Disruption of poly (3-hydroxyalkanoate) depolymerase gene and overexpression of three poly (3-hydroxybutyrate) biosynthetic genes improve poly (3-hydroxybutyrate) production from nitrogen rich medium by Rhodobacter sphaeroides.

BACKGROUND: Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production. RESULTS: We constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l-1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively. CONCLUSION: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.

Anaerobic poly-3-D-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase.

BACKGROUND: Poly-3-D-hydroxybutyrate (PHB) that is a promising precursor for bioplastic with similar physical properties as polypropylene, is naturally produced by several bacterial species. The bacterial pathway is comprised of the three enzymes ß-ketothiolase, acetoacetyl-CoA reductase (AAR) and PHB synthase, which all together convert acetyl-CoA into PHB. Heterologous expression of the pathway genes from Cupriavidus necator has enabled PHB production in the yeast Saccharomyces cerevisiae from glucose as well as from xylose, after introduction of the fungal xylose utilization pathway from Scheffersomyces stipitis including xylose reductase (XR) and xylitol dehydrogenase (XDH). However PHB titers are still low. RESULTS: In this study the acetoacetyl-CoA reductase gene from C. necator (CnAAR), a NADPH-dependent enzyme, was replaced by the NADH-dependent AAR gene from Allochromatium vinosum (AvAAR) in recombinant xylose-utilizing S. cerevisiae and PHB production was compared. A. vinosum AAR was found to be active in S. cerevisiae and able to use both NADH and NADPH as cofactors. This resulted in improved PHB titers in S. cerevisiae when xylose was used as sole carbon source (5-fold in aerobic conditions and 8.4-fold under oxygen limited conditions) and PHB yields (4-fold in aerobic conditions and up to 5.6-fold under oxygen limited conditions). Moreover, the best strain was able to accumulate up to 14% of PHB per cell dry weight under fully anaerobic conditions. CONCLUSIONS: This study reports a novel approach for boosting PHB accumulation in S. cerevisiae by replacement of the commonly used AAR from C. necator with the NADH-dependent alternative from A. vinosum. Additionally, to the best of our knowledge, it is the first demonstration of anaerobic PHB synthesis from xylose.

Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply.

BACKGROUND: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. RESULTS: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q3HB = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L(-1), which was 50% higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L(-1) and a productivity of 0.42 g L(-1) h(-1), which is comparable to maximum values found in recombinant E. coli. CONCLUSIONS: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

Expression of His-tagged NADPH-dependent acetoacetyl-CoA reductase in recombinant Escherichia coli BL-21(DE3).

Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E. coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4 h, induction time of 12 h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 U mg-1 protein and total protein concentration of 0.518 mg mg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 U mg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.

Integrated analysis of Whole genome sequencing and life cycle assessment for polyhydroxyalkanoates production by Cupriavidus sp. ISTL7.

The current study demonstrates the enhanced production capability of strain Cupriavidus sp. ISTL7 for polyhydroxyalkanoates (PHA) using acetate and glucose (4.93 ± 0.4571 g L-1) which was characterised analytically by GC-MS, FTIR and NMR analysis. Whole genome sequencing of strain ISTL7 unveiled an array of PHA metabolism genes which included phaA, phaB and phaC. Life cycle assessment of the protocol established that the production was most sustainable with the carbon source acetate. + Glucose as compared to acetate/glucose alone. It also concluded that solvent extraction of PHA and energy consumption during the process requires optimization to sustain the production on ecological fronts. Additionally, acetoacetyl-CoA reductase (phaB) gene was molecularly cloned, expressed and purified (27 KDa, 2.63 mg/ml). Conclusively, Cupriavidus sp. ISTL7 is a potential strain for PHA production with a scope of improvement on energy fronts which would transform the production environmentally and economically appealing.

An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli.

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.

Crystal structure of acetoacetyl-CoA reductase from Rickettsia felis.

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

Production of Poly-3-Hydroxybutyrate (P3HB) with Ultra-High Molecular Weight (UHMW) by Mutant Strains of Azotobacter vinelandii Under Microaerophilic Conditions.

Poly-3-hydroxybutyrate (P3HB) is a biopolymer, which presents characteristics similar to those of plastics derived from the petrochemical industry. The thermomechanical properties and biodegradability of P3HB are influenced by its molecular weight (MW). The aim of the present study was to evaluate the changes of the molecular weight of P3HB as a function of oxygen transfer rate (OTR) in the cultures using two strains of Azotobacter vinelandii, a wild-type strain OP, and PhbZ1 mutant with a P3HB depolymerase inactivated. Both strains were grown in a bioreactor under different OTR conditions. An inverse relationship was found between the average molecular weight of P3HB and the OTRmax, obtaining a polymer with a maximal MW (8000-10,000 kDa) from the cultures developed at OTRmax of 5 mmol L-1 h-1 using both strains, with respect to the cultures conducted at 8 and 11 mmol L-1 h-1, which produced a P3HB between 4000 and 5000 kDa. The increase in MW of P3HB was related to the activity of enzymes involved in the synthesis and depolymerization. Overall, our results show that it is possible to modulate the average molecular weight of P3HB by manipulating oxygen transfer conditions with both strains (OP and PhbZ1 mutant) of A. vinelandii.

