Membrane-bound Transhydrogenase Research Articles

Metabolic engineering of Corynebacterium glutamicum for fatty alcohol production from glucose and wheat straw hydrolysate

BackgroundFatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable.ResultsTo engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L−1 from 20 g glucose L−1 with a product yield of 0.054 ± 0.001 Cmol Cmol−1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h−1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(Ptac-pntAB-TrrnB) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L−1 with a product yield of 0.054 ± 0.005 Cmol Cmol−1 and a volumetric productivity of 0.109 ± 0.005 gFAL L−1 h−1 in a pulsed fed-batch cultivation using wheat straw hydrolysate.ConclusionThe combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock.

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L−1l-lysine from mannitol at a yield of 0.26 mol mol−1 and a maximum productivity of 2.1 g L−1 h−1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol−1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol−1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.

Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri.

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

Production of scyllo-Inositol: Conversion of Rice Bran into a Promising Disease-Modifying Therapeutic Agent for Alzheimer's Disease.

scyllo-Inositol (SI) is one of the inositol stereoisomers, rare in the nature, and expected as a promising disease-modifying therapeutic agent for Alzheimer's disease. On the other hand, myo-inositol (MI) is another inositol stereoisomer most abundant in nature and thus supplied from agricultural byproducts including rice bran. Bacillus subtilis was genetically modified in its inositol metabolism and phytase secretion, to develope the bioconversion processes to produce SI from rice bran. Phytase, an enzyme that degrades phytate in rice bran into MI, was secreted in a B. subtilis strain with the optimized signal peptide. Another B. subtilis strain was constructed with the constitutive and simultaneous overexpression of IolG and IolW, which are the two inositol dehydrogenases responsible for the conversion, to demonstrate an efficient conversion of MI into SI with a rate up to 10 g/L/48 h. In order to devise further elevation in the conversion efficiency, we attempted to improve the substrate uptake by overexpressing iolT for the major MI transporter. In addition, Escherichia coli pntAB encoding the membrane-bound transhydrogenase was introduced aiming at enhanced supply of NADPH required for the rate-limiting IolW reaction. These additional modifications successfully elevated the conversion efficiency with an improved rate up to almost 30 g/L/48 h. Together with the improved phytase secretion, technological infrastructure for social implementation of SI production from rice bran is on the way.

RNA accumulation in Candida tropicalis based on cofactor engineering.

Redox cofactors play an important role in biosynthetic and catabolic reactions and the transfer of energy for the cell. Therefore, studying the relationship between cofactor perturbation and metabolism is a useful approach to improve the yield of target products. To study RNA accumulation and metabolism when intracellular cofactor balance was impaired, the water-forming NADH oxidase (NoxE) from Lactococcus lactis and membrane-bound transhydrogenase (PntAB) from Escherichia coli were expressed in Candidatropicalis no. 121, respectively. Expression of noxE significantly decreased the intracellular NADH/NAD+ ratio, but the NADPH/NADP+ ratio did not differ significantly. PntAB increased the intracellular NADH pool, while the NADPH/NADP+ ratio decreased. The perturbation of the cofactors caused a large redistribution of metabolic fluxes. The biomass and RNA content decreased by 11.0% and 10.6% in pAUR-noxE strain, respectively, while the RNA content increased by 5.5% and the biomass showed no signification difference in pAUR-pntAB strain. Expression of noxE and pntAB led to decreases and increases in the ATP concentration and yield of RNA, respectively, which also indicated that ATP plays an important role in the RNA biosynthesis.

13C metabolic flux analysis-guided metabolic engineering of Escherichia coli for improved acetol production from glycerol

BackgroundBioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. 13C metabolic flux analysis (13C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied 13C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.ResultsWe performed 13C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed nadK encoding NAD kinase or pntAB encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP+ and the NADPH/NADP+ ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, 13C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.ConclusionsThis research demonstrated how 13C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.

Metabolic engineering of Corynebacterium glutamicum for improved L-arginine synthesis by enhancing NADPH supply.

Corynebacterium glutamicum SNK 118 was metabolically engineered with improved L-arginine titer. Considering the crucial role of NADPH level in L-arginine production, pntAB (membrane-bound transhydrogenase) and ppnK (NAD+ kinase) were co-expressed to increase the intracellular NADPH pool. Expression of pntAB exhibited significant effects on NADPH supply and L-arginine synthesis. Furthermore, argR and farR, encoding arginine repressor ArgR and transcriptional regulator FarR, respectively, were removed from the genome of C. glutamicum. The competitive branch pathway gene ldh was also deleted. Eventually, an engineered C. glutamicum JML07 was obtained for L-arginine production. Fed-batch fermentation in 5-L bioreactor employing strain JML07 allowed production of 67.01gL-1L-arginine with productivity of 0.89gL-1h-1 and yield of 0.35gg-1 glucose. This study provides a productive L-arginine fermentation strain and an effective cofactor manipulating strategy for promoting the biosynthesis of NADPH-dependent metabolites.

