Microbial production of polyhydroxyalkanoate from soy sauce oil, a byproduct of soy sauce manufacturing.

See accompanying articles by Jieun Kim et al. DOI 10.1002/biot.201600648 & Yeo-Jin Kim et al. DOI 10.1002/biot.201600649 It has been my great pleasure to read the two papers entitled ”Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms“ by Kim et al. 1, and ”Structure and function of the N-terminal domain of Ralstonia eutropha polyhydroxyalkanoate synthase, and the proposed structure and mechanisms of the whole enzyme“ by Kim et al. 2. Polyhydroxyalkanoates (PHAs) are diverse biopolyesters accumulated by many bacteria as energy and carbon storage materials. PHAs have been exploited as biodegradable and biocompatible thermal plastics (also called Bioplastics). PHA synthase plays important roles in determining the general characteristics of PHAs produced, including molecular weight, polydispersity, monomer composition, and productivity. Global efforts have been made to understand PHA synthase via revealing its crystal structure (short as PhaC) without success over the last 30 years; however, Prof. Sang Yup Lee and his colleagues unveil the mystery in these two studies. Kim et al. 1 firstly demonstrate the crystal structure of PHA synthase from Ralstonia eutropha, the best studied bacterium for PHA production, and report the structural basis for the detailed molecular mechanisms of PHA biosynthesis. Kim et al. 2 then for the first time report the 3D reconstructed model of full length PHA synthase from R. eutropha, and performed several biochemical studies. These two studies provide feasibility for rational engineering of PHA synthase toward more efficient production of bioplastics, more broad PHA structure diversity and possibly controllable PHA molecular weights. If the mechanism is better understood, we may even be able to develop artificial synthases for synthesis of other non-PHA polyesters. With the PHA synthase structure revealed now, we are moving more close to tailor-made PHA to meet various needs. The author declares no financial or commercial conflict of interest.

ObjectiveSoy sauce oil, a byproduct of whole soybean processing by the soy sauce industry, was evaluated as a source of linoleic acid for dairy cows for the purpose of manipulating the composition of milk.MethodsEight dairy Holstein cows fitted with rumen cannulas were used for ruminal administration of soy sauce oil for a 28-day period using a 4×4 Latin square study design with 4 doses (0, 200, 400, and 600 g soy sauce oil/d).ResultsAlthough dry matter intake and milk yield were not affected by soy sauce oil administration, ruminal concentrations of total volatile fatty acids and acetate were decreased, specifically at 600 g/d administration. While milk fat percentage was decreased with administration of soy sauce oil, proportions of linoleic, vaccenic and conjugated linoleic acids in the rumen, blood and milk were increased with increasing soy sauce oil dose.ConclusionThese results suggest that soy sauce oil feeding could be useful for improving milk functionality without adverse effects on animal production performance when fed at less than 400 g/d.

Oleic acid as a substrate for poly-3-hydroxyalkanoate formation in Alcaligenes eutrophus and Pseudomonas putida

Chimeric enzymes composed of polyhydroxyalkanoate (PHA) synthases from Ralstonia eutropha (Cupriavidus necator) (PhaC(Re)) and Aeromonas caviae (PhaC(Ac)) were constructed. PhaC(Re) is known for its potent enzymatic activity among the characterized PHA synthases. PhaCAc has broad substrate specificity and synthesizes short-chain-length (SCL)/medium-chain-length (MCL) PHA. We attempted to create chimeric enzymes inheriting both of the advantageous properties. Among eight chimeras, AcRe12, with 26% of the N-terminal of PhaC(Ac) and 74% of the C-terminal of PhaC(Re), exhibited comparable P(3-hydroxybutyrate) accumulation as parental enzymes in Escherichia coli JM109. Thus, AcRe12 was applied to SCL/MCL PHA production using E. coli LS5218 as the host. AcRe12 accumulated higher amount of PHA (50 wt %) than the parental enzymes. Furthermore, the PHA consisted of 2 mol % 3-hydroxyhexanoate as well as 3-hydroxybutyrate. Therefore, the chimeric PHA synthase, AcRe12, inherited the character of both of the parental enzymes and thus exhibits improved enzymatic properties.

