Affiliation:
1. Institut für Biotechnologie, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany
Abstract
ABSTRACT
d
-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in
Corynebacterium glutamicum
and determined specific activities ranging from 0.00014 to 0.001 μmol/min mg (protein)
−1
. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that
panBC
constitutes an operon. By using
panC
, an assay system was developed to quantify
d
-pantothenate. The wild type of
C. glutamicum
was found to accumulate 9 μg of this vitamin per liter. A strain was constructed (i) to abolish
l
-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to
d
-pantothenate. The best resulting strain has
ilvA
deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (
ilvBNCD
) and
d
-pantothenate (
panBC
) synthesis. With this strain a
d
-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10
5
-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the
d
-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for
d
-pantothenate formation.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
65 articles.
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