Carbon competition between cell growth and product synthesis is the bottleneck in efficient N-acetyl glucosamine (GlcNAc) production in microbial cell factories. In this study, a xylose-induced T7 RNA polymerase−P T7 promoter system was introduced in Escherichia coli W3110 to control the GlcNAc synthesis. Meanwhile, an arabinose-induced CRISPR interference (CRISPRi) system was applied to adjust cell growth by attenuating the transcription of key growth-related genes. By designing proper sgRNAs, followed by elaborate adjustment of the addition time and concentration of the two inducers, the carbon flux between cell growth and GlcNAc synthesis was precisely redistributed. Comparative metabolomics analysis results confirmed that the repression of pf kA and zwf significantly attenuated the TCA cycle and the synthesis of related amino acids, saving more carbon for the GlcNAc synthesis. Finally, the simultaneous repression of pf kA and zwf in strain GLA-14 increased the GlcNAc titer by 47.6% compared with that in E. coli without the CRISPRi system in a shake flask. GLA-14 could produce 90.9 g/L GlcNAc within 40 h in a 5 L bioreactor, with a high productivity of 2.27 g/L/h. This dynamic strategy for rebalancing cell growth and product synthesis could be applied in the fermentative production of other chemicals derived from precursors synthesized via central carbon metabolism.
Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/ yield, the presence of the byproduct ectoine, and the requirement of high salinity.Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based autoregulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.
A strain of Penicilliurn nigricans, which produces both the antifungal antibiotic griseofulvin and tremorgenic penitrem mycotoxins concurrently in static liquid culture, also elaborated both metabolites in submerged culture when stimulated by calcium chloride to sporulate. Maximum yield of penitrems (60 mg 1-l ) occurred within 5 d in a 60 1 stirred fermenter, thus constituting the first significant process for penitrem production in submerged culture.
Currently, microbial production is becoming a competitive method for N-acetyl-glucosamine production. As the biosynthesis of N-acetyl-glucosamine originating from fructose-6-P directly competes with central carbon metabolism for precursor supply, the consumption of glucose for cell growth and cellular metabolism severely limits the yield of N-acetyl-glucosamine. In this study, appropriate catabolic division of labor in the utilization of mixed carbon sources was achieved by deleting the pf kA gene and enhancing the utilization of glycerol by introducing the glpK mutant. Glycerol thus mainly contributed to cell growth and cellular metabolism, and more glucose was saved for efficient N-acetyl-glucosamine synthesis. By optimizing the ratio of glycerol to glucose, the balancing of cell growth/cellular metabolism and N-acetyl-glucosamine synthesis was achieved. The resulting strain GLALD-7 produced 179.7 g/L N-acetyl-glucosamine using mixed glycerol/glucose (1:8, m/m) carbon sources in a 5 L bioreactor, with a yield of 0.458 g/g total carbon sources (0.529 g/g glucose) and a productivity of 2.57 g/L/h. Coherent high titer/yield/productivity was obtained, with the highest values ever reported, suggesting that an appropriate catabolic division of labor using mixed glycerol/ glucose carbon sources is a useful strategy for facilitating the microbial production of chemicals originating from glucose or metabolites upstream of glycolysis.
Evolution is a powerful tool for the breeding of microorganisms, while the connection between the changes of intracellular metabolism and different evolution directions is still unclear, which once clarified, will greatly expand the application of evolutionary engineering. We aim to clarify the correlation between metabolism changes and evolution directions in two Corynebacterium glutamicum strains for l-valine and l-leucine overproducing originated from the same parental strain by repeated random mutagenesis and selection. GC-MS metabolomics was performed to identify and quantify intracellular metabolites of the evolved and wild-type C. glutamicum strains. Time-series comparison of the fermentation processes was performed. The metabolism differences of three strains mainly exist in central carbon metabolism and the stress-resisting modes. C. glutamicum XV developed an overall "pyruvate-saving" mode for l-valine synthesis, and adopted a trehalose accumulating strategy to resist environmental stresses. C. glutamicum CP depended on an enhanced "pyruvate-producing" mode, together with certain "pyruvate-saving" strategies, for efficient l-leucine synthesis, and accumulated proline, myinositol, and inositol as the stress-resisting measure. These elaborate regulation strategies could be used in future metabolic engineering, making evolution more informative and applicable. KeywordsBranched-chain amino acid • Corynebacterium glutamcium • Metabolomics • Multivariate statistical analysis * Xixian Xie
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