d -Pantothenate Synthesis in Corynebacterium glutamicum and Use of panBC and Genes Encoding l -Valine Synthesis for d -Pantothenate Overproduction

Author:

Sahm Hermann1,Eggeling Lothar1

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

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