Glucose-xylose Mixture Research Articles

Metabolic engineering of an E. coli ndh knockout strain for PHB production from mixed glucose–xylose feedstock

AbstractBACKGROUNDPoly(3‐hydroxybutyrate) (PHB), which is completely biodegradable, is considered a potential candidate to replace a number of petroleum‐derived polymers due to similar mechanical properties. In a previous study, inactivation of ndh gene in E. coli, which encodes the NDH‐II dehydrogenase, resulted in significantly increased PHB production from either glucose or xylose as substrate.RESULTSIn this study, the xylose isomerase (EC:5.3.1.5), xylulokinase (EC:2.7.1.17) and the arabinose/xylose transport protein from Bacillus subtilis 168 (encoded by xylA, xylB and araE, respectively) were co‐expressed in the ndh knockout strain, E. coli LJ03(pBHR68), which harbors the PHB biosynthesis genes from Ralstonia eutropha. The resulting strain E. coli LJ03(pBHR68+pM‐ABE) was able to simultaneously utilize glucose and xylose to accumulate PHB. In flask cultivation, 3.67 g L−1 PHB was produced from a glucose–xylose mixture (10 g L−1 glucose and 5 g L−1 xylose), which was 2.09‐fold higher than the production of the control strain E. coli JM109(pBHR68+pM‐ABE). Ultimately, PHB production in fed‐batch fermentation reached a maximum titer of 21.0 g L−1, representing a 1.93‐fold increase relative to the control strain.CONCLUSIONResults indicated that the engineered E. coli LJ03 strain is significantly more efficient than the parent strain E. coli JM109 in producing PHB from mixed glucose–xylose feedstock. To the best of our knowledge, this is the first study describing the implementation of an exogenous xylose utilization pathway in E. coli for the production of PHB from mixed glucose–xylose feedstock – a model of lignocellulosic hydrolysate. © 2017 Society of Chemical Industry

A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures

Efficient production of poly-3-hydroxybutyrate (P(3HB)) based on glucose-xylose mixtures simulating different types of lignocellulosic hydrolysate (LCH) was addressed using Burkholderia sacchari, a wild strain capable of metabolizing both sugars and producing P(3HB). Carbon catabolite repression was avoided by maintaining glucose concentration below 10g/L. Xylose concentrations above 30g/L were inhibitory for growth and production. In fed-batch cultivations, pulse size and feed addition rate were controlled in order to reach high productivities and efficient sugar consumptions. High xylose uptake and P(3HB) productivity were attained with glucose-rich mixtures (glucose/xylose ratio in the feed=1.5w/w) using high feeding rates, while with xylose-richer feeds (glucose/xylose=0.8w/w), a lower feeding rate is a robust strategy to avoid xylose build-up in the medium. Xylitol production was observed with xylose concentrations in the medium above 30–40g/L. With sugar mixtures featuring even lower glucose/xylose ratios, i.e. xylose-richer feeds (glucose/xylose=0.5), xylonic acid (a second byproduct) was produced. This is the first report of the ability of Burkholderia sacchari to produce both xylitol and xylonic acid.

Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose-xylose mixture.

Background4-Hydroxymandelic acid (4-HMA) is a valuable aromatic fine chemical and widely used for production of pharmaceuticals and food additives. 4-HMA is conventionally synthesized by chemical condensation of glyoxylic acid with excessive phenol, and the process is environmentally unfriendly. Microbial cell factory would be an attractive approach for 4-HMA production from renewable and sustainable resources.ResultsIn this study, a biosynthetic pathway for 4-HMA production was constructed by heterologously expressing the fully synthetic 4-hydroxymandelic acid synthase (shmaS) in our l-tyrosine-overproducing Escherichia coli BKT5. The expression level of shmaS was optimized to improve 4-HMA production by fine tuning of four promoters of different strength combined with three plasmids of different copy number. Furthermore, two genes aspC and tyrB in the competitive pathway were deleted to block the formation of byproduct to enhance 4-HMA biosynthesis. The final engineered E. coli strain HMA15 utilized glucose and xylose simultaneously and produced 15.8 g/L of 4-HMA by fed-batch fermentation in 60 h.ConclusionsMetabolically engineered E. coli strain for 4-HMA production was designed and constructed, and efficiently co-fermented glucose and xylose, the major components in the hydrolysate mixture of agricultural biomass. Our research provided a promising biomanufacturing route to produce 4-HMA from lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0489-4) contains supplementary material, which is available to authorized users.

