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Background: Terpenes are remarkably diverse natural structures, which can be formed via two different pathways leading to two common intermediates. Among those, sesquiterpenes represent a variety of industrially relevant products. One important industrially produced product is β-farnesene as a precursor for a jet fuel additive. So far, microbial terpene production has been mostly limited to known production hosts, which are only able to grow on heterotrophic substrates. Results: In this paper, we for the first time describe β-farnesene production by the versatile bacterial host Cupriavidus necator on fructose, which is known to grow hetero- and autotrophically and even in bioelectrochemical systems. We were able to show a growth-dependent production of β-farnesene by expressing the β-farnesene synthase from Artemisia annua in C. necator H16 PHB-4. Additionally, we performed a scale-up in a parallel reactor system with production titers of 26.3 ± 1.3 µM β-farnesene with a fed-batch process. Conclusions: The β-farnesene production titers reported in this paper are not in the same range as titers published with known heterotrophic producers E. coli or S. cerevisiae. However, this proof-of-principle study with C. necator as production host opens new synthesis routes toward a sustainable economy and leaves room for further optimizations, which have been already performed with the known production strains. [ABSTRACT FROM AUTHOR]

We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase is used for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis-Menten parameters from in vitro experiments with isolated enzymes and used these values to simulate the change in the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate-determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically. [ABSTRACT FROM AUTHOR]

Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli ( E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone-dependent aldolase (Fsa1-A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3 S,4 R)-1,3,4-trihydroxy-5-phenylpentan-2-one, in 70 % yield within short reaction times. [ABSTRACT FROM AUTHOR]

An immense number of chemical reactions are carried out simultaneously in living cells. Nature's optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man-made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor-made microbial cell factories. [ABSTRACT FROM AUTHOR]

The Cover shows a biochemical cell factory (Escherichia coli) for the synthesis of chiral polyhydroxylated compounds via an artificial enzyme cascade.In their Communication, T. Bayer et al. presented a rational approach for tuning the concentration of toxic aldehyde intermediates in living cells. By combining an alcohol dehydrogenase and a carboxylic acid reductase, the redox equilibrium between alcohol, aldehyde and carboxylic acid species can be adjusted, thus maintaining the aldehyde concentration in a ‘hidden reservoir’ and therefore below the toxicity level, yet freely available for subsequent aldolase mediated reaction. More information can be found in the Communication by T. Bayer et al. on page 2919 in Issue 15, 2017 (DOI: 10.1002/cctc.201700469). [ABSTRACT FROM AUTHOR]

Review: [217 refs. [ABSTRACT FROM AUTHOR]

This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. [ABSTRACT FROM AUTHOR]