James M. Clomburg

Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals

Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon–carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including β-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases. Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (β-ketoacyl-ACP synthases) attracting the most attention in recent years. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids, can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the β-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ2-carboxylic acids. The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n-alcohols (C ≥ 4) and extracellular long-chain fatty acids (C > 10) at higher efficiency than previously reported. The ubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology.

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.

Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals

To ensure the long-term viability of biorefineries, it is essential to go beyond the carbohydrate-based platform and develop complementing technologies capable of producing fuels and chemicals from a wide array of available materials. Glycerol, a readily available and inexpensive compound, is generated during biodiesel, oleochemical, and bioethanol production processes, making its conversion into value-added products of great interest. The high degree of reduction of carbon atoms in glycerol confers the ability to produce fuels and reduced chemicals at higher yields when compared to the use of carbohydrates. This review focuses on current engineering efforts as well as the challenges involved in the utilization of glycerol as a carbon source for the production of fuels and chemicals.

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed ∼95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK‐GlpD/GlpABC) and fermentative (GldA‐DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate‐lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl‐CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co‐production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by‐products succinate, acetate, and lactate. Co‐production of hydrogen required the use of acidic pH while formate co‐production was facilitated by inactivation of the enzyme formate‐hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements. Biotechnol. Bioeng. 2009;103: 148–161.

Metabolic engineering of Escherichia coli for the production of succinate from glycerol.

Glycerol has become an ideal feedstock for the microbial production of bio-based chemicals due to its abundance, low cost, and high degree of reduction. We have previously reported the pathways and mechanisms for the utilization of glycerol by Escherichia coli in minimal salts medium under microaerobic conditions. Here we capitalize on such results to engineer E. coli for the production of value-added succinate from glycerol. Through metabolic engineering of E. coli metabolism, succinate production was greatly elevated by (1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate and (2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). As such, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). Our biocatalysts demonstrated a maximum specific productivity of approximately 400 mg succinate/g cell/h and a yield of 0.69 g succinate/g glycerol, on par with the use of glucose as a feedstock.

Escherichia coli strains engineered for homofermentative production of D-lactic acid from glycerol.

ABSTRACT Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.

Metabolic engineering of Escherichia coli for the production of 1,2‐propanediol from glycerol

Due to its availability, low‐price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2‐propanediol (1,2‐PDO). A functional 1,2‐PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP‐dependent dihydroxyacetone kinase (DHAK) with an ATP‐dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2‐PDO production. Analysis of the major fermentative pathways indentified ethanol as a required co‐product while increases in 1,2‐PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2‐PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by‐product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2‐PDO from common sugars. Biotechnol. Bioeng. 2011; 108:867–879.

