Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene |
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| Authors: | M A G Koffas R Ramamoorthi W A Pine A J Sinskey G Stephanopoulos |
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| Institution: | (1) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA02139, USA e-mail: gregstep@mit.edu Tel.: +1-617-253-4583 Fax: +1-617-253-3122, US;(2) Department of Chemical Engineering, Massachusetts Institute of techNology, Cambridge, MA02139, USA, US |
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| Abstract: | Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth,
or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase
sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction
(PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds
of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified.
Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998 |
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