Dataset Information

Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review.

ABSTRACT: Some strains of Pseudomonas corrugata (Pco) and P. mediterranea (Pme) efficiently synthesize medium-chain-length polyhydroxyalkanoates elastomers (mcl-PHA) and extracellular products on related and unrelated carbon sources. Yield and composition are dependent on the strain, carbon source, fermentation process, and any additives. Selected Pco strains produce amorphous and sticky mcl-PHA, whereas strains of Pme produce, on high grade and partially refined biodiesel glycerol, a distinctive filmable PHA, very different from the conventional microbial mcl-PHA, suitable for making blends with polylactide acid. However, the yields still need to be improved and production costs lowered. An integrated process has been developed to recover intracellular mcl-PHA and extracellular bioactive molecules. Transcriptional regulation studies during PHA production contribute to understanding the metabolic potential of Pco and Pme strains. Data available suggest that pha biosynthesis genes and their regulations will be helpful to develop new, integrated strategies for cost-effective production.

SUBMITTER: Licciardello G

PROVIDER: S-EPMC6955742 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review.

Licciardello Grazia G Catara Antonino F AF Catara Vittoria V

Bioengineering (Basel, Switzerland) 20191114 4

Some strains of Pseudomonas corrugata (Pco) and P. mediterranea (Pme) efficiently synthesize medium-chain-length polyhydroxyalkanoates elastomers (mcl-PHA) and extracellular products on related and unrelated carbon sources. Yield and composition are dependent on the strain, carbon source, fermentation process, and any additives. Selected Pco strains produce amorphous and sticky mcl-PHA, whereas strains of Pme produce, on high grade and partially refined ...[more]

PMID: 31739507

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Project description:Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

Project description:Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.

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Dataset Information

Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review.

Publications

Production of Polyhydroxyalkanoates and Extracellular Products Using <i>Pseudomonas Corrugata</i> and <i>P. Mediterranea</i>: A Review.

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