Lifecome Biochemistry Co., Ltd.

Streptomyces fradiae is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in S. fradiae . Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor P tipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of nagB , replacement of P frr with P ermE *, and integration of exogenous vgbS , with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae . Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae , paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces .

This study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in Streptomyces fradiae for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of “off-target effect.” Our work not only provides a facile editing tool for constructing S. fradiae strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.

Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes . Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C . Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD + or NADPH/NADP + . These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD + -favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD +in vivo . Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.

Avilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes , which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C . More importantly, AviZ1 exhibits a unique NADH/NAD + preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD + in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.