The bacterial strain isolated from soil was identified as Cupriavidus necator IBP/SFU-1 and investigated as a PHA producer. The strain was found to be able to grow and synthesize PHAs under autotrophic conditions and showed a broad organotrophic potential towards different carbon sources: sugars, glycerol, fatty acids, and plant oils. The highest cell concentrations (7–8 g/L) and PHA contents were produced from oleic acid (78%), fructose, glucose, and palm oil (over 80%). The type of the carbon source influenced the PHA chemical composition and properties: when grown on oleic acid, the strain synthesized the P(3HB-co-3HV) copolymer; on plant oils, the P(3HB-co-3HV-co-3HHx) terpolymer, and on the other substrates, the P(3HB) homopolymer. The type of the carbon source influenced molecular-weight properties of PHAs: P(3HB) synthesized under autotrophic growth conditions, from CO2, had the highest number-average (290 ± 15 kDa) and weight-average (850 ± 25 kDa) molecular weights and the lowest polydispersity (2.9 ± 0.2); polymers synthesized from organic carbon sources showed increased polydispersity and reduced molecular weight. The carbon source was not found to affect the degree of crystallinity and thermal properties of the PHAs. The type of the carbon source determined not only PHA composition and molecular weight but also surface microstructure and porosity of the polymer films. The new strain can be recommended as a promising P(3HB) producer from palm oil, oleic acid, and sugars (fructose and glucose) and as a producer of P(3HB-co-3HV) from oleic acid and P(3HB-co-3HV-co-3HHx) from palm oil.

ObjectiveTo evaluate soy sauce oil (a by-product of making whole soybean soy sauce) as a new dietary lipid source, a large amount of soy sauce oil was administered into the rumen of dairy cows.MethodsFour Holstein dairy cows fitted with rumen cannulae were used in a 56-day experiment. Ruminal administration of soy sauce oil (1 kg/d) was carried out for 42 days from day 8 to day 49 to monitor nutritional, physiological and production responses.ResultsDry matter intake and milk yield were not affected by soy sauce oil administration, whereas 4% fat-corrected milk yield and the percentage of milk fat decreased. Although ruminal concentration of total volatile fatty acids (VFA) and the proportion of individual VFA were partially affected by administration of soy sauce oil, values were within normal ranges, showing no apparent inhibition in rumen fermentation. Administration of soy sauce oil decreased the proportions of milk fatty acids with a carbon chain length of less than 18, and increased the proportions of stearic, oleic, vaccenic and conjugated linoleic acids. Conjugated linoleic acid content in milk became 5.9 to 8.8 times higher with soy sauce oil administration. Blood serum concentrations of non-esterified fatty acid, 3-hydroxybutyric acid, total cholesterol, free cholesterol, esterified cholesterol, triglyceride and phospholipid increased with administration of soy sauce oil, suggesting a higher energy status of the experimental cows.ConclusionThe results suggest that soy sauce oil could be a useful supplement to potentially improve milk functionality without adverse effects on ruminal fermentation and animal health. More detailed analysis is necessary to optimize the supplementation level of this new lipid source in feeding trials.

Effect of glycerol and its analogs on polyhydroxyalkanoate biosynthesis by recombinant Ralstonia eutropha: A quantitative structure–activity relationship study of chain transfer agents

Development of novel polyhydroxyalkanoate materials

Location of functional region at N-terminus of polyhydroxyalkanoate (PHA) synthase by N-terminal mutation and its effects on PHA synthesis

BackgroundPhasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1Re locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil.ResultsConsidering the high expression level of phaP1Re, phaJAc [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1Re on chromosome 1 of R. eutropha strain NSDG harboring phaCNSDG (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJAc within pha operon. Insertion of the second phaCNSDG along with phaJAc at the phaP1Re locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1Re with phaPAc (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester.ConclusionsThe downstream of phaP1Re was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0380-8) contains supplementary material, which is available to authorized users.

Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats

Polyhydroxyalkanoates (PHA) have received attention owing to their biodegradability and biocompatibility, with studies exploring PHA-producing bacterial strains. As vegetable oil provides carbon and monomer precursors for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), oil-utilizing strains may facilitate PHA production. Herein, Cupriavidus necator BM3-1, which produces 11.1 g/L of PHB with 5% vegetable oil, was selected among various novel Cupriavidus necator strains. This strain exhibited higher preference for vegetable oils over sugars, with soybean oil and tryptone determined to be optimal sources for PHA production. BM3-1 produced 33.9 g/L of exopolysaccharides (EPS), which was three-fold higher than the amount produced by H16 (10.1 g/L). EPS exhibited 59.7% of emulsification activity (EI24), higher than that of SDS and of EPS from H16 with soybean oil. To evaluate P(3HB-co-3HHx) production from soybean oil, BM3-1 was engineered with P(3HB-co-3HHx) biosynthetic genes (phaCRa, phaARe, and phaJPa). BM3-1/pPhaCJ produced 3.5 mol% of 3HHx and 37.1 g/L PHA. BM3-1/pCB81 (phaCAJ) produced 32.8 g/L PHA, including 5.9 mol% 3HHx. Physical and thermal analyses revealed that P(3HB-co-5.9 mol% 3HHx) was better than PHB. Collectively, we identified a novel strain with high vegetable oil utilization capacity for the production of EPS, with the option to engineer the strain for P(3HB-co-3HHx).

Volatile fatty acids (VFA) may serve as building blocks for Polyhydroxyalkanoates (PHA) production and can be derived from waste streams. Ideal streams for PHA production contain a high Chemical Oxygen Demand (COD) to nutrient ratio, such as (waste)water from a paper-mill factory or candy-bar factory. The (waste)water generated by these companies are usually treated anaerobically with the final product being methane containing biogas. Usually, the methane is burned to produce either heat or electricity. Potentially, more value can be added to these streams by producing VFA and/or PHA. PHA can be produced using microbial enrichment cultures that can be established by cultivation in a selective environment that favours the growth of PHA producing microorganisms. Some advantages of using open cultures are that no sterilization and expensive equipment is required compared to pure culture biotechnology. Open culture biotechnology can be effectively applied when the right selection pressure for a specific microbial trait is identified. The microorganism that is most effective in the given conditions will win the competition, i.e. the strongest will survive. A selection criteria for PHA productis is consuming substrate very fast by first making a storage polymer (in this case PHA) from the supplied substrate. The PHA producers prefer VFA as substrate, hence it is important to maximize the VFA content in the substrate stream. For the production of VFA in the product chain towards PHA it is important to minimize the solid content in the feedstock for PHA production. The objective of the research described in this thesis was to gain more insight in the two-step upstream process for PHA production from agricultural waste streams. The first step concerns the maximization of the VFA concentration in the feedstock. Optimization of VFA production was investigated using the granular sludge process in order to maximize the volumetric VFA production capacity and to minimize the solids concentration in the effluent. Two process variables were investigated regarding the PHA production process. Firstly, the influence of the presence of nutrients on PHA production was investigated using PHA producing enrichment cultures. Secondly, the production of PHA was investigated using the leachate of the organic fraction of municipal solid waste (OFMSW) at pilot scale.

Waste paper as a resource for polyhydroxyalkanoate (PHA) production through anaerobic digestion is a low-cost strategy to produce bioplastic. In this study, volatile fatty acids (VFAs) produced from waste paper, one of the significant constituents of municipal solid waste, was utilized as a feedstock for polyhydroxyalkanoate (PHA) production. PHA production from synthetic VFAs by Cupriavidus necator was initially optimized under different VFAs concentrations, VFAs ratios, and nitrogen sources. VFAs concentration of 10 g/L, 5:1:4 ratio of acetic, propionic, and butyric acids (HAc:HPr:HBu) and NaNO3 as nitrogen source were considered the optimum conditions with 56.98% PHA and 0.31 g/g yield. Anaerobic digestion of shredded office paper (OP/S) produced the maximum VFAs (521.50 mg/L) after 15 days of incubation and were utilized for PHA synthesis. Almost 2.24-fold increase in the yield of PHA was achieved with limited nutrient medium compared to nutrient contained medium with a PHA content of 53.50 and 23.88%, respectively. PHA production using anaerobic effluent of waste paper is a promising approach where a series of pretreatment processes, the expensive enzymatic hydrolysis, and detoxification were no longer required, suggesting an environmentally friendly way of biopolymer production.

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.