氧化葡萄糖酸杆菌中硫辛酸合成模块对维生素C一步混菌发酵的影响
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摘要
在由氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌构建的维生素C两菌一步发酵体系中,为了强化氧化葡萄糖酸杆菌对普通生酮古龙酸杆菌生长和产酸的促进作用,文中在氧化葡萄糖酸杆菌中构建硫辛酸合成功能模块。由含硫辛酸功能模块的氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌组成的两菌一步体系,能减轻普通生酮古龙酸杆菌单菌培养时的生长抑制,强化两菌的互作关系,使维生素C前体(2-酮基-L-古龙酸,2-KGA)的产量提高到73.34 g/L(对照组为59.09 g/L),醇酸转化率提高到86.0%。研究结果为进一步优化维生素C两菌一步发酵体系提供了新思路。
In a one-step fermentation system of vitamin C production with Gluconobacter oxydans and Ketogulonicigenium vulgare,a functional module of a-lipoic acid biosynthesis was constructed in G.oxydans.The engineered G.oxydans was co-cultured with K.vulgare to enhance the growth and 2-keto-L-gulonic acid(2-KGA)production of K.vulgare.This one-step fermentation system alleviated the growth inhibition during the mono-culture of K.vulgare and strengthened the interaction between the two bacteria.Moreover,the yield of vitamin C precursor(2-KGA)increased to 73.34 g/L(the control group was59.09 g/L),and the conversion of D-sorbitol to 2-KGA increased to 86.0%.This study provides a new idea for further optimizing the one-step fermentation system of vitamin C production.
In a one-step fermentation system of vitamin C production with Gluconobacter oxydans and Ketogulonicigenium vulgare,a functional module of a-lipoic acid biosynthesis was constructed in G.oxydans.The engineered G.oxydans was co-cultured with K.vulgare to enhance the growth and 2-keto-L-gulonic acid(2-KGA)production of K.vulgare.This one-step fermentation system alleviated the growth inhibition during the mono-culture of K.vulgare and strengthened the interaction between the two bacteria.Moreover,the yield of vitamin C precursor(2-KGA)increased to 73.34 g/L(the control group was59.09 g/L),and the conversion of D-sorbitol to 2-KGA increased to 86.0%.This study provides a new idea for further optimizing the one-step fermentation system of vitamin C production.
引文
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[25]Du J,Bai W,Song H,et al.Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium.Metab Eng,2013,19:50-56.
[26]Wang PP,Xia Y,Li JH,et al.Overexpression of pyrroloquinoline quinone biosynthetic genes affects L-sorbose production in Gluconobacter oxydans WSH-003.Biochem Eng J,2016,112:70-77.
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[28]Zou W,Liu LM,Zhang J,et al.Reconstruction and analysis of a genome-scale metabolic model of the vitamin C producing industrial strain Ketogulonicigenium vulgare WSH-001.J Biotechnol,2012,161(1):42-48.
[29]Jia N,Ding MZ,Du J,et al.Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies.Sci Rep,2016,6:23068.
[30]Xu Y,Zhou X,Shi CL,et al.α-Lipoic acid protects against the oxidative stress and cytotoxicity induced by cadmium in HepG2 cells through regenerating glutathione regulated by glutamate-cysteine ligase.Toxicol Mech Methods,2015,25(8):596-603.
[31]Moini H,Packer L,Saris NEL.Antioxidant and prooxidant activities ofα-lipoic acid and dihydrolipoic acid.Toxicol Appl Pharmacol,2002,182(1):84-90.
[32]Packer L,Kraemer K,Rimbach G.Molecular aspects of lipoic acid in the prevention of diabetes complications.Nutrition,2001,17(10):888-895.
[33]Ruan LJ,Hu ZC,Zheng YG.Research advances in biosynthesis of alpha-lipoic acid for antioxidant.Fine Specialty Chem,2012,20(7):49-53(in Chinese).阮丽娟,胡忠策,郑裕国.抗氧化剂α-硫辛酸的生物合成研究进展.精细与专用化学品,2012,20(7):49-53.
[34]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408.
[35]Xiong XH,Zhang WC,Wang JH,et al.Method for promoting growth and acid production of Ketogulonigenium vulgare:CN,CN102321698B.2013-05-08(in Chinese).熊向华,张惟材,汪建华,等.一种促进酮古龙酸菌生长和产酸的方法:中国,CN102321698B.2013-05-08.