Sorbitol production and optimization of photosynthetic supply in the cyanobacterium Synechocystis PCC 6803

Biochemicals production is a major theme in the application of photosynthesis to address global warming and organic-resource problems. Among biochemicals, sugar alcohols have attracted research attention because they are directly derived from two photosynthetic products, sugars and reductants. Here, we produced sorbitol photosynthetically by using cyanobacteria and modified the supply of its substrates through genetic engineering. Expression of an NADPH-dependent enzyme that generates sorbitol-6-phosphate, S6PDH, was highly toxic to cyanobacteria likely due to the sorbitol production, whereas expression of an NADH-dependent enzyme, SrlD2, yielded no sorbitol. The toxicity was partly overcome by introducing a theophylline-inducible riboswitch for S6PDH expression and optimizing induction, but sorbitol production was still low and severely inhibited growth. Co-expression of fructose-1,6-bisphosphatase drastically alleviated the growth inhibition, but did not increase short-term sorbitol production. The NADPH/NADP+ ratio decreased during sorbitol production. Overexpression of a membrane-bound transhydrogenase for NADPH generation from NADH elevated the short-term sorbitol production, but only partly alleviated the growth inhibition. Notably, a strain overexpressing all three enzymes exhibited sustainable sorbitol production at 312 mg/L, which was nearly 27-fold higher than the yield of the initial S6PDH-overexpressing strain. We discuss these results in relation to the optimization of photosynthetic supply for sorbitol production in cyanobacteria.

Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase

The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across anarrow pore and a hydrophobic "dry" region inthe middle of the membrane channel, with key residues His42α2 (chain A) being protonated and Thr214β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.

Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural.

Furfural is a toxic by-product formulated from pretreatment processes of lignocellulosic biomass. In order to utilize the lignocellulosic biomass on isobutanol production, inhibitory effect of the furfural on isobutanol production was investigated and combinatorial application of two oxidoreductases, FucO and YqhD, was suggested as an alternative strategy. Furfural decreased cell growth and isobutanol production when only YqhD or FucO was employed as an isobutyraldehyde oxidoreductase. However, combinatorial overexpression of FucO and YqhD could overcome the inhibitory effect of furfural giving higher isobutanol production by 110% compared with overexpression of YqhD. The combinatorial oxidoreductases increased furfural detoxification rate 2.1-fold and also accelerated glucose consumption 1.4-fold. When it compares to another known system increasing furfural tolerance, membrane-bound transhydrogenase (pntAB), the combinatorial aldehyde oxidoreductases were better on cell growth and production. Thus, to control oxidoreductases is important to produce isobutanol using furfural-containing biomass and the combinatorial overexpression of FucO and YqhD can be an alternative strategy.

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillum rubrum.

Different genes encoding pyridine nucleotide transhydrogenases (pntAB, udhA) and acetoacetyl-CoA reductases (phaB) were heterologously overexpressed in Rhodospirillum rubrum S1. A recombinant strain, which harbored the gene encoding the membrane-bound transhydrogenase PntAB from Escherichia coli MG1655 and the phaB1 gene coding for an NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha H16, accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [Poly(3HB-co-3HV)] with a 3HV fraction of up to 13 mol% from fructose. This was a 13-fold increase of the 3HV content when compared to the wild-type strain. Higher contents of 3HV are known to reduce the brittleness of this polymer, which is advantageous for most applications. The engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO from artificial synthesis gas (syngas) with a 3HV content of 56 mol%. The increased incorporation of 3HV was attributed to an excess of propionyl-CoA, which was generated from threonine and related amino acids to compensate for the intracellular redox imbalance resulting from the transhydrogenase reaction. Thereby, our study presents a novel, molecular approach to alter the composition of bacterial PHAs independently from external precursor supply. Moreover, this study also provides a promising production strain for syngas-derived second-generation biopolymers.

Transhydrogenase promotes the robustness and evolvability of E. coli deficient in NADPH production.

Metabolic networks revolve around few metabolites recognized by diverse enzymes and involved in myriad reactions. Though hub metabolites are considered as stepping stones to facilitate the evolutionary expansion of biochemical pathways, changes in their production or consumption often impair cellular physiology through their system-wide connections. How does metabolism endure perturbations brought immediately by pathway modification and restore hub homeostasis in the long run? To address this question we studied laboratory evolution of pathway-engineered Escherichia coli that underproduces the redox cofactor NADPH on glucose. Literature suggests multiple possibilities to restore NADPH homeostasis. Surprisingly, genetic dissection of isolates from our twelve evolved populations revealed merely two solutions: (1) modulating the expression of membrane-bound transhydrogenase (mTH) in every population; (2) simultaneously consuming glucose with acetate, an unfavored byproduct normally excreted during glucose catabolism, in two subpopulations. Notably, mTH displays broad phylogenetic distribution and has also played a predominant role in laboratory evolution of Methylobacterium extorquens deficient in NADPH production. Convergent evolution of two phylogenetically and metabolically distinct species suggests mTH as a conserved buffering mechanism that promotes the robustness and evolvability of metabolism. Moreover, adaptive diversification via evolving dual substrate consumption highlights the flexibility of physiological systems to exploit ecological opportunities.

