Production of 2,3‐butanediol by Klebsiella oxytoca from various sugars in microalgal hydrolysate

A new fermentation process using a mixed sugar medium is proposed in this study for 2,3‐butanediol (2,3‐BDO) production. The medium contained seven different monosugars known to be present in Nannochloropsis oceanica hydrolysate. The performance of each sugar when existing alone or together with glucose was evaluated. All the sugars except fucose were successfully metabolized for 2,3‐BDO production. A 2,3‐BDO yield of 0.31g/g was achieved with the mixed sugar medium, which was very close to that with the glucose‐only medium. However, the 2,3‐BDO productivity (0.28 g L^?1 h^?1) was found to be about 30% lower than that with glucose, implying, as expected, the existence of glucose repression on the uptake of other sugars. Strain development is in need to remove such negative effect of glucose for improved process efficiency. Fucose with the lowest uptake rate and no contribution to 2,3‐BDO production can be a high value‐added byproduct, once recovered and purified. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1669–1675, 2015     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

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.     

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Enhanced direct ethanol production by cofactor optimization of cell surface‐displayed xylose isomerase in yeast

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d ‐xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC‐displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co²⁺) especially enhanced the activity by 46‐fold. Co²⁺ also contributed to d ‐xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0‐ and 2.7‐fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC‐displaying yeast showed the faster d ‐xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co‐culture system. A xylan‐degrading yeast strain was established by displaying two kinds of xylanases; endo‐1,4‐β‐xylanase (Xyn11B) from Saccharophagus degradans, and β‐xylosidase (XlnD) from Aspergillus niger. The yeast co‐culture system enabled fine‐tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)‐displaying yeast and XylC‐displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10¹³: 1.39 × 10¹³: 2.78 × 10¹³ molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co‐culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068–1076, 2017     

Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains

ABSTRACT: BACKGROUND: The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from Escherichia coli C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent simultaneous utilization of the sugars. RESULTS: In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain E. coli C ptsG manZ glk crr xylA araA) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three E. coli strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the crr deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15 % compared to the wild-type. Similarly, the araA deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption. CONCLUSIONS: Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.     

Lactobacillus pentosus CECT 4023 T co-utilizes glucose and xylose to produce lactic acid from wheat straw hydrolysate: Anaerobiosis as a key factor

Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analyzed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate. The highest lactic acid yields based on present sugar, around 0.80 g g⁻¹, were obtained from glucose in presence of 25%, 50%, and 75% v v⁻¹ of prehydrolysate in strict anaerobiosis. Lactic fermentation of wheat straw hydrolysate obtained after enzymatic hydrolysis of the prehydrolysate yielded 0.39 g of lactic acid per gram of released sugars, which demonstrated the high potential of L. pentosus to produce lactic acid from hemicellulosic hydrolysates. Results presented herein not only corroborated the ability of L. pentosus to grow using mixtures of sugars, but also demonstrated the suitability of this strain to be applied as an efficient lactic acid producer in a lignocellulosic biorefinery approach. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2739, 2019     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

Construction of industrial xylose-fermenting Saccharomyces cerevisiae strains through combined approaches

Saccharomyces cerevisiae strain with excellent xylose-fermenting capacity and inhibitor tolerance is crucial for lignocellulosic ethanol production. In this study, a combined strategy including site-directed mutagenesis, mating, evolutionary engineering, and haploidization was applied to obtain strains with ideal xylose fermentabilities. Haploid industrial strain KFG4-6B was engineered to overexpress endogenous xylulokinase (XK) and heterologous native or mutated xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis. The XR-mutated strain HX57D showed over 12% increase in both xylose consumption rate and ethanol yield compared with the XR-native strain. To improve the xylose uptake, the HX57D-derived diploids were subjected to evolutionary engineering. In comparison with HX57D, evolved diploid Z4X-21-18 achieved 4.5-fold increases in rates of xylose consumption and ethanol production when fermenting xylose. When fermenting mixed sugars, the glucose and xylose uptake rates were 1.4-fold and 8.3-fold, respectively, higher. H18s28, a haploid of Z4X-21-18, enabled a further 10% increase in xylose consumption rate when fermenting xylose only. However, it was inferior to its diploid parent when fermenting mixed sugars. In the presaccharification-simultaneous saccharification and fermentation (P-SSF) of the whole pretreated wheat straw slurry with high contents of multiple inhibitors, Z4X-21-18 produced approximately 42 g/L ethanol with a yield of 0.38 g/g total sugars.     

A substrate-selective co-fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.     

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr⁻¹. The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr⁻¹, strain QU 50 produced 42.6 g L⁻¹ of l -LA with a yield of 0.912 g g⁻¹-consumed sugars, LA yield of 0.655 g g⁻¹ based on mixed sugar-loaded, and a productivity of 4.31 g L⁻¹ hr⁻¹ from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L⁻¹ with LA yield of 0.944 g g⁻¹-consumed sugar and LA productivity of 6.49 ± 0.357 g L⁻¹ hr⁻¹. Besides this, a dramatic increase in LA yield of 0.927 g g⁻¹ based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.