Nangle, S. N. | bioRxiv Science

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↗

bioRxiv2020

Valorization of CO2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H2 and CO2 and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO2 under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20-60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO2. And, we engineered C. necator to convert CO2 into the LCO, a plant growth enhancer, with titers of [~]1.4 mg/L--equivalent to yields in its native source, Bradyrhizobium. We applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding f how a gas-utilizing bacteria can promote sustainable production.

synthetic biology↗

bioRxiv2019

Synthetic gene circuits enable systems-level biosensor discovery at the host-microbe interface

The composition and function of the gut microbiota are strongly associated with human health, and dysbiosis is linked to an array of diseases, ranging from obesity and diabetes to infection and inflammation. Engineering synthetic circuits into gut bacteria to sense, record and respond to in vivo signals is a promising new approach for the diagnosis, treatment and prevention of disease. Here, we repurpose a synthetic bacterial memory circuit to rapidly screen for and discover new in vivo-responsive biosensors in commensal gut Escherichia coli. We develop a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably robust identification of responsive biosensors from pooled libraries. By testing both genome-wide and curated libraries of potential biosensor triggers--each consisting of a promoter and ribosome binding site (RBS)--and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensors with increased response in the inflamed gut through comparative screening of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensors in the gut and provide a platform for disease-specific screening using synthetic circuits, capable of contributing to both the understanding and clinical management of intestinal illness. IMPORTANCEThe gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the design and construction of effective strains that respond to the in vivo environment is hindered by our limited understanding of bacterial behavior in the gut. Our work expands the pool of potential biosensors for the healthy and diseased gut, providing insight into host-microbe interactions and enabling future development of increasingly complex synthetic circuits. This method also provides a framework for rapid prototyping of engineered systems and for application across bacterial strains and disease models.

synthetic biology↗