该研究以产肌苷的大肠杆菌INO4-5为出发菌,针对其发酵过程中副产物含量高、肌苷产量低以及需要额外添加腺嘌呤等问题,对肌苷酸节点代谢流进行了优化。首先使用不同启动子和核糖体结合位点对枯草芽孢杆菌来源的purAbsu基因进行了表达,以强化腺苷合成支路,使菌株5 L发酵罐上的肌苷产量达到了29.5 g/L,且无需额外添加腺嘌呤。接着通过替换guaB基因的原有启动子以弱化鸟苷合成支路,并过表达guaC基因促使鸟苷酸回流至肌苷酸,使菌株副产物含量显著降低且肌苷产量提升至35.1 g/L;最后对不同5'-核苷酸酶进行了筛选和强化,发现引入来自酿酒酵母的特异性5'-核苷酸酶基因(introduction of specific 5'-nucleotidase gene,ISN1)可有效促进肌苷生产。最终所得菌株INO7-6在5 L发酵罐上发酵,肌苷产量为41.2 g/L,转化率为0.17 g/g葡萄糖,生产强度为0.86 g/(L·h),为目前报道的基因工程菌株的最高产量。
In this study, an inosine-producing strain Escherichia coli INO4-5 was used as the starting strain, and the metabolic flow of inosinic acid (IMP) node was optimized in view of the high content of byproducts, low inosine production and the requirement of additional adenine during fermentation. Firstly, the purAbsu gene from Bacillus subtilis was expressed using different promoters and ribosome binding sites to strengthen the adenosine synthesis branch, which enabled the strain to produce 29.5 g/L inosine in 5 L fermentor without extra addition of adenine. Then, the original promoter of guaB gene was replaced to weaken the guanosine synthesis branch and guaC gene was overexpressed to promote guanylic acid reflux to IMP, which significantly reduced the byproduct content of the strain and increased the inosine production to 35.1 g/L. Finally, different 5'-nucleotidases were screened and enhanced, and the introduction of specific 5'-nucleotidase gene (ISN1) from Saccharomyces cerevisiae was found to effectively promote inosine production. The resulting strain INO7-6 produced 41.2 g/L inosine with yield of 0.17 g/g glucose and productivity of 0.86 g/(L·h) in 5 L fermentor. The inosine production of this strain is the highest within the genetically engineered strains reported so far, laying a good foundation for the industrial production of inosine.
[1] HASKÓ G, SITKOVSKY M V, SZABÓ C.Immunomodulatory and neuroprotective effects of inosine[J].Trends in Pharmacological Sciences, 2004, 25(3):152-157.
[2] LEDESMA-AMARO R, JIMÉNEZ A, SANTOS M A, et al.Biotechnological production of feed nucleotides by microbial strain improvement[J].Process Biochemistry, 2013, 48(9):1263-1270.
[3] ZHANG Y, MORAR M, EALICK S E.Structural biology of the purine biosynthetic pathway[J].Cellular and Molecular Life Sciences, 2008, 65(23):3699-3724.
[4] ASAHARA T, MORI Y, ZAKATAEVA N P, et al.Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production[J].Applied Microbiology and Biotechnology.2010, 87(6):2195-2207.
[5] SHIMAOKA M, TAKENAKA Y, KURAHASHI O, et al.Effect of amplification of desensitized purF and prs on inosine accumulation in Escherichia coli[J].Journal of Bioscience and Bioengineering, 2007, 103(3):255-261.
[6] LI H J, ZHANG G Q, DENG A H, et al.De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis[J].Biotechnology Letters, 2011, 33(8):1575-1580.
[7] PEIFER S, BARDUHN T, ZIMMET S, et al.Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine[J].Microbial Cell Factories, 2012,11:138.
[8] WANG X Y, WANG G L, LI X L, et al.Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering[J].Journal of Biotechnology, 2016, 231:115-121.
[9] 朱彦凯, 刘铁重, 伍法清, 等.代谢工程改造大肠杆菌生产肌苷[J].食品与发酵工业, 2022, 48 (24):1-9.
ZHU Y K, LIU T Z, WU F Q, et al.Metabolic engineering of Escherichia coli for inosine production[J].Food and Fermentation Industries, 2022, 48 (24):1-9.
[10] LI Y F, LIN Z Q, HUANG C, et al.Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing[J].Metabolic Engineering, 2015, 31:13-21.
[11] WU M Y, SUNG L Y, LI H, et al.Combining CRISPR and CRISPRi systems for metabolic engineering of E.coli and 1,4-BDO biosynthesis[J].ACS Synthetic Biology, 2017, 6(12):2350-2361.
[12] MATSUI H, KAWASAKI H, SHIMAOKA M, et al.Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110[J].Bioscience Biotechnology and Biochemistry, 2001, 65(3):570-578.
[13] ZHANG Q, YAO R, CHEN X L, et al.Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors[J].Metabolic Engineering, 2018, 47:314-322.
[14] LEDESMA-AMARO R, BUEY R M, REVUELTA J L.Increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya gossypii[J].Microbial Cell Factories, 2015, 14(1):1-8.
[15] KUZNETSOVA E, PROUDFOOT M, GONZALEZ C F, et al.Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family[J].Journal of Biological Chemistry, 2006, 281(47):36149-36161.
[16] PROUDFOOT M, KUZNETSOVA E, BROWN G, et al.General enzymatic screens identify three new nucleotidases in Escherichia coli.Biochemical characterization of SurE, YfbR, and YjjG[J].Journal of Biological Chemistry, 2004, 279(52):54687-54694.
[17] KUNST F, OGASAWARA N, MOSZER I, et al.The complete genome sequence of the gram-positive bacterium Bacillus subtilis[J].Nature, 1997, 390(6657):249-256.
[18] ITOH R, SAINT-MARC C, CHAIGNEPAIN S, et al.The yeast ISN1 (YOR155c) gene encodes a new type of IMP-specific 5'-nucleotidase[J].BMC Biochemistry, 2003, 4:4.
