Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin.

Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.

Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor.

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. RESULTS: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. CONCLUSIONS: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by 'silent' BGCs.

Enriching intracellular macrolides in Escherichia coli improved the sensitivity of bioluminescent sensing systems.

A repressor protein MphR and an enhanced green fluorescent protein (eGFP) were used to construct a bioluminescent sensing system for macrolide analysis in Escherichia coli host cells. We deleted TolC, an efflux pump for macrolides in E. coli, to promote the intracellular accumulation of macrolides. The binding constant (K1/2) of the sensing system constructed in an E. coli strain was decreased up to 33-fold with deleted TolC, and its sensitivity to the macrolides erythromycin, azithromycin, roxithromycin, and pikromycin was increased. The limit of detection of the bioluminescent sensing system for serum azithromycin was 4.1 nM. The ability to detect serum azithromycin concentrations was confirmed by analyzing photographs using ImageJ software. We also developed a novel sensing system for the immune suppressor FK506, another macrolide that is frequently prescribed. Deleting TolC also significantly improved the sensitivity of this sensing system. Bioluminescent sensing systems constructed in TolC mutants were sensitive to various macrolides, indicating their potential for clinical application with hand-held devices.

Improved 2,3-butanediol yield and productivity from lignocellulose biomass hydrolysate in metabolically engineered Enterobacter aerogenes.

We previously engineered Enterobacter aerogenesfor glucose and xylose co-utilization and 2,3-butanediol production. Here, strain EMY-22 was further engineered to improve the 2,3-butanediol titer, productivity, and yield by reducing the production of byproducts. To reduce succinate production, the budABC operon and galP gene were overexpressed, which increased 2,3-butanediol production. For further reduction of succinate and 2-ketogluconate production, maeA was selected and overexpressed in EMY-22. The optimally engineered strain produced 2,3-butanediol for a longer time and showed reduced byproduct formation from sugarcane bagasse hydrolysate under flask cultivation conditions. The engineered strain displayed 66.6, 13.4, and 16.8% improvements in titer, yield, productivity of 2,3-butanediol, respectively, compared to its parental strain under fed-batch fermentation conditions. The data demonstrate that the metabolic engineering to reduce byproduct formation is a promising strategy to improve 2,3-butanediol production from lignocellulosic biomass.

Metabolic engineering of Enterobacter aerogenes for 2,3-butanediol production from sugarcane bagasse hydrolysate.

The pathway engineering of Enterobacter aerogenes was attempted to improve its production capability of 2,3-butanediol from lignocellulosic biomass. In the medium containing glucose and xylose mixture as carbon sources, the gene deletion of pflB improved 2,3-butanediol carbon yield by 40%, while the deletion of ptsG increased xylose consumption rate significantly, improving the productivity at 12 hr by 70%. The constructed strain, EMY-22-galP, overexpressing glucose transporter (galP) in the triple gene knockout E. aerogenes, ldhA, pflB, and ptsG, provided the highest 2,3-butanediol titer and yield at 12 hr flask cultivation. Sugarcane bagasse was pretreated with green liquor, a solution containing Na2CO3 and Na2SO3 and was hydrolyzed by enzymes. The resulting hydrolysate was used as a carbon source for 2,3-butanediol production. After 72 hr in fermentation, the yield of 0.395g/g sugar was achieved, suggesting an economic production of 2,3-butanediol was possible from lignocellulosic biomass with the metabolically engineered strain.