谷氨酸棒杆菌代谢工程高效生产L-缬氨酸
作者:
基金项目:

江苏省农业科技自主创新资金项目(CX(22)1012);国家自然科学基金(22122806, 22208124, 32000037);江苏省自然科学基金(BK20211529, BK20200614)


Highly efficient production of L-valine by multiplex metabolic engineering of Corynebacterium glutamicum
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [45]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5 L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110 g/L、0.51 g/g和2.29 g/(L·h)。

    Abstract:

    As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L·h), respectively.

    参考文献
    [1] OLDIGES M, EIKMANNS BJ, BLOMBACH B. Application of metabolic engineering for the biotechnological production of L-valine[J]. Applied Microbiology and Biotechnology, 2014, 98(13):5859-5870.
    [2] KARAU A, GRAYSON I. Amino acids in human and animal nutrition[M]//Advances in Biochemical Engineering/Biotechnology. Berlin, Heidelberg:Springer Berlin Heidelberg, 2014:189-228.
    [3] BISHOP CA, SCHULZE MB, KLAUS S, WEITKUNAT K. The branched-chain amino acids valine and leucine have differential effects on hepatic lipid metabolism[J]. The FASEB Journal, 2020, 34(7):9727-9739.
    [4] CHEN XH, LIU SR, PENG B, LI D, CHENG ZX, ZHU JX, ZHANG S, PENG YM, LI H, ZHANG TT, PENG XX. Exogenous L-valine promotes phagocytosis to kill multidrug-resistant bacterial pathogens[J]. Frontiers in Immunology, 2017, 8:207.
    [5] WEN J, HELMBRECHT A, ELLIOT MA, THOMSON J, PERSIA ME. Evaluation of the valine requirement of smalL-framed first cycle laying hens[J]. Poultry Science, 2019, 98(3):1272-1279.
    [6] LIAQAT U, DITTA Y, NAVEED S, KING A, PASHA T, ULLAH S, MAJEED KA. Effects of L-valine in layer diets containing 0.72% isoleucine[J]. PLoS One, 2022, 17(4):e0258250.
    [7] D'ESTE M, ALVARADO-MORALES M, ANGELIDAKI I. Amino acids production focusing on fermentation technologies-a review[J]. Biotechnology Advances, 2018, 36(1):14-25.
    [8] WANG XY, ZHANG HL, QUINN PJ. Production of L-valine from metabolically engineered Corynebacterium glutamicum[J]. Applied Microbiology and Biotechnology, 2018, 102(10):4319-4330.
    [9] GAO H, TUYISHIME P, ZHANG X, YANG TW, XU MJ, RAO ZM. Engineering of microbial cells for L-valine production:challenges and opportunities[J]. Microbial Cell Factories, 2021, 20(1):1-16.
    [10] ZHAO K, LIU JM, GAO C, LIU J, CHEN XL, LIU LM, GUO L. Advances in microbial production of feed amino acid[M]//Advances in Applied Microbiology. Amsterdam:Elsevier, 2022:1-33.
    [11] PARK JH, LEE SY. Fermentative production of branched chain amino acids:a focus on metabolic engineering[J]. Applied Microbiology and Biotechnology, 2010, 85(3):491-506.
    [12] LIU J, XU JZ, WANG BB, RAO ZM, ZHANG WG. L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering:progress and prospects[J]. Amino Acids, 2021, 53(9):1301-1312.
    [13] van der REST ME, LANGE C, MOLENAAR D. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA[J]. Applied Microbiology and Biotechnology, 1999, 52(4):541-545.
    [14] JIANG Y, QIAN FH, YANG JJ, LIU YM, DONG F, XU CM, SUN BB, CHEN B, XU XS, LI Y, WANG RX, YANG S. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J]. Nature Communications, 2017, 8:15179.
    [15] REED JL, VO TD, SCHILLING CH, PALSSON BO. An expanded genome-scale model of Escherichia coli K-12(iJR904 GSM/GPR)[J]. Genome Biology, 2003, 4(9):R54.
