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Applied and Environmental Microbiology, December 2006, p. 7652-7660, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01132-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Engineered Cyanophycin Synthetase (CphA) from Nostoc ellipsosporum Confers Enhanced CphA Activity and Cyanophycin Accumulation to Escherichia coli{triangledown}

Tran Hai, Kay M. Frey, and Alexander Steinbüchel*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany

Received 16 May 2006/ Accepted 25 September 2006

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-ß-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.

* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstrasse 3, D-48149 Münster, Germany. Phone: 49 251 8339821. Fax: 49 251 8338388. E-mail: steinbu{at}uni-muenster.de.

{triangledown} Published ahead of print on 29 September 2006.

Applied and Environmental Microbiology, December 2006, p. 7652-7660, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01132-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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