Background Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds—which are often utilised for anti-microbial and anti-fungal functions in their host plant. Results In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed. Conclusion In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production. Electronic supplementary material The online version of this article (10.1186/s12934-019-1130-0) contains supplementary material, which is available to authorized users.

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There is an increasing realization that structure-based drug design may show improved success by understanding the ensemble of conformations and sub-states accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson's disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear and routes to drugging this target would be valuable. Evidence of a radical in either the transition state or resting state of MAO-B is present throughout the literature, and is suggested to be a flavin semiquinone, a tyrosyl radical or both. Here we see evidence of a resting state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. Based on enzyme kinetic studies, enzyme variants and molecular dynamics simulations we find evidence for the importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity, protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these targets.

Medium chain esters produced from fruits and flowering plants have a number of commercial applications including use as flavour and fragrance ingredients, biofuels, and in pharmaceutical formulations. These esters are typically made via the activity of an alcohol acyl transferase (AAT) enzyme which catalyses the condensation of an alcohol and an acyl-CoA. Developing a microbial platform for medium chain ester production using AAT activity presents several obstacles, including the low product specificity of these enzymes for the desired ester and/or low endogenous substrate availability. In this study, we engineered Escherichia coli for the production of butyl octanoate from endogenously produced octanoyl-CoA. This was achieved through rational protein engineering of an AAT enzyme from Actinidia chinensis for improved octanoyl-CoA substrate specificity and metabolic engineering of E. coli fatty acid metabolism for increased endogenous octanoyl-CoA availability. This resulted in accumulation of 3.3 + 0.1 mg/L butyl octanoate as the sole product from E. coli after 48 h. This study represents a preliminary examination of the feasibility of developing E. coli platforms for the synthesis single medium chain esters from endogenous fatty acids.

The engineering of xylo-oligosaccharide-consuming Saccharomyces cerevisiae strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in S. cerevisiae strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by S. cerevisiae strains. The suitability of S. cerevisiae for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.