D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.
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 micromol/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 micrograms 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 beta-alanine is essential for D-pantothenate formation.
Sahm
H
H
Institut für Biotechnologie, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Eggeling
L
L
D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D045782:Hemiterpenes; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D010205:Pantothenic Acid / Q000096:biosynthesis*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000096:biosynthesis*
D017931:DNA Primers; D045782:Hemiterpenes; D007651:Keto Acids; D010205:Pantothenic Acid; C001505:alpha-ketoisovalerate; D019877:Hydroxymethyl and Formyl Transferases; C021477:3-methyl-2-oxobutanoate hydroxymethyltransferase; D010453:Peptide Synthases; C022318:pantothenate synthetase; D014633:Valine