Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening

Abstract

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages.

Synthesis and Physical Properties of Polyhydroxyalkanoate Polymers with Different Monomer Compositions by Recombinant Pseudomonas putida LS46 Expressing a Novel PHA SYNTHASE (PhaC116) Enzyme

“…Characteristics References P. putida LS46 P. putida isolated from waste water [13] P. putida LS461 phaC1-phaZ-phaC2 deletion mutant of P. putida LS46 This study P. putida LS46123 P. putida LS461 carrying plasmid pJC123; Tc R This study E. coli DH5α ∆lacZ, ∆M15, ∆(lacZYA-argF), U169, recA1, endA1, hsdR17(rK-mK+), supE44, thi-1, gyrA96, relA1 Qiagen, Hilden, Germany pRK7813 IncP oriT cos lacZα, Tc R [30] pJC123 Derivative of pRK7813 carrying phaC1 16 gene from clone 16; Tc R [31] pK18mobsacB Narrow host-range cloning vector; sacB, Km R [32] pRK2013 Helper plasmid pRK290 derivative; Km R [33] pPHAC1C2 pK18mobsacB carrying 840 bp from phaC1 and 857 bp from phaC2 for operon knockout This study The PHA synthesis gene operon of P. putida LS46 was identified from the complete genome sequence (http//www.ncbi.nlm.nih.gov/nucore/ALPV02000008) coordinate 202310-197359; 13) and encoded the following genes: phaC1phaZphaC2phaD. A deletion mutant, designated P. putida LS461, was constructed by deleting the 3 -phaC1, phaZ and 5 -phaC2 genes from the P. putida LS46 pha operon.…”

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases

“…Pseudomonas strains were grown at 30°C in LB or 0.1 N M63 minimal medium (Escapa et al 2011) supplemented with 0.5 % sodium octanoate (w/v), 0.5 % nonanoic acid (v/v), or 1 % gluconic acid (w/v). S. meliloti was grown in LBmc (Finan et al 1986) or YM medium (Schallmey et al 2011). Antibiotics were used at the following concentrations: streptomycin, 200 μg/ml for S. meliloti and 100 μg/ml for E. coli; kanamycin, 100 μg/ml for Pseudomonas and 50 μg/ml for E. coli; neomycin, 200 μg/ml; rifampicin, 100 μg/ml; gentamicin, 10 μg/ml for E. coli and 100 for P. putida; and tetracycline 20 μg/ml for E. coli or 40 μg/ml for P. putida.…”

“…In previous work, functional metagenomics was used to isolate new class I PhaC from soil metagenomic clones by Nile red staining and phenotypic screening in α-Proteobacteria Sinorhizobium meliloti (Schallmey et al 2011). In another study, phaC genes encoding both classes I and II PhaC proteins were PCR amplified from oil-contaminated soil library clones (Cheema et al 2012).…”

“…MCL-PHA is generally more useful than SCL-PHA due to it being less brittle and more flexible. To produce PHA with versatile properties cost-effectively, strategies have involved mining new PHA synthase enzymes (PhaC), engineering PhaC proteins, and modifying the metabolic pathways of the production strains (Keshavarz and Roy 2010;Schallmey et al 2011;Cheema et al 2012;Park et al 2012;Tripathi et al 2013;Meng et al 2014).…”

Seleção de genes codificadores de PHA sintases para a construção de recombinantes em Burkholderia sacchari e Pseudomonas sp e avaliação da produção de polihidroxialcanoatos com diferentes composições monoméricas.

“…Quando o gene da PHA sintase está presente no vetor, o fenótipo da bactéria é restabelecido, e ela se torna capaz de produzir PHA comprovando a existência do gene na biblioteca. Utilizando essa metodologia obteve-se sucesso em encontrar novos tipos de PHA sintase (ANEJA et al, 2004;SCHALLMEY et al, 2011 Outro dado importante, com relação às PHA sintases de classe IV (descritas em Bacillus) é que apenas recentemente foi reportada sua expressão na bactéria Gram negativa Ralstonia eutropha, mostrando que, dependendo da origem bacteriana da enzima, especificidades mais relaxadas podem ser observadas e também a formação do polímero alvo mencionado (HYAKUTAKE et al, 2011). Dentro desse contexto foi proposto o presente trabalho.…”