Principles and practice of designing microbial biocatalysts for fuel and chemical production.

The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, nonfood carbohydrates, such as lignocellulosic biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other nonethanol fuels like butanol. However, currently there is no native microorganism that can ferment all lignocellulosic sugars to fuel molecules. Thus, research is focused on engineering improved microbial biocatalysts for production of liquid fuels at high productivity, titer, and yield. A clear understanding and application of the basic principles of microbial physiology and biochemistry are crucial to achieve this goal. In this review, we present and discuss the construction of microbial biocatalysts that integrate these principles with ethanol-producing Escherichia coli as an example of metabolic engineering. These principles also apply to fermentation of lignocellulosic sugars to other chemicals that are currently produced from petroleum.

Combinatorial effects of trans-cinnamaldehyde with fluoride and chlorhexidine on Streptococcus mutans.

AIMS: The aim of this study was to investigate the effects of trans-cinnamaldehyde (TC) and its synergistic activity with chlorhexidine (CHX) and fluoride against Streptococcus mutans. METHODS AND RESULTS: Streptococcus mutans UA159 was treated with TC alone and in combination with CHX or sodium fluoride. The synergy profile was analysed using the Zero Interaction Potency model. TC showed strong synergism (synergy score of 21·697) with CHX, but additive effect (synergy score of 5·298) with fluoride. TC and the combinations were tested for acid production (glycolytic pH drop) and biofilm formation by S. mutans, and nitric oxide production in macrophages. TC significantly inhibited sucrose-dependent biofilm formation and acid production by S. mutans. Mechanistic studies were carried out by qRT-PCR-based transcriptomic studies which showed that TC acts by impairing genes related to metabolism, quorum sensing, bacteriocin expression, stress tolerance and biofilm formation. CONCLUSIONS: trans-Cinnamaldehyde potentiates CHX and sodium fluoride in inhibiting S. mutans biofilms and virulence through multiple mechanisms. This study sheds significant new light on the potential to develop TC as an anti-caries treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral diseases were classified as a 'silent epidemic' in the US Surgeon General's Report on Oral Health. Two decades later, >4 billion people are still affected worldwide by caries, having significant effects on the quality of life. There is an urgent need to develop novel compounds and strategies to combat dental caries. Here, we prove that TC downregulates multiple pathways and potentiates the CHX and fluoride to prevent S. mutans biofilms and virulence. This study sheds significant new light on the potential to develop TC in combination with CHX or fluoride as novel treatments to arrest dental caries.

Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products.

Methane can be converted to triose dihydroxyacetone (DHA) by chemical processes with formaldehyde as an intermediate. Carbon dioxide, a by-product of various industries including ethanol/butanol biorefineries, can also be converted to formaldehyde and then to DHA. DHA, upon entry into a cell and phosphorylation to DHA-3-phosphate, enters the glycolytic pathway and can be fermented to any one of several products. However, DHA is inhibitory to microbes due to its chemical interaction with cellular components. Fermentation of DHA to d-lactate by Escherichia coli strain TG113 was inefficient, and growth was inhibited by 30 g⋅L-1 DHA. An ATP-dependent DHA kinase from Klebsiella oxytoca (pDC117d) permitted growth of strain TG113 in a medium with 30 g⋅L-1 DHA, and in a fed-batch fermentation the d-lactate titer of TG113(pDC117d) was 580 ± 21 mM at a yield of 0.92 g⋅g-1 DHA fermented. Klebsiella variicola strain LW225, with a higher glucose flux than E. coli, produced 811 ± 26 mM d-lactic acid at an average volumetric productivity of 2.0 g-1⋅L-1⋅h-1 Fermentation of DHA required a balance between transport of the triose and utilization by the microorganism. Using other engineered E. coli strains, we also fermented DHA to succinic acid and ethanol, demonstrating the potential of converting CH4 and CO2 to value-added chemicals and fuels by a combination of chemical/biological processes.

Prognostic value of MACC1 and proficient mismatch repair status for recurrence risk prediction in stage II colon cancer patients: the BIOGRID studies.

