Microbial cell factories offer an attractive approach for production of biobased products.Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 ± 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

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Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse β-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL−1), as well as hexanoic acid (3.06 ± 0.03 gL−1) and 1-hexanol (1.0 ± 0.1 gL−1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL−1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.

An efficient taurine-catalyzed green multicomponent approach has been described for the first time to synthesize densely substituted therapeutic core dihydropyrano­[2,3-c]­pyrazoles. Applications of the developed synthetic strategies and technologies revealed the synthesis of a series of newly designed 1,4-dihydropyrano­[2,3-c]­pyrazoles containing isonicotinamide, spirooxindole, and indole moieties. Detailed in silico analysis of the synthesized analogues revealed their potential to bind wild-type and antibiotic-resistant variants of dihydrofolate reductase, a principal drug target enzyme for emerging antibiotic-resistant pathogenic Staphylococcus aureus strains. Hence, the synthesized dihydropyrano­[2,3-c]­pyrazole derivatives presented herein hold immense promise to develop future antistaphylococcal therapeutic agents.

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy-to-use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desired the metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

Convergence of market drivers such as abundant availability of inexpensive natural gas and increasing awareness of its global warming effects have created new opportunities for the development of small-scale gas-to-liquid (GTL) conversion technologies that can efficiently utilize methane, the primary component of natural gas. Leveraging the unique ability of methanotrophs that use methane as carbon and energy source, biological GTL platforms can be envisioned that are readily deployable at remote petroleum drilling sites where large chemical GTL infrastructure is uneconomical to set-up. Methylomicrobium buryatense, an obligate methanotroph, has gained traction as a potential industrial methanotrophic host because of availability of genetic tools and recent advances in its metabolic engineering. However, progress is impeded by low strain performance and lack of an industrial medium. In this study, we first established a small-scale cultivation platform using Hungate tubes for growth of M. buryatense at medium-to-high-throughput that also enabled 2X faster growth compared to that obtained in traditional glass serum bottles. Then, employing a synthetic biology approach we engineered M. buryatense with varying promoter (inducible and constitutive) and ribosome-binding site combinations, and obtained a strain capable of producing L-lactate from methane at a flux 14-fold higher than previously reported. Finally, we demonstrated L-lactate production in an industrial medium by replacing nitrate with less-expensive ammonium as the nitrogen source. Under these conditions, L-lactate was synthesized at a flux approximately 50-fold higher than that reported previously in a bioreactor system while achieving a titer of 0.6 g/L. These findings position M. buryatense closer to becoming an industrial host strain of choice, and pave new avenues for accelerating methane-to-chemical conversion using synthetic biology.

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