两阶段溶氧控制及FeSO_4添加对谷氨酸棒杆菌合成4-羟基异亮氨酸的影响
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摘要
优化4-羟基异亮氨酸发酵过程中溶氧水平及FeSO_4添加量,以提高其发酵水平。结合菌株Corynebacterium glutamicum HIL18及4-羟基异亮氨酸合成特点,首先考察不同溶氧水平及两阶段溶氧控制对4-羟基异亮氨酸发酵的影响。然后考察FeSO_4添加对其发酵的作用。20%溶氧有利于4-羟基异亮氨酸的合成。利用两阶段溶氧控制工艺(0～20 h、20%溶氧;20～64 h、30%溶氧)经64 h发酵,4-羟基异亮氨酸产量达到38.7 g/L,较未使用该工艺提高11.2%。采用两阶段FeSO_4添加策略(初始浓度为65μmol/L、20 h添加30μmol/L),4-羟基异亮氨酸的产量达到43.4 g/L。采用优化后的工艺使得4-羟基异亮氨酸产量较优化前提高27.3%。获得了4-羟基异亮氨酸发酵过程中两阶段溶氧控制及FeSO_4添加工艺。该结果为4-羟基异亮氨酸的高效发酵合成提供参考。
Effects of DO levels and two-stage DO control strategy on 4-hydroxyisoleucine production were investigated,followed by analyzing the effects of two-stage FeSO_4 addition.The results indicated that 20% DO was suitable for 4-hydroxyisoleucine synthesis during one-stage DO control process.During two-stage DO control process(20% DO for 0-20 h and 30% for 20-64 h),38.7 g/L 4-hydroxyisoleucine was obtained,which was 11.2% higher than that at 20%DO.By using two-stage FeSO_4 addition strategy(65μmol/L for primary concentration and 30μmol/L FeSO_4 added at 20 h),the production of 4-hydroxyisoleucine increased to 43.4 g/L,which was 27.3% higher than the control.In conclusion,the strategy of two-stage DO control and FeSO_4 addition provides a reference for highly efficient production of 4-hydroxyisoleucine.
Effects of DO levels and two-stage DO control strategy on 4-hydroxyisoleucine production were investigated,followed by analyzing the effects of two-stage FeSO_4 addition.The results indicated that 20% DO was suitable for 4-hydroxyisoleucine synthesis during one-stage DO control process.During two-stage DO control process(20% DO for 0-20 h and 30% for 20-64 h),38.7 g/L 4-hydroxyisoleucine was obtained,which was 11.2% higher than that at 20%DO.By using two-stage FeSO_4 addition strategy(65μmol/L for primary concentration and 30μmol/L FeSO_4 added at 20 h),the production of 4-hydroxyisoleucine increased to 43.4 g/L,which was 27.3% higher than the control.In conclusion,the strategy of two-stage DO control and FeSO_4 addition provides a reference for highly efficient production of 4-hydroxyisoleucine.
引文
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[26]SHI F,NIU T,FANG H.4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp.lactofermentum under optimal corn steep liquor limitation[J].Applied Microbiology and Biotechnology,2015,99(9):3 851-3 863.
[2]AHMAD A,ALGHAMDI S S,MAHMOOD K,et al.Fenugreek a multipurpose crop:Potentialities and improvements[J].Saudi Journal of Biological Sciences,2016,23(2):300-310.
[3]HAERI M R,LIMAKI H K,WHITE C J B,et al.Noninsulin dependent anti-diabetic activity of(2S,3R,4S)-4-hydroxyisoleucine of fenugreek(Trigonella foenum-graecum)in streptozotocin-induced type I diabetic rats[J].Phytomedicine,2012,19(7):571-574.
[4]JAISWAL N,MAURYA C K,VENKATESWARLU K,et al.4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway[J].European Journal of Nutrition,2012,51(7):893-898.
[5]MAURYA C K,SINGH R,JAISWAL N,et al.4-Hydroxyisoleucine ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells[J].Molecular and Cellular Endocrinology,2014,395(1-2):51-60.
[6]FULLER S,STEPHENS J M.Diosgenin,4-hydroxyisoleucine,and fiber from Fenugreek:Mechanisms of actions and potential effects on metabolic syndrome[J].Advances in Nutrition,2015,6(2):189-197.
[7]FOWDEN L,PRATT H M,SMITH A.4-Hydroxyisoleucine from seed of Trigonella foenum-graecum[J].Phytochemistry,1973,12(7):1 707-1 711.
