Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

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Fuelling the future: microbial engineering for the production of sustainable biofuels

Production of industrial chemicals using renewable biomass feedstock is becoming increasingly important to address limited fossil resources, climate change and other environmental problems. To develop high-performance microbial cell factories, equivalent to chemical plants, microorganisms undergo systematic metabolic engineering to efficiently convert biomass-derived carbon sources into target chemicals. Over the past two decades, many engineered microorganisms capable of producing natural and non-natural chemicals have been developed. This Review details the current status of representative industrial chemicals that are produced through biological and/or chemical reactions. We present a comprehensive bio-based chemicals map that highlights the strategies and pathways of single or multiple biological reactions, chemical reactions and combinations thereof towards production of particular chemicals of interest. Future challenges are also discussed to enable production of even more diverse chemicals and more efficient production of chemicals from renewable feedstocks.

A comprehensive metabolic map for production of bio-based chemicals

Tolerance engineering in bacteria for the production of advanced biofuels and chemicals

The last few years have witnessed an unprecedented increase in the number of novel bacterial species that hold potential to be used for metabolic engineering. Historically, however, only a handful of bacteria have attained the acceptance and widespread use that are needed to fulfil the needs of industrial bioproduction - and only for the synthesis of very few, structurally simple compounds. One of the reasons for this unfortunate circumstance has been the dearth of tools for targeted genome engineering of bacterial chassis, and, nowadays, synthetic biology is significantly helping to bridge such knowledge gap. Against this background, in this review, we discuss the state of the art in the rational design and construction of robust bacterial chassis for metabolic engineering, presenting key examples of bacterial species that have secured a place in industrial bioproduction. The emergence of novel bacterial chassis is also considered at the light of the unique properties of their physiology and metabolism, and the practical applications in which they are expected to outperform other microbial platforms. Emerging opportunities, essential strategies to enable successful development of industrial phenotypes, and major challenges in the field of bacterial chassis development are also discussed, outlining the solutions that contemporary synthetic biology-guided metabolic engineering offers to tackle these issues.

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Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non-traditional microorganisms.

Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, to achieve lycopene overproduction in S. cerevisiae, both host cell and heterologous pathway should be delicately engineered. In this study, lycopene biosynthesis pathway was constructed by integration of CrtE, CrtB and CrtI in S. cerevisiae CEN.PK2. When YPL062W, a distant genetic locus, was deleted, little acetate was accumulated and approximately 100 % increase in cytosolic acetyl-CoA pool was achieved relative to that in parental strain. Through screening CrtE, CrtB and CrtI from diverse species, an optimal carotenogenic enzyme combination was obtained, and CrtI from Blakeslea trispora (BtCrtI) was found to have excellent performance on lycopene production as well as lycopene proportion in carotenoid. Then, the expression level of BtCrtI was fine-tuned and the effect of cell mating types was also evaluated. Finally, potential distant genetic targets (YJL064W, ROX1, and DOS2) were deleted and a stress-responsive transcription factor INO2 was also up-regulated. Through the above modifications between host cell and carotenogenic pathway, lycopene yield was increased by approximately 22-fold (from 2.43 to 54.63 mg/g DCW). Eventually, in fed-batch fermentation, lycopene production reached 55.56 mg/g DCW, which is the highest reported yield in yeasts. Saccharomyces cerevisiae was engineered to produce lycopene in this study. Through combining host engineering (distant genetic loci and cell mating types) with pathway engineering (enzyme screening and gene fine-tuning), lycopene yield was stepwise improved by 22-fold as compared to the starting strain. The highest lycopene yield (55.56 mg/g DCW) in yeasts was achieved in 5-L bioreactors. This study provides a good reference of combinatorial engineering of host cell and heterologous pathway for microbial overproduction of pharmaceutical and chemical products.

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Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering

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 simila...

Microbial Synthesis of Pinene

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.

Microbial synthesis of medium-chain chemicals from renewables.

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 15N-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...

Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production

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...

Pterin-Dependent Mono-oxidation for the Microbial Synthesis of a Modified Monoterpene Indole Alkaloid.

Provided herein are GPCR-based chemical biosensors that can have a sensing unit, a processing unit, and a response unit that can be used to detect a chemical of interest. Also provided herein are methods of making and using the GPCR-based chemical biosensors.

Stephen Sarria

Papers

Microbial Synthesis of Pinene

Microbial synthesis of medium-chain chemicals from renewables.

Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production

Pterin-Dependent Mono-oxidation for the Microbial Synthesis of a Modified Monoterpene Indole Alkaloid.

G-protein coupled receptor (gpcr)-based biosensors and uses thereof