Ethanol production from xylose by Fusarium oxysporum and the optimization of culture conditions

Bioethanol is the most commonly used renewable biofuel as an alternative to fossil fuels. Many microbial strains can convert lignocellulosics into bioethanol. However, very few natural strains with a high capability of fermenting pentose sugars and simultaneously utilizing various sugars have been reported. In this study, fermentation of sugar by Fusarium oxysporum G was performed for the production of ethanol to improve the performance of the fermentation process. The influences of pH, substrate concentration, temperature, and rotation speed on ethanol fermentation are investigated. The three significant factors (pH, substrate concentration, and temperature) are further optimized by quadratic orthogonal rotation regression combination design and response surface methodology (RSM). The optimum conditions are pH 4, 40 g/L of xylose, 32 °C, and 110 rpm obtained through single factor experiment design. Finally, it is found that the maximum ethanol production (10.0 g/L) can be achieved after 7 d of fermentation under conditions of pH 3.87, 45.2 g/L of xylose, and 30.4 °C. Glucose is utilized preferentially for the glucose–xylose mixture during the initial fermentation stage, but glucose and xylose are synchronously consumed without preference in the second period. These findings are significant for the potential industrial application of this strain for bioethanol production.

In silico analysis of bioethanol overproduction by genetically modified microorganisms in coculture fermentation.

Lignocellulosic biomass is an attractive sustainable carbon source for fermentative production of bioethanol. In this context, use of microbial consortia consisting of substrate-selective microbes is advantageous as it eliminates the negative impacts of glucose catabolite repression. In this study, a detailed in silico analysis of bioethanol production from glucose-xylose mixtures of various compositions by coculture fermentation of xylose-selective Escherichia coli strain ZSC113 and glucose-selective wild-type Saccharomyces cerevisiae is presented. Dynamic flux balance models based on available genome-scale metabolic networks of the microorganisms have been used to analyze bioethanol production and the maximization of ethanol productivity is addressed by computing optimal aerobic-anaerobic switching times. A set of genetic engineering strategies for ethanol overproduction by E. coli strain ZSC113 have been evaluated for their efficiency in the context of batch coculture process. Finally, simulations are carried out to determine the pairs of genetically modified E. coli strain ZSC113 and S. cerevisiae that significantly enhance ethanol productivity in batch coculture fermentation.

Evaluation of Paecilomyces variotii potential in bioethanol production from lignocellulose through consolidated bioprocessing.

The ascomycete Paecillomyces variotii was evaluated for the first time as a candidate species for the production of bioethanol from lignocellulose through consolidated bioprocessing (CBP) approaches. The examined strain (ATHUM 8891) revealed all the necessary phenotypic characteristics required for 2nd generation biofuel production. The fungus is able to efficiently ferment glucose and xylose to ethanol, with yields close to the theoretical maximum. Nitrogen supplementation greatly affected ethanol production with nitrate-nitrogen presenting the best results. Notably, ethanol yield on xylose fermentation was higher than that of glucose, while in co-fermentation of glucose-xylose mixtures no distinguished diauxic behavior was observed. Furthermore, the fungus seems to possess the necessary enzyme factory for the degradation of lignocellulosic biomass, as it was able to grow and produce ethanol on common agro-industrial derivatives. Overall, the results of our study indicate that P. variotii is a new and possibly powerful candidate for CBP applications.