[8]AOUADI K,JEANNEAU E,MSADDEK M,et al.1,3-dipolar cycloaddition of a chiral nitrone to(E)-1,4-dichloro-2-butene:A new efficient synthesis of(2S,3S,4R)-4-hydroxyisoleucine[J].Tetrahedron Letters,2012,53(23):2 817-2 821.
[9]KODERA T,SMIRNOV S V,SAMSONOVA N N,et al.A novel L-isoleucine hydroxylating enzyme,L-isoleucine dioxygenase from Bacillus thuringiensis,produces(2S,3R,4S)-4-hydroxyisoleucine[J].Biochemical and Biophysical Research Communication,2009,390(3):506-510.
[10]SHI X,MIYAKAWA T,NAKAMURA A,et al.Engineering a short-chain dehydrogenase/reductase for the stereoselective production of(2S,3R,4S)-4-hydroxyisoleucine with three asymmetric centers[J].Scientific Reports,2017,7(1):1-13.
[11]SMIRNOV S V,KODERA T,SAMSONOVA N N,et al.Metabolic engineering of Escherichia coli to produce(2S,3R,4S)-4-hydroxyisoleucine[J].Applied Microbiology and Biotechnology,2010,88(3):719-726.
[12]OGAWA J,KODERA T,SMIRNOV S V.A novel L-isoleucine metabolism in Bacillus thuringiensis generating(2S,3R,4S)-4-hydroxyisoleucine,a potential insulinotropic and anti-obesity amino acid[J].Applied Microbiology and Biotechnology,2011,89(6):1 929-1 938.
[13]KIVERO A D,NOVIKOVA A E,SMIRNOV S V.Modification of E.coli central metabolism to optimize the biotransformation of L-isoleucine into 4-hydroxyisoleucine by enzymatic hydroxylation[J].Applied Biochemistry and Microbiology,2012,48(7):639-644.
[14]ZHANG C,LI Y,MA J,et al.High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering[J].Metabolic Engineering,2018,49:287-298.
[15]SHI F,FANG H,NIU T,et al.Overexpression of ppc and lys C to improve the production of 4-hydroxyisoleucine and its precursor L-isoleucine in recombinant Corynebacterium glutamicum ssp.lactofermentum[J].Enzyme and Microbial Technology,2016,87:79-85.
[16]SHI F,ZHANG M,LI Y,et al.Sufficient NADPH supply and pkn G deletion improve 4-hydroxyisoleucine production in recombinant Corynebacterium glutamicum[J].Enzyme and Microbial Technology,2018,115:1-8.
[17]DIAO J J,LI X R,PEI G S,et al.Comparative metabolomic analysis of Crypthecodinium cohnii in response to different dissolved oxygen levels during docosahexaenoic acid fermentation[J].Biochemical and Biophysical Research Communications,2018,499(4):941-947.
[18]WEI Z H,CHEN N,LI Y J,et al.Glucose fed-batch integrated dissolved oxygen control strategy enhanced polysaccharide,total triterpenoids and inotodiol production in fermentation of a newly isolated Inonotus obliquus strain[J].Process Biochemistry,2018,66:1-6.
[19]ZHANG X L,CHEN J X,WU D,et al.Economical lipid production from Trichosporon oleaginosus via dissolved oxygen adjustment and crude glycerol addition[J].Bioresource Technology,2019,273:288-296.
[20]白亚磊,徐庆阳,谢希贤,等.溶氧控制对黄色短杆菌YILW合成L-异亮氨酸的影响[J].天津科技大学学报,2011,26(1):5-9;30.
[21]张成林,刘远,薛宁,等.苏云金芽孢杆菌重组L-异亮氨酸羟化酶的酶学性质及其在4-羟基异亮氨酸合成中的应用[J].微生物学报,2014,54(8):889-896.
[22]ZHANG C,MA J,LI Z,et al.A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells[J].Bioengineered,2017,9(1):72-79.
[23]张成林,龙辉,温冰,等.双底物指数流加和双阶段溶氧控制对谷氨酸棒状杆菌生产L-异亮氨酸的影响[J].食品与发酵工业,2014,40(4):1-6.
[24]LI Y,SUN L,FENG J,et al.Efficient production ofα-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase p H and biotin control strategy[J].Bioprocess and Biosystems Engineering,2016,39(6):967-976.
[25]HAUSINGER R P.Fe II/alpha-ketoglutarate-dependent hydroxylases and related enzymes[J].Critical Reviews in Biochemistry and Molecular Biology,2004,39(1):21-68.
[26]SHI F,NIU T,FANG H.4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp.lactofermentum under optimal corn steep liquor limitation[J].Applied Microbiology and Biotechnology,2015,99(9):3 851-3 863.