Engineering of recombinant Escherichia coli cells co-expressing poly-γ-glutamic acid (γ-PGA) synthetase and glutamate racemase for differential yielding of γ-PGA.
Bottom Line: The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing γ-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria-Bertani medium containing glucose or L-glutamate.However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon γ-PGA production.Furthermore, RacE integration could enhance γ-PGA yield through improving the preferred d-glutamate content.
Affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.Show MeSH
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Mentions: The sequence of pgsBCA was amplified from genome DNA of NK-03 and LL3 strains as before (Cao et al., 2010; 2011), which was about 3000 bp and contained three open-reading frames (ORFs), designated as pgsB, pgsC and pgsA. After alignment by BLAST (Basic Local Alignment Search Tool) and DNAMAN, the synthetase constituents PgsB and PgsC showed great similarity above 93%, while the PgsA showed only about 78.5% homology (Table S1). Based on the consensus of PgsBCA components and their amino acids sequences (Fig. 1), the feature of each element have been analysed and validated as that reported (Ashiuchi and Misono, 2003). It was revealed that PgsB conserves the consensus sequences found in enzymes of an amide ligase superfamily (Eveland et al., 1997). It harbours an ATP-binding motif at the N-teminal residues of 37–42 (GIRGKS), which is responsible for catalysing the hydrolysis of essential ATP, providing the energy for γ-PGA synthesis. PgsC, the most conserved component of PgsBCA complex, is a hydrophobic and a membrane-bound protein containing four transmembrane helices regions. The active site of PgsBCA, which was supposed to be constituted by PgsB and PgsC, was assumed to display ATPase activity and assimilate both isomers of γ-DL-PGA as the substrate (Urushibata et al., 2002). As the core structure for membrane integration and the transporter of γ-PGA, PgsA consisted mainly of hydrophobic and cationic amino acid residues, and showed the most variable part of synthetase to generate the differential elongation (molecular weight) of γ-PGA (Candela and Fouet, 2006). However, to elucidate the precise function of each membrane-associated component in γ-PGA polymerization and transportation, it should be recurred to three-dimensional modelling and crystal structure resolution.
Affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.