A novel gene (Aa-accA) encoding acetyl-CoA carboxyltransferase α-subunit of Alkalimonas amylolytica N10 enhances salt and alkali tolerance of Escherichia coli and tobacco BY-2 cells

Abstract:

Abstract:［Objective］Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis．In most bacteria，ACC is composed of four subunits encoded by accA，accB，accC，and accD. of them，accA encodes acetyl-CoA carboxyltransferase α-subunit．Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress． This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance.［Methods］The Aa-accA was amplified by PCR from A．amylolytica N10 and expressed in E． coli K12 host．The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions． Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation． The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock．［Results］The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases．It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E.coli. Compared to the wild-type strains，transgenic E．coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels． The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9，respectively． Complementary expression of Aa-accA in an accA-depletion E.coli can recover the tolerance of K12ΔaccA to salt and alkali stresses to some extent． Similar to the transgenic E.coli，transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition.［Conclusion］ We found that Aa-accA from A． amylolytica N10 overexpression enhances the tolerance of both transgenic E.coli and tobacco BY-2 cells to NaCl and alkali stresses．