利用定向进化提高基因工程大肠杆菌的甲醇利用能力
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摘要
甲醇是一种重要的有机化工原料,价格低廉,碳还原度高,在生物化工中是糖质原料的理想替代原料。但是常用的工业微生物宿主如大肠杆菌不能利用甲醇作为碳源,这限制了甲醇在生物化工领域的应用。通过表达甲醇脱氢酶、3-己酮糖-6-磷酸合成酶和6-磷酸-3-己酮糖异构酶在大肠杆菌中构建可同化甲醇的核酮糖单磷酸途径。以基因工程菌作为出发菌株,通过连续传代和定向进化引入随机突变,突变株进行以甲醇为碳源的压力筛选,得到了2株可以利用甲醇生长的突变株。在以甲醇为辅助碳源的培养基中,25113Δfrm A/p ZWM1-13号突变株较原始菌株25113Δfrm A/p ZWM1菌体生长量增加27.6%。对25113Δfrm A/p ZWM1-13号突变株进行~(13)C示踪分析检测蛋白质合成氨基酸,结果表明氨基酸中~(13)C比例有明显提高。其中,甲硫氨酸~(13)C标记含量增加7.236%。因此,定向进化有效地提高了基因工程大肠杆菌的甲醇利用效率。
Methanol is an important organic chemical raw material as well as an attractive alternative of sugar for biochemical industrybecause of its cost advantage and higher degree of carbon reduction. However,commonly used industrial microorganisms such as Escherichia coli cannot utilize methanol as a carbon source,which has become a bottleneck for the application of methanol in bio-production processes.In this study,a methanol utilization pathway(ribulose monophosphate pathway)was constructed in E. coli using an engineering geneticallyengineered E. coli as a starting strain,the random mutation occurred by continuous passage and direct evolution. Then the mutants werescreened using methanol as the carbon source,and 2 mutants that grew well on methanol were obtained. When methanol used as an accessorycarbon source,biomass of the mutant 25113Δfrm A/p ZWM1-13 increased by 27.6% compared to the starting strain 25113Δfrm A/p ZWM1. 13 Clabeling of proteinogenic amino acids detection showed that ~(13)C labeled amino acids increased obviously in the mutant 25113Δfrm A/p ZWM1-13. Among them,~(13)C labeled methionine increased by 7.236%. Therefore,directed evolution improved methanol utilization efficiency of thegenetically engineered E. coli.
Methanol is an important organic chemical raw material as well as an attractive alternative of sugar for biochemical industrybecause of its cost advantage and higher degree of carbon reduction. However,commonly used industrial microorganisms such as Escherichia coli cannot utilize methanol as a carbon source,which has become a bottleneck for the application of methanol in bio-production processes.In this study,a methanol utilization pathway(ribulose monophosphate pathway)was constructed in E. coli using an engineering geneticallyengineered E. coli as a starting strain,the random mutation occurred by continuous passage and direct evolution. Then the mutants werescreened using methanol as the carbon source,and 2 mutants that grew well on methanol were obtained. When methanol used as an accessorycarbon source,biomass of the mutant 25113Δfrm A/p ZWM1-13 increased by 27.6% compared to the starting strain 25113Δfrm A/p ZWM1. 13 Clabeling of proteinogenic amino acids detection showed that ~(13)C labeled amino acids increased obviously in the mutant 25113Δfrm A/p ZWM1-13. Among them,~(13)C labeled methionine increased by 7.236%. Therefore,directed evolution improved methanol utilization efficiency of thegenetically engineered E. coli.
引文
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[19]Kalyuzhnaya MG,Puri AW,Lidstrom ME.Metabolic engineering inmethanotrophic bacteria[J].Metabolic Engineering,2015,29:142-152.
[20]Leβmeier L,Pfeifenschneider J,Carnicer M,et al.Production ofcarbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as cosubstrate[J].Applied Microbiology and Biotechnology,2015,99(23):10163-76.
