Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs. Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE. Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS, involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis. In this work, we have devised a useful platform for future engineering steps.

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.

Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH3). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date.Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.

The need for sustainable, low-cost production of bioenergy and commodity chemicals is increasing. Unfortunately, the engineering potential of whole-cell catalysts to address this need can be hampered by cellular toxicity. When such bottlenecks limit the commercial feasibility of whole-cell fermentation, cell-free, or in vitro, based approaches may offer an alternative. Here, we assess the impact of three classes of growth toxic compounds on crude extract-based, cell-free chemical conversions. As a model system, we test a metabolic pathway for conversion of glucose to 2,3-butanediol (2,3-BDO) in lysates of Escherichia coli. First, we characterized 2,3-BDO production with different classes of antibiotics and found, as expected, that the system is uninhibited by compounds that prevent cell growth by means of cell wall replication and DNA, RNA, and protein synthesis. Second, we considered the impact of polar solvent addition (e.g., methanol, n-butanol). We observed that volumetric productivities (g/L/h) were slowed with increasing hydrophobicity of added alcohols. Finally, we investigated the effects of using pretreated biomass hydrolysate as a feed stock, observing a 25% reduction in 2,3-BDO production as a result of coumaroyl and feruloyl amides. Overall, we find the cell-free system to be robust to working concentrations of antibiotics and other compounds that are toxic to cell growth, but do not denature or inhibit relevant enzymes.