前期研究构建了1株组成型表达山梨糖脱氢酶(sorbose dehydrogenase, SDH)的大肠杆菌工程菌,该菌株能利用L-山梨糖生产维生素C前体2-酮基-L-古龙酸(2-keto-L-gulonic acid, 2-KLG),但对底物L-山梨糖的耐受性较差。为解决这一问题,对该菌株进行适应性进化,并强化了进化菌株发酵生产2-KLG的能力。首先,应用基于微流控技术的全自动高通量微生物液滴培养系统,将出发菌株在不同浓度梯度的L-山梨糖培养基中生长、传代,获得能够耐受高浓度L-山梨糖的进化菌株。在摇瓶上进一步验证,最终获得了1株能耐受高浓度L-山梨糖的进化菌株2-F6。然后在2-F6中共表达了能促进2-酮基-L-古龙酸积累的山梨酮脱氢酶(sorbosone dehydrogenase, SNDH),并在摇瓶水平上对接种量、发酵温度、SNDH诱导时间、IPTG诱导浓度以及L-山梨糖添加量进化了优化,在最优条件下,2-KLG的产量达6.05 g/L。最终,将摇瓶发酵条件放大至5 L发酵罐后,2-KLG的产量为5.70 g/L。研究结果为一菌一步发酵法生产维生素C前体2-KLG提供了参考。
An engineered Escherichia coli constitutively expressing sorbose dehydrogenase (SDH) was previously constructed for producing the vitamin C precursor 2-keto-L-gulonic acid (2-KLG) with L-sorbose as substrate. However, this strain has a poor tolerance to L-sorbose. In order to solve this problem, adaptive laboratory evolution was carried out. Firstly, to enhance the tolerance of L-sorbose, the starting strain was grown and subcultured in the medium with different gradient concentration of L-sorbose by applying Microbial Microdroplet Culture System designed based on microfluidic technology. An evolutionary strain 2-F6 that could tolerate high concentration of L-sorbose was obtained after further verifying on the shaking flasks. Then, the sorbosone dehydrogenase (SNDH) was expressed in 2-F6 to promote the strength of 2-KLG production. The inoculation amount, fermentation temperature, induction time for SNDH, IPTG concentration and L-sorbose addition were optimized in the shaking flasks, and the titer of 2-KLG reached 6.05 g/L under the optimized optimal conditions. Finally, after scaling up in a 5 L fermenter, 2-KLG of 5.70 g/L was obtained. The results obtained could provide references for the production of vitamin C precursor 2-KLG with one-step fermentation process.
[1] PAPPENBERGER G,HOHMANN H P.Industrial production of L-ascorbic acid (vitamin C) and D-isoascorbic acid[J].Advances in Biochemical Engineering/Biotechnology,2014,143:143-188.
[2] CAMARENA V,WANG G.The epigenetic role of vitamin C in health and disease[J].Cellular and Molecular Life Sciences,2016,73(8):1 645-1 658.
[3] KAZMIERCZAK-BARANSKA J,BOGUSZEWSKA K,ADAMUS-GRABICKA A,et al.Two faces of vitamin C-Antioxidative and pro-oxidative agent[J].Nutrients,2020,12(5):1 501.
[4] TIMOSHNIKOV V A,KOBZEVA T V,POLYAKOV N E,et al.Redox interactions of vitamin C and iron:Inhibition of the pro-oxidant activity by deferiprone[J].International Journal of Molecular Sciences,2020,21(11):3 967.
[5] 万慧,康振,李江华,等.高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响[J].微生物学报,2016,56(10):1 656-1 663.
WAN H,KANG Z,LI J,et al.Effect of high 2-KLG concentration on expression of pivotal genes involved in 2-KLG synthesis in Gluconobacter oxydans WSH-003[J].Acta Microbiologica Sinica,2016,56(10):1 656-1 663.
