过程优化提高解脂亚洛酵母积累α-酮戊二酸
|
摘要
目前,应用解脂亚洛酵母发酵生产α-酮戊二酸由于产量和底物转化率低、生产周期长等问题,仍未大规模工业化生产。为了解决这些问题,以研究室诱变选育获得的1株高产α-酮戊二酸的解脂亚洛酵母Yarrowia lipolytica WSH-Z06 C3为出发菌株,考察了该菌株在50 L发酵罐中转速、碳酸钙浓度、溶氧以及补料方式(多节点补料、恒速补料)等因素对α-酮戊二酸积累的影响。结果表明,当转速为300 r/min时,α-酮戊二酸和丙酮酸的产量分别为32.4 g/L和19.66 g/L;碳酸钙质量浓度为20 g/L时,α-酮戊二酸的产量提高至38.55 g/L,丙酮酸降低至8.28 g/L;控制溶氧水平在50%时,α-酮戊二酸产量为42.39 g/L,此时丙酮酸为6.22 g/L。比较高初始甘油浓度和不同的补料发酵策略,发现恒速补料效果最好,发酵144 hα-酮戊二酸产量达到66.27 g/L,丙酮酸产量为20.82 g/L。通过上述发酵过程参数的优化,α-酮戊二酸的产量和底物的转化率比未优化前分别提高了67.3%和4.56%,为解脂亚洛酵母工业化生产α-酮戊二酸提供一定参考。
Currently, problems of low output, low substrate conversion rate and long fermentation cycle restricted the industrial production of α-ketoglutaric acid(α-KG) by Yarrowia lipolytica in large scale. A mutant strain, Y. lipolytica WSH-Z06 C3, with capacity of high output of α-KG, obtained in previous research was taken as starting strain to solve these three problems. Accumulation effects of rotating speed, calcium carbonate concentration, dissolved oxygen and feeding methods(multi-node feeding and constant speed feeding) and other factors on α-KG with Y. lipolytica WSH-Z06 C3 were investigated in 50 L fermentor. The results showed that when the rotating speed was set at 300 r/min, the concentration of α-KG and pyruvate reached 32.4 g/L and 19.66 g/L respectively. The output of α-KG was increased to 38.55 g/L, and pyruvate decreased to 8.28 g/L; when the concentration of calcium carbonate was at 20 g/L. The output of α-KG was further increased to 42.39 g/L with pyruvate at 6.22 g/L by controlling the level of dissolved oxygen at 50%. With the strategies of high initial glycerol concentration and different feeding methods investigated, it was found that the constant feeding mode had the best effect. The production of α-KG and pyruvate reached 66.27 g/L and 20.82 g/L, respectively. The output of α-KG and pyruvate increased 67.3% and 4.56%, respectively, as compared with the process before optimization, and provided considerable reference for industrial production of α-KG with Y. lipolytica.
Currently, problems of low output, low substrate conversion rate and long fermentation cycle restricted the industrial production of α-ketoglutaric acid(α-KG) by Yarrowia lipolytica in large scale. A mutant strain, Y. lipolytica WSH-Z06 C3, with capacity of high output of α-KG, obtained in previous research was taken as starting strain to solve these three problems. Accumulation effects of rotating speed, calcium carbonate concentration, dissolved oxygen and feeding methods(multi-node feeding and constant speed feeding) and other factors on α-KG with Y. lipolytica WSH-Z06 C3 were investigated in 50 L fermentor. The results showed that when the rotating speed was set at 300 r/min, the concentration of α-KG and pyruvate reached 32.4 g/L and 19.66 g/L respectively. The output of α-KG was increased to 38.55 g/L, and pyruvate decreased to 8.28 g/L; when the concentration of calcium carbonate was at 20 g/L. The output of α-KG was further increased to 42.39 g/L with pyruvate at 6.22 g/L by controlling the level of dissolved oxygen at 50%. With the strategies of high initial glycerol concentration and different feeding methods investigated, it was found that the constant feeding mode had the best effect. The production of α-KG and pyruvate reached 66.27 g/L and 20.82 g/L, respectively. The output of α-KG and pyruvate increased 67.3% and 4.56%, respectively, as compared with the process before optimization, and provided considerable reference for industrial production of α-KG with Y. lipolytica.