NADH-driven poly-3-hydroxybutyrate accumulation in Escherichia coli: Data from enzymatic assays and oxygen-limited continuous cultures.

Biosynthesis of poly-3-hydroxybutyrate (PHB) as a fermentation product enables the coupling of growth and product generation. Moreover, the reduction of oxygen supply should reduce operative cost and increase product yield. Generation of PHB as a fermentation product depends on the in vivo activity of an NADH-preferring acetoacetyl-CoA reductase. Proof of this concept requires (i) quantification of the cofactor preference, in physiologically relevant conditions, of a putative NADH-preferring acetoacetyl-CoA reductase and (ii) verification of PHB accumulation using an NADH-preferring acetoacetyl-CoA reductase in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains kinetic data obtained by spectrophotometry and data from a continuous culture of an engineered E. coli strain accumulating PHB under oxygen-limiting conditions. In this dataset it is possible to find (1) enzyme stability assays; (2) initial rates and progress curves from reactions catalyzed by two acetoacetyl-CoA reductases; (3) estimations of the relative use of NADH and NADPH by two acetoacetyl-CoA reductases; (4) estimations of the flux capacity of the reaction catalyzed by an acetoacetyl-CoA reductase; (5) biomass composition of an engineered E. coli strain transformed with a plasmid; (6) calculation of reconciled specific rates of this engineered strain growing on sucrose as the sole carbon source under oxygen limitation and (7) metabolic fluxes distributions during the continuous growth of this engineered strain. Because a relatively small number of acetoacetyl-CoA reductases have been kinetically characterized, data and scripts here provided could be useful for further kinetic characterizations. Moreover, the procedure described to estimate biomass composition could be interesting to estimate plasmid and protein burden in other strains. Application of data reconciliation to fermentations should help to obtain specific rates consistent with the principle of mass and electron conservation. All the required data and scripts to perform these analyses are deposited in a Mendeley Data repository. This article was co-submitted with the manuscript entitled "An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichiasia. coli".

Two NADH-dependent (S)-3-hydroxyacyl-CoA dehydrogenases from polyhydroxyalkanoate-producing Ralstonia eutropha.

Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C4 to C8. The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse ß-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.

Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans.

(R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, ß-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L-1 (R)-3-HB, at a rate of 0.023 g L-1 h-1 over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L-1 of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L-1 h-1 which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.

FabG can function as PhaB for poly-3-hydroxybutyrate biosynthesis in photosynthetic cyanobacteria Synechocystis sp. PCC 6803.

The production of poly-3-hydroxybutyrate (PHB) by photosynthetic cyanobacteria is a potentially sustainable production method for the biodegradable plastics industry. ß-Ketoacyl-ACP reductase (FabG), from the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (SpFabG), is the first NADPH-dependent reductase in the fatty acid biosynthesis pathway. Its structure is similar to that of acetoacetyl-CoA reductase (SpPhaB), which is critical for PHB synthesis and can replace SpPhaB for acetoacetyl-CoA reduction in vitro. However, the specific function of SpFabG in fatty acid synthesis and whether SpFabG could participate in PHB synthesis in vivo were not yet clear. In this study, the role of SpFabG in fatty acid synthesis was first verified in vivo by knocking down and overexpressing of fabG. It was shown that SpFabG was essential yet not rate-limiting for fatty acid biosynthesis. The biochemical characterization of SpFabG using acetoacetyl-CoA as the substrate showed that the optimum temperature, optimum pH, Km and kcat were 30°C, 7, 2.30 mM, and 19.85 s-1, respectively, which exemplified the ability of SpFabG to reduce acetoacetyl-CoA with a relatively low affinity and weak catalytic efficiency. Functional analysis of SpFabG in vivo indicated that SpFabG was able to partially complement SpPhaB under nitrogen-deprived conditions, and overexpression of fabG led to the diversion of partial carbon flux from fatty acid toward PHB synthesis.

Generation of poly-ß-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

It has been reported that Poly-ß-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: ß-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

Structure-directed construction of a high-performance version of the enzyme FabG from the photosynthetic microorganism Synechocystis sp. PCC 6803.

PhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrier-protein reductase) catalyzes the ß-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a high-performance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA.

Production of (S)-3-hydroxybutyrate by metabolically engineered Saccharomyces cerevisiae.

(S)-3-Hydroxybutyrate (S-3HB) can be used as a precursor for the synthesis of biodegradable polymers such as polyhydroxyalkanoate and stereo-specific fine chemicals such as antibiotics, pheromones, and drugs. For the production of S-3HB in yeast, the biosynthetic pathway of S-3HB from acetyl-CoA, consisting of the three enzymes, acetyl-CoA C-acetyltransferase (ACCT), acetoacetyl-CoA reductase (ACR), and 3-hydroxybutyryl-CoA thioesterase (HBT), was introduced into Saccharomyces cerevisiae. An engineered yeast strain overexpressing ERG10, hbd, and tesB genes not only exhibited enzyme activities of AACT, ACR, and HBT, but also produced S-3HB from ethanol. In order to increase the titer of S-3HB, a fed-batch fermentation based on pulse feeding of ethanol as a carbon source was performed, and a final S-3HB titer of 12.0g/L was achieved. This is the first report on the production of 3HB by engineered yeast, utilizing ethanol as the carbon source, suggesting that the industrially preferred S. cerevisiae can be a promising host for producing S-3HB.