Engineered Cyanophycin Synthetase (CphA) from Nostoc ellipsosporum Confers Enhanced CphA Activity and Cyanophycin Accumulation to Escherichia coli
2006
Hai, Tran | Frey, Kay M. | Steinbüchel, A.
The cyanophycin (CGP) synthetase gene (cphANE₁) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphANE₁ gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-β-D-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphANE₁del₉₆) or 59 (CphANE₁del₁₈₀) amino acids of the C-terminal region, were derived from cphANE₁ by deleting 96 or 180 bp from its 3' region through the introduction of stop codons. In comparison to the wild-type gene, cphANE₁del₉₆ conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphANE₁del₁₈₀ was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphANE₁del₉₆ but absent in CphANE₁del₁₈₀. In addition, CphANE₁ and CphANE₁del₉₆ are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.
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