BACKGROUND: We assessed the novel MACC1 gene to further stratify stage II colon cancer patients with proficient mismatch repair (pMMR). PATIENTS AND METHODS: Four cohorts with 596 patients were analyzed: Charité 1 discovery cohort was assayed for MACC1 mRNA expression and MMR in cryo-preserved tumors. Charité 2 comparison cohort was used to translate MACC1 qRT-PCR analyses to FFPE samples. In the BIOGRID 1 training cohort MACC1 mRNA levels were related to MACC1 protein levels from immunohistochemistry in FFPE sections; also analyzed for MMR. Chemotherapy-naïve pMMR patients were stratified by MACC1 mRNA and protein expression to establish risk groups based on recurrence-free survival (RFS). Risk stratification from BIOGRID 1 was confirmed in the BIOGRID 2 validation cohort. Pooled BIOGRID datasets produced a best effect-size estimate. RESULTS: In BIOGRID 1, using qRT-PCR and immunohistochemistry for MACC1 detection, pMMR/MACC1-low patients had a lower recurrence probability versus pMMR/MACC1-high patients (5-year RFS of 92% and 67% versus 100% and 68%, respectively). In BIOGRID 2, longer RFS was confirmed for pMMR/MACC1-low versus pMMR/MACC1-high patients (5-year RFS of 100% versus 90%, respectively). In the pooled dataset, 6.5% of patients were pMMR/MACC1-low with no disease recurrence, resulting in a 17% higher 5-year RFS [95% confidence interval (CI) (12.6%-21.3%)] versus pMMR/MACC1-high patients (P = 0.037). Outcomes were similar for pMMR/MACC1-low and deficient MMR (dMMR) patients (5-year RFS of 100% and 96%, respectively). CONCLUSIONS: MACC1 expression stratifies colon cancer patients with unfavorable pMMR status. Stage II colon cancer patients with pMMR/MACC1-low tumors have a similar favorable prognosis to those with dMMR with potential implications for the role of adjuvant therapy.

Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals.

Pretreatments such as dilute acid at elevated temperature are effective for the hydrolysis of pentose polymers in hemicellulose and also increase the access of enzymes to cellulose fibers. However, the fermentation of resulting syrups is hindered by minor reaction products such as furfural from pentose dehydration. To mitigate this problem, four genetic traits have been identified that increase furfural tolerance in ethanol-producing Escherichia coli LY180 (strain W derivative): increased expression of fucO, ucpA, or pntAB and deletion of yqhD. Plasmids and integrated strains were used to characterize epistatic interactions among traits and to identify the most effective combinations. Furfural resistance traits were subsequently integrated into the chromosome of LY180 to construct strain XW129 (LY180 ΔyqhD ackA::PyadC'fucO-ucpA) for ethanol. This same combination of traits was also constructed in succinate biocatalysts (Escherichia coli strain C derivatives) and found to increase furfural tolerance. Strains engineered for resistance to furfural were also more resistant to the mixture of inhibitors in hemicellulose hydrolysates, confirming the importance of furfural as an inhibitory component. With resistant biocatalysts, product yields (ethanol and succinate) from hemicellulose syrups were equal to control fermentations in laboratory media without inhibitors. The combination of genetic traits identified for the production of ethanol (strain W derivative) and succinate (strain C derivative) may prove useful for other renewable chemicals from lignocellulosic sugars.

Removing chiral contamination of lactate solutions by selective metabolism of the D-enantiomer.

OBJECTIVE: A bio-based process is appealing for purification of L-lactic acid, the major enantiomer of polylactic acid syrup, generated by thermochemical processes at the end of life of PLA-based plastics, from its chiral impurity, D-lactic acid, before reuse. RESULTS: Polylactic acid (PLA), a renewable alternative to petroleum-derived plastics, contains a mixture of L- and D-lactic acid (LA) isomers with the L-isomer dominating (up to 95 %). A novel bio-based process was developed to produce chirally pure L-LA from syrup produced during recycling of PLA-plastics. This process utilizes an engineered Escherichia coli (strain DC1001) containing novel gene deletions (lld, ykg) that eliminated the oxidative metabolism of L-lactate, leaving the membrane-bound D-lactate dehydrogenases to selectively metabolize the D-isomer. Strain DC1001 removed 8.7 g D-lactate l(-1) from a PLA-syrup containing 135 g total lactic acid l(-1) in 24 h. Average rates of removal of D-lactic acid were 0.25 g D-lactate h(-1) (g cell dry weight)(-1) and 0.36 g D-lactate l(-1) h(-1). CONCLUSION: Bio-based purification of PLA-syrup utilizing E. coli strain DC1001 is an attractive process step during recycling of PLA-plastics. This selective oxidation process can also be used to remove chiral contamination of L-lactate in medical applications.

