葡萄糖脱氢酶基因的缺失对Acetobacter sp.碳源代谢及3-羟基丙酸合成的影响
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摘要
3-羟基丙酸(3-HP)是一种新兴的高附加值平台化合物,醋酸杆菌(Acetobacter sp.)可高效催化1,3-丙二醇(1,3-PDO)合成3-HP,但其膜上脱氢酶亦可将葡萄糖氧化使培养液酸化,菌体生长受限导致生物量较低.利用同源重组技术对葡萄糖脱氢酶(GDH)基因gdh进行敲除,并考察该基因缺失对细胞生长、碳源代谢及3-HP合成的影响. gdh基因敲除后混合(葡萄糖、甘油)碳源培养条件下菌体量较野生菌提高了1.72倍;碳流分析显示葡萄糖在膜上被GDH氧化生成葡萄糖酸,大部分葡萄糖酸最终被氧化为2-酮基葡萄糖酸,少量经戊糖磷酸途径(PPP)途径被分解,而甘油经磷酸化后进入中心代谢途径或糖异生途径.本研究表明gdh基因敲除后混合碳源培养可大幅度提高菌体量且可以保证较高的催化合成3-HP性能,可为改善醋酸菌碳源利用及催化性能提供理论基础.
3-Hydroxypropionic acid(3-HP) is an emerging platform chemical with a high added-value. Resting cells of Acetobacter sp. can efficiently catalyze 1,3-propanediol(1,3-PDO) to 3-HP. Glucose is oxidized by the membrane-bound dehydrogenase, resulting in an acidic environment that inhibits cell growth and reduces the biomass. We deleted the gdh gene for glucose dehydrogenase(GDH), and investigated the effects on cell growth, carbon metabolism, and 3-HP production. The gdh gene knocked-out showed a 1.72-fold increase in biomass in the mixed medium containing glucose and glycerol. A carbon flux analysis showed that glucose was converted to gluconic acid by GDH, followed by an oxidation to 2-ketogluconic acid. In addition, a small percentage of the gluconic acid was degraded via the pentose phosphate pathway. Glycerol was phosphorylated and entered the central pathway(gluconeogenesis). Results indicate that the deletion of gdh can effectively promote higher cell densities and improve the catalytic performance for the production of 3-HP, and thus provide a theoretical reference for improving the carbon source utilization and the catalytic performance of acetic acid bacteria.
3-Hydroxypropionic acid(3-HP) is an emerging platform chemical with a high added-value. Resting cells of Acetobacter sp. can efficiently catalyze 1,3-propanediol(1,3-PDO) to 3-HP. Glucose is oxidized by the membrane-bound dehydrogenase, resulting in an acidic environment that inhibits cell growth and reduces the biomass. We deleted the gdh gene for glucose dehydrogenase(GDH), and investigated the effects on cell growth, carbon metabolism, and 3-HP production. The gdh gene knocked-out showed a 1.72-fold increase in biomass in the mixed medium containing glucose and glycerol. A carbon flux analysis showed that glucose was converted to gluconic acid by GDH, followed by an oxidation to 2-ketogluconic acid. In addition, a small percentage of the gluconic acid was degraded via the pentose phosphate pathway. Glycerol was phosphorylated and entered the central pathway(gluconeogenesis). Results indicate that the deletion of gdh can effectively promote higher cell densities and improve the catalytic performance for the production of 3-HP, and thus provide a theoretical reference for improving the carbon source utilization and the catalytic performance of acetic acid bacteria.
