQ.ChemInform abstract:a microbial Polyhydroxyalkanoates(PHA)based Bio-and materials industry.Chem Soc Rev,2009,38(8):2434-2446.
[5]Drumright RE,Gruber PR,Henton DE.Polylactic acid technology.Adv Mater,2000,12(23):1841-1846.
[6]Chen GQ,Jiang XR,Yao H,et al.Polyhydroxyalkanoates:Microbial Synthesis and Applications.Hoboken:John Wiley&Sons,Inc,2017:21-27.
[7]S?derg?rd A,Stolt M.Properties of lactic acid based polymers and their correlation with composition.Prog Polym Sci,2002,27(6):1123-1163.
[8]Yin J,Che XM,Chen GQ.Progress on polyhydroxyalkanoates(PHA).Chin J Biotech,2016,32(6):726-737(in Chinese).尹进,车雪梅,陈国强.聚羟基脂肪酸酯的研究进展.生物工程学报,2016,32(6):726-737.
[9]Chen GQ,Wang Y.Medical applications of biopolyesters polyhydroxyalkanoates.Chin J Polym Sci,2013,31(5):719-736.
[10]Auras R,Harte B,Selke S.An overview of polylactides as packaging materials.Macromol Biosci,2004,4(9):835-864.
[11]Yamada M,Matsumoto K,Uramoto S,et al.Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli.J Biotechnol,2011,154(4):255-260.
[12]Nduko JM,Matsumoto K,Ooi T,et al.Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate)using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme.Metab Eng,2013,15(1):159-166.
[13]Taguchi S,Yamada M,Matsumoto K,et al.Amicrobial factory for lactate-based polyester using a lactate-polymerizing enzyme.Proc Natl Acad Sci USA,2008,105(45):17323-17327.
[14]Yamada M,Matsumoto K,Nakai T,et al.Microbial production of lactate-enriched poly[(R)-lactate-co-(R)-3-hydroxybutyrate]with novel thermal properties.Biomacromolecules,2009,10(4):677-681.
[15]Yang TH,Kim TW,Kang HO,et al.Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase.Biotechnol Bioeng,2010,105(1):150-160.
[16]Wu H,Tuli L,Bennett GN,et al.Metabolic transistor strategy for controlling electron transfer chain activity in Escherichia coli.Metab Eng,2015,28:159-168.
[17]Datsenko KA,Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci USA,2000,97(12):6640-6645.
[18]Gao X.Optimization and chromosomal expression of polyhydroxyalkanoates related genes in Escherichia coli[D].Beijing:Tsinghua University,2013(in Chinese).高雪.大肠杆菌中聚羟基脂肪酸酯合成基因的优化和染色体表达[D].北京:清华大学,2013.
[19]Cheng Z,Jiang JQ,Wu H,et al.Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli.Bioresource Technol,2016,200:897-904.
[20]Shozui F,Matsumoto K,Motohashi R,et al.Biosynthesis of a lactate(LA)-based polyester with a96 mol%LA fraction and its application to stereocomplex formation.Polym Degrad Stabil,2011,96(4):499-504.
[21]Choi SY,Park SJ,Kim WJ,et al.One-step fermentative production of poly(lactate-co-glycolate)from carbohydrates in Escherichia coli.Nat Biotechnol,2016,34(4):435-440.