BackgroundAcetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production.ResultsHere, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain.ConclusionsTyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5′ untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1106-0) contains supplementary material, which is available to authorized users.
Background: Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate. Results: As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13 C metabolic flux analysis (13 C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants. Conclusions: The elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.
Resveratrol, a phytoalexin produced by plants, has several beneficial effects in humans. It can be produced using Escherichia coli by introducing only three heterologous genes: TAL, 4CL, and STS. However, the resveratrol synthesis pathway requires two precursors, tyrosine and acetyl-CoA, which are produced by two branched central metabolic pathways. Therefore, overexpression of these genes in E. coli results in the production of only trace amounts of resveratrol. In this study, we attempted to produce resveratrol via coculture of two engineered strains in which the two metabolic pathways are activated. The first strain was engineered to produce p-coumaric acid using tyrosine as a precursor, which can be synthesized by the pentose phosphate pathway. The second strain produced resveratrol by combining p-coumaric acid from the first strain and malonyl-CoA synthesized from acetyl-CoA, which is produced by the glycolytic pathway. In total, 55.7 mg/L of resveratrol was produced from 20 g/L of glucose via coculture of these two strains in glucose minimal medium without any supplements. The metabolic fluxes in each of the strains producing resveratrol were successfully investigated by 13 C metabolic flux analysis. The results showed that the balance between the citric acid cycle and the malonyl-CoA supply node was important for resveratrol production.
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