Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai.
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ABSTRACT:
SUBMITTER: Balakrishna Pillai A
PROVIDER: S-EPMC5498450 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.
| S-EPMC8123457 | biostudies-literature
Project description:Currently, the selection of materials for tissue engineering scaffolds is still limited because some tissues require flexible and compatible materials with human cells. Medium-chain-length polyhydroxyalkanoate (MCL-PHA) synthesized in microorganisms is an interesting polymer for use in this area and has elastomeric properties compatible with the human body. MCL-PHAs are elastomers with biodegradability and cellular compatibility, making them an attractive material for fabricating soft tissue that requires high elasticity. In this research, MCL-PHA was produced by fed-batch fermentation that Pseudomonas Putida ATCC 47054 was cultured to accumulate MCL-PHA by using glycerol and sodium octanoate as carbon sources. The amounts of dry cell density, MCL-PHA product per dry cells, and MCL-PHA productivity were at 15 g/L, 27%, and 0.067 g/L/h, respectively, and the components of MCL-PHA consisting of 3-hydroxydecanoate (3HD) 64.5%, 3-hydroxyoctanoate (3HO) 32.2%, and 3-hydroxyhexanoate (3HHx) 3.3%. The biosynthesized MCL-PHA terpolyester has a relatively low melting temperature, low crystallinity, and high ductility at 52 °C, 15.7%, and 218%, respectively, and considering as elastomeric polyester. The high-resolution scaffold of MCL-PHA terpolyester biomaterial-ink (approximately 0.36 mm porous size) could be printed in a selected condition with a 3D printer, similar to the optimum pore size for cell attachment and proliferation. The rheological characteristic of this MCL-PHA biomaterial-ink exhibits shear-thinning behavior, leading to good shape fidelity. The study results yielded a condition capable of fabricating an elastomer scaffold of the MCL-PHA terpolyester, giving rise to the ideal soft tissue engineering application.
| S-EPMC8309464 | biostudies-literature
Project description:Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.
| S-EPMC1828677 | biostudies-literature
Project description:Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases.
| S-EPMC3675958 | biostudies-literature
Project description:Environmental contamination by phenol has been reported in both aquatic and atmospheric environments. This study aimed to separate and purify the peroxidase enzyme from bacteria that degrade phenol from wastewater sources. An enrichment culture of MSM was used to screen 25 bacterial isolates from different water samples for peroxidase production, six of the isolates exhibited high levels of peroxidase enzyme activity. Qualitative analysis of peroxidase revealed that isolate No. 4 had the highest halo zones (Poly-R478: 14.79 ± 0.78 mm, Azure B: 8.81 ± 0.61 mm). The promising isolate was identified as Bacillus aryabhattai B8W22 by 16S rRNA gene sequencing with accession number OP458197. As carbon and nitrogen sources, mannitol and sodium nitrate were utilized to achieve maximum peroxidase production. A 30-h incubation period was used with pH 6.0, 30 °C, mannitol, and sodium nitrate, respectively, for maximal production of peroxidase. Purified peroxidase enzyme showed 0.012 U/mg specific activity, and SDS-PAGE analysis indicated a molecular weight of 66 kDa. The purified enzyme exhibits maximum activity and thermal stability at pH values of 4.0 and 8.0, respectively, with maximum activity at 30 °C and complete thermal stability at 40 °C. In the purified enzyme, the Km value was 6.942 mg/ml and the Vmax value was 4.132 mol/ml/hr, respectively. The results demonstrated that Bacillus aryabhattai B8W22 has promising potential for degrading phenols from various phenol-polluted wastewater sources.
| S-EPMC10148497 | biostudies-literature
Project description:Waste cooking oil is a common byproduct in the culinary industry, often posing disposal challenges. This study explores its conversion into the valuable bioplastic material, medium-chain-length polyhydroxyalkanoate (mcl-PHA), through microbial biosynthesis in controlled bioreactor conditions. Twenty-four bacterial isolates were obtained from oil-contaminated soil and waste materials in Mahd Ad-Dahab, Saudi Arabia. The best PHA-producing isolates were identified via 16S rDNA analysis as Neobacillus niacini and Metabacillus niabensis, with the sequences deposited in GenBank (accession numbers: PP346270 and PP346271). This study evaluated the effects of various carbon and nitrogen sources, as well as environmental factors, such as pH, temperature, and shaking speed, on the PHA production titer. Neobacillus niacini favored waste cooking oil and yeast extract, achieving a PHA production titer of 1.13 g/L, while Metabacillus niabensis preferred waste olive oil and urea, with a PHA production titer of 0.85 g/L. Both strains exhibited optimal growth at a neutral pH of 7, under optimal shaking -flask conditions. The bioreactor performance showed improved PHA production under controlled pH conditions, with a final titer of 9.75 g/L for Neobacillus niacini and 4.78 g/L for Metabacillus niabensis. Fourier transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) confirmed the biosynthesized polymer as mcl-PHA. This research not only offers a sustainable method for transforming waste into valuable materials, but also provides insights into the optimal conditions for microbial PHA production, advancing environmental science and materials engineering.
| S-EPMC11314287 | biostudies-literature
Project description:Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.
| S-EPMC3457088 | biostudies-literature
Project description:The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition.Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.
| S-EPMC5068169 | biostudies-other
Project description:Pseudomonas putida's versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs' metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.
| S-EPMC7801951 | biostudies-literature
Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway.
2015-05-01 | GSE65029 | GEO