d-Pantothenate Synthesis inCorynebacterium glutamicum and Use of panBC and Genes Encoding l-Valine Synthesis ford-Pantothenate Overproduction

摘要

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 inCorynebacterium 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 usingpanC, an assay system was developed to quantifyd-pantothenate. The wild type of C. glutamicumwas 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 105-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 thed-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for d-pantothenate formation.