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 clone d 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 th e PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-Coa re ductase (phaB) genes. They show striking homology to the Ralstonia eut ropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way. The only major differ ence is the replacement of 560 nucleotides by 160 non-homologous nucle otides in the 5' region of phaC in A. latus. The phaC ORF lacks 29 ami no acids at the N-terminus, compared to that of R. eutropha, and start s 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 investigat ed. Both operons gave rise to less than 5% P(3HB) formation during exp onential growth. At the end of the growth phase, the P(3HB) content re ached approximately 20% of cell dry mass. Under nitrogen-depleted cond itions, 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 de tectable in A. latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth. (C) 1998 Elsevier Science B.V. All rights reserved.