Increasing available NADH supply during succinic acid production by Corynebacterium glutamicum.

A critical factor in the biotechnological production of succinic acid with Corynebacterium glutamicum is the sufficient supply of NADH. It is conceivable that cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the succinic acid yield. PntAB genes from Escherichia coli can directly catalyze the reversible hydride transfer and adjust the dynamic balance between NADP(H) and NAD(H). Hence, we studied the physiological effect of coenzyme systems by expressing the membrane-bound transhydrogenase pntAB genes. We have shown experimentally that the pntAB genes could function as an alternative source of NADH. In an anaerobic fermentation with C. glutamicum NC-3-pntAB, a 16% higher succinic acid yield and a 57% higher production from glucose were obtained by pntAB expression. Moreover, the formation of by-products was significantly decreased. The concomitant increase in the consumption of intracellular NADPH from 0.6 to 1.2 mmol/g CDW and the increased NADH/NAD(+) ratio resulted from introduction of pntAB, suggesting that the membrane-bound transhydrogenase converted excess NADPH to NADH for succinic acid production. Finally, we explored whether the transhydrogenase had different effects on the succinic acid formation on different carbon sources. The succinic acid yield was increased in the presence of pntAB by 16% on glucose, 7% on sucrose, and without large influence on fructose and xylose. The results of this study demonstrated that the effectiveness of cofactor manipulation could be a promising strategy applied in metabolic engineering.

Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13C‐metabolic flux analysis

We engineered a fatty acid overproducing Escherichia coli strain through overexpressing tesA (“pull”) and fadR (“push”) and knocking out fadE (“block”). This “pull-push-block” strategy yielded 0.17 g of fatty acids (C12–C18) per gram of glucose (equivalent to 48% of the maximum theoretical yield) in batch cultures during the exponential growth phase under aerobic conditions. Metabolic fluxes were determined for the engineered E. coli and its control strain using tracer ([1,2-13C]glucose) experiments and 13C-metabolic flux analysis. Cofactor (NADPH) and energy (ATP) balances were also investigated for both strains based on estimated fluxes. Compared to the control strain, fatty acid overproduction led to significant metabolic responses in the central metabolism: (1) Acetic acid secretion flux decreased 10-fold; (2) Pentose phosphate pathway and Entner–Doudoroff pathway fluxes increased 1.5- and 2.0-fold, respectively; (3) Biomass synthesis flux was reduced 1.9-fold; (4) Anaplerotic phosphoenolpyruvate carboxylation flux decreased 1.7-fold; (5) Transhydrogenation flux converting NADH to NADPH increased by 1.7-fold. Real-time quantitative RT-PCR analysis revealed the engineered strain increased the transcription levels of pntA (encoding the membrane-bound transhydrogenase) by 2.1-fold and udhA (encoding the soluble transhydrogenase) by 1.4-fold, which is in agreement with the increased transhydrogenation flux. Cofactor and energy balances analyses showed that the fatty acid overproducing E. coli consumed significantly higher cellular maintenance energy than the control strain. We discussed the strategies to future strain development and process improvements for fatty acid production in E. coli.

Anaerobic Obligatory Xylitol Production inEscherichia coliStrains Devoid of Native Fermentation Pathways

Anaerobic glucose oxidation was coupled to xylose reduction in a nonfermentative Escherichia coli strain expressing NADPH-dependent xylose reductase. Xylitol production serves as the primary means of NAD(P)(+) regeneration, as glucose is converted primarily to acetate and CO(2). The membrane-bound transhydrogenase PntAB is required to achieve the maximum theoretical yield of four moles of xylitol per mole of glucose consumed.

Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism

To sustain growth, the catabolic formation of the redox equivalent NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on 13C-detected intracellular fluxes, metabolite concentrations, and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH production in Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, and Zymomonas mobilis. For six species, the estimated NADPH production from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions were P. fluorescens, with balanced rates, and E. coli, with insufficient catabolic production, in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB. P. versutus and B. subtilis were the only species that appear to rely on transhydrogenases for balancing NADPH overproduction during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, except E. coli and B. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+ dependent.

Analysis of NADPH supply during xylitol production by engineered Escherichia coli

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.

Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum

Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol) −1, whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol) −1. Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol) −1. A further increase is possible by the application of additional C 1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C 1 carbon and sulfur (0.91 (C-mol) (C-mol) −1). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g −1 h −1, as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g −1 h −1. The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer.

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.