Enhancement of essential cofactors for in vivo biocatalysis.

A scarcity of cofactors, necessary metabolites or substrates for in vivo enzymatic reactions, is among the major barriers for product synthesis in metabolically engineered cells. This work compares our recently developed cofactor-boosting strategy, which uses xylose reductase (XR) and lactose to increase the intracellular levels of reduced or oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), adenosine triphosphate (ATP) and acetyl coenzymeA (acetyl-CoA), with other previously reported methods. We demonstrated that the XR/lactose approach enhances levels of sugar alcohols and sugar phosphates, which leads to elevated levels of crucial cofactors required by specific metabolic pathways. The patterns of cofactor enhancement are not uniform and depend upon the specific pathway components that are overexpressed. We term this model the "user-pool" model. Here, we investigated metabolite alteration in the fatty-alcohol-producing system in the presence of XR/lactose within an early time frame (5 min after the bioconversion started). All metabolite data were analyzed using untargeted metabolomics. We found that the XR/lactose system could improve fatty-alcohol production as early as 5 min after the bioconversion started. The enhancement of key cofactors and intermediates, such as hexitol, NAD(P)H, ATP, 3-phosphoglycerate, acetyl-CoA, 6-phosphogluconate (6-PG) and glutathione, was consistent with those previously reported on a longer time scale (after 1 h). However, measurements performed at the early time reported here showed detectable differences in metabolite enhancement patterns, such as those of ATP, NADPH, acetyl-CoA and glutathione. These data could serve as a basis for future analysis of metabolic flux alteration by the XR/lactose system. Comparative analysis of the cofactor enhancement by XR and other methods suggests that XR/lactose can serve as a simple tool to increase levels of various cofactors for microbial cell factories.

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

Unusual aldehyde reductase activity for the production of full-length fatty alcohol by cyanobacterial aldehyde deformylating oxygenase.

Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes the deformylation of aldehydes to generate alkanes/alkenes. In this study, we report for the first time that under anaerobic or limited oxygen conditions, Prochlorococcus marinus (PmADO) can generate full-length fatty alcohols from fatty aldehydes without eliminating a carbon unit. In contrast to ADO's native activity, which requires electrons from the Fd/FNR electron transfer complex, ADO's aldehyde reduction activity requires only NAD(P)H. Our results demonstrated that the yield of alcohol products could be affected by oxygen concentration and the type of aldehyde. Under strictly anaerobic conditions, yields of octanol were up to 31%. Moreover, metal cofactors are not involved in the aldehyde reductase activity of PmADO because the yields of alcohols obtained from apoenzyme and holoenzyme treated with various metals were similar under anaerobic conditions. In addition, PmADO prefers medium-chain aldehydes, specifically octanal (kcat/Km around 15 × 10-3 µM-1min-1). The findings herein highlight a new activity of PmADO, which may be applied as a biocatalyst for the industrial synthesis of fatty alcohols.

Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability.

Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO2 and CH4) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

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.

Addition of formate dehydrogenase increases the production of renewable alkane from an engineered metabolic pathway.

An engineered metabolic pathway consisting of reactions that convert fatty acids to aldehydes and eventually alkanes would provide a means to produce biofuels from renewable energy sources. The enzyme aldehyde-deformylating oxygenase (ADO) catalyzes the conversion of aldehydes and oxygen to alkanes and formic acid and uses oxygen and a cellular reductant such as ferredoxin (Fd) as co-substrates. In this report, we aimed to increase ADO-mediated alkane production by converting an unused by-product, formate, to a reductant that can be used by ADO. We achieved this by including the gene (fdh), encoding formate dehydrogenase from Xanthobacter sp. 91 (XaFDH), into a metabolic pathway expressed in Escherichia coli Using this approach, we could increase bacterial alkane production, resulting in a conversion yield of ∼50%, the highest yield reported to date. Measuring intracellular nicotinamide concentrations, we found that E. coli cells harboring XaFDH have a significantly higher concentration of NADH and a higher NADH/NAD+ ratio than E. coli cells lacking XaFDH. In vitro analysis disclosed that ferredoxin (flavodoxin):NADP+ oxidoreductase could use NADH to reduce Fd and thus facilitate ADO-mediated alkane production. As formic acid can decrease the cellular pH, the addition of formate dehydrogenase could also maintain the cellular pH in the neutral range, which is more suitable for alkane production. We conclude that this simple, dual-pronged approach of increasing NAD(P)H and removing extra formic acid is efficient for increasing the production of renewable alkanes via synthetic biology-based approaches.