[36]Pan CH.The engineered Ketogulonigenium vulgare enhanced interaction with companion Bacillus strain[D].Tianjin:Tianjin University,2017(in Chinese).潘才慧.工程化改造酮古龙酸杆菌强化混菌间的互作关系[D].天津:天津大学,2017.
[37]Cronan JE.Biotin and lipoic acid:synthesis,attachment,and regulation.EcoSal Plus,2008,3(1),doi:10.1128/ecosalplus.3.6.3.5.
[2]Cimmino L,Dolgalev I,Wang YB,et al.Restoration of TET2 function blocks aberrant self-renewal and leukemia progression.Cell,2017,170(6):1079-1095.e20.
[3]Wei DZ,Yuan WK,Yin GL,et al.Studies on kinetic model of vitamin C two-step fermentation process.Chin J Biotech,1992,8(3):277-282(in Chinese).魏东芝,袁渭康,尹光琳,等.维生素C二步发酵过程动力学模型的研究.生物工程学报,1992,8(3):277-282.
[4]Ma Q,Zhou J,Zhang WW,et al.Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C.PLoSONE,2011,6(10):e26108.
[5]Zhou J,Ma Q,Yi H,et al.Metabolome profiling reveals metabolic cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during induced swarm motility.Appl Environ Microbiol,2011,77(19):7023-7030.
[6]Ding MZ,Zou Y,Song H,et al.Metabolomic analysis of cooperative adaptation between co-cultured Bacillus cereus and Ketogulonicigenium vulgare.PLoS ONE,2014,9(4):e94889.
[7]Zhou J,Yi H,Wang LL,et al.Metabolomic analysis of the positive effects on Ketogulonigenium vulgare growth and 2-keto-L-gulonic acid production by reduced glutathione.OMICS,2012,16(7/8):387-396.
[8]Bai L.Mechanisms of the mutualism between Bacillus endophyticus and Ketogulonicigenium vulgare[D].Shenyang:Shenyang Agricultural University,2017(in Chinese).白玲.内生芽孢杆菌与普通生酮基古龙酸杆菌互作机制的研究[D].沈阳:沈阳农业大学,2017.
[9]LüSX,Liao L,Zhang YH.Research progress on the oxidative stress relieving of acid-producing strain by companion strain in vitamin C mixed cultures fermentation.JShenyang Agric Univ,2017,48(6):641-646(in Chinese).吕淑霞,廖林,张云鹤.Vc混菌发酵中伴生菌解除产酸菌氧化胁迫的研究进展.沈阳农业大学学报,2017,48(6):641-646.
[10]Zhu YB.Mechanisms in the mutualism between Bacillus megaterium and Ketogulonigenium vulgare[D].Wuxi:Jiangnan University,2012(in Chinese).朱益波.巨大芽孢杆菌与普通生酮基古龙酸菌互生作用研究[D].无锡:江南大学,2012.
[11]Liao L.Mechanisms on the oxidative stress relieving of Ketogulonicigenium vulgare by Rhodotorula mucilaginosa in vitamin C mixed culture fermentation[D].Shenyang:Shenyang Agricultural University,2018(in Chinese).廖林.Vc混菌发酵中胶红酵母解除普通生酮基古龙酸杆菌氧化胁迫的研究[D].沈阳:沈阳农业大学,2018.
[12]Yang Y,Gao M,Yu XD,et al.Optimization of medium composition for two-step fermentation of vitamin Cbased on artificial neural network-genetic algorithm techniques.Biotechnol Biotechnol Equip,2015,29(6):1128-1134.
[13]Wang T,Sun JB,Yuan JQ.Modeling and parameters identification of 2-keto-L-gulonic acid fed-batch fermentation.Bioprocess Biosyst Eng,2015,38(4):605-614.
[14]Zhang ZX,Sun JW,Yuan JQ.Investigating the interaction between Gluconobacter oxydans and Bacillus megaterium for 2-keto-L-gulonic acid biosynthesis in the two-step vitamin C fermentation.JShanghai Jiaotong Univ(Sci),2015,20(3):281-285.
[15]Zhu YB,Liu J,Du GC,et al.Sporulation and spore stability of Bacillus megaterium enhance Ketogulonigenium vulgare propagation and 2-keto-L-gulonic acid biosynthesis.Bioresour Technol,2012,107:399-404.
[16]Liu J,Hu SB,Chang F,et al.Effects of the accompany strain on the fermentation performance of Ketogulonigenium vulgare.J Taizhou Polytech Coll,2013,13(2):76-78(in Chinese).刘杰,胡少斌,常芳,等.伴生菌对普通生酮基古龙酸菌发酵的影响.泰州职业技术学院学报,2013,13(2):76-78.
