초록
<P><B>Abstract</B></P> <P>A <I>Bacillus amyloliquefaciens</I> strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the <I>epsA-O</I> (responsible for extracellular polysaccharide synthesis), <I>sac</I> (responsible for levan synthesis), <I>lps</I> (responsible for lipopolysaccharide synthesis) and <I>pta</I> (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15g/L. The γ-PGA degrading genes <I>pgdS</I> (encoding poly-gamma-glutamate depolymerase) and <I>cwlO</I> (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18g/L. The autoinducer AI-2 synthetase gene <I>luxS</I> was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54g/L). In addition, we overexpressed the <I>pgsBCA</I> genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the <I>rocG</I> gene (encoding glutamate dehydrogenase) and the <I>glnA</I> gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The <I>rocG</I>-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a <I>B. amyloliquefaciens</I> strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Improve γ-PGA production by a systematic modular pathway engineering strategy. </LI> <LI> The by-product pathways were blocked to increase γ-PGA production and purity. </LI> <LI> The degrading enzyme genes were deleted to improve γ-PGA production. </LI> <LI> The synthetic small regulatory RNAs were used for <I>B. amyloliquefaciens</I> engineering. </LI> <LI> γ-PGA production increased 2.91-fold in shake flask and 5.34-fold in 5-L fermenter. </LI> </UL> </P>