Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 ₇₁₂₀ gene encoding a cyanophycin (CGP) synthetase of Anabaena sp. PCC7120 in plasmids pVLT31::cphA1 ₇₁₂₀ and pBBR1MCS-3::cphA1 ₇₁₂₀ was expressed in the wild-type S. meliloti 1021 and in a phbC-negative mutant generated in this study. Expression of cphA1 ₇₁₂₀ and accumulation of CGP in cells were studied in various media. Yeast mannitol broth (YMB) and pBBR1MCS-3::cphA1 ₇₁₂₀ yielded the highest CGP contents in both S. meliloti 1021 strains. Supplying the YMB medium with isopropyl-β-D-thiogalactopyranoside, aspartic acid, and arginine enhanced CGP contents about 2.5- and 2.8-fold in S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀), respectively. Varying the nitrogen-to-carbon ratio in the medium enhanced the CGP content further to 43.8% (w/w) of cell dry weight (CDW) in recombinant cells of S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀). Cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) accumulated CGP up to 39.6% in addition to 12.1% PHB (w/w, of CDW). CGP from the S. meliloti strains consisted of equimolar amounts of aspartic acid and arginine and contained no other amino acids even if the medium was supplemented with glutamic acid, citrulline, ornithine, or lysine. CGP isolated from cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weights between 20 and 25 kDa, whereas CGP isolated from Escherichia coli S17-1 (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weight between 22 and 30 kDa. Co-expression of cyanophycinase from Anabaena sp. PCC7120 encoded by cphB1 ₇₁₂₀ in cphA1 ₇₁₂₀-positive E. coli S17-1, S. meliloti 1021, and its phbC-negative mutant gave cyanophycinase activities in crude extracts, and no CGP granules occurred. A higher PHB content in S. meliloti 1021 (pBBR1MCS-3::cphB1 ₇₁₂₀::cphA1 ₇₁₂₀) in comparison to the control indicated that the cells used CGP degradation product (β-aspartate-arginine dipeptide) to fuel PHB biosynthesis.