The volumetric heating values of today’s biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chain fatty acyl-ACP thioesterase for improved MCFA production in Escherichia coli. Electrostatically matching the interface between the heterologous medium-chain Acinetobacter baylyi fatty acyl-ACP thioesterase (AbTE) and the endogenous E. coli fatty acid ACP ( E. coli AcpP) by replacing small nonpolar amino acids on the AbTE surface for positively charged ones increased secreted MCFA titers more than 3-fold. Nuclear magnetic resonance titration of E. coli N-octanoyl-AcpP with a single AbTE point mutant and the best double mutant showed a progressive and significant increase in the number of interactions when compared to AbTE wildtype. The best AbTE mutant produced 131 mg/L of MCFAs, with MCFAs being 80% of all secreted fatty acid chain lengths after 72 h. To enable the future screening of larger numbers of AbTE variants to further improve MCFA titers, we show that a previously developed G-protein coupled receptor (GPCR)-based MCFA sensor differentially detects MCFAs secreted by E. coli expressing different AbTE variants. This work demonstrates that engineering the interface of heterologous enzymes to better couple with endogenous host proteins is a useful strategy to increase the titers of microbially produced chemicals. Further, this work shows that GPCR-based sensors are producer microbe agnostic and can detect chemicals directly in the producer microbe supernatant, setting the stage for the sensor-guided engineering of MCFA producing microbes.

Monoterpene indole alkaloids (MIAs) have important therapeutic value, including as anticancer and antimalarial agents. Because of their chemical complexity, therapeutic MIAs, or advanced intermediates thereof, are often isolated from the native plants. The microbial synthesis of MIAs would allow for the rapid and scalable production of complex MIAs and MIA analogues for therapeutic use. Here, we produce the modified MIA hydroxystrictosidine from glucose and the monoterpene secologanin via a pterin-dependent mono-oxidation strategy. Specifically, we engineered the yeast Saccharomyces cerevisiae for the high-level synthesis of tetrahydrobiopterin to mono-oxidize tryptophan to 5-hydroxytryptophan, which, after decarboxylation to serotonin, is coupled to exogenously fed secologanin to produce 10-hydroxystrictosidine in an eight-enzyme pathway. We selected hydroxystrictosidine as our synthetic target because hydroxylation at the 10' position of the alkaloid core strictosidine provides a chemical handle for the future chemical semisynthesis of therapeutics. We show the generality of the pterin-dependent mono-oxidation strategy for alkaloid synthesis by hydroxylating tyrosine to L-DOPA-a key intermediate in benzylisoquinoline alkaloid (BIA) biosynthesis-and, thereafter, further converting it to dopamine. Together, these results present the first microbial synthesis of a modified alkaloid, the first production of tetrahydrobiopterin in yeast, and the first use of a pterin-dependent mono-oxidation strategy for the synthesis of L-DOPA. This work opens the door to the scalable production of MIAs as well as the production of modified MIAs to serve as late intermediates in the semisynthesis of known and novel therapeutics. Further, the microbial strains in this work can be used as plant pathway discovery tools to elucidate known MIA biosynthetic pathways or to identify pathways leading to novel MIAs.

Amorpha‐4,11‐diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2/qglucose led to a 6.7‐fold increase in product titers and a 6.5‐fold increase in carbon yield.