Dataset Information

N-terminal truncation of PhaC_BP-M-CPF4 and its effect on PHA production.

ABSTRACT:

Background

Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaC_BP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaC_BP-M-CPF4 through N-terminal truncation.

Results

The N-terminal truncated mutants of PhaC_BP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaC_BP-M-CPF4 mutants were evaluated in C. necator mutant PHB^-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaC_BP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaC_BP-M-CPF4 G8. PhaC_BP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (M_w) of PHA produced due to lower expression of the truncated PhaC_BP-M-CPF4. Transformants harbouring PhaC_BP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaC_BP-M-CPF4 G8 produced higher M_w PHA in mostly single larger PHA granules with comparable production as the full-length PhaC_BP-M-CPF4.

Conclusion

This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, M_w, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher M_w in mostly single larger granules.

SUBMITTER: Neoh SZ

PROVIDER: S-EPMC10867992 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Publications

N-terminal truncation of PhaC<sub>BP-M-CPF4</sub> and its effect on PHA production.

Neoh Soon Zher SZ Tan Hua Tiang HT Trakunjae Chanaporn C Chek Min Fey MF Vaithanomsat Pilanee P Hakoshima Toshio T Sudesh Kumar K

Microbial cell factories 20240215 1

<h4>Background</h4>Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaC<sub>BP-M-CPF4</sub>) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to fur ...[more]

PMID: 38360657

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Project description:BackgroundSeveral members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains.ResultsCobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains.ConclusionsCobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Project description:The inexhaustible nature and biodegradability of bioplastics like polyhydroxyalkanoates (PHAs) make them suitable assets to replace synthetic plastics. The eventual fate of these eco-friendly and non-toxic bioplastics relies upon the endeavors towards satisfying cost and, in addition, execution necessity. In this study, we utilized and statistically optimized different food (kitchen-/agro-) waste as a sole carbon/nitrogen source for the production of PHA at a reduced cost, indicating a proficient waste administration procedure. Seven different types of kitchen-/agro-waste were used as unique carbon source and four different types of nitrogen source were used to study their impact on PHA production by Bacillus subtilis MTCC 144. Among four different studied production media, mineral salt medium (MSM) (biomass: 37.7 g/L; cell dry weight: 1.8 g/L; and PHA: 1.54 g/L) was found most suitable for PHA production. Further, carbon and nitrogen components of MSM were optimized using one-factor-at-a-time experiments, and found that watermelon rind (PHA = 12.97 g/L) and pulse peel (PHA = 13.5 g/L) were the most suitable carbon and nitrogen sources, respectively, in terms of PHA (78.60%) recovery. The concentrations of these factors (sources) were statistically optimized using response surface methodology coupled with the genetic algorithm approach. Additionally, in order to enhance microbial PHA production, the interaction of citrate synthase, a key enzyme in the TCA cycle, with different known inhibitors was studied using in silico molecular docking approach. The inhibition of citrate synthase induces the blockage of the tricarboxylic cycle (TCA), thereby increasing the concentration of acetyl-CoA that helps in enhanced PHA production. Molecular docking of citrate synthase with different inhibitors of PubChem database revealed that hesperidin (PubChem compound CID ID 10621), generally present in citrus fruits, is the most efficient inhibitor of the TCA cycle with the binding score of -11.4 and warrants experimental validation. Overall, this study provides an efficient food waste management approach by reducing the production cost and enhancing the production of PHA, thereby lessening our reliance on petroleum-based plastics.

Dataset Information

N-terminal truncation of PhaC_BP-M-CPF4 and its effect on PHA production.

Background

Results

Conclusion

Publications

N-terminal truncation of PhaC<sub>BP-M-CPF4</sub> and its effect on PHA production.

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Dataset Information

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

Background

Results

Conclusion

Publications

N-terminal truncation of PhaC<sub>BP-M-CPF4</sub> and its effect on PHA production.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

N-terminal truncation of PhaC_BP-M-CPF4 and its effect on PHA production.