Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

Abstract

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Whole-Cell Biosensor and Producer Co-cultivation-Based Microfludic Platform for Screening Saccharopolyspora erythraea with Hyper Erythromycin Production

“…Iterative mutagenesis and HTS were efficient strategies to breed better microorganisms. − To further improve erythromycin production, we started from mutant 1-A2, which was the best mutant in the first-round screening, and conducted the second round of ARTP mutagenesis and FADS screening in a similar way. After the second-round WELCOME, three best mutants, B4, B5, and C2, were determined to produce 1.391 ± 0.050, 1.344 ± 0.044, and 1.345 ± 0.073 g/L of erythromycin in 24-well plate fermentation, exhibiting 21.5 ± 4.4, 17.4 ± 3.8, and 17.5 ± 6.4% improved titer compared to the starting strain 1-A2, respectively (Figure a).…”

Streptomyces clavuligerus: The Omics Era

“…S1B). Improved CA-producing strains have been obtained by increasing the glyceraldehyde-3-P pool through genetic engineering of the glycolytic pathway (Li & Townsend, 2006 ), by increasing the copy number of the glpK1D1 glycerol biosynthesis genes and by addition of glycerol to the cultures (Baños et al, 2009 ; Shin et al, 2021 ). The gene cluster for arginine and the arginine gene cluster regulation in Streptomyces have been thoroughly studied using genetic and transcriptomic tools and this might be useful to improve CA production (Botas et al, 2018 ; Pérez-Redondo et al, 2012 ; Rodríguez-García et al, 2000 ).…”

Sequence and origin of the Streptomyces intergenetic-conjugation helper plasmid pUZ8002

“…These silent BGCs may be the key to finding new antimicrobials to overcome the rise of antimicrobial resistance [ 4 7 ]. Genetic manipulation can help both express these silent BGCs and improve the robustness or yield of production strains of Streptomyces currently used in industrial fermentations [ 8 10 ].…”