Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.     

Engineering Escherichia coli for xylitol production from glucose-xylose mixtures

The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (approximately 275 mM in LB cultures, approximately 180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, DeltaxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of approximately 250 mM xylitol (38 g/L), with concomitant utilization of approximately 150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to > 3.7:1 using metabolically active "resting" cells.     

Role of xylose transporters in xylitol production from engineered Escherichia coli

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.     

Anaerobic obligatory xylitol production in Escherichia coli strains 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.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose

Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD(+) during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L(-1) biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L(-1) biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-(13)C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.     

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg?1 protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding α-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway.     

Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05

Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose ? 2 acetyl-CoA + 4 NADH ? 2 ethanol) in the engineered strain, Escherichia coli SZ420 (?frdBC ?ldhA ?ackA ?focA-pflB ?pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (Y_RPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual Y_RPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.     

Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol⁻¹, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.     

Production of xylitol from D-xylose by recombinant Lactococcus lactis

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.     

Heterologous expression of D-xylulokinase from Pichia stipitis enables high levels of xylitol production by engineered Escherichia coli growing on xylose

Deletion of the Escherichia coli xylulokinase gene (xylB) is essential for achieving high xylitol titers from xylitol-producing E. coli strains growing on glucose in the presence of xylose. Our study suggests that this is due to XylB-catalyzed toxic synthesis of xylitol-phosphate. This activity prohibits the use of xylose as the sole carbon source during xylitol production by E. coli. To overcome this limitation we turned to the yeast Pichia stipitis, which naturally produces xylitol, as a source of xylulokinase (Xyl3). We examined the effects of plasmid-based expression of Xyl3 versus XylB on growth and xylitol production by engineered E. coli strains. Xylulokinase activity assays show similar levels of functional expression of both enzymes (determined as activity on xylulose), and reveal significantly more activity on xylitol by XylB compared to Xyl3. (31)P NMR confirms the production of xylitol-phosphate from in vitro reactions with XylB. Lastly, the replacement of xylB with XYL3 results in drastically enhanced xylitol titers from E. coli strains co-expressing xylose reductase during growth on xylose.     

Metabolic engineering and transhydrogenase effects on NADPH availability in escherichia coli

The synthesis of several industrially useful compounds are cofactor‐dependent, requiring reducing equivalents like NADPH in enzymatic reactions leading up to the synthesis of high‐value compounds like polymers, chiral alcohols, and antibiotics. However, NADPH is costly and has limited intracellular availability. This study focuses on the study of the effect of the two transhydrogenase enzymes of Escherichia coli, PntAB and UdhA (SthA) on reducing equivalents‐dependent biosynthesis. The production of (S)‐2‐chloropropionate from 2‐chloroacrylate is used as a model system for monitoring NADPH availability because 2‐haloacrylate reductase, the enzyme catalyzing the one‐step conversion to (S)‐2‐chloropropionate in the synthesis pathway, requires NADPH as a cofactor. Results suggest that the presence of UdhA increases product yield and NADPH availability while the presence of PntAB has the opposite effect. A maximum product yield of 1.4 mol product/mol glucose was achieved aerobically in a pnt‐deletion strain with udhA overexpression, a 150% improvement over the wild‐type control strain. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1124–1130, 2013     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Adaptation yields a highly efficient xylose-fermenting Zymomonas mobilis strain

Zymomonas mobilis is a superb ethanol producer with productivity exceeding yeast strains by several fold. Although metabolic engineering was successfully applied to expand its substrate range to include xylose, xylose fermentation lagged far behind glucose. In addition, xylose fermentation was often incomplete when its initial concentration was higher than 5%. Improvement of xylose fermentation is therefore necessary. In this work, we applied adaptation to improve xylose fermentation in metabolically engineered strains. As a result of adaptation over 80 days and 30 serial transfers in a medium containing high concentration of xylose, a strain, referred as A3, with markedly improved xylose metabolism was obtained. The strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, a total of 9% (w/v) ethanol was obtained from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Further investigation reveals evidence for an altered xylitol metabolism in A3 with reduced xylitol formation. Additionally xylitol tolerance in A3 was increased. Furthermore, xylose isomerase activity was increased by several times in A3, allowing cells to channel more xylose to ethanol than to xylitol. Taken together, these results strongly suggest that altered xylitol metabolism is key to improved xylose metabolism in adapted A3 strain. This work further demonstrates that adaptation and metabolic engineering can be used synergistically for strain improvement.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

辅酶工程在酿酒酵母木糖代谢工程中的研究进展

辅酶工程（cofactor engineering）是代谢工程的一个重要分支,它通过改变辅酶的再生途径,达到改变细胞内代谢产物构成的目的。介绍了酿酒酵母(Saccharomyces cerevisiae)木糖代谢工程中,利用辅酶工程解决氧化还原平衡问题的研究进展,包括引入转氢酶系统,增加代谢中可利用的NADPH,实现NADH的厌氧氧化等策略。同时介绍了改变XR、XDH辅酶偏好的研究进展。     

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.