引文
[1] Chernyavskaya OG,Shishkanova NV,Il′chenko AP,et al.Synthesis of alpha-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol[J].Appl Microbiol Biotechnol,2000,53(2):152-158.
[2] Stottmeister U,Aurich A,Wilde H,et al.White biotechnology for green chemistry:fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses[J].Journal of Industrial Microbiology and Biotechnology,2005,32(11-12):651-664.
[3] Coudraylucas C,Le BH,Cynober L,et al.Ornithine alpha-ketoglutarate improves wound healing in severe burn patients:a prospective randomized double-blind trial versus isonitrogenous controls[J].Critical Care Medicine,2000,28(6):1772-1776.
[4] Tjener K,Stahnke LH,Andersen L,et al.Addition of alpha-ketoglutarate enhances formation of volatiles by Staphylococcus carnosus during sausage fermentation[J].Meat Science,2004,67(4):711-719.
[5] Cooper AJL,Ginos JZ,Meister A.Synthesis and properties of the α-keto acids[J].Chemical Reviews,1983,83(3):321-358.
[6] Verseck S,Karau A,Weber M.Fermentative production of alpha-ketoglutaric acid:DE,WO/2009/053489 [P].2009.
[7] Finogenova TV,Morgunov IG,Kamzolova SV,et al.Organic Acid Production by the Yeast Yarrowia lipolytica:A Review of Prospects[J].Applied Biochemistry and Microbiology,2005,41(5):418-425.
[8] Otto C,Yovkova V,Barth G.Overproduction and secretion of α-ketoglutaric acid by microorganisms[J].Applied Microbiology & Biotechnology,2011,92(4):689-695.
[9] Liu L,Hossain GS,Shin HD,et al.One-step production of α-ketoglutaric acid from glutamic acid with an engineered l-amino acid deaminase from Proteus mirabilis[J].Journal of Biotechnology,2013,164(1):97-104.
[10] Groenewald M,Boekhout T,Neuvéglise C,et al.Yarrowia lipolytica:safety assessment of an oleaginous yeast with a great industrial potential[J].Critical reviews in microbiology,2014,40(3):187-206.
[11] Madzak C.Engineering Yarrowia lipolytica for Use in Biotechnological Applications:A Review of Major Achievements and Recent Innovations[J].Molecular Biotechnology,2018,60(3):1-15.
[12] Zhou JW,Yin XX,Madzak C,et al.Enhanced alpha-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism[J].Journal of Biotechnology,2012,161(3):257-264.
[13] Krzysztof C,Ludwika TH,Magdalena R,et al.The bioconversion of waste products from rapeseed processing into keto acids by Yarrowia lipolytica[J].Industrial Crops and Products,2018,119:102-110.
[14] Yin X,Madzak C,Du G,et al.Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway[J].Applied Microbiology & Biotechnology,2012,96(6):1527-1537.
[15] Kamzolova SV,Chiglintseva MN,Lunina JN,et al.α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation[J].Applied microbiology and biotechnology,2012,96(3):783-791.
[16] Zhang H L,Fang F,Song L,et al.The pH Regulation Strategy for Boosting Production of α-Ketoglutaric Acid by Yarrowia lipolytica[J].Chinese Journal of Process Engineering,2017,17(1):183-187.
[17] Zeng WZ,Du GC,Chen J,et al.A high-throughput screening procedure for enhancing alpha-ketoglutaric acid production in Yarrowia lipolytica by random mutagenesis[J].Process Biochemistry,2015,50(10):1516-1522.
[18] Zhou JW,Zhou HY,Du GC,et al.Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction[J].Letters in Applied Microbiology,2010,51(3):264-271.
[19] Liu LM,Li Y,Du GC,et al.CaCO3 stimulates alpha-ketoglutarate accumulation during pyruvate fermentation by Torulopsis glabrata[J].Chinese Journal of Biotechnologr,2003,19(6):745-749.
[20] Braga A,Mesquita DP,Amaral AL,et al.Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media[J].Journal of Biotechnology,2016,217:22-30.
[21] Bellou S,Makri A,Triantaphyllidou IE,et al.Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment[J].Microbiology,2014,160(4):807-817.
[22] 张海林,方芳,刘松,等.强化解脂亚洛酵母菌体积累产α-酮戊二酸的pH调控策略[J].过程工程学报,2017,17(1):183-187
[23] 刘立明,李寅,堵国成,等.碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成[J].生物工程学报,2003,19(6):745-749.