    [16] SEGRÈ D, VITKUP D, CHURCH GM. Analysis of optimality in natural and perturbed metabolic networks[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(23):15112-15117.
    [17] CHEN C, LI YY, HU JY, DONG XY, WANG XY. Metabolic engineering of Corynebacterium glutamicum ATCC 13869 for L-valine production[J]. Metabolic Engineering, 2015, 29:66-75.
    [18] HAO YN, PAN XW, XING RF, YOU JJ, HU MK, LIU ZF, LI XF, XU MJ, RAO ZM. High-level production of L-valine in Escherichia coli using multi-modular engineering[J]. Bioresource Technology, 2022, 359:127461.
    [19] LI YJ, WEI HB, WANG T, XU QY, ZHANG CL, FAN XG, MA Q, CHEN N, XIE XX. Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives[J]. Bioresource Technology, 2017, 245:1588-1602.
    [20] GUO L, DING S, LIU YD, GAO C, HU GP, SONG W, LIU J, CHEN XL, LIU LM. Enhancing tryptophan production by balancing precursors in Escherichia coli[J]. Biotechnology and Bioengineering, 2022, 119(3):983-993.
    [21] LIU JH, LI HL, XIONG H, XIE XX, CHEN N, ZHAO GR, CAIYIN Q, ZHU HJ, QIAO JJ. Two-stage carbon distribution and cofactor generation for improving L-threonine production of Escherichia coli[J]. Biotechnology and Bioengineering, 2019, 116(1):110-120.
    [22] PARK JH, LEE KH, KIM TY, LEE SY. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(19):7797-7802.
    [23] PARK JH, KIM TY, LEE KH, LEE SY. 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.
    [24] SAVRASOVA EA, STOYNOVA NV. Application of leucine dehydrogenase Bcd from Bacillus subtilis for L-valine synthesis in Escherichia coli under microaerobic conditions[J]. Heliyon, 2019, 5(4):e01406.
    [25] HAO YN, MA Q, LIU XQ, FAN XG, MEN JX, WU HY, JIANG S, TIAN DG, XIONG B, XIE XX. High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation[J]. Metabolic Engineering, 2020, 62:198-206.
    [26] PARK JH, JANG YS, LEE JW, LEE SY. Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering[J]. Biotechnology and Bioengineering, 2011, 108(5):1140-1147.
    [27] YUKAWA H, OMUMASABA CA, NONAKA H, KÓS P, OKAI N, SUZUKI N, SUDA M, TSUGE Y, WATANABE J, IKEDA Y, VERTÈS AA, INUI M. Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R[J]. Microbiology, 2007, 153(4):1042-1058.
    [28] LIU J, LIU MS, SHI T, SUN GN, GAO N, ZHAO XJ, GUO X, NI XM, YUAN QQ, FENG JH, LIU ZM, GUO YM, CHEN JZ, WANG Y, ZHENG P, SUN JB. CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction[J]. Nature Communications, 2022, 13:891.
    [29] WOLF S, BECKER J, TSUGE Y, KAWAGUCHI H, KONDO A, MARIENHAGEN J, BOTT M, WENDISCH VF, WITTMANN C. Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and welL-being[J]. Essays in Biochemistry, 2021, 65(2):197-212.
    [30] HOU XH, CHEN XD, ZHANG Y, QIAN H, ZHANG WG. L-valine production with minimization of by-products' synthesis in Corynebacterium glutamicum and Brevibacterium flavum[J]. Amino Acids, 2012, 43(6):2301-2311.
    [31] DENINA I, PAEGLE L, PROUZA M, HOLÁTKO J, PÁTEK M, NEŠVERA J, RUKLISHA M. Factors enhancing L-valine production by the growth-limited L-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13(pECKAilvBNC)[J]. Journal of Industrial Microbiology & Biotechnology, 2010, 37(7):689-699.
    [32] HOLÁTKO J, ELIŠÁKOVÁ V, PROUZA M, SOBOTKA M, NEŠVERA J, PÁTEK M. Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation[J]. Journal of Biotechnology, 2009, 139(3):203-210.
    [33] HAN GQ, XU N, SUN XP, CHEN JZ, CHEN C, WANG Q. Improvement of L-valine production by atmospheric and room temperature plasma mutagenesis and high-throughput screening in Corynebacterium glutamicum[J]. ACS Omega, 2020, 5(10):4751-4758.
    [34] SCHWENTNER A, FEITH A, MÜNCH E, BUSCHE T, RÜCKERT C, KALINOWSKI J, TAKORS R, BLOMBACH B. Metabolic engineering to guide evolution-creating a novel mode for L-valine production with Corynebacterium glutamicum[J]. Metabolic Engineering, 2018, 47:31-41.
    [35] BUCHHOLZ J, SCHWENTNER A, BRUNNENKAN B, GABRIS C, GRIMM S, GERSTMEIR R, TAKORS R, EIKMANNS BJ, BLOMBACH B. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate[J]. Applied and Environmental Microbiology, 2013, 79(18):5566-5575.
    [36] HASEGAWA S, UEMATSU K, NATSUMA Y, SUDA M, HIRAGA K, JOJIMA T, INUI M, YUKAWA H. 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.
    [37] ELIŠÁKOVÁ V, PÁTEK M, HOLÁTKO J, NEŠVERA J, LEYVAL D, GOERGEN JL, DELAUNAY S. Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2005, 71(1):207-213.
    [38] BLOMBACH B, SCHREINER ME, HOLÁTKO J, BARTEK T, OLDIGES M, EIKMANNS BJ. L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2007, 73(7):2079-2084.
    [39] BLOMBACH B, SCHREINER ME, BARTEK T, OLDIGES M, EIKMANNS BJ. Corynebacterium glutamicum tailored for high-yield L-valine production[J]. Applied Microbiology and Biotechnology, 2008, 79(3):471-479.
    [40] YU SZ, ZHENG B, CHEN ZY, HUO YX. Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids[J]. Microbial Cell Factories, 2021, 20(1):1-14.
    [41] HASEGAWA S, SUDA M, UEMATSU K, NATSUMA Y, HIRAGA K, JOJIMA T, INUI M, YUKAWA H. Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions[J]. Applied and Environmental Microbiology, 2013, 79(4):1250-1257.
    [42] BARTEK T, BLOMBACH B, ZÖNNCHEN E, MAKUS P, LANG S, EIKMANNS BJ, OLDIGES M. Importance of NADPH supply for improved L-valine formation in Corynebacterium glutamicum[J]. Biotechnology Progress, 2010, 26(2):361-371.
    [43] BRINKMANN-CHEN S, FLOCK T, CAHN JKB, SNOW CD, BRUSTAD EM, McINTOSH JA, MEINHOLD P, ZHANG L, ARNOLD FH. General approach to reversing ketoL-acid reductoisomerase cofactor dependence from NADPH to NADH[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(27):10946-10951.
    [44] RADMACHER E, VAITSIKOVA A, BURGER U, KRUMBACH K, SAHM H, EGGELING L. Linking central metabolism with increased pathway flux:L-valine accumulation by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2002, 68(5):2246-2250.
    [45] CHEN XL, GAO C, GUO L, HU GP, LUO QL, LIU J, NIELSEN J, CHEN J, LIU LM. DCEO biotechnology:tools to design, construct, evaluate, and optimize the metabolic pathway for biosynthesis of chemicals[J]. Chemical Reviews, 2018, 118(1):4-72.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

赵阔,程金宇,郭亮,高聪,宋伟,吴静,刘佳,柳亚迪,刘立明,陈修来. 谷氨酸棒杆菌代谢工程高效生产L-缬氨酸[J]. 生物工程学报, 2023, 39(8): 3253-3272

复制
分享
文章指标
  • 点击次数:443
  • 下载次数: 1642
  • HTML阅读次数: 766
  • 引用次数: 0
历史
  • 收稿日期:2022-12-13
  • 最后修改日期:2023-02-17
  • 在线发布日期: 2023-08-10
  • 出版日期: 2023-08-25
文章二维码
您是第5993090位访问者
生物工程学报 ® 2025 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司