Enhancing surfactin production in B. velezensis Bs916 combined cumulative mutagenesis and expression key enzymes.

Surfactin is a lipopeptide which has attracted massive attention due to its versatile bioactive properties, although it has less commercial application due to its low yield in wild strains. The B. velezensis Bs916 has enable commercial production of surfactin due to its outstanding capacity to synthesize lipopeptides and amenable to genetically engineering. In this study, 20 derivatives with high surfactin production were obtained firstly by transposon mutagenesis and knockout techniques, and the surfactin yield of the derivative H5 (△GltB) was increased approximately 7-folds, reaching to 1.48 g/L. The molecular mechanism of high yielding surfactin in △GltB was investigated by the transcriptomic and KEGG pathway analysis. The results indicated that △GltB enhanced its ability to synthesize surfactin mainly by promoting transcription of the srfA gene cluster and inhibiting degradation of some key precursors such as fatty acid. Secondly, we obtained a triple mutant derivative BsC3 by cumulative mutagenesis of the negative genes GltB, RapF, and SerA, and it could increase the surfactin titer by twofold, reaching to 2.98 g/L. Thirdly, we achieved overexpression of two key rate-limiting enzyme genes, YbdT, and srfAD, and the derivative BsC5 which further increased the surfactin titer by 1.3-fold, reaching to 3.79 g/L. Finally, the yield of surfactin by derivatives was significantly increased under the optimal medium, particularly the BsC5 increased the surfactin titer to 8.37 g/L. To the best of our knowledge, this is one of the highest yields that have been reported. Our work may pave way for large scale production of surfactin by B. velezensis Bs916. KEY POINTS: • Elucidation of the molecular mechanism of surfactin high-yielding transposon mutant. • Genetically engineering of B. velezensis Bs916 surfactin titer to 8.37 g/L for large scale preparation.

Transcriptome investigation on the multicellular behavior of Bacillus velezensis Bs916 anchoring surfactin.

AIMS: To provide valuable information for a comprehensive understanding of the multicellular behavior of Bacillus velezensis Bs916 regulated by surfactin and other natural signals by Transcriptome. METHODS AND RESULTS: Transcriptomics revealed a distinct effect on gene expression alterations caused by disruption of the surfactin gene cluster(Δsrf) and 100 µg/ml surfactin addition(Δsrf + SRF). A total of 1573 differential expression genes were identified among Bs916, Δsrf, and Δsrf + SRF and grouped into eight categories based on their expression profiles. RT-qPCR analysis of 30 candidate genes showed high consistency with those of transcriptome. Additionally, the expression of eight candidate genes regulated by surfactin in a dose-dependent manner was revealed by lacZ fusion. Based on the above evidence, we proposed that surfactin can act as an extracellular signal for monitoring biofilm formation in Bs916 by directly regulating the expression of AbrB, DegS-degU, and SinI-SinR, and indirectly regulating the phosphorylation of ComA and Spo0A. CONCLUSIONS: The biofilm of Δsrf was unable to restore significantly by surfactin addition, combined inclusion of surfactin (SRF), exopolysaccharide (EPS), and γ-poly-dl-glutamic acid (γ-PGA), results in significant restoration of Δsrf biofilm formation, thereby a preliminary model was presented about the molecular mechanism by which the signaling molecule surfactin regulates Bs916 multicellular behavior.

Extracellular lipopeptide bacillomycin L regulates serial expression of genes for modulating multicellular behavior in Bacillus velezensis Bs916.

In wild strains of Bacillus, a handful of extracellular natural products act as signals that can regulate multicellular behavior, but relatively little is known about molecular mechanisms' detail. We proposed a previously unreported molecular mechanism for triggering multicellularity in B. velezensis Bs916 by an endogenous cyclic lipopeptide, bacillomycin L. The genome-wide effect on gene expression was caused by the disruption of bacillomycin L gene cluster, and 100 µg/mL bacillomycin L was revealed by quantitative transcriptomics. A total of 878 differentially expressed genes among Bs916, Δbl, and Δbl + 100BL were identified and grouped into 9 functional categories. The transcription levels of 40 candidate genes were further evaluated by RT-qPCR analysis. The expression of eight candidate genes regulated by bacillomycin L in a dose-dependent manner was revealed by LacZ fusion experiment. Although the addition of bacillomycin L could not completely restore the expression levels of the differentially regulated genes in △bl, our results strongly suggest that bacillomycin L acts as a tuning signal of swarming motility and complex biofilm formation by indirectly regulating the expression levels of some two-component systems (TCSs) connector genes, particularly including several Raps that potentially regulate the phosphorylation levels of three major regulators ComA, DegU, and Spo0A.Key points• Proposed model for bacillomycin L regulation in B. velezensis Bs916.• Bacillomycin L can act as an extracellular signal to regulate the phosphorylation levels of three major regulators, ComA, DegU, and Spo0A and control the multicellular processes of vegetative growth, competent, motility, matrix production, sporulation, and autolysis.

Structural Insights into the Trans-Acting Enoyl Reductase in the Biosynthesis of Long-Chain Polyunsaturated Fatty Acids in Shewanella piezotolerans.

