Identification of two gene loci involved in poly-beta-hydroxybutyrate production in Rhodobacter sphaeroides FJ1.
OBJECTIVE
Polyhydroxyalkanoates (PHAs), biopolyesters of hydroxyl fatty acids, are synthesized and deposited as cytoplasmic inclusions in many bacteria. We isolated a poly-beta-hydroxybutyrate (PHB)-producing bacterium designated Rhodobacter sphaeroides FJ1. To characterize PHB biosynthesis in this organism, we isolated the genes encoding proteins involved in PHB metabolism.


METHODS
The genes responsible for the synthesis, accumulation, and degradation of PHB in R. sphaeroides FJ1 were cloned and characterized.


RESULTS
Genes involved in the biosynthesis and metabolism of PHB were found to be located in 2 different loci in the genome of R. sphaeroides FJ1. One locus contained genes encoding PHB depolymerase (phbZ), PHB synthase (phbC), phasin (phbP) and the regulator protein (phbR). The other locus contained the beta-ketothiolase gene (phbA) and the acetoactyl-CoA reductase gene (phbB). The phbZ gene was orientated in an opposite direction to that of phbC, phbP and phbR genes that were located in the same cluster. R. sphaeroides FJ1 was able to grow in wastewater released from the human waste treatment plant of Fu-Jen University. Optimal growth and PHB production were achieved when R. sphaeroides FJ1 was grown in tryptic soy broth containing 50% wastewater. PHB production by R. sphaeroides FJ1 varied in media with different carbon to nitrogen ratios, but the level of PHB synthase was constant, suggesting that PHB production depends mainly on substrate supply.


CONCLUSIONS
Six genes encoding proteins related to PHB metabolism are clustered in 2 separate loci, phbZCPR and phbAB, in a PHB-producing bacterium R. sphaeroides FJ1 isolated from wastewater. PHB synthase, the key enzyme for PHB synthesis, is constitutively expressed, and its expression level is not affected by different growth conditions.
Yang
Mei Kwei
MK
Department of Life Science, Fu-Jen University, Taipei, Taiwan. mkyang@mails.fju.edu.tw
Lin
Yu Chih
YC
Shen
Che Hung
CH
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D005618:Fresh Water / Q000382:microbiology; D006885:Hydroxybutyrates / Q000378:metabolism*; D009876:Operon; D010957:Plasmids; D011091:Polyesters / Q000378:metabolism*; D000091402:Prohibitins; D012242:Rhodobacter sphaeroides / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014865:Waste Disposal, Fluid / Q000379:methods; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D001426:Bacterial Proteins; D003470:Culture Media; D006885:Hydroxybutyrates; D011091:Polyesters; D000091402:Prohibitins; C000717328:PHB protein, human; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C060970:poly-beta-hydroxybutyrate polymerase