Xanthosine, also known as 9-ribofuranosyl-3,9-dihydro-1H-purine-2,6-dione, is a nucleoside composed of xanthine and a ribosyl group.Currently, xanthosine is synthesized through plant extraction or chemical synthesis, both of which suffer from low yields and high production costs.To develop a plasmid-free xanthosine-producing strain, this study systematically engineered the metabolic pathways of the wild-type Escherichia coli W3110.Initial strategies aimed at enhancing precursor supply by overexpressing the glycine hydroxymethyltransferase gene (glyA), the aspartase gene (aspA), and the carbonic anhydrase gene (can) to increase intracellular glycine, L-aspartate, and CO2, and blocking the conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP) by deleting the guaA gene did not lead to detectable xanthosine production.Significant production was first achieved by overexpressing a feedback inhibition-resistant mutant of phosphoribosyl pyrophosphate (PRPP) synthetase (prsEcoD128A) and deleting the purine repressor gene (purR), resulting in 1.05 g/L xanthosine.Introducing a feedback-resistant PRPP amidotransferase mutant (purFBazK326Q) from Bacillus amyloliquefaciens alleviated a key rate-limiting step in de novo purine biosynthesis and increased production to 1.43 g/L.Further enhancement of the pentose phosphate pathway via overexpression of the glucose-6-phosphate dehydrogenase gene (zwf) and transketolase gene (tktA) elevated xanthosine titer to 2.28 g/L.Expression of the heterologous purine operon (purEKBCSQLFMNHD) further boosted production to 2.85 g/L.To reduce xanthosine degradation, the genes ppnP and deoD, both encoding purine nucleoside phosphorylases, were knocked out, resulting in a final titer of 4.00 g/L.After 26 hours of fed-batch fermentation in a 5 L bioreactor, the engineered strain XL12 achieved a xanthosine titer of 10.25 g/L, representing the highest reported production of xanthosine via microbial fermentation to date.
LENG Yanlin
,
SUN Xiaojia
,
LU Yujiao
,
WANG Feiao
,
TIAN Siyu
,
LI Yanjun
. Metabolic engineering of Escherichia coli for xanthosine production[J]. Food and Fermentation Industries, 2026
, 52(5)
: 10
-16
.
DOI: 10.13995/j.cnki.11-1802/ts.043150
[1] 刘芳, 陈少红, 刘青波, 等.黄嘌呤核苷促进骨髓间质干细胞体外快速扩增的实验研究[J].中国病理生理杂志, 2010, 26(12):2389-2393.
LIU F, CHEN S H, LIU Q B, et al.Rapid proliferation of bone marrow mesenchymal stem cells enhanced by xanthosine[J].Chinese Journal of Pathophysiology, 2010, 26(12):2389-2393.
[2] 闫振, 黄健垚, 高路, 等.茶树体内咖啡碱生物代谢研究进展[J].中国茶叶, 2020, 42(7):1-7.
YAN Z, HUANG J Y, GAO L, et al.Research progress of caffeine biological metabolism in tea plant[J].China Tea, 2020, 42(7):1-7.
[3] LIU M, FU Y X, GAO W J, et al.Highly efficient biosynthesis of hypoxanthine in Escherichia coli and transcriptome-based analysis of the purine metabolism[J].ACS Synthetic Biology, 2020, 9(3):525-535.
[4] 刘铁重, 高志强, 侯德欣, 等.大肠杆菌肌苷酸节点代谢优化高效合成肌苷[J].食品与发酵工业, 2024, 50(4):44-50.
LIU T Z, GAO Z Q, HOU D X, et al.Inosinic acid node metabolism optimization for efficient synthesis of inosine in Escherichia coli[J].Food and Fermentation Industries, 2024, 50(4):44-50.
[5] 邓爱华, 温廷益, 陈振翔, 等.发酵生产嘌呤核苷的重组菌及其构建方法与应用:中国, CN113583929A[P].2021-11-02.
DENG A H, WEN T Y, CHEN Z X, et al.Recombinant strain for fermentative production of purine nucleosides, construction method and application thereof:China, CN113583929A[P].2021-11-02.
[6] EBBOLE D J, ZALKIN H.Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis[J].The Journal of Biological Chemistry, 1987, 262(17):8274-8287.
[7] 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.
[8] 梅漫莉. 腺苷工程菌的构建及其发酵过程控制[D].天津:天津科技大学, 2021.
MEI M L.Construction of adenosine engineering bacteria and its fermentation process control[D].Tianjin:Tianjin University of Science and Technology, 2021.
[9] RODIONOVA I A, GAO Y, SASTRY A, et al.Identification of a transcription factor, PunR, that regulates the purine and purine nucleoside transporter punC in E.coli[J].Communications Biology, 2021, 4:991.
[10] WILSON H R, TURNBOUGH C L Jr.Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12[J].Journal of Bacteriology, 1990, 172(6):3208-3213.
[11] 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.
[12] 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.
[13] 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.
[14] 刘万红, 刘爱福.glyA基因及其编码的丝氨酸羟甲基转移酶[J].氨基酸和生物资源, 2001, 23(2):13-16;21.
LIU W H, LIU A F.GlyA gene and serine hydroxymethyltransferase encoded by GlyA gene[J].Amino Acids & Biotic Resources, 2001, 23(2):13-16;21.
[15] 潘富军, 周作明.碳酸酐酶固定化技术研究与应用进展[J].化学工程, 2014, 42(3):11-17;58.
PAN F J, ZHOU Z M.Research and application progress of carbonic anhydrase-immobilized technology[J].Chemical Engineering (China), 2014, 42(3):11-17;58.
[16] 刘铁重. 优化大肠杆菌中肌苷酸节点提升肌苷产量[D].天津:天津科技大学, 2023.
LIU T Z.Optimization of inosinic acid node in Escherichia coli to increase inosine production[D].Tianjin:Tianjin University of Science and Technology, 2023.
[17] 徐庆阳, 李良文, 陈志超, 等.一种鸟苷生产菌株及其构建方法与应用:中国, 202410465797.6[P].2024-07-09.
XU Q Y, LI L W, CHEN Z C, et al.A guanosine-producing strain and its construction method and application:China, 202410465797.6[P].2024-07-09.
[18] 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.
