VHY18是之前构建的可在限氧条件下高产缬氨酸的菌株,但在发酵过程中会大量积累琥珀酸和乙酸,生长活力也会显著衰退,限制其发酵性能。为此,该研究首先敲除了VHY18中丙酮酸氧化酶和乙酸激酶编码基因poxB和ackA,构建了VHY19,使乙酸浓度降低了43.7%。接着敲除了VHY19的苹果酸脱氢酶编码基因mdh,构建了VHY20,使琥珀酸浓度降低了50.8%。最后优化了VHY20的双阶段发酵工艺,主要摸索了限氧发酵阶段的供氧条件、2个阶段的转换时机及葡萄糖流加速率。优化后的VHY20双阶段发酵工艺为,发酵开始时先进行好氧发酵,14 h后将好氧发酵转换至限氧发酵,并将供氧条件设置为搅拌转速400 r/min、风量2 L/min,补糖速率控制在25 g/h。菌株和发酵过程优化后,缬氨酸产量为90.4 g/L,转化率为0.53 g/g葡萄糖,分别较优化前提高了7.8%和30.5%;乙酸和琥珀酸的积累量分别为2.7和1.2 g/L,较优化前降低了76.9%和95.4%。
The VHY18 strain capable of high-yield valine production under oxygen-limited conditions was previously constructed. However, a large amount of succinate and acetate were accumulated during the fermentation process and the strain growth declined significantly, limiting the fermentation performance. To this end, the genes encoding the pyruvate oxidase and acetate kinase, poxB and ackA, were firstly knocked out from VHY18 in this study, generating VHY19, and enabled acetate concentration to decrease by 43.7%. Then the mdh gene encoding the malate dehydrogenase of WHY19 was knocked out to construct VHY20, contribute to decrease of acetate concentration by 43.7%. Finally, the two-stage fermentation process of VHY20 was optimized, mainly exploring the oxygen supply conditions in the oxygen-limited fermentation stage, the switch timing between the two stages, and the supplementation rate of glucose. The optimized two-stage fermentation process of VHY20 was as follows: aerobic fermentation was carried out at the beginning of the fermentation, and after 14 h, the aerobic fermentation was switched to oxygen-limited fermentation, and the oxygen supply conditions were set as stir speed of 400 r/min, air flow of 2 L/min and the glucose supplementation rate was controlled at 25 g/h. Through the optimization of the strain and fermentation process, the valine titer was up to 90.4 g/L with the yield of 0.53 g/g glucose, increasing by 7.8% and 30.5% respectively and the succinate and acetate titers were down to 2.7 and 1.2 g/L, decreasing by 76.9% and 95.4% respectively compared with results before optimization.
[1] KARAU A, GRAYSON I.Amino acids in human and animal nutrition[J].Advances in Biochemical Engineering/Biotechnology, 2014, 143:189-228.
[2] DUNSHEA F R, BAUMAN D E, NUGENT E A, et al.Hyperinsulinaemia, supplemental protein and branched-chain amino acids when combined can increase milk protein yield in lactating sows[J].The British Journal of Nutrition, 2005, 93(3):325-332.
[3] TAKPHO N, WATANABE D, TAKAGI H.High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae[J].Metabolic Engineering, 2018, 46:60-67.
[4] PARK J H, LEE S Y.Fermentative production of branched chain amino acids:A focus on metabolic engineering[J].Applied Microbiology and Biotechnology, 2010, 85(3):491-506.
[5] 张学礼, 郭恒华, 刘萍萍, 等.生产L-缬氨酸的重组大肠杆菌、其构建方法及其应用:中国, CN113278641A[P].2021-08-20.
ZHANG X L, GUO H H, LIU P P, et al.Producing L-valine in recombinant Escherichia coli and the application with the method of its construction:China, CN113278641A[P].2021-08-20.
[6] PARK J H, KIM T Y, LEE K H, et al.Fed-batch culture of Escherichia coli for L-valine production based on in silico flux response analysis[J].Biotechnology and Bioengineering, 2011, 108(4):934-946.
[7] HASEGAWA S, UEMATSU K, NATSUMA Y, et al.Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions[J].Applied and Environmental Microbiology, 2012, 78(3):865-875.
[8] HAO Y N, MA Q, LIU X Q, et al.High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation[J].Metabolic Engineering, 2020, 62:198-206.
[9] 周景文, 高松, 刘延峰, 等.新一代发酵工程技术:任务与挑战[J].食品与生物技术学报, 2021, 40(1):1-11.
ZHOU J W, GAO S, LIU Y F, et al.Next generation fermentation engineering:Missions and challenges[J].Journal of Food Science and Biotechnology, 2021, 40(1):1-11.
