A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Visualizing the complexity of proteins in living cells with genetic code expansion.

Genetic code expansion has emerged as an enabling tool to provide insight into functions of understudied proteinogenic species, such as small proteins and peptides, and to probe protein biophysics in the cellular context. Here, we discuss recent technical advances and applications of genetic code expansion in cellular imaging of complex mammalian protein species, along with considerations and challenges on using the method.

Carboxylic Acid Reductase Can Catalyze Ester Synthesis in Aqueous Environments.

Most of the well-known enzymes catalyzing esterification require the minimization of water or activated substrates for activity. This work reports a new reaction catalyzed by carboxylic acid reductase (CAR), an enzyme known to transform a broad spectrum of carboxylic acids into aldehydes, with the use of ATP, Mg2+ , and NADPH as co-substrates. When NADPH was replaced by a nucleophilic alcohol, CAR from Mycobacterium marinum can catalyze esterification under aqueous conditions at room temperature. Addition of imidazole, especially at pH 10.0, significantly enhanced ester production. In comparison to other esterification enzymes such as acyltransferase and lipase, CAR gave higher esterification yields in direct esterification under aqueous conditions. The scalability of CAR catalyzed esterification was demonstrated for the synthesis of cinoxate, an active ingredient in sunscreen. The CAR esterification offers a new method for green esterification under high water content conditions.

Enzymatic reactions and pathway engineering for the production of renewable hydrocarbons.

Hydrocarbons such as alkanes and alkenes are extensively used as organic compounds for combustion reactions and as building block components for the synthesis of numerous materials. Various synthetic enzymatic cascades and engineered metabolic pathways can be used to produce alkanes and alkenes from bio-based materials. An understanding of the native reactions and pathways used by various organisms to synthesize these compounds together with novel approaches in biocatalysis and synthetic biology have been instrumental in the development of methods to produce alkanes and alkenes with reasonable yield. This article discusses the present state of knowledge regarding hydrocarbon biosynthetic pathways and discusses current mechanistic understanding of relevant enzymatic reactions in cyanobacteria, aerobic bacteria, insects, algae, and plants. Recent advancements in metabolic engineering and process scale up for production of hydrocarbons from fatty acids are also discussed. This technology is important for sustainability, as it provides a clean and eco-friendly method for the future production of fuels and industrial materials. Further development towards whole cell biocatalysts that are able to provide good yield with a low production cost may allow countries without big oil reserves to be capable of producing precursors for the materials industries in the future.