[2] Stottmeister U,Aurich A,Wilde H,et al.White biotechnology for green chemistry:fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses[J].Journal of Industrial Microbiology and Biotechnology,2005,32(11-12):651-664.
[3] Coudraylucas C,Le BH,Cynober L,et al.Ornithine alpha-ketoglutarate improves wound healing in severe burn patients:a prospective randomized double-blind trial versus isonitrogenous controls[J].Critical Care Medicine,2000,28(6):1772-1776.
[4] Tjener K,Stahnke LH,Andersen L,et al.Addition of alpha-ketoglutarate enhances formation of volatiles by Staphylococcus carnosus during sausage fermentation[J].Meat Science,2004,67(4):711-719.
[5] Cooper AJL,Ginos JZ,Meister A.Synthesis and properties of the α-keto acids[J].Chemical Reviews,1983,83(3):321-358.
[6] Verseck S,Karau A,Weber M.Fermentative production of alpha-ketoglutaric acid:DE,WO/2009/053489 [P].2009.
[7] Finogenova TV,Morgunov IG,Kamzolova SV,et al.Organic Acid Production by the Yeast Yarrowia lipolytica:A Review of Prospects[J].Applied Biochemistry and Microbiology,2005,41(5):418-425.
[8] Otto C,Yovkova V,Barth G.Overproduction and secretion of α-ketoglutaric acid by microorganisms[J].Applied Microbiology & Biotechnology,2011,92(4):689-695.
[9] Liu L,Hossain GS,Shin HD,et al.One-step production of α-ketoglutaric acid from glutamic acid with an engineered l-amino acid deaminase from Proteus mirabilis[J].Journal of Biotechnology,2013,164(1):97-104.
[10] Groenewald M,Boekhout T,Neuvéglise C,et al.Yarrowia lipolytica:safety assessment of an oleaginous yeast with a great industrial potential[J].Critical reviews in microbiology,2014,40(3):187-206.
[11] Madzak C.Engineering Yarrowia lipolytica for Use in Biotechnological Applications:A Review of Major Achievements and Recent Innovations[J].Molecular Biotechnology,2018,60(3):1-15.
[12] Zhou JW,Yin XX,Madzak C,et al.Enhanced alpha-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism[J].Journal of Biotechnology,2012,161(3):257-264.
[13] Krzysztof C,Ludwika TH,Magdalena R,et al.The bioconversion of waste products from rapeseed processing into keto acids by Yarrowia lipolytica[J].Industrial Crops and Products,2018,119:102-110.
[14] Yin X,Madzak C,Du G,et al.Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway[J].Applied Microbiology & Biotechnology,2012,96(6):1527-1537.
[15] Kamzolova SV,Chiglintseva MN,Lunina JN,et al.α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation[J].Applied microbiology and biotechnology,2012,96(3):783-791.
[16] Zhang H L,Fang F,Song L,et al.The pH Regulation Strategy for Boosting Production of α-Ketoglutaric Acid by Yarrowia lipolytica[J].Chinese Journal of Process Engineering,2017,17(1):183-187.
[17] Zeng WZ,Du GC,Chen J,et al.A high-throughput screening procedure for enhancing alpha-ketoglutaric acid production in Yarrowia lipolytica by random mutagenesis[J].Process Biochemistry,2015,50(10):1516-1522.
[18] Zhou JW,Zhou HY,Du GC,et al.Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction[J].Letters in Applied Microbiology,2010,51(3):264-271.
[19] Liu LM,Li Y,Du GC,et al.CaCO3 stimulates alpha-ketoglutarate accumulation during pyruvate fermentation by Torulopsis glabrata[J].Chinese Journal of Biotechnologr,2003,19(6):745-749.
[20] Braga A,Mesquita DP,Amaral AL,et al.Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media[J].Journal of Biotechnology,2016,217:22-30.
[21] Bellou S,Makri A,Triantaphyllidou IE,et al.Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment[J].Microbiology,2014,160(4):807-817.
[22] 张海林,方芳,刘松,等.强化解脂亚洛酵母菌体积累产α-酮戊二酸的pH调控策略[J].过程工程学报,2017,17(1):183-187
[23] 刘立明,李寅,堵国成,等.碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成[J].生物工程学报,2003,19(6):745-749.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |