Identification of key genes involved in polysaccharide bioflocculant synthesis in Bacillus licheniformis

Abstract

The present study reports the sequenced genome of Bacillus licheniformis CGMCC 2876, which is composed of a 4,284,461 bp chromosome that contains 4,188 protein-coding genes, 72 tRNA genes, and 21 rRNA genes. Additional analysis revealed an eps gene cluster with 16 open reading frames. Conserved Domains Database analysis combined with qPCR experiments indicated that all genes in this cluster were involved in polysaccharide bioflocculant synthesis. Phosphoglucomutase and UDP-glucose pyrophosphorylase were supposed to be key enzymes in polysaccharide secretion in B. licheniformis. A biosynthesis pathway for the production of polysaccharide bioflocculant involving the integration of individual genes was proposed based on functional analysis. Overexpression of epsDEF from the eps gene cluster in B. licheniformis CGMCC 2876 increased the flocculating activity of the recombinant strain by 90% compared to the original strain. Similarly, the crude yield of polysaccharide bioflocculant was enhanced by 27.8%. Overexpression of the UDP-glucose pyrophosphorylase gene not only increased the flocculating activity by 71% but also increased bioflocculant yield by 13.3%. Independent of UDP-N-acetyl-D-mannosamine dehydrogenase gene, flocculating activity, and polysaccharide yield were negatively impacted by overexpression of the UDP-N-acetylglucosamine 2-epimerase gene. Overall, epsDEF and gtaB2 were identified as key genes for polysaccharide bioflocculant synthesis in B. licheniformis. These results will be useful for further engineering of B. licheniformis for industrial bioflocculant production. Biotechnol. Bioeng. 2017;114: 645-655. © 2016 Wiley Periodicals, Inc.

Effect of glucose on poly-γ-glutamic acid metabolism in Bacillus licheniformis

“…In our previous studies, B . licheniformis CGMCC2876 was observed to produce extracellular polysaccharides when using glucose as the sole carbon source [ 13 , 14 ], whereas poly-γ-glutamic acid (γ-PGA) was secreted when trisodium citrate and glycerol were used as the carbon sources [ 15 ]. Both of the extracellular polymeric substances exhibited high flocculating activities.…”

Increasing the bioflocculant production and identifying the effect of overexpressing epsB on the synthesis of polysaccharide and γ-PGA in Bacillus licheniformis

“…In our previous study, we found that EpsB contained the structural domain of the tyrosine kinase CpsD, which had a negative regulatory role on the formation of capsular polysaccharides in Streptococcus aureus [ 15 ] according to an analysis of the NCBI Conserved Domains. Therefore, we inferred that epsB had a negative regulatory effect on the synthesis of polysaccharides in B. licheniformis and thereby decreased the flocculating activity [ 11 ]. However, although the most recent results showed that overexpressing epsB indeed decreased the polysaccharide content synthesized by B. licheniformis , the flocculating activity of the bioflocculant was greatly improved compared with that of the original strain because overexpressing epsB produced a marked increase in the γ-PGA content.…”

“…In our previous research, a bioflocculant-producing bacterium was isolated and identified as B. licheniformis [China General Microbiological Culture Collection Centre (CGMCC) 2876] [ 10 ], and it could produce polysaccharide and γ-PGA under different culture conditions. When using glucose as the sole carbon source, B. licheniformis mainly produces extracellular polysaccharides [ 11 ], whereas when using trisodium citrate and glycerol as the carbon sources, this strain mainly secretes γ-PGA [ 12 ]. Both polysaccharides and γ-PGA have shown high flocculating activities in industrial applications, so the ability to increase the flocculating activities of bioflocculants produced by B. licheniformis has huge commercial potential [ 13 ].…”

“…In this study, we mainly addressed bioflocculants that use glucose as the sole carbon source, and we used genetic engineering as a means to improve the flocculating activity and crude yield of the bioflocculants. In our previous research, we suggested the hypothetical metabolic pathways of polysaccharides in B. licheniformis and speculated on the key genes involved in polysaccharide synthesis; these genes are distributed in different locations in the genome and are responsible for the conversion of glucose into the subunits of polysaccharides [ 11 ]. Among the genes, crr encodes the protein component of glucose-specific phosphotransferase, whose function is to catalyse d -glucose to glucose-6-phosphate; pgcA encodes phosphoglucomutase, which is involved in converting glucose-6-phosphate to glucose-1-phosphate; then, glucose-1P is converted into UDP-glucose by UTP-glucose-1-phosphate uridylyltransferase, which is encoded by gtaB ; further, epsA and epsB are two gene segments of the eps gene cluster, which encodes a series of glycosyltransferases that have different substrate specificities and are all important in the process of synthesizing polysaccharide repeating units.…”

“…In Streptococcus pneumoniae , galU (glucose-1-phosphate uridylyltransferase gene) knockout mutants are unable to synthesize a detectable capsule polysaccharide [ 26 ]. Further, our previous research also indicated that the overexpression of UDP-glucose pyrophosphorylase gene gtaB2 in B. licheniformis could markedly increase both the yield of the polysaccharide bioflocculant and its flocculating activities [ 11 ]. In addition, many researchers have reported the importance of the eps gene cluster during polysaccharide synthesis, and it exhibits conspicuous homology across different strains [ 27 , 28 ].…”