Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity

Synthetic materials are integral components of consumables and durable goods and indispensable in our modern world. Polyesters are the most versatile bulk- and specialty-polymers, but their production is not sustainable, and their fate at end-of-life of great concern. Bioplastics are highly regarded alternatives but have shortcomings in material properties and commercial competitiveness with conventional synthetic plastics. These constraints have limited the success in global markets. Enabling bio-production of advanced bioplastics with superior properties from waste-derived feedstocks could change this. We have created microbial cell factories that can produce a range of aliphatic and aromatic polyesters. A DphaC1 mutant of Cupriavidus necator H16 was complemented with hydroxyacyl-CoA transferases from either Clostridium propionicum (pct540) or Clostridium difficile (hadA), respectively. These were combined with a mutant PHA synthase (phaC1437) from Pseudomonas sp. MBEL 6 19, which rescued the PHA- phenotype of the knock-out mutant and allowed polymerization of various hydroxy carboxylates, including phloretic acid. This is the first-time, incorporation of an aromatic ring in the backbone of a biological polyester was achieved. Polymers contain para-hydroxyphenyl subunits are structurally analogous to synthetic aromatic polyesters like PET and high-strength polyarylates. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters from H2 and O2 generated in situ, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility of de novo production of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of high-performance thermoplastics and thermosets.

Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources. A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes. Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources. A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes. Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

Artificial polyhydroxyalkanoate poly[2-hydroxybutyrate-block-3-hydroxybutyrate] elastomer-like material

The first polyhydroxyalkanoate (PHA) block copolymer poly(2-hydroxybutyrate-b-3-hydroxybutyrate) [P(2HB-b-3HB)] was previously synthesized using engineered Escherichia coli expressing a chimeric PHA synthase PhaCAR with monomer sequence-regulating capacity. In the present study, the physical properties of the block copolymer and its relevant random copolymer P(2HB-ran-3HB) were evaluated. Stress–strain tests on the P(88 mol% 2HB-b-3HB) film showed an increasing stress value during elongation up to 393%. In addition, the block copolymer film exhibited slow contraction behavior after elongation, indicating that P(2HB-b-3HB) is an elastomer-like material. In contrast, the P(92 mol% 2HB-ran-3HB) film, which was stretched up to 692% with nearly constant stress, was stretchable but not elastic. The differential scanning calorimetry and wide-angle X-ray diffraction analyses indicated that the P(2HB-b-3HB) contained the amorphous P(2HB) phase and the crystalline P(3HB) phase, whereas P(2HB-ran-3HB) was wholly amorphous. Therefore, the elasticity of P(2HB-b-3HB) can be attributed to the presence of the crystalline P(3HB) phase and a noncovalent crosslinked structure by the crystals. These results show the potential of block PHAs as elastic materials.

Biotechnological Conversion of Grape Pomace to Poly(3-Hydroxybutyrate) by Moderately Thermophilic Bacterium Tepidimonas taiwanensis

Polyhydroxyalkanoates (PHA) are microbial polyesters that have recently come to the forefront of interest due to their biodegradability and production from renewable sources. A potential increase in competitiveness of PHA production process comes with a combination of the use of thermophilic bacteria with the mutual use of waste substrates. In this work, the thermophilic bacterium Tepidimonas taiwanensis LMG 22826 was identified as a promising PHA producer. The ability to produce PHA in T. taiwanensis was studied both on genotype and phenotype levels. The gene encoding the Class I PHA synthase, a crucial enzyme in PHA synthesis, was detected both by genome database search and by PCR. The microbial culture of T. taiwanensis was capable of efficient utilization of glucose and fructose. When cultivated on glucose as the only carbon source at 50°C, the PHA titers reached up to 3.55 g/L, and PHA content in cell dry mass was 65%. The preference of fructose and glucose opens the possibility to employ T. taiwanensis for PHA production on various food wastes rich in these abundant sugars. In this work, PHA production on grape pomace extracts was successfully tested.

Direct carbon capture for production of high-performance biodegradable plastic by cyanobacterial cell factory

Plastic pollution has become one of the most pressing environmental issues today, leading to an urgent need to develop biodegradable plastics1-3. Polylactic acid (PLA) is one of the most promising biodegradable materials because of its potential applications in disposable packaging, agriculture, medicine, and printing filaments for 3D printers4-6. However, current biosynthesis of PLA entirely uses edible biomass as feedstock, which leads to competition for resources between material production and food supply7,8. Meanwhile, excessive emission of CO2 that is the most abundant carbon source aggravates global warming, and climate instability. Herein, we first developed a cyanobacterial cell factory for the de novo biosynthesis of PLA directly from CO2, using a combinational strategy of metabolic engineering and high-density cultivation (HDC). Firstly, the heterologous pathway for PLA production, which involves engineered D-lactic dehydrogenase (LDH), propionate CoA-transferase (PCT), and polyhydroxyalkanoate (PHA) synthase, was introduced into Synechococcus elongatus PCC7942. Subsequently, different metabolic engineering strategies, including pathway debottlenecking, acetyl-CoA self-circulation, and carbon-flux redirection, were systematically applied, resulting in approximately 19-fold increase to 15 mg/g dry cell weight (DCW) PLA compared to the control. In addition, HDC increased cell density by 10-fold. Finally, the PLA titer of 108 mg/L (corresponding to 23 mg/g DCW) was obtained, approximately 270 times higher than that obtained from the initially constructed strain. Moreover, molecular weight (Mw, 62.5 kDa; Mn, 32.8 kDa) of PLA produced by this strategy was among the highest reported levels. This study sheds a bright light on the prospects of plastic production from CO2 using cyanobacterial cell factories.

Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov., Two Acidophilic Bacteria Isolated from Decaying Wood, Hydrolyzing Cellulose and Producing Poly-3-hydroxybutyrate

Two novel strains, HW T2.11T and HW T5.17T, were isolated from decaying wood (forest of Champenoux, France). Study of the 16S rRNA sequence similarity indicated that the novel strains belong to the genus Acidisoma. The sequence similarity of the 16S rRNA gene of HW T2.11T with the corresponding sequences of A. tundrae and A. sibiricum was 97.30% and 97.25%, while for HW T5.17T it was 96.85% and 97.14%, respectively. The DNA G+C contents of the strains were 62.32–62.50%. Cells were Gram-negative coccobacilli that had intracellular storage granules (poly-3-hydroxybutyrate (P3HB)) that confer resistance to environmental stress conditions. They were mesophilic and acidophilic organisms growing at 8–25 °C, at a pH of 2.0–6.5, and were capable of using a wide range of organic compounds and complex biopolymers such as starch, fucoidan, laminarin, pectin and cellulose, the latter two being involved in wood composition. The major cellular fatty acid was cyclo C19:0ω8c and the major quinone was Q-10. Overall, genome relatedness indices between genomes of strains HW T2.11T and HW T5.17T (Orthologous Average Nucleotide Identity (OrthoANI) value = 83.73% and digital DNA-DNA hybridization score = 27.5%) confirmed that they belonged to two different species. Genetic predictions indicate that the cyclopropane fatty acid (CFA) pathway is present, conferring acid-resistance properties to the cells. The two novel strains might possess a class IV polyhydroxyalcanoate (PHA) synthase operon involved in the P3HB production pathway. Overall, the polyphasic taxonomic analysis shows that these two novel strains are adapted to harsh environments such as decaying wood where the organic matter is difficult to access, and can contribute to the degradation of dead wood. These strains represent novel species of the genus Acidisoma, for which the names Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov. are proposed. The type strains of Acidisoma silvae and Acidisomacellulosilytica are, respectively, HW T2.11T (DSM 111006T; UBOCC-M-3364T) and HW T5.17T (DSM 111007T; UBOCC-M-3365T).

Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using High-Performance Liquid Chromatography

A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The cultured cells containing benzyl alcohol-capped PHA were decomposed by alkaline treatment, and the peaks of the decomposed monomer and benzyl alcohol were detected using HPLC. The DP of PHA could be determined from the peak ratio of the decomposed monomer to terminal benzyl alcohol. The measured DP was validated by other instrumental analyses using purified PHA samples. Using this system, mutants of PHA synthase from Bacillus cereus YB-4 (PhaRCYB4) were screened, and some enzymes capable of producing PHA with higher DP than the wild-type enzyme were obtained. The PHA yields of two of these enzymes were equivalent to the yield of the wild-type enzyme. Therefore, this screening method is suitable for the selection of beneficial mutants that can produce high molecular weight PHAs.

Artificial polyhydroxyalkanoate poly[2-hydroxybutyrate-block-3-hydroxybutyrate] elastomer-like material

The first polyhydroxyalkanoate (PHA) block copolymer poly(2-hydroxybutyrate-b-3-hydroxybutyrate) [P(2HB-b-3HB)] was previously synthesized using engineered Escherichia coli expressing a chimeric PHA synthase PhaCAR with monomer sequence-regulating capacity. In the present study, the physical properties of the block copolymer and its relevant random copolymer P(2HB-ran-3HB) were evaluated. Stress–strain tests on the P(88 mol% 2HB-b-3HB) film showed an increasing stress value during elongation up to 393%. In addition, the block copolymer film exhibited slow contraction behavior after elongation, indicating that P(2HB-b-3HB) is an elastomer-like material. In contrast, the P(92 mol% 2HB-ran-3HB) film, which was stretched up to 692% with nearly constant stress, was stretchable but not elastic. The differential scanning calorimetry and wide-angle X-ray diffraction analyses indicated that the P(2HB-b-3HB) contained the amorphous P(2HB) phase and the crystalline P(3HB) phase, whereas P(2HB-ran-3HB) was wholly amorphous. Therefore, the elasticity of P(2HB-b-3HB) can be attributed to the presence of the crystalline P(3HB) phase. These results show the potential of block PHAs as elastic materials.