[2]Muller JE,Meyer F,Litsanov B,et al.Engineering Escherichia colifor methanol conversion[J].Metabolic engineering,2015,28:190-201.
[3]Schrader J,Schilling M,Holtmann D,et al.Methanol-based industrial biotechnology:current status and future perspectives of methylotrophic bacteria[J].Trends in Biotechnology,2009,27(2):107-115.
[4]Kalyuzhnaya M,Puri AW,Lidstrom ME.Metabolic engineering inmethanotrophic bacteria[J].Metabolic Engineering,2015,29:142-152.
[5]Kato N,Yurimoto H,Thauer RK.The physiological role of theribulose monophosphate pathway in bacteria and archaea[J].Bioscience Biotechnology&Biochemistry,2006,70(1):10-21.
[6]晁红军,宋修鹏,孙继华,等.甲基营养菌的研究进展[J].微生物学通报,2009,36(11):1727-1737.
[7]Whitaker WB,Sandoval NR,Bennett RK,et al.Syntheticmethylotrophy:engineering the production of biofuels andchemicals based on the biology of aerobic methanol utilization[J].Current Opinion in Biotechnology,2015,33:165-175.
[8]宋中邦,陈丽梅,李昆志,等.细菌的核酮糖单磷酸途径与甲醛同化作用[J].微生物学报,2007,47(1):168-172.
[9]Witthoff S,Schmitz K,Niedenfuhr S,et al.Metabolic engineeringof Corynebacterium glutamicum for methanol metabolism[J].Applied and environmental microbiology,2015,81(6):2215-2225.
[10]Whitaker WB,Jones JA,Bennett K,et al.Engineering thebiological conversion of methanol to specialty chemicals inEscherichia coli[J].Metabolic Engineering,2017,39:49-59.
[11]Ochsner AM,Muller JE,Mora CA,et al.In vitro activation of NADdependent alcohol dehydrogenases by Nudix hydrolases is morewidespread than assumed[J].Febs Letters,2014,588(17):2993-2999.
[12]Wu TY,Chen CT,Liu TJ,et al.Characterization and evolution ofan activator-independent methanol dehydrogenase from Cupriavidus necator N-1[J].Applied Microbiology and Biotechnology,2016,100(11):1-15.
[13]Price JV,Chen L,Whitaker WB,et al.Scaffoldless engineeredenzyme assembly for enhanced methanol utilization[J].Proceedings of the National Academy of Sciences of the UnitedStates of America,2016,113(45):12691-12696.
[14]Luan GD,Cai Z,Li Y,et al.Genome replication engineeringassisted continuous evolution(GREACE)to improve microbialtolerance for biofuels production[J].Biotechnology for Biofuels,2013,6(1):1-11.
[15]Amann E,Ochs B,Abel KJ.Tightly regulated tac promotervectors useful for the expression of unfused and fused proteins inEscherichia coli[J].Gene,1988,69(2):301-15.
[16]Ishikawa H,Yoshihara M,Baba A,et al.Formation of zincprotoporphyrin IX form myoglobin with pork loin extract[J].Journal of the Faculty of Agriculture Kyushu University,2006,51(1):93-97.
[17]Wahl SA,Dauner M,Wiechert W.New tools for mass isotopomerdata evaluation in 13C flux analysis:Mass isotope correction,data consistency checking,and precursor relationships[J].Biotechnology&Bioengineering,2004,85(3):259-68.
[18]You L,Page L,Feng X,et al.Metabolic pathway confirmation anddiscovery through C-labeling of proteinogenic amino acids[J].Journal of Visualized Experiments,2012,59(59):e3583-3689.
[19]Kalyuzhnaya MG,Puri AW,Lidstrom ME.Metabolic engineering inmethanotrophic bacteria[J].Metabolic Engineering,2015,29:142-152.
[20]Leβmeier L,Pfeifenschneider J,Carnicer M,et al.Production ofcarbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as cosubstrate[J].Applied Microbiology and Biotechnology,2015,99(23):10163-76.