Dynamic flux balance analysis of batch fermentation: effect of genetic manipulations on ethanol production

In silico optimization of bioethanol production from lignocellulosic biomasses is investigated by combining process systems engineering approach and systems biology approach. Lignocellulosic biomass is an attractive sustainable carbon source for fermentative production of bioethanol. For enhanced ethanol production, metabolic engineering of wild-type strains-that can metabolize both hexose and pentose sugars or microbial consortia consisting of substrate-selective microbes-may be advantageous. This study presents a detailed in silico analysis of bioethanol production from glucose-xylose mixtures of various compositions by batch mono-culture and co-culture fermentation of specialized microbes. Dynamic flux balance models based on available genome-scale reconstructions of the microorganisms have been used to analyze bioethanol production, and the maximization of ethanol productivity is addressed by computing optimal aerobic-anaerobic switching times. Effects of ten metabolic engineering strategies that have been suggested in the literature for ethanol overproduction, have been evaluated for their efficiency in enhancing the ethanol productivity in the context of batch mono-culture and co-culture processes.

Systematic Approach To Engineer Escherichia coli Pathways for Co-utilization of a Glucose–Xylose Mixture

Glucose and xylose are two major sugars of lignocellulosic hydrolysate. The regulatory program of catabolite repression in Escherichia coli dictates the preferred utilization of glucose over xylose, which handicaps the development of the lignocellulose-based fermentation process. To co-utilize a glucose-xylose mixture, the E. coli strain was manipulated by pathway engineering in a systematic way. The approach included (1) blocking catabolite repression, (2) enhancing glucose transport, (3) increasing the activity of the pentose phosphate pathway, and (4) eliminating undesirable pathways. Moreover, the ethanol synthetic pathway from Zymomonas mobilis was introduced into the engineered strain. As a consequence, the resulting strain was able to simultaneously metabolize glucose and xylose and consume all sugars (30 g/L each) in 16 h, leading to 97% of the theoretical ethanol yield. Overall, this indicates that this approach is effective and straightforward to engineer E. coli for the desired trait.

Genetic engineering of yeasts to improve ethanol production from xylose

Saccharomyces cerevisiae, expressing xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes from Pichia stipitis, has the ability to produce ethanol from xylose. To improve ethanol production, three recombinant strains of F102X (fps1Δ), F104X (fps1Δ gpd2Δ) and F106X (fps1Δ gpd2Δ GLN1) with TKL1, TAL1, RKI1, RPE1, XYL1, XYL2 and XKS1 overexpression were constructed in this study. Compared to the control strain FB3X, ethanol production from xylose in strains F102X, F104X and F106X was increased by 4.3, 9.5 and 17.2%, respectively. F106X produced 18.2% less glycerol from a glucose–xylose mixture medium than FB3X, whereas no glycerol production was detected in these four strains when grown on a xylose medium. In addition, the xylitol production and ethanol yield in strain F106X did not differ significantly. These results suggested that the improvement in ethanol production in strain F106X was mainly due to increased xylose consumption.

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose-xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1h(-1). With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.

Bioconversion of wheat straw lignocellulosic sugars to ethanol by recombinant Escherichia coli

Recombinant microorganisms are a promising alternative for production of bioethanol from sugars produced from lignocellulosic materials. In the present work, recombinant Escherichia coli FBR16 has been utilized to produce bioethanol from simulated glucose-xylose mixtures and wheat straw hydrolysates. Hydrolysates were produced by sequential treatment of dilute acid pretreatment at 180 °C for 7 min using 0.5% (v/v) H2SO4 and enzymatic saccharification using cellulase from Trichoderma reesei and β-glucosidase from Aspergillus niger. With increased concentration of glucose-xylose sugar mixtures, ethanol yield and volumetric ethanol productivity decreased. At 22 g/l, ethanol yield of 0.34 g/g and volumetric ethanol productivity of 0.36 g/l·h were obtained which reduced to only 0.19 g/g and 0.17 g/l·h, respectively, at 160 g/l glucose-xylose sugar mixture. Fermentation kinetic parameters were also estimated and it was found that values of parameters were highly dependent on initial sugar concentration. Furthermore, it was observed that E. coli FBR16 is capable of producing bioethanol from almost all lignocellulosic monomeric sugars, especially glucose and xylose. At 16.4 g/l lignocellulosic hydrolysate concentration, ethanol yield of 0.32 g/g and productivity of 0.24 g/l·h were obtained. In order to see the effect of lignocellulosic sugar concentration on ethanol production, hydrolysates were concentrated to 50 g/l from the original concentration of 16.4 g/l. E. coli FBR16 was able to ferment the increased sugar concentration as well; however decreased ethanol yield of 0.29 g/g and volumetric ethanol productivity of 0.17 g/l·h were obtained.

Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production

The present work deals with the improvement of multiple stress tolerance in a glucose-xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L(-1) furfural and 8 g L(-1) acetic acid. The mutant also showed good growth at high temperature (39-40°C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39°C) and inhibitor stress (0.25 g L(-1) vanillin, 0.5 g L(-1) furfural and 4 g L(-1) acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L(-1)), thermal (39°C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L(-1) ethanol by utilizing 162.42 g L(-1) of glucose-xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L(-1) h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L(-1) ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L(-1) h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast.

Improved Bioethanol Production Using Fusants of Saccharomyces cerevisiae and Xylose-Fermenting Yeasts

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose-xylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8 ± 0.31 g L(-1)), ethanol productivity (1.06 g L(-1) h(-1)) and ethanol yield (0.458 g g(-1)) by fermentation of glucose-xylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08 ± 0.142 g L(-1), ethanol yield of 0.44 g g(-1), productivity of 0.45 g L(-1) h(-1) and biomass yield of 0.40 g g(-1).

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose–xylose mixtures

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

Adaptive Evolution of Escherichia coli Inactivated in the Phosphotransferase System Operon Improves Co-utilization of Xylose and Glucose Under Anaerobic Conditions

Modification of the phosphoenolpyruvate/sugar phosphotransferase system (PTS) has shown improvement in sugar coassimilation in Escherichia coli production strains. However, in preliminary experiments under anaerobic conditions, E. coli strains with an inactive PTS and carrying pLOI1594, which encodes pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, were unable to grow. These PTS⁻ strains were previously evolved under aerobic conditions to grow rapidly in glucose (PTS⁻ Glucose+ phenotype). Thus, in this work, applying a continuous culture strategy under anaerobic conditions, we generate a new set of evolved PTS⁻ Glucose+ mutants, VH30N1 to VH30N6. Contrary to aerobically evolved mutants, strains VH30N2 and VH30N4 carrying pLOI1594 grew in anaerobiosis; also, their growth capacity was restored in a 100%, showing specific growth rates (μ ~ 0.12 h⁻¹) similar to the PTS+ parental strain (μ = 0.11 h⁻¹). In cultures of VH30N2/pLOI1594 and VH30N4/pLOI1594 using a glucose-xylose mixture, xylose was totally consumed and consumption of sugars occurred in a simultaneous manner indicating that catabolic repression is alleviated in these strains. Also, the efficient sugar coassimilation by the evolved strains caused an increment in the ethanol yields.

Multi-stage continuous culture fermentation of glucose–xylose mixtures to fuel ethanol using genetically engineered Saccharomyces cerevisiae 424A

Multi-stage continuous (chemostat) culture fermentation (MCCF) with variable fermentor volumes was carried out to study the utilization of glucose and xylose for ethanol production via mixed sugar fermentation (MSF). Variable fermentor volumes were used to enable enhanced sugar utilization, accounting for differences in glucose and xylose utilization rates. Saccharomyces cerevisiae 424A-LNH-ST was used for fermentation of glucose–xylose mixtures. The dilution rates employed for continuous fermentation were based on earlier batch kinetic studies of ethanol production and sugar utilization. With a feed containing approximately 30 g L −1 glucose and 15 g L −1 xylose, cell washout was observed at a dilution rate of 0.8 h −1. At dilution rates below 0.5 h −1, complete glucose utilization was observed. Xylose consumption in the first-stage 1 L reactor was only 37% at the lowest dilution rate studied, 0.05 h −1. At this same flow rate, xylose consumption rose to 69% after subsequently passing through 3 and 1 L reactors in series, primarily due to the longer residence time in the 3 L reactor (0.0167 h −1 dilution rate).

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-basedSaccharomyces cerevisiaestrain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose-xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L(-1) acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L(-1)) in all cultures. Xylose fermentation in acetic -acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.

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.

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 (Δ 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 (Δ 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*.

Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on d-xylose

Overexpression of d-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Δ mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Δ mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose–xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Δ mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.