Cost-competitive γ-PGA production from low costs feedstock using an engineered Bacillus subtilis lab strain

Poly-γ-glutamic acid (γ-PGA) represents one of the most promising biomaterial naturally secreted by microorganisms mainly belonging to the Bacillales order. This uncommon homo-polyamide is formed by D-/L- glutamic acid units polymerized by γ-amide linkages. The polymer, resistant to common proteases, is endowed with characteristics that are suitable for many biotechnological applications, e.g. as metal flocculant, drug carrier, food additive and many more. However it is crucial to improve the economic viability of its production for γ-PGA industrial exploitation. Nowadays valorization of agro-food wastes is also a crucial issue. This work aims at improving the cellulolytic capabilities of a B. subtilis strain to obtain γ-PGA exploiting the abundant and low-cost organic fraction present in rice straw as feedstock. Unfortunately rice straw is rich in silica which inhibits bacterial growth. However, an alkali treatment was established in which hemicelluloses are collected in the liquid fraction and silica can be extracted from the solid lignocellulosic part, concurrently enhancing cellulose bioavailability. The liquid hemicellulose-rich fraction and the remaining solid cellulose-rich part can then be used for bacterial fermentation. Preliminary results demonstrate that B. subtilis can grow on such a substrate as sole carbon source. Conveniently, the strain carrying the degU32(Hy) mutation, necessary for hyper-production of γ-PGA, shows more efficient hydrolysis of cellobiose and xylan than the wild type. To further improve the strain cellulolytic potential genome and transcriptome data analyses were performed to select endogenous genes coding for enzymes important for saccharification of lignocellulose matrixes that could be modified. Genes bglC and xynA (encoding an endo-1,3,(4)-beta-glucanase and an endo-1,4-beta-xylanase, respectively) were chosen as targets, since their expression is poor and limited to the transition phase. An allelic-exchange approach was set up to increase xynA and bglC expression levels by optimizing the endogenous promoters and translation signals as well as by inserting a promoter UP element. The cellulolytic properties and γ-PGA yield of the engineered strains will be presented and discussed.