Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in Bacillus licheniformis.

Extracellular polymeric substances (EPSs) are extracellular macromolecules in bacteria, which function in cell growth and show potential for mechanism study and biosynthesis application. However, the biosynthesis mechanism of EPS is still not clear. We herein chose Bacillus licheniformis CGMCC 2876 as a target strain to investigate the EPS biosynthesis. epsK, a member of eps cluster, the predicted polysaccharide synthesis cluster, was overexpressed and showed that the overexpression of epsK led to a 26.54% decrease in the production of EPS and resulted in slenderer cell shape. Transcriptome analysis combined with protein-protein interactions analysis and protein modeling revealed that epsK was likely responsible for the synthesis of teichuronic acid, a substitute cell wall component of teichoic acid when the strain was suffering phosphate limitation. Further cell cultivation showed that either phosphate limitation or the overexpression of teichuronic acid synthesis genes, tuaB and tuaE could similarly lead to EPS reduction. The enhanced production of teichuronic acid induced by epsK overexpression triggered the endogenous phosphate starvation, resulting in the decreased EPS synthesis and biomass, and the enhanced bacterial chemotaxis. This study presents an insight into the mechanism of EPS synthesis and offers the potential in controllable synthesis of target products.

Functions of PKS Genes in Lipid Synthesis of Schizochytrium sp. by Gene Disruption and Metabolomics Analysis.

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.

Characterization of a beta-glucosidase from Bacillus licheniformis and its effect on bioflocculant degradation.

Bacillus licheniformis CGMCC 2876, an aerobic spore-forming bacterium, produces a polysaccharide bioflocculant that is biodegradable and harmless. The present study determined that ß-glucosidase played a negative role in bioflocculant synthesis. The gene encoding ß-glucosidase was cloned and expressed in Escherichia coli BL21. This gene consists of 1437 bp and encodes 478 amino acid residues. The recombinant ß-glucosidase (Bgl.bli1) was purified and showed a molecular mass of 53.4 kDa by SDS-PAGE. The expression and reaction conditions of Bgl.bli1 were optimized; the activity of ß-glucosidase reached a maximum at 45.44 U/mL. Glucose clearly inhibited the activity of ß-glucosidase. The purified recombinant Bgl.bli1 hydrolysed polysaccharide bioflocculant in vitro and synergised with other cellulases. The ability of Bgl.bli1 to hydrolyse polysaccharide bioflocculant was the reason for the decrease in flocculating activity and indicated the utility of this enzyme for diverse industrial processes.

Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance.

A Clostridium beijerinckii mutant RT66 with considerable inhibitor-tolerance obtained by continuous culture was used for butanol production from non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). In fed-batch fermentation, 1.8L of diluted SAHHC containing 10 g/L of reducing sugar was provided during the acidogenic phase and 0.2L of concentrated SAHHC containing 300 g/L of reducing sugar was provided during the solventogenic phase. The mutant produced a total amount of solvents of 12.9 g/L, which consisted of 3.1 g/L of acetone, 9.3 g/L of butanol and 0.5 g/L of ethanol. A solvent yield of 0.35 g/g sugar and a productivity of 0.18 g/L h in 72 h were achieved. The remarkable inhibitor-tolerance of C. beijerinckii RT66 demonstrates that this may be an excellent strain for butanol production from ligocellulosic materials.

Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation.

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l(-1) of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g(-1). When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l(-1) of ABE with a yield of 0.34 g g(-1), including 2.2 g l(-1) acetone, 6.8 g l(-1) butanol, and 0.5 g l(-1) ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.

Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields.

With 30 g glucose/l as carbon source, Clostridium beijerinckii ART124, a mutant created by atmospheric pressure glow discharge, produced 13.7 g total solvent/l (containing 3.1 g acetone/l, 10.4 g butanol/l and 0.2 g ethanol/l) in 72 h. The mutant could also use sucrose or xylose or a mixture of glucose/xylose/arabinose with nearly equal yields.