Production enhancement of the extracellular lipase LipA in Bacillus subtilis: Effects of expression system and Sec pathway components.

Lipases are among the most versatile biocatalysts, and are used in a range of industrially relevant bioconversion reactions. However, the production of LipA in recombinant Bacillus subtilis is still limited, due to unresolved issues surrounding the regulation of the expression and secretion systems. In this study, the gene encoding LipA from B. subtilis 168 was expressed in BNA under the control of the P43 and the PAE promoter. The extracellular lipase activity of the resulting strains BNACL and BNAAL was 7.8 U ml-1 and 12.6 U ml-1, respectively. To further enhance the expression of LipA, pHP13L was constructed by inserting the PAE-lip into the shuttle vector pHP13, which produced an extracellular lipase activity of 180.5 U ml-1 of BNA/pHP13L. The strain BNAY8 described in Supplement data which lacks eight extracellular proteins was constructed and the deletion a few of the much weaker secreting proteins had no significant effect on the secretion of LipA. Moreover, the four Sec pathway components, secA-prfB, secDF, secYEG, prsA, were individually overexpressed in BNA. The overexpression of secDF and prsA enhanced the production of LipA by 28% and 49%, respectively. Furthermore, the co-overexpression of secDF with prsA improved the extracellular amount of LipA by 59% over that of BNA/pHP13L, reaching 287.8 U ml-1. It can therefore be said that both regulatory elements and secretion pathway had an impact on the production of secreted LipA. Their optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in B. subtilis.

[Response of Microbial Community to Natural Organic Matter Input in Lake Sediments].

Increased loads of biogenic and terrestrial natural organic matter into freshwater lakes are projected to be a major consequence of global climate change and cyanobacterial blooms. In this study, the effects of phytoplankton-derived organic matter (POM) and terrestrial humic acid (HA) on the activity, composition, and function of the microbial community in Lake Taihu sediments were investigated. Fluorescent spectra demonstrated that POM with high chemodiversity was composed of labile protein-like components (50%) and recalcitrant humic-like components (50%), while humic-like substances accounted for 97% of HA. Over two-month periods, the organic carbon mineralization in sediments was enhanced owing to increasing NOM concentrations; however, the carbon consumption in POM-amended sediments was significantly higher than that in sediments amended with the same concentrations of HA. Analysis of extracellular polymeric substances indicated that NOM input improved the microbial secretion of proteins and polysaccharides, increasing the aggregation and stability of the microbial community. The amendment of POM also stimulated the activity of organic matter metabolic enzymes, promoting microbial activity. Moreover, 16S rRNA gene sequencing suggested that the mineralization of NOM (especially POM) increased the diversity of the microbial community, favored the survival of Proteobacteria and Bacteroidetes, and upregulated the function genes of organic matter metabolism. These results suggest that the composition and function of microbial community in sediments were associated with the origin, composition, and concentration of NOM input.