Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.

Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.

Dual cytoplasmic-peroxisomal compartmentalization engineering and multiple metabolic engineering strategies for high yield non-psychoactive cannabinoid in Saccharomyces cerevisiae.

CBG (Cannabigerol), a nonpsychoactive cannabinoid, has garnered attention due to its extensive antimicrobial and anti-inflammatory properties. However, the natural content of CBG in Cannabis sativa L. is minimal. In this study, we developed an engineered cell factory for CBG production using Saccharomyces cerevisiae. We introduced the CBGA biosynthetic pathway into S. cerevisiae and employed several strategies to enhance CBGA production. These strategies included dynamically inhibiting the competitive bypass of key metabolic pathways regulated by Erg20p. Additionally, we implemented a dual cytoplasmic-peroxisomal compartmentalization approach to further increase CBGA production. Furthermore, we ensured efficient CBGA production by optimizing NADPH and acetyl-CoA pools. Ultimately, our engineered strain achieved a CBG titer of 138 mg L-1 through fed-batch fermentation in a 5 L bioreactor, facilitated by microwave decarboxylation extraction. These findings underscore the significant potential of yeast cell factories for achieving higher yields in cannabinoid production.

Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS.

In pharmaceutical industry, isepamicin is mainly manufactured from gentamicin B, which is produced by Micromonospora echinospora as a minor component of the gentamicin complex. Improvement of gentamicin B production through metabolic engineering is therefore important to satisfy the increasing demand for isepamicin. We hypothesized that gentamicin B was generated from gentamicin JI-20A via deamination of the C2' amino group. Using kanJ and kanK as the gene probes, we identified the putative deamination-related genes, genR and genS, through genome mining of the gentamicin B producing strain M. echinospora CCTCC M 2018898. Interestingly, genR and genS constitute a gene cassette located approximately 28.7 kb away from the gentamicin gene cluster. Gene knockout of genR and genS almost abolished the production of gentamicin B in the mutant strain, suggesting that these two genes, which are responsible for the last steps in gentamicin B biosynthesis, constitute the missing part of the known gentamicin biosynthetic pathway. Based on these finding, we successfully constructed a gentamicin B high-yielding strain (798 mg/L), in which an overexpression cassette of genR and genS was introduced. Our work fills the missing piece to solve the puzzle of gentamicin B biosynthesis and may inspire future metabolic engineering efforts to generate gentamycin B high-yielding strains that could eventually satisfy the need for industrial manufacturing of isepamicin.

Systematic Metabolic Engineering of Saccharomyces cerevisiae for Lycopene Overproduction.

Lycopene is widely used in foods, cosmetics, nutritional supplements, and pharmaceuticals. Microbial production of lycopene has been intensively studied. However, there are few systematic engineering studies on Saccharomyces cerevisiae aimed at achieving high-yield lycopene production. In the current study, by employing a systematic optimization strategy, we screened the key lycopene biosynthetic genes, crtE, crtB, and crtI, from diverse organisms. By adjusting the copy number of these three key genes, knocking out endogenous bypass genes, increasing the supply of the precursor acetyl-CoA, balancing NADPH utilization, and regulating the GAL-inducible system, we constructed a high-yield lycopene-producing strain BS106, which can produce 310 mg/L lycopene in shake-flask fermentation, with gene expression controlled by glucose. In optimized two-stage fed-batch fermentation, BS106 produced 3.28 g/L lycopene in a 7 L fermenter, which is the highest concentration achieved in S. cerevisiae to date. It will decrease the consumption of tomatoes for lycopene extraction and increase the market supply of lycopene.