Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei.
D-2-Hydroxyisocaproic acid dehydrogenase (D-HicDH) from Lactobacillus casei was purified and partially sequenced. A 65-mer oligodeoxyribonucleotide probe corresponding to the N-terminal 23 amino acids was synthesized and a physical map was made of the genomic region which hybridized most strongly. A strongly hybridising restriction fragment was highly purified and eventually cloned at low frequency in pBR322. The original clones spontaneously produced D-HicDH at about 0.05% of total protein and showed viability problems in that 10- to 12-h growth-lag periods occurred after diluting stationary cultures into fresh medium. Subcloning into pGEM3 plasmids for sequencing with concomitant ExoIII deletion led to clones which no longer exhibited the growth inhibition characteristics but now made D-HicDH as 3 to 5% of total protein. Subcloning downstream from a double PL PR promoter in plasmid pJLA601 gave a highly inducible clone that builds large inclusion bodies of largely denatured D-HicDH. The gene transcript was mapped for L. casei and Escherichia coli hosts. The promoter, terminator and Shine-Dalgarno sequence are functional in both organisms. The gene encodes a protein subunit of 38 kDa, whereby 67% of the sequence could be checked by correlation with partial peptide sequences from the original enzyme. So far no Lactobacillus gene has been found to utilize the Arg codons AGG and AGA.
Lerch
H P
HP
Gesellschaft für Biotechnologische Forschung, Braunschweig, F.R.G.
Blöcker
H
H
Kallwass
H
H
Hoppe
J
J
Tsai
H
H
Collins
J
J
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger; D015183:Restriction Mapping
D004269:DNA, Bacterial; D012333:RNA, Messenger; D000429:Alcohol Oxidoreductases; C047961:2-hydroxyisocaproate dehydrogenase