Industrial biomanufacturing: The future of chemical production

The next era of chemical manufacturing Producing mass quantities of chemicals has its roots in the industrial revolution. But industrial synthesis leads to sizeable sustainability and socioeconomic challenges. The rapid advances in biotechnology suggest that biological manufacturing may soon be a feasible alternative, but can it produce chemicals at scale? Clomburg et al. review the progress made in industrial biomanufacturing, including the tradeoffs between highly tunable biocatalysts and units of scale. The biological conversion of single-carbon compounds such as methane, for example, has served as a testbed for more sustainable, decentralized production of desirable compounds. Science, this issue p. 10.1126/science.aag0804 BACKGROUND Environmental, geopolitical, and economic factors are reshaping our view of global energy and manufacturing demands. Addressing these challenges may require a shift from the current model of industrial chemical manufacturing, which employs large-scale megafacilities that benefit from economies of unit scale. Contrary to the traditional approach, a model based on economies of unit number is proposed to reduce capital costs per unit capacity. Industrial biomanufacturing, which exploits biological processes for manufacturing, offers one way to address these changing global factors while using the economies of unit number model. This model employs both facility-level mass production of small-scale, modular units and improvements to process design resulting from repetition in a “learning-by-doing” approach. The lower investment and financial risk associated with smaller-scale, lower–capital cost facilities allow a larger number and more diverse group of technology players to be involved, in turn enabling faster innovation and novel technology adoption as well as a faster response to market drivers. ADVANCES In contrast to current chemical manufacturing methods, characteristics inherent to bioconversion processes—such as the ability to operate at mild temperatures and pressures and achieve high carbon- and energy-conversion efficiencies in single-unit operations—result in more streamlined and less technologically complex processes. These characteristics enable flexible, smaller-scale, and capital expenditure–efficient operation that can both support and benefit from a large number of facilities, according to the economies of unit number model. For example, the capital expenditure entry-level cost of corn-grain ethanol facilities, the most widely developed current example of a bioconversion process, has substantially decreased as the number of plants has increased over the past few decades. This has facilitated rapid, small-scale, and widespread deployment resulting in a more than 10-fold increase in U.S. ethanol production from 1995 to 2015. Advances in metabolic engineering, synthetic biology, genomics, and industrial process design have pushed industrial biomanufacturing closer to more widespread adoption. Particular emphasis on single-carbon feedstocks, such as methane or CO2, in applications where large-scale chemical manufacturing is infeasible or too costly can leverage both economies of unit number for mass production of facilities and the benefits of manufacturing automation to reduce capital expenditure per unit scale. Current research efforts focused on the design of carbon- and energy-efficient metabolic pathways have been particularly beneficial. Advances in tool development for metabolic pathway design have included in silico organism design strategies, genome mining techniques, and computational enzyme design efforts. The integration of these with systems biology techniques, such as next-generation sequencing and high-sensitivity “omics” methods, has allowed for the design and potential development of millions of chemical production pathways. The use of genome engineering technologies—such as clustered regulatory interspaced short palindromic repeats (CRISPR)–associated protein Cas9 systems and multiplex automated genomic engineering—and recent advances in screening and selecting for edited organisms using biosensors have reduced the time required to complete genomic editing to a fraction of the traditional time requirements. Automation-based process design improvements of these biotechnology advances have also facilitated rapid advances in industrial biomanufacturing. OUTLOOK Continued development of industrial biomanufacturing in the 21st century will require further advances in biocatalyst design and process design engineering. To facilitate a future based on industrial biomanufacturing, further development of genomic tools and industrial design automations suited to these purposes is paramount. Furthermore, reducing the time required from concept design to industrial relevance is essential. Specific developments in the area of one-carbon feedstocks stand to exploit the opportunity presented by currently wasted, distributed methane through the increased adoption of an economy of unit numbers approach in industrial biomanufacturing. Although much work remains, the future of industrial biomanufacturing holds great promise in meeting the evolving demands of chemical production in the current century and beyond. Methane-based industrial biomanufacturing for fuel and chemical production. Exploiting biological processes can enable the conversion of single-carbon feedstocks, like methane, to the array of chemical products currently produced through industrial chemical manufacturing with considerable economic, environmental, and societal advantages. The current model for industrial chemical manufacturing employs large-scale megafacilities that benefit from economies of unit scale. However, this strategy faces environmental, geographical, political, and economic challenges associated with energy and manufacturing demands. We review how exploiting biological processes for manufacturing (i.e., industrial biomanufacturing) addresses these concerns while also supporting and benefiting from economies of unit number. Key to this approach is the inherent small scale and capital efficiency of bioprocesses and the ability of engineered biocatalysts to produce designer products at high carbon and energy efficiency with adjustable output, at high selectivity, and under mild process conditions. The biological conversion of single-carbon compounds represents a test bed to establish this paradigm, enabling rapid, mobile, and widespread deployment, access to remote and distributed resources, and adaptation to new and changing markets.

Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli

BackgroundDue to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate).ResultsEscherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity.ConclusionsThis study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by‐product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions. Biotechnol. Bioeng. 2012;109: 187–198.