Criddle, C. S. | Search bioRxiv

bioRxiv2024

Isolation and Characterization of a Halomonas Species for Non-Axenic Growth-Associated Production of Bio-Polyesters from Sustainable Feedstocks

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01". Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strains exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon-feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further also glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to [~]60% of its biomass [dry w/w] in poly(3-hydroxbutyrate), while reaching a doubling time of 1.7 h at 30{whitebullet}C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but can also facilitate the release of intracellular products through osmolysis. Development of a minimal medium also allowed the estimation of maximum PHB production rates, which were projected to exceed 5 gPHB/h. Finally, also the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid-vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCEThe urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing. Especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

microbiology↗

bioRxiv2021

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 consumable and durable goods and are indispensable in the modern world. Polyesters are the most versatile bulk- and specialty-polymers but their production is not sustainable and their fate at end-of-life is of great concern. Bioplastics, though highly regarded, often lag behind conventional plastics due to poor material properties and a competitive market. This has limited the success of sustainable replacements at scale. Enabling the production of bioplastics with superior properties from waste-derived feedstocks could change that. To this end, we created a synthetic entry into the metabolic pathway of bio-polyester synthesis of Cupriavidus necator H16 employing heterologous hydroxyacyl-CoA transferase and mutant PHA synthase. The resulting microbial cell factories enabled the co-polymerization of a range of aliphatic and aromatic hydroxy carboxylates, including a hydroxyphenylic and a hydroxyfuranoic acid, for the first time incorporating aromatic rings in the backbone of biological polyesters. The latter were structurally analogous to synthetic polyesters like PET and PEF, as well as PBST and PBAT. The obtained bio-polyesters underwent characterization for specific physicochemical properties, complemented by the prediction of uncharacterized physicochemical and mechanical properties. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters with in situ generated H2+O2, while assimilating CO2. Employing elementary flux-mode analysis, metabolic modeling confirmed the feasibility of producing an extended range of aliphatic, arylatic, and aromatic PHAs de novo from various feedstocks. This comprehensive study paves the way toward sustainable bio-production of advanced high-performance thermoplastics and thermosets. Significance StatementBiomaterials can help the chemical industry transition to a carbon-neutral and circular economy, reducing the accumulation of greenhouse gases and plastic waste by developing biological replacements for fossil carbon-based plastics and implementing end-of-life strategies. Accomplished via the genetic engineering of a microbe that can assimilate carbon dioxide, this work demonstrates the first biocatalytic polymerization and incorporation of aromatic building blocks into the backbone of polyhydroxyalkanoates (PHAs). Employing a bio-electrochemical system for cultivation of the microbes, oxyhydrogen is formed and consumed in situ, thus avoiding explosive gas mixtures. The obtained aromatic PHAs are structurally similar and functionally analogs to synthetic bulk- and high-performance polymers such as PET (polyethylene terephthalate) and PEF (polyethylene furanoate) as well as PBST (polybutylene succinate-co-terephthalate) and PBAT (polybutylene adipate-co-terephthalate). Graphical AbstractThe biosynthesized novel aromatic polyhydroxyalkanoates described in this study are structurally similar to incumbent synthetic polyesters such as poly(ethylene terephthalate) (PET, orange box) and poly(ethylene furan-dicarboxylate) (PEF, yellow box). While PET and PEF may be bio-based to varying degrees, they are always synthesized chemically. In contrast, not only the monomers of poly(hydroxybutyrate-co-phloretate) and poly(hydroxybutyrate-co-hydroxymethylfuranoate) (green box) are fully bio-derived, but also the entire polymers are synthesized biologically. This was demonstrated by employing the autotrophic bacterium Cupriavidus necator H16. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/472320v3_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@20714borg.highwire.dtl.DTLVardef@1ee1c06org.highwire.dtl.DTLVardef@171cee7org.highwire.dtl.DTLVardef@c6bc21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst-ever biocatalytic formation of polyesters with aromatic rings in the backbone C_LIO_LIMicrobial production of aromatic PHAs in a bio-electrochemical system on CO2 and H2+O2 C_LIO_LIExpression-level of PHA synthase and molecular weight of polyesters are inversely correlated C_LIO_LIIn silico design and pathway analysis of bio-polyesters from low-cost carbon-feedstocks C_LIO_LIPrediction of material properties for novel aromatic co-polyesters and structural analogs C_LI

synthetic biology↗

bioRxiv2018

Ethane-dependent synthesis of polyhydroxyalkanoates by the obligate methanotroph Methylocystis parvus OBBP

Under conditions of nutrient-limited growth, Type II obligate methanotrophs oxidize C1 compounds, such as methane or methanol and accumulate intracellular granules of poly(3-hydroxybutyrate) (P3HB). Here, we report that, under same nutrient-limited conditions, the Type II obligate methanotroph Methylocystis parvus OBBP can use ethane as its sole carbon and energy source for synthesis P3HB granules, accumulating up to 35 {+/-} 4 wt% P3HB. 13C-NMR spectra of the P3HB confirmed incorporation of 13C from [13C2]ethane. Moreover, when valerate was added as a co-substrate with ethane, oxidation of the ethane supported synthesis of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).\n\nIMPORTANCEThe presence of ethane in natural gas is often considered undesirable for methanotroph-based biotechnology due to the C1 specialization of obligate methanotrophs and concerns about inhibitory byproducts arising from methane monooxygenase-mediated cometabolism of ethane. This work establishes that co-oxidation of ethane and further metabolism in the absence of methane can support synthesis of the valuable polyhydroxyalkanoate bioplastics P3HB and PHBV.

microbiology↗