Efficient production of guanosine in Escherichia coli by combinatorial metabolic engineering

Abstract

Background: Guanosine is a purine nucleoside that is widely used as a raw material for food additives and pharmaceutical products. Microbial fermentation is the main production method of guanosine. However, the guanosine-producing strains possess multiple metabolic pathway interactions and complex regulatory mechanisms. The lack of strains with efficiently producing-guanosine greatly limited industrial application.

Results: We attempted to efficiently produce guanosine in Escherichia coli using systematic metabolic engineering. First, we overexpressed the purine synthesis pathway from Bacillus subtilis and the prs gene, and deleted three genes involved in guanosine catabolism to increase guanosine accumulation. Subsequently, we attenuated purA expression and eliminated feedback and transcription dual inhibition. Then, we modified the metabolic flux of the glycolysis and Entner-Doudoroff (ED) pathways and performed redox cofactors rebalancing. Finally, transporter engineering and enhancing the guanosine synthesis pathway further increased the guanosine titre to 134.9 mg/L. After 72 h of the fed-batch fermentation in shake-flask, the guanosine titre achieved 289.8 mg/L.

Conclusions: Our results reveal that the guanosine synthesis pathway was successfully optimized by combinatorial metabolic engineering, which could be applicable to the efficient synthesis of other nucleoside products.

Keywords: Escherichia coli; Guanosine; Integration expression; Metabolic engineering; Metabolic flux.

Fig. 1

An overview of engineering strategies to increase guanosine production in E. coli. Blue and green arrows indicate overexpression and attenuation of the target genes. The red X indicates deletion of the corresponding gene. Dashed arrows indicate repression by regulatory protein. Bs indicates Bacillus subtilis. Ba indicates Bacillus amyloliquefaciens. Abbreviations: pfkA, 6-phosphofructokinase I; BsglpX, fructose-1,6-bisphosphatase II; edd, phosphogluconate dehydratase; eda, KHG/KDPG aldolase; Baprs, ribose-phosphate diphosphokinase; BspurF, glutamine PRPP amidotransferase; BspurD, phosphoribosylglycinamide synthetase; BspurN, THFA-dependent phosphoribosylglycinamide transformylases; BspurQLS, phosphoribosylformyl-glycinamidine synthetases I, II, and III; BspurM, phosphoribosylaminoimidazole synthetase; BspurEK, phosphoribosylaminoimidazole carboxylases I and II; BspurC, phosphoribosylaminoimidazolesuccinocarboxamide synthetase; BsPurB, adenylosuccinate lyase; BspurH, phosphoribosylaminoimidazole carboxamide formyltransferase and IMP cyclohydrolase; purR, DNA-binding transcriptional repressor; purA, adenylosuccinate synthase; guaA, GMP synthase; guaB, IMP dehydrogenase; guaC, GMP reductase; deoD, purine nucleoside phosphorylase; ppnP, nucleoside phosphorylase; gsk, inosine/guanosine kinase; nupG, nucleoside: H⁺ symporter; nepI, purine ribonucleoside exporter

Fig. 2

Overexpressing the purine synthesis operon from B. subtilis. (a) The native metabolic pathway of purine biosynthesis in E. coli; (b) Structural gene diagram of the purine operon in B. subtilis; (c) Integrated expression cassettes of the purine operon; (d) Production of hypoxanthine and inosine by overexpressing the purine synthesis operon. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; ** p value < 0.01, *** p value < 0.001)

Fig. 3

Overexpressing the prs gene for guanosine production. (a) Schematic of the PRPP synthesis pathway in E. coli; (b) Integrated expression cassettes of the prs gene; (c) Production of hypoxanthine and inosine by overexpressing the prs gene. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; ** p value < 0.01, *** p value < 0.001)

Fig. 4

Disruption of guanosine catabolism-related genes. (a) The related pathways of guanosine catabolism in E. coli. (b) Production of guanosine by deleting guanosine catabolism-related genes. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; *** p value < 0.001)

Fig. 5

Adjusting the metabolic flux from adenosine synthesis and the feedback inhibition of PurR. The intrinsic pathway of adenosine synthesis (a) and the feedback inhibition mediated by PurR (b); (c) Production of guanosine in the engineered strains. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; ** p value < 0.01, *** p value < 0.001, ns represents no significant difference)

Fig. 6

Redistributing the metabolic flux of EMP and ED and redox cofactor rebalancing for the accumulation of guanosine. (a) Schematic of the EMP and ED pathways; (b) Schematic of redox cofactor rebalancing; (c) Increased guanosine production by downregulating the metabolic flux of EMP and ED and redox cofactor rebalancing. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; ns represents no significant difference)

Fig. 7

Engineering transporters for guanosine production. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; *p value < 0.05)

Fig. 8

The effect of engineering the guanosine synthesis pathway. (a) Schematic of guanosine synthesis pathway; (b) Effects of strengthening the guanosine synthesis pathway on guanosine accumulation. All data represent the mean ± s.d. (n = 4 biologically independent samples). Error bars were analysed by Student’s t test (two-sample, two-tailed; *p value < 0.05)

Fig. 9

Assessment of guanosine production using the strain MQ39. Fed-batch fermentation was performed in shake flasks. Four biological replicates were performed, and the error bars indicate the standard deviation