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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 [...]

The fermentative metabolism of D-glucuronic acid (glucuronate) in Escherichia coil was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh--) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh--were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : [NAD.sup.+] ratio supported the proposed role of PDH. Moreover, the NADH : [NAD.sup.+] ratio in a strain deficient in both PDH and the ox-PPP (Pdh--Zwf--) was even lower than that observed for Pdh--. Strain Pdh--Zwf--also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh--. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh--mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : [NAD.sup.+] ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules. DOI 10.1099/mic.0.036251-0

Summary: Most microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2, complementing the previously reported role of 1,2‐propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol‐3‐phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase‐dependent H2 evolution. Despite the importance of the GK‐G3PDH route under alkaline conditions, we found that the NADH‐generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)‐dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system‐wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA‐DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose‐1,6‐bisphosphatases and the fructose phosphate bypass. [ABSTRACT FROM AUTHOR]

Escherichia coli was engineered for the efficient conversion of glycerol to D-lactic acid (D-lactate) and understand the pathways mediating the metabolism of glycerol in wild-type strains. The strain exhibited maximum volumetric and specific productivities for D-lactate production and the engineered homolactic route was observed to generate 1 to 2 mol of ATP per mol of D-lactate and is redox balanced and hence represents a viable metabolic pathway.

Prenylated aromatics (PAs) are an important class of natural products with valuable pharmaceutical applications. To address current limitations of their sourcing from plants, here, we present a microbial platform for the in vivo synthesis of PAs based on the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. As proof of concept, we targeted the prenylation of phenolic/phenolcarboxylic acids, including orsellinic (OSA), divarinolic (DVA), and olivetolic (OLA) acids, whose prenylated products have important biopharmaceutical applications. Although the ability of wild‐type NphB to catalyze the prenylation reaction with each acid was validated by in vitro characterization, improvement of product titers in vivo required protein modeling and rational design to engineer NphB variants with increased activity and product selectivity. When a designed NphB variant with eightfold improved catalytic efficiency toward OSA was expressed in an Escherichia coli host engineered to generate geranyl pyrophosphate at high flux through the mevalonate pathway, we observed up to 300 mg/L prenylated products by exogenously supplying OSA. The improved properties of engineered NphB were also utilized to demonstrate the diversification of this in vivo platform by using both different aromatic acceptors and different prenyl donors to generate various PA compounds, including medicinally important compounds such as cannabigerovarinic, cannabigerolic, and grifolic acids. A microbial platform for the synthesis of prenylated aromatic compounds was developed based on functional expression of the soluble aromatic prenyltransferase NphB. Key to this approach was engineering NphB through protein modeling and rational design, resulting in significant improvement to its catalytic efficiency. Expression of an engineered NphB variant coupled with exogenous addition of aromatic substrates and mevalonate pathway engineering for pyrophosphate supply enabled production of various prenylated aromatics, including medicinally important compounds such as cannabigerovarinic, cannabigerolic, and grifolic acids. [ABSTRACT FROM AUTHOR]

We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541-554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli. [ABSTRACT FROM AUTHOR]

Background: Due 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). Results: Escherichia 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. Conclusions: This 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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 ?-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 (?-ketoacyl-ACP synthases) attracting the most attention in recent years. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the ?-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 ?-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 ?-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-?2-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 ?-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms. [ABSTRACT FROM AUTHOR]

The article discusses a study on the fermentation of glycerol to produce 1,2 propanediol. An abundant by-product of biodiesel and bioethanol production is glycerol. 1,2-propanediol (1,2-PDO), which is used as an anti-freeze fluid, as plasticizers and in cosmetics, is extracted from the fermentation of glucose by several microorganisms such as Saccharomyces cerevisiae and Escherichia coli. It was found that with the use of key pathways and the right environmental conditions, E. coli can ferment glycerol under anaerobic conditions to produce 1,2-PDO.

Type III polyketide synthases (PKS IIIs) contribute to the synthesis of many economically important natural products, most of which are currently produced by direct extraction from plants or through chemical synthesis. Olivetolic acid (OLA) is a plant secondary metabolite sourced from PKS III catalysis, which along with its prenylated derivatives has various pharmacological activities. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for OLA, here we utilize a synthetic approach to engineer Escherichia coli for the production of OLA. In vitro characterization of polyketide synthase and cyclase enzymes, OLA synthase and OLA cyclase, respectively, validated their requirement as enzymatic components of the OLA pathway and confirmed the ability for these eukaryotic enzymes to be functionally expressed in E. coli. This served as a platform for the combinatorial expression of these enzymes with auxiliary enzymes aimed at increasing the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units, respectively. Through combining OLA synthase and OLA cyclase expression with the required modules of a β-oxidation reversal for hexanoyl-CoA generation, we demonstrate the in vivo synthesis of olivetolic acid from a single carbon source. The integration of additional auxiliary enzymes to increase hexanoyl-CoA and malonyl-CoA, along with evaluation of varying fermentation conditions enabled the synthesis of 80 mg/L OLA. This is the first report of OLA production in E. coli, adding a new example to the repertoire of valuable compounds synthesized in this industrial workhorse.

While we have recently constructed a functional reversal of the β-oxidation cycle as a platform for the production of fuels and chemicals by engineering global regulators and eliminating native fermentative pathways, the system-level approach used makes it difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and prevents the transfer of the engineered pathway to other organisms. In the work reported here, we overcome the aforementioned limitations by using a synthetic biology approach to construct and functionally characterize a reversal of the β-oxidation cycle. This was achieved through the in vitro kinetic characterization of each functional unit of the core and termination pathways, followed by their in vivo assembly and functional characterization. With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized in vitro. When these four functional units were assembled in vivo in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional reversal of the β-oxidation cycle was realized. The individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis was demonstrated by overexpressing a long-chain thiolase that enables the operation of multiple turns of the reversal of the β-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The well-defined and self-contained nature of each functional unit makes the engineered reversal of the β-oxidation cycle "chassis neutral" and hence transferrable to the host of choice for efficient fuel or chemical production.

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of (13)C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional (13)C,(1)H NMR spectroscopy. Metabolite balancing, biosynthetic (13)C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO(2) and the calculated CO(2) evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO(2) for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO(2), thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO(2) and H(2)) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO(2) generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.