[6] SONOYAMA T,TANI H,MATSUDA K,et al.Production of 2-keto-L-gulonic acid from D-glucose by two-stage fermentation[J].Applied and Environmental Microbiology,1982,43(5):1 064-1 069.
[7] ZHANG J,LIU J,SHI Z,et al.Manipulation of B.megaterium growth for efficient 2-KLG production by K.vulgare[J].Process Biochemistry,2010,45(4):602-606.
[8] WANG C Y,LI Y,GAO Z W,et al.Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters[J].Microbial Cell Factories,2018,17(1):81.
[9] QIAN M,YAN H B,EN X W,et al.Integrated proteomic and metabolomic analysis of a reconstructed three-species microbial consortium for one-step fermentation of 2-keto-L-gulonic acid,the precursor of vitamin C[J].Journal of Industrial Microbiology & Biotechnology,2019,46(1):21-31.
[10] ANDERSON S,MARKS C B,LAZARUS R,et al.Production of 2-keto-L-gulonate,an intermediate in L-ascorbate synthesis,by a genetically modified Erwinia herbicola[J].Science,1985,230(4 722):144-149.
[11] SAITO Y,ISHII Y,HAYASHI H,et al.Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate,a precursor of L-ascorbic acid,in a recombinant G.oxydans strain[J].Applied and Environmental Microbiology,1997,63(2):454-460.
[12] GAO L,HU Y,LIU J,et al.Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol[J].Metablic Engneering,2014,24:30-37.
[13] 杨燕花,吕永坤,陈吉铭,等.2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化[J].食品与发酵工业,2016,42(7):60-64.
YANG Y,LV Y,CHEN J,et al.Bioprocess optimization for the production of 2-keto-L-gulonic acid by a one-step fermentation strain[J].Food and Fermentation Industries,2016,42(7):60-64.
[14] WANG P,XIA Y,LI J,et al.Overexpression of pyrroloquinoline quinone biosynthetic genes affects L-sorbose production in Gluconobacter oxydans WSH-003[J].Biochemical Engineering Journal,2016,112:70-77.
[15] KIM T S,HUI G,LI J,et al.Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose[J].Scientific Reports,2019,9(1):815.
[16] CHEN Y,LIU L,SHAN X,et al.High-throughput screening of a 2-keto-L-gulonic acid-producing Gluconobacter oxydans strain based on related dehydrogenases[J].Frontiers in Bioengineering and Biotechnology,2019,7:385.
[17] SHAN X,LIU L,ZENG W,et al.High throughput screening platform for a FAD-dependent L-sorbose sehydrogenase[J].Frontiers in Bioengineering and Biotechnology,2020,8:194.
[18] MCCLOSKEY D,XU S,SANDBERG T E,et al.Adaptive laboratory evolution resolves energy depletion to maintain high aromatic metabolite phenotypes in Escherichia coli strains lacking the Phosphotransferase System[J].Metabolic Engineering,2018,48:233-242.
[19] ZENG W,GUO L,XU S,et al.High-throughput screening technology in industrial biotechnology[J].Trends in Biotechnology,2020,38(8):888-906.
[20] JIAN X,GUO X,WANG J,et al.Microbial microdroplet culture system (MMC):An integrated platform for automated,high-throughput microbial cultivation and adaptive evolution[J].Biotechnology and Bioengineering,2020,117(6):1 724-1 737.
[21] TAN Z L,ZHENG X,WU Y,et al.In vivo continuous evolution of metabolic pathways for chemical production[J].Microbial Cell Factories,2019,18(1):82.
[22] TRAVICA N,RIED K,SALI A,et al.Vitamin C status and cognitive function:A systematic review[J].Nutrients,2017,9(9):960.
[23] KIM E E.Vitamin C in human health and disease:Effects,mechanisms of action,and new guidance on intake[J].Journal of Nuclear Medicine,2020,61(4):623-623.
[24] WANG P,ZENG W,XU S,et al.Current challenges facing one-step production of L-ascorbic acid[J].Biotechnology Advances,2018,36(7):1 882-1 899.