[10] YU Y, ZHU X N, XU H T, et al.Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli[J].Metabolic Engineering, 2019, 56:181-189.
[11] 刘剑, 赵策, 储炬, 等.溶氧对谷氨酸棒杆菌发酵产谷氨酸代谢的影响[J].华东理工大学学报(自然科学版), 2012, 38(4):459-464.
LIU J, ZHAO C, CHU J, et al.Influence of dissolved oxygen on metabolism of glutamic acid biosynthesis by Corynebacterium glutamicum[J].Journal of East China University of Science and Technology (Natural Science Edition), 2012, 38(4):459-464.
[12] 焦策. 溶氧对Candida glycerinogenes产甘油发酵的调控机理研究[D].无锡:江南大学, 2012.
JIAO C.Regulate mechanism of dissolved oxygen on the glycerol fermentation by Candida glycerinogenes[D].Wuxi:Jiangnan University, 2012.
[13] BOECKER S, SLAVIERO G, SCHRAMM T, et al.Deciphering the physiological response of Escherichia coli under high ATP demand[J].Molecular Systems Biology, 2021, 17(12):e10504.
[14] HASEGAWA S, SUDA M, UEMATSU K, et al.Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions[J].Applied Environmental Microbiology, 2013, 79(4):1 250-1 257.
[15] STEINSIEK S, FRIXEL S, STAGGE S, et al.Characterization of E.coli MG1655 and frdA and sdhC mutants at various aerobiosis levels[J].Journal of Biotechnology, 2011, 154(1):35-45.
[16] ZOU S P, ZHAO K, TANG H, et al.Improved production of D-pantothenic acid in Escherichia coli by integrated strain engineering and fermentation strategies[J].Journal of Biotechnology, 2021, 339:65-72.
[17] LI Y F, LIN Z Q, HUANG C, et al.Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing[J].Metabolic Engineering, 2015, 31:13-21.
[18] 杜丽红, 熊海波, 徐达, 等.利用谷氨酸棒杆菌CRISPRi系统构建L-缬氨酸高产菌株[J].食品与发酵工业, 2020, 46(17):1-8.
DU L H, XIONG H B, XU D, et al.Construction of L-valine production strain by CRISPRi system of Corynebacterium glutamicum[J].Food and Fermentation Industries, 2020, 46(17):1-8.
[19] SCHÜTZE A, BENNDORF D, PÜTTKER S, et al.The impact of ackA, pta, and ackA-pta mutations on growth, gene expression and protein acetylation in Escherichia coli K-12[J].Frontiers in Microbiology, 2020, 11:233.
[20] WANG J, SHANG Q, ZHAO C, et al.Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway[J].Letters in Applied Microbiology, 2020, 70(2):64-70.
[21] PARIMI N S, DURIE I A, WU X H, et al.Eliminating acetate formation improves citramalate production by metabolically engineered Escherichia coli[J].Microbial Cell Factories, 2017, 16(1):114.
[22] KIM W J, AHN J H, KIM H U, et al.Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering[J].Biotechnology Journal, 2017, 12(2):1600701.
[23] 刘嵘明, 梁丽亚, 吴明科, 等.微生物发酵生产丁二酸研究进展[J].生物工程学报, 2013, 29(10):1 386-1 397.
LIU R M, LIANG L Y, WU M K, et al.Progress in microbial production of succinic acid[J].Chinese Journal of Biotechnology, 2013, 29(10):1 386-1 397.
[24] MA J F, JIANG M, CHEN K Q, et al.Strategies for efficient repetitive production of succinate using metabolically engineered Escherichia coli[J].Bioprocess and Biosystems Engineering, 2011, 34(4):411-418.
[25] 王莉. 乙醛酸循环途径对谷氨酸棒杆菌两阶段发酵生产丁二酸的影响[D].杭州:浙江大学, 2013.
WANG L.Effects of glyoxylate cycle on succinic acid dual-phase production in Corynebacterium glutamicum ATCC 13032[D].Hangzhou:Zhejiang University, 2013.
[26] BETTENBROCK K, BAI H, EDERER M, et al.Towards a systems level understanding of the oxygen response of Escherichia coli[J].Advances in Microbial Physiology, 2014, 64:65-114.
[27] PATIL V, SANTOS C N S, AJIKUMAR P K, et al.Balancing glucose and oxygen uptake rates to enable high amorpha-4, 11-diene production in Escherichia coli via the methylerythritol phosphate pathway[J].Biotechnology and Bioengineering, 2021, 118(3):1 317-1 329.
