Identification and characterization of extracellular mcl-PHA depolymerase and lipases in members of the Proteobacteria

Degradation of extracellular Polyhydroxyalkanoates (PHAs) in the environment depends on the secretion of specific extracellular PHA depolymerases and lipases. Compared to extracellular scl-PHA degrading enzymes, extracellular enzymes that can degrade mcl-PHAs are poorly investigated. In this project, Proteobacteria members that produce extracellular mcl-PHAs depolymerases/ lipases were screened on mcl-PHA agar plates. Then, genes involved in the production and regulation of mcl-PHA depolymerases/lipases were identified using transposon mutagenesis, insertional mutagenesis, and bioinformatics prediction tools. Once the genes were identified, the mcl-PHA depolymerase/lipase encoding genes were cloned and characterized. Burkholderia thailandensis E264, Stenotrophomonas maltophilia K279a, S. maltophilia D457, and S. maltophilia NCPPB1683 were found to produce extracellular mcl-PHA depolymerases/lipases. In Pseudomonas chlororaphis PA23, transposon mutagenesis showed that disruption of certain metabolic pathways positively affects mcl-PHA depolymerase/lipase production. Transposon mutagenesis of B. cepacia ATCC 25416 indicated that disruption of the APZ15_13320 gene negatively affected the production of pigmented metabolites and positively affected mcl-PHA depolymerase/lipase production. A novel S. maltophilia strain that can produce mcl-PHA depolymerase/lipase was discovered during the screening process as a contaminant strain of the T. caryophylli DSMZ16666. Using whole-genome sequencing and bioinformatics, a putative extracellular mcl-PHA depolymerase/lipase known as fig|40323.255.peg.2780 was identified in the novel S. maltophilia HT01 strain. The in-silico prediction of a putative extracellular mcl-PHA depolymerase/lipase in S. maltophilia K279a resulted in twenty-five protein-coding sequences. Five of the twenty-five putative depolymerase/lipases were cloned in E. coli. The E. coli clones expressing these genes were screened for degradation of polyhydroxyoctanaoate