Decarbonizing the air transportation sector remains one of the most challenging hurdles to mitigating climate change. Lignocellulosic biomass-derived jet fuel blendstocks can contribute to the shift ...toward renewable, low-carbon energy sources for aircrafts. Producing these renewable jet fuel molecules from biomass requires advanced pathways with the potential for efficient and affordable conversion routes. This paper presents a detailed techno-economic analysis and sensitivity analysis, including estimated minimum selling price (MSP), and life-cycle greenhouse gas (GHG) mitigation costs for five routes to four potential bio-jet fuel molecules – limonane via limonene, limonane via 1,8-cineole, tetrahydromethylcyclopentadiene dimer (RJ-4), bisabolane, and epi -isozizaane. The simulated biorefineries utilize biomass sorghum and an integrated high-gravity ionic liquid-based biomass deconstruction process. We present results reflecting the current state of the technology and potential future scenarios with improved yields. Among the conversion pathways and the fuel molecules evaluated in this study, limonane, bisabolane, and epi -isozizaane could reach an MSP of $0.73–$0.91 per L-Jet A ($2.75–$3.45 per gal-Jet A) in optimized future cases, without a hypothetical lignin-derived co-product. RJ-4 requires a more costly upgrading process and catalysts, resulting in a comparatively higher MSP ($1.33 per L-Jet A or $5.04 per gal-jet A). Based on the GHG footprints of each fuel, the minimum achievable carbon mitigation cost relative to conventional Jet-A is $29 per metric ton CO 2e , which is just under double the current cap-and-trade market price in California. In the absence of any policy support, the economics could be improved through high-value uses for lignin. To reach a target selling price of $0.66 per L-Jet A ($2.50 per gal), lignin-derived products would need to be sold for at least $1.9 per kg. However, the higher energy density of these bio-based blendstocks offers valuable improvements in aircraft efficiency/range; we find that commercial airlines may be willing to pay a 4–14 cent per L premium for these bio-jet fuels. Our results highlight the need for improvements beyond currently-reported yields for the biologically produced intermediates, identification of ideal microbial hosts, selection of metabolic pathways to achieve competitive production costs, and a focus on fuels with attractive properties that increase their value.
Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels is biofuels produced by engineered ...microorganisms that use a renewable carbon source. Two biofuels, ethanol and biodiesel, have made inroads in displacing petroleum-based fuels, but their uptake has been limited by the amounts that can be used in conventional engines and by their cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure but have had limited uptake due to costs. In this Review, we discuss engineering metabolic pathways to produce advanced biofuels, challenges with substrate and product toxicity with regard to host microorganisms and methods to engineer tolerance, and the use of functional genomics and machine learning approaches to produce advanced biofuels and prospects for reducing their costs.
Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be ...produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.
•We report limonene production in E. coli using a heterologous mevalonate pathway.•Perillyl alcohol was produced in E. coli from endogenously generated limonene.•Limonene and perillyl alcohol titers were highly improved by metabolic engineering.•An anion exchange resin was used to improve specific recovery of perillyl alcohol.
Rising petroleum costs, trade imbalances and environmental concerns have stimulated efforts to advance the microbial production of fuels from lignocellulosic biomass. Here we identify a novel ...biosynthetic alternative to D2 diesel fuel, bisabolane, and engineer microbial platforms for the production of its immediate precursor, bisabolene. First, we identify bisabolane as an alternative to D2 diesel by measuring the fuel properties of chemically hydrogenated commercial bisabolene. Then, via a combination of enzyme screening and metabolic engineering, we obtain a more than tenfold increase in bisabolene titers in Escherichia coli to >900 mg l(-1). We produce bisabolene in Saccharomyces cerevisiae (>900 mg l(-1)), a widely used platform for the production of ethanol. Finally, we chemically hydrogenate biosynthetic bisabolene into bisabolane. This work presents a framework for the identification of novel terpene-based advanced biofuels and the rapid engineering of microbial farnesyl diphosphate-overproducing platforms for the production of biofuels.
Covering: 2005 to 2015
Although natural products are best known for their use in medicine and agriculture, a number of fatty acid-derived and isoprenoid natural products are being developed for use ...as renewable biofuels and bio-based chemicals. This review summarizes recent work on fatty acid-derived compounds (fatty acid alkyl esters, fatty alcohols, medium- and short-chain methyl ketones, alkanes, α-olefins, and long-chain internal alkenes) and isoprenoids, including hemiterpenes (
e.g.
, isoprene and isopentanol), monoterpenes (
e.g.
, limonene), and sesquiterpenes (
e.g.
, farnesene and bisabolene).
We review recent progress in the development of fatty acid-derived and isoprenoid natural products for use as renewable biofuels and bio-based chemicals.
The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous ...amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.
One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the ...properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.
As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. ...Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors.
Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number.
The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.
Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a ...heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.
CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking ...down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway. To identify genomic knockdown targets in competing pathways, we first designed a single-gRNA library with 15 individual targets, where 3 gRNA cassettes targeting gene asnA, prpE, and gldA increased isopentenol titer by 18–24%. We then combined the 3 single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression. Our strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite, while the repression level needs to be adjusted to balance the metabolic network and achieve the maximum titer improvement.
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