Molecular cloning, sequencing and expression in Escherichia coli of the poly(3-hydroxyalkanoate) synthesis genes from Alcaligenes latus DSM1124.
Fragments of chromosomal DNA from Alcaligenes latus DSM1124 were cloned into Escherichia coli and transformants were screened for poly(D(-)-3-hydroxybutyrate) [P(3HB)] production during excess carbon supply. A plasmid harboring a 5.5-kb insert of A. latus DNA was isolated from a P(3HB)-producing bacterial colony. The insert was partially sequenced and three major open reading frames (ORFs) were found, representing the PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) genes. They show striking homology to the Ralstonia eutropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way. The only major difference is the replacement of 560 nucleotides by 160 non-homologous nucleotides in the 5' region of phaC in A. latus. The phaC ORF lacks 29 amino acids at the N-terminus, compared to that of R. eutropha, and starts with a GTG codon. The transcription start points of the operon were determined. P(3HB) production of recombinant E. coli strains harboring the pha operons of A. latus DSM1124 or R. eutropha H16 was investigated. Both operons gave rise to less than 5% P(3HB) formation during exponential growth. At the end of the growth phase, the P(3HB) content reached approximately 20% of cell dry mass. Under nitrogen-depleted conditions, the A. latus pha genes gave rise to 50-52% P(3HB), compared to 33-38% for the R. eutropha pha genes. No NADH oxidase activity was detectable in A. latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth.
Genser
K F
KF
Institut für Biotechnologie, Technische Universität Graz, Austria.
Renner
G
G
Schwab
H
H
D000217:Acyltransferases / Q000378:metabolism; D000421:Alcaligenes / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D006885:Hydroxybutyrates; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase