Synthase Gene phaC Research Articles

Engineering Pseudomonas chlororaphis HT66 for the Biosynthesis of Copolymers Containing 3-Hydroxybutyrate and Medium-Chain-Length 3-Hydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are promising alternatives to petroleum-based plastics, owing to their biodegradability and superior material properties. Here, the controllable biosynthesis of scl-co-mcl PHA containing 3-hydroxybutyrate (3HB) and mcl 3-hydroxyalkanoates was achieved in Pseudomonas chlororaphis HT66. First, key genes involved in fatty acid β-oxidation, the de novo fatty acid biosynthesis pathway, and the phaC1-phaZ-phaC2 operon were deleted to develop a chassis strain. Subsequently, an acetoacetyl-CoA reductase gene phaB and a PHA synthase gene phaC with broad substrate specificity were heterologously expressed for producing and polymerizing the 3HB monomer with mcl 3-hydroxyalkanoates under the assistance of native β-ketothiolase gene phaA. Furthermore, the monomer composition of scl-co-mcl PHA was regulated by adjusting the amount of glucose and dodecanoic acid supplemented. Notably, the cell dry weight and scl-co-mcl PHA content reached 14.2 g/L and 60.1 wt %, respectively, when the engineered strain HT11Δ::phaCB was cultured in King's B medium containing 5 g/L glucose and 5 g/L dodecanoic acid. These results demonstrated that P. chlororaphis can be a platform for producing scl-co-mcl PHA and has the potential for industrial application.

Inhibition of Rumen Methanogens by a Novel Archaeal Lytic Enzyme Displayed on Tailored Bionanoparticles.

Methane is a potent greenhouse gas, 25 times more efficient at trapping heat than carbon dioxide. Ruminant methane emissions contribute almost 30% to anthropogenic sources of global atmospheric methane levels and a reduction in methane emissions would significantly contribute to slowing global temperature rises. Here we demonstrate the use of a lytic enyzme, PeiR, from a methanogen virus that infects Methanobrevibacter ruminantium M1 as an effective agent inhibiting a range of rumen methanogen strains in pure culture. We determined the substrate specificity of soluble PeiR and demonstrated that the enzyme is capable of hydrolysing the pseudomurein cell walls of methanogens. Subsequently, peiR was fused to the polyhydroxyalkanoate (PHA) synthase gene phaC and displayed on the surface of PHA bionanoparticles (BNPs) expressed in Eschericia coli via one-step biosynthesis. These tailored BNPs were capable of lysing not only the original methanogen host strain, but a wide range of other rumen methanogen strains in vitro. Methane production was reduced by up to 97% for 5 days post-inoculation in the in vitro assay. We propose that tailored BNPs carrying anti-methanogen enzymes represent a new class of methane inhibitors. Tailored BNPs can be rapidly developed and may be able to modulate the methanogen community in vivo with the aim to lower ruminant methane emissions without impacting animal productivity.

Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.

Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions

Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co²⁺-deficient conditions accumulated copolyesters of 3HB and a C₅-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co²⁺ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1C(Ac) in which the original PHA synthase gene phaC(Me) had been replaced by phaC(Ac), encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C₆-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1C(Ac) were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C(Ac) showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding β-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.

Escherichia coli NemA Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (kcat/KM = 1.1×105 M−1s−1 with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation.

Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity

The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.

Metabolic engineering for microbial production of polyhydroxyalkanoates consisting of high 3-hydroxyhexanoate content by recombinant Aeromonas hydrophila

Polyhydroxyalkanoate synthase gene phaCah in Aeromonas hydrophila strain 4AK4 was deleted and its function was replaced by phaC1ps cloned from Pseudomonas stutzeri strain 1317 which favors 3-hydroxyhexanoate (3HHx) and longer chain length monomers. Genes fadD and fadL encoding Escherichia coli acyl-CoA synthase and Pseudomonas putida KT2442 fatty acid transport protein, respectively, were introduced into the recombinant with new phaC1ps. Accumulation of a series of novel medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 80–94mol% 3HHx were observed. The recombinant accumulated 54% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in cell dry weight consisting of 94.5mol% 3HHx or 51% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) consisting of 82mol% 3HHx and 16mol% of 3HO during a two-step cultivation process under nitrogen limitation when grown on sodium hexanoate or sodium octanoate. The two polyesters containing high percentage of 3HHx are physically characterized. They could be used as biodegradable pressure sensitive adhesives, coatings, polymer binding agents in organic-solvent-free paints or a source for chiral R-3-hydroxyhexanoate.