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In situ extraction offers an effective way to recover products during microbial production of fatty alcohols, which can be operated in multi-cycle mode. Workup steps may be required to increase the driving force for the extraction or to minimize by-product accumulation. Different workup steps, including intermittent glucose addition, dodecane change, and buffer change, were investigated in this study. We demonstrated that reconfiguration of these workup steps (e.g. rare intermittent workup steps) between bioconversion cycles led to significantly improved productivity and sustainability. The optimized reconfiguration enhanced the yield (0.071 g fatty alcohol per g glucose) while reducing the E-factor to 34.98, which was 120-fold lower than that of the control condition without in situ extraction. The results from this work shed light on the sustainable process development of the fatty alcohol production.

Immobilization methods have emerged as feasible solutions for increasing the reusability of biocatalysts and for simplifying their separation from the desired products. Immobilized biocatalysts can directly be applied to a continuous-flow system, such as tubular or packed bed reactors. Such continuous-flow systems with immobilized biocatalysts are promising tools for enhancing the sustainability of bioprocessing. This minireview discusses important considerations for successfully developing biocatalyst-based continuous-flow systems, namely immobilization techniques, mass transfer and reactor design. The key sustainability aspects of these systems are highlighted along with recent examples.

Fatty alcohols are widely used for various applications and are found in products ranging from detergents to cosmetics and cleaning agents Currently, over 3 million tonnes of fatty alcohols are produced per year from bio-based and petroleum-based feedstocks through chemical conversion and synthetic processes However, the environmental impact of current fatty alcohol production processes has become a major concern, and several bioprocesses have emerged as alternative methods for synthesis These biological processes are environmentally friendly due to their metal-free nature, low consumption of energy, and mild reaction conditions This review overviews the current status of fatty alcohol production ? both chemical and biological approaches Our focus is on biological synthesis approaches using biocatalysis and metabolic engineering due to their proven efficacy Potential downstream processing for these bioprocesses is also presented ? 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

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 (k

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.

Background: Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes 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. Results: Unlike the native activity of ADO which requires electrons from the Fd/FNR electron transfer complex, the aldehyde reduction activity of ADO requires only NADPH. Our results demonstrated that yield of alcohol products can be affected by oxygen concentration and type of aldehyde. Under O2-scant conditions (10-15%), yields of octanol and dodecanol were around 40-60% and could be increased up to 80% under strict anaerobic conditions (>0.0004%). Unexpectedly, Fe2+ cofactor is not involved in the aldehyde reductase activity of PmADO because yields of alcohols obtained from holo- and apo-enzymes were similar under anaerobic conditions. The direct hydride transfer activity of PmADO is highly specific to substrates; NADPH not NADH can be used as a reductant to reduce medium-chain fatty aldehydes (C6-C10) with decanal as the most preferred substrate (the highest kcat/Km value with 98% bioconversion yield). Molecular dynamics (MD) simulations was used to identify a binding site of NADPH which is located close to the aldehyde binding site. In the metabolic engineered cells containing PmADO, dual activities of alkane and alcohol production could be detected. Conclusion: The findings reported herein highlight a new activity of PmADO which may be applied as a biocatalyst for industrial synthesis of fatty alcohols in the future.

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, β-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces β-FAD to β-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of β-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from β-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.