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 µmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

Amino acid substitutions at glutamate-354 in dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH.

Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) for pyruvate or NAD(+) for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.

Increased furan tolerance in Escherichia coli due to a cryptic ucpA gene.

Expression arrays were used to identify 4 putative oxidoreductases that were upregulated (>3-fold) by furfural (15 mM, 15 min). Plasmid expression of one (ucpA) increased furan tolerance in ethanologenic strain LY180 and wild-type strain W. Deleting ucpA decreased furfural tolerance. Although the mechanism remains unknown, the cryptic ucpA gene is now associated with a phenotype: furan resistance.

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes.

Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

Safety and efficacy of siddha medicine preparation in the management of COVID-19: A prospective randomised open label study.

Background: The use of complementary and alternative medicine (CAM) therapies has surged since the spread of COVID-19 pandemic. However, the efficacy and safety of these CAM therapies remains majorly unexplored. Objective: To understand the efficacy and safety of Nochi Kudineer Chooranam (5 gm), Mahasudarsan Chooranam (3 gm) , Adathodai Manapagu (10 ml), Omatheeneer (10 ml), Maldevi chenduram (100 mg) with honey in management of COVID 19 patients. Methods: We conducted a randomised, controlled, open label trial in patients hospitalized with SARS-CoV-2 infection who had an oxygen saturation of 90% or more while breathing ambient air. Patients were randomized into two groups in a 1:1 ratio to either intervention group, receiving seven days of siddha medicine (Intervention group; n = 50) or standard care (control group; n = 50). The primary end point was clinical markers and patient recovery status on day 8. Results: A total of 100 patients with confirmed COVID-19 with average age of 37 yrs (interquartile range, 28-49) participated in the study. There was no statistically difference between groups at baseline (P > 0.05). After intervention, patients in the intervention group had statistically (P < 0.05) significant reduction in the symptoms when compared to standard care. By end of the intervention period, 6 patients (12%) were hospitalized in the control group and none of them were reported for intervention group. Conclusion: Among patients with mild to moderate COVID-19, 7 days of siddha medicine showed a significant reduction in the clinical symptoms and requirement of hospitalisation, with no adverse events. Therefore, the particular siddha medicine preparation could be used safely and effectively for the management of COVID-19 patients.

L-malate production by metabolically engineered Escherichia coli.

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts.

Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate.

Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms.

L: (+)-Lactic acid production from non-food carbohydrates by thermotolerant Bacillus coagulans.

Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150-180 g l(-1)) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l(-1) and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to L: (+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the process and a reduction in contamination of large-scale fermentations.

Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.

YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance.

Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria.

Fermentation of glycerol to succinate by metabolically engineered strains of Escherichia coli.

The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD(+) ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.

Metabolic flux control at the pyruvate node in an anaerobic Escherichia coli strain with an active pyruvate dehydrogenase.

During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y(ATP)) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y(ATP) suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology.

Production of fuels and chemicals through microbial fermentation of plant material is a desirable alternative to petrochemical-based production. Fermentative production of biorenewable fuels and chemicals requires the engineering of biocatalysts that can quickly and efficiently convert sugars to target products at a cost that is competitive with existing petrochemical-based processes. It is also important that biocatalysts be robust to extreme fermentation conditions, biomass-derived inhibitors, and their target products. Traditional metabolic engineering has made great advances in this area, but synthetic biology has contributed and will continue to contribute to this field, particularly with next-generation biofuels. This work reviews the use of metabolic engineering and synthetic biology in biocatalyst engineering for biorenewable fuels and chemicals production, such as ethanol, butanol, acetate, lactate, succinate, alanine, and xylitol. We also examine the existing challenges in this area and discuss strategies for improving biocatalyst tolerance to chemical inhibitors.