引文
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15 Kostner D,Luchterhand B,Junker A,Volland S,Daniel R,Buchs J,Liebl W,Ehrenreich A.The consequence of an additional NADHdehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504[J].Appl Microbiol Biotechnol,2015,99(1):375-386
16 Pronk JT,Levering PR,Olijve W,Dijken JPV.Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans[J].Enzyme Microb Technol,2012,11(3):160-164
17 Hanke T,N?h K,Noack S,Polen T,Bringer S,Sahm H.Combined f luxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans621H[J].Appl Environ Microbiol,2013,79(7):2336-2348
18 Yuan J,Wu M,Lin J,Yang L.Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation[J].BMC Biotechnol,2016,16(1):16-42
2 Zhu B,Li J,He Y,Yamane H,Kimura Y,Nishida H.Effect of steric hindrance on hydrogen-bonding interaction between polyesters and natural polyphenol catechin[J].J Appl Polym Sci,2010,91(6):3565-3573
3 Werpy TA,Holladay JE.White JF.Top Value Added Chemicals from Biomass:I.Results of Screening for Potential Candidates from Sugars and Synthesis Gas[M].Richland,WA(US):Pacific Northwest National Laboratory(PNNL),2004:28-36
4 Jiang X,Meng X,Xian M.Biosynthetic pathways for 3-hydroxypropionic acid production[J].Appl Microbiol Biotechnol,2009,82(6):995-1003
5 Adachi O,Yakushi T.Membrane-Bound Dehydrogenases of Acetic Acid Bacteria[M].Japan:Springer,2016
6 Arai H,Sakurai K,Ishii M.Metabolic Features of Acetobacter aceti[M].Japan:Springer,2016
7吴金鑫,宗红,陆信曜,诸葛斌,方慧英,宋健.高效催化合成3-羟基丙酸的菌株特性[J].应用与环境生物学报,2014,20(5):804-808[Wu JX,Zong H,Lu XY,Zhuge B,Fang HY,Song J.Characterization of a strain catalyzing biosynthesis of 3-hydroxypropionic acid[J].Chin JAppl Environ Biol,2014,20(5):804-808]
8 Li J,Zong H,Zhuge B,Lu X,Fang H,Sun J.Immobilization of Acetobacter sp.CGMCC 8142 for efficient biocatalysis of 1,3-propanediol to 3-hydroxypropionic acid[J].Biotechnol Bioproc Eng,2016,21(4):523-530
9李俊,宗红,诸葛斌,陆信曜,方慧英,宋健.Acetobacter sp.驯化选育及固定化生产3-羟基丙酸[J].食品与发酵工业,2016,42(9):40-44[Li J,Zong H,Zhuge B,Lu XY,Fang HY,Song J.Domestication and immobilization of Acetobacter sp.for 3-hydroxypropionic acid bioproduction[J].Food Fermemt Ind,2016,42(9):40-44]
10 Wei L,Zhu D,Zhou J,Zhang J,Zhu K,Du L,Hua Q.Revealing in vivo glucose utilization of Gluconobacter oxydans 621HΔmgdh strain by mutagenesis[J].Microbiol Res,2014,169(5-6):469-475
11 Zhu K,Lu L,Wei L,Wei D,Imanaka T,Hua Q.Modification and evolut ion of Gluconobacter ox ydans for en hanced g row th and biotransformation capabilities at low glucose concentration[J].Mol Biotechnol,2011,49(1):56-64
12 Krajewski V,Simic P,Mouncey NJ,Bringer S,Sahm H,Bott M.Metabolic engineering of Gluconobacter oxydans for improved growth rate and growth yield on glucose by elimination of gluconate formation[J].Appl Environ Microbiol,2010,76(13):4369-4376
13 Silberbach M,Maier B,Zimmermann M,Büchs J.Glucose oxidation by Gluconobacter oxydans:characterization in shaking-flasks,scale-up and optimization of the pH profile[J].Appl Microbiol Biotechnol,2003,62(1):92-98
14 Richhardt J,Luchterhand B,Bringer S,Büchs J,Bott M.Evidence for a key role of cytochrome bo3 oxidase in respiratory energy metabolism of Gluconobacter oxydans[J].J Bacteriol,2013,195(18):4210-4220
15 Kostner D,Luchterhand B,Junker A,Volland S,Daniel R,Buchs J,Liebl W,Ehrenreich A.The consequence of an additional NADHdehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504[J].Appl Microbiol Biotechnol,2015,99(1):375-386
16 Pronk JT,Levering PR,Olijve W,Dijken JPV.Role of NADP-dependent and quinoprotein glucose dehydrogenases in gluconic acid production by Gluconobacter oxydans[J].Enzyme Microb Technol,2012,11(3):160-164
17 Hanke T,N?h K,Noack S,Polen T,Bringer S,Sahm H.Combined f luxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans621H[J].Appl Environ Microbiol,2013,79(7):2336-2348
18 Yuan J,Wu M,Lin J,Yang L.Combinatorial metabolic engineering of industrial Gluconobacter oxydans DSM2343 for boosting 5-keto-D-gluconic acid accumulation[J].BMC Biotechnol,2016,16(1):16-42