[17]Jia N,Du J,Ding MZ,et al.Genome sequence of Bacillus endophyticus and analysis of its companion mechanism in the Ketogulonigenium vulgare-Bacillus strain consortium.PLoS ONE,2015,10(8):e0135104.
[18]Wang EX,Ding MZ,Ma Q,et al.Reorganization of a synthetic microbial consortium for one-step vitamin Cfermentation.Microb Cell Fact,2016,15:21.
[19]Gao LL,Hu YD,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.Metab Eng,2014,24:30-37.
[20]Anderson S,Marks CB,Lazarus R,et al.Production of2-keto-L-gulonate,an intermediate in L-ascorbate synthesis,by a genetically modified Erwinia herbicola.Science,1985,230(4722):144-149.
[21]Huang Z,Zou W,Liu J,et al.Glutathione enhances2-keto-L-gulonic acid production based on Ketogulonicigenium vulgare model i WZ663.JBiotechnol,2013,164(4):454-460.
[22]Ma Q,Zhang WW,Zhang L,et al.Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection.PLoS ONE,2012,7(2):e32156.
[23]Leduc S,De Troostembergh JC,Lebeault JM.Folate requirements of the 2-keto-L-gulonic acid-producing strain Ketogulonigenium vulgare LMP P-20356 in l-sorbose/CSL medium.Appl Microbiol Biotechnol,2004,65(2):163-167.
[24]Pan CH,Wang EX,Jia N,et al.Reconstruction of amino acid biosynthetic pathways increases the productivity of2-keto-L-gulonic acid in Ketogulonicigenium vulgare-Bacillus endophyticus consortium via genes screening.J Ind Microbiol Biotechnol,2017,44(7):1031-1040.
[25]Du J,Bai W,Song H,et al.Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium.Metab Eng,2013,19:50-56.
[26]Wang PP,Xia Y,Li JH,et al.Overexpression of pyrroloquinoline quinone biosynthetic genes affects L-sorbose production in Gluconobacter oxydans WSH-003.Biochem Eng J,2016,112:70-77.
[27]Miyazaki T,Tomiyama N,Shinjoh M,et al.Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO3255,which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E.coli.Biosci,Biotechnol,Biochem,2002,66(2):262-270.
[28]Zou W,Liu LM,Zhang J,et al.Reconstruction and analysis of a genome-scale metabolic model of the vitamin C producing industrial strain Ketogulonicigenium vulgare WSH-001.J Biotechnol,2012,161(1):42-48.
[29]Jia N,Ding MZ,Du J,et al.Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies.Sci Rep,2016,6:23068.
[30]Xu Y,Zhou X,Shi CL,et al.α-Lipoic acid protects against the oxidative stress and cytotoxicity induced by cadmium in HepG2 cells through regenerating glutathione regulated by glutamate-cysteine ligase.Toxicol Mech Methods,2015,25(8):596-603.
[31]Moini H,Packer L,Saris NEL.Antioxidant and prooxidant activities ofα-lipoic acid and dihydrolipoic acid.Toxicol Appl Pharmacol,2002,182(1):84-90.
[32]Packer L,Kraemer K,Rimbach G.Molecular aspects of lipoic acid in the prevention of diabetes complications.Nutrition,2001,17(10):888-895.
[33]Ruan LJ,Hu ZC,Zheng YG.Research advances in biosynthesis of alpha-lipoic acid for antioxidant.Fine Specialty Chem,2012,20(7):49-53(in Chinese).阮丽娟,胡忠策,郑裕国.抗氧化剂α-硫辛酸的生物合成研究进展.精细与专用化学品,2012,20(7):49-53.
[34]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408.
[35]Xiong XH,Zhang WC,Wang JH,et al.Method for promoting growth and acid production of Ketogulonigenium vulgare:CN,CN102321698B.2013-05-08(in Chinese).熊向华,张惟材,汪建华,等.一种促进酮古龙酸菌生长和产酸的方法:中国,CN102321698B.2013-05-08.
[36]Pan CH.The engineered Ketogulonigenium vulgare enhanced interaction with companion Bacillus strain[D].Tianjin:Tianjin University,2017(in Chinese).潘才慧.工程化改造酮古龙酸杆菌强化混菌间的互作关系[D].天津:天津大学,2017.
[37]Cronan JE.Biotin and lipoic acid:synthesis,attachment,and regulation.EcoSal Plus,2008,3(1),doi:10.1128/ecosalplus.3.6.3.5.