Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109

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Abstract

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaACo gene, 1182bp), acetoacetyl-CoA reductase (phaBCo gene, 738bp) and PHA synthase, class I (phaCCo gene, 1694bp) were identified. Sequence analysis of the phaACo, phaBCo and phaCCo genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaCCo and phaABCo) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaCCoABRe and pGEM'-phaCReABCo were shown to be functionally active synthesising 33wt.% and 17wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCABCo) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCABCo could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively. © 2012 Elsevier GmbH.

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Yee, L. N., Chuah, J. A., Chong, M. L., Phang, L. Y., Raha, A. R., Sudesh, K., & Hassan, M. A. (2012). Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109. Microbiological Research, 167(9), 550–557. https://doi.org/10.1016/j.micres.2011.12.006

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