Two long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), play vital roles in human health. Similarly, two biosynthetic pathways, based on desaturase/elongase and polyketide synthase, have been implicated in the synthesis of microbial LC-PUFA. Up to now, only several microalgae, no bacteria, have been used in the commercial production of oils rich in DHA and/or EPA. Fully understanding the enzymatic mechanism in the biosynthesis of LC-PUFA would contribute significantly to produce EPA and/or DHA by the bacteria. In this study, we report a 1.998 Å-resolution crystal structure of trans-acting enoyl reductase (ER), SpPfaD, from Shewanella piezotolerans. The SpPfaD model consists of one homodimer in the asymmetric unit, and each subunit contains three domains. These include an N-terminal, a central domain forming a classic TIM barrel with a single FMN cofactor molecule bound atop the barrel, and a C-terminal domain with a lid above the TIM barrel. Furthermore, we docked oxidized nicotinamide adenine dinucleotide phosphate (NADP) and an inhibitor 2-(4-(2-((3-(5-(pyridin-2-ylthio)thiazol-2-yl)ureido)methyl)-1H-imidazole-4-yl)phenoxy)acetic acid (TUI) molecule into the active site and analyzed the inhibition and catalytic mechanisms of the enoyl reductase SpPfaD. To the best of our knowledge, this is the first crystal structure of trans-ER in the biosynthesis of bacterial polyketides.

Two Trichome Birefringence-Like Proteins Mediate Xylan Acetylation, Which Is Essential for Leaf Blight Resistance in Rice.

Acetylation is a ubiquitous modification on cell wall polymers, which play a structural role in plant growth and stress defenses. However, the mechanisms for how crop plants accomplish cell wall polymer O-acetylation are largely unknown. Here, we report on the isolation and characterization of two trichome birefringence-like (tbl) mutants in rice (Oryza sativa), which are affected in xylan O-acetylation. ostbl1 and ostbl2 single mutant and the tbl1 tbl2 double mutant displayed a stunted growth phenotype with varied degree of dwarfism. As shown by chemical assays, the wall acetylation level is affected in the mutants and the knock-down and overexpression transgenic plants. Furthermore, NMR spectroscopy analyses showed that all those mutants have varied decreases in xylan monoacetylation. The divergent expression levels of OsTBL1 and OsTBL2 explained the chemotype difference and indicated that OsTBL1 is a functionally dominant gene. OsTBL1 was found to be Golgi-localized. The recombinant OsTBL1 protein incorporates acetyl groups onto xylan. By using xylopentaose, a preferred acceptor substrate, OsTBL1 can transfer up to four acetyl residues onto xylopentaose, and this activity showed saturable kinetics. 2D-NMR spectroscopy showed that OsTBL1 transfers acetate to both 2-O and 3-O sites of xylosyl residues. In addition, ostbl1 and tbl1 tbl2 displayed susceptibility to rice blight disease, indicating that this xylan modification is required for pathogen resistance. This study identifies the major genes responsible for xylan acetylation in rice plants.

Investigation on artificial blood vessels prepared from bacterial cellulose.

BC (bacterial cellulose) exhibits quite distinctive properties than plant cellulose. The outstanding properties make BC a promising material for preparation of artificial blood vessel. By taking advantage of the high oxygen permeability of PDMS (polydimethylsiloxane) as a tubular template material, a series of BC tubes with a length of 100 mm, a thickness of 1mm and an outer diameter of 4 or 6mm were biosynthesized with the help of Gluconacetobacter xylinum. Through characterization by SEM (scanning electron microscope), tensile testing and thermal analysis, it is demonstrated that BC tubes are good enough for artificial blood vessel with elaborated nano-fiber architecture, qualified mechanical properties and high thermal stability. In addition, measurement of biocompatibility also shows that BC tubes are greatly adaptable to the in vivo environment. The results indicate that BC tubes have great potential for being utilized as tubular scaffold materials in the field of tissue engineering.

Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins.

Cephalosporins constitute a large class of ß-lactam antibiotics clinically used as antimicrobial drugs. New Dehli metallo-ß-lactamase (NDM-1) poses a global threat to human health as it confers on bacterial pathogen resistance to almost all ß-lactams, including penicillins, cephalosporins, and carbapenems. Here we report the first crystal structures of NDM-1 in complex with cefuroxime and cephalexin, as well as NMR spectra monitoring cefuroxime and cefixime hydrolysis catalyzed by NDM-1. Surprisingly, cephalosporoate intermediates were captured in both crystal structures determined at 1.3 and 2.0 Å. These results provide detailed information concerning the mechanism and pathways of cephalosporin hydrolysis. We also present the crystal structure and enzyme assays of a D124N mutant, which reveals that D124 most likely plays a more structural than catalytic role.

Study of osteogenic differentiation of human adipose-derived stem cells (HASCs) on bacterial cellulose.

Bacterial cellulose (BC) has been proposed as a biomaterial applied in biomedical scope due to its good biocompatibility. Recent reports showed that human adipose-derived stem cells (HASCs) have become a new choice to be used as seeding cells in tissue engineering. The objective of this study is to explore the potential of using BC and HASCs as scaffold and seeding cells in bone tissue engineering. The osteogenic differentiation was investigated by Von Kossa, Alizarin Red, ALP cellular staining and RT-PCR. The results showed that HASCs took a successful osteogenic differentiation on BC. Moreover, the in vivo animal test also provided the confirmation of the repair ability of BC on damaged bone. In conclusion, the author demonstrates the osteogenic differentiation of HASCs on BC and the feasibility of using BC and HASCs as scaffold and seeding cells in bone tissue engineering.