Xylose Consumption Rate Research Articles

Experimental evolution reveals an effective avenue for d-lactic acid production from glucose-xylose mixtures via enhanced Glk activity and a cAMP-independent CRP mutation.

d-Lactic acid holds significant industrial importance due to its versatility and serves as a crucial component in the synthesis of environmentally friendly and biodegradable thermal-resistant poly-lactic acid. This polymer exhibits promising potential as a substitute for nonbiodegradable, petroleum-based plastics. The production of d-lactic acid from lignocellulosic biomass, a type of biorenewable and nonfood resources, can lower costs and improve product competitiveness. Glucose and xylose are the most abundant sugar monomers in lignocellulosic biomass materials. Despite Escherichia coli possessing native xylose catabolic pathways and transport, their ability to effectively utilize xylose is often hindered in the presence of glucose. Here, the E. coli strain Rec1.0, previously engineered to overcome carbon catabolite repression, was selected as the initial strain for reengineering to produce d-lactic acid. An adaptive evolution approach was employed to achieve highly efficient fermentation of glucose-xylose mixtures. The resulting strain, QJL010, could produce d-lactic acid of 87.5 g/L with a carbon yield of 0.99 mol/mol. Notably, the consumption rates of glucose and xylose reached 0.75 and 0.82 g/gDCW/h, respectively. Further analysis revealed that increased Glk activity, resulting from glk mutations (A142V and R188H), along with their upregulated expression, contributed to an elevated glucose consumption rate. Additionally, a CRP G141D mutation, cAMP-independent, stimulated the expression of the xylR, xylE, and galABC* genes, resulting in an accelerated xylose consumption rate. These findings provide valuable support for the utilization of E. coli platform strains in the production of value-added chemicals from lignocellulosic biomass.

Construction and characterization of a promoter library with varying strengths to enhance acetoin production from xylose in Serratia marcescens.

Serratia marcescens is utilized as a significant enterobacteria in the production of various high-value secondary metabolites. Acetoin serves as a crucial foundational compound of development and finds application in a broad range of fields. Furthermore, S. marcescens HBQA-7 is capable of utilizing xylose as its exclusive carbon source for acetoin production. The objective of this study was to utilize a constitutive promoter screening strategy to enhance both xylose utilization and acetoin production in S. marcescens HBQA-7. By utilizing RNA-seq, we identified the endogenous constitutive promoter P6 that is the most robust, which facilitated the overexpression of the sugar transporter protein GlfL445I , α-acetyl lactate synthase, and α-acetyl lactate decarboxylase, respectively. The resultant recombinant strains exhibited enhanced xylose utilization rates and acetoin yields. Subsequently, a recombinant plasmid, denoted as pBBR1MCS-P6-glfL445I alsSalsD, was constructed, simultaneously expressing the aforementioned three genes. The resulting recombinant strain, designated as S3, demonstrated a 1.89-fold boost in xylose consumption rate compared with the original strain during shake flask fermentation. resulting in the accumulation of 7.14g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75g/L, corresponding to a xylose-to-acetoin conversion yield of 0.375g/g.

Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

Construction of Bacillus subtilis for efficient production of fengycin from xylose through CRISPR-Cas9.

Fengycin is a multifunctional peptide antibiotic produced mainly by Bacillus species and the purpose of this research was to construct a Bacillus subtilis strain that can produce fengycin with the xylose as the substrate with CRSIPR-Cas9. Hence, at the beginning of this study, functional sfp and degQ were expressed in B. subtilis 168 strain to give the strain the ability to produce the fengycin with the titer of 71.21 mg/L. Subsequently, the native promoter PppsA of the cluster responsible for the fengycin synthesis was replaced by the Pveg promoter, resulting in a further 5.22-fold increase in fengycin titer. To confer xylose utilization capacity to B. subtilis, deletion of araR and constitutive overexpression of araE were performed, and the xylose consumption rate of the engineered strain BSUY06 reached 0.29 g/L/h, which is about 6.25-fold higher than that of the parent strain BSUY04-1. In the final phase of this study, the fermentation characteristics were observed and the initial xylose concentration was optimized. In this study, 40 g/L xylose was proved to be the most suitable initial concentration for growth and fengycin fermentation, which leading to a fengycin titer of 430.86 mg/L. This study demonstrated that lignocellulose, the clean and sustainable substrate with xylose as the second largest sugar, is a potential substrate for the production of fengycin.

Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

Isolation of xylose-utilizing yeasts from oil palm waste for xylitol and ethanol production

The energy crisis triggers the use of energy sources that are renewable, such as biomass made from lignocellulosic materials, to produce various chemical compounds for food ingredients and biofuel. The efficient conversion of lignocellulosic biomass into products with added value involves the activity of microorganisms, such as yeasts. For the conversion, microorganisms must be able to use various sugars in lignocellulosic biomass, including pentose sugars, especially xylose. This study aims to isolate xylose-utilizing yeasts and analyze their fermentation activity to produce xylitol and ethanol, as well as their ability to grow in liquid hydrolysate produced from pretreated lignocellulosic biomass. Nineteen yeast isolates could grow on solid and liquid media using solely xylose as a carbon source. All isolates can grow in a xylose medium with incubation at 30 °C, 37 °C, 42 °C, and 45 °C. Six isolates, namely SLI (1), SL3, SL6, SL7, R5, and OPT4B, were chosen based on their considerable growth and high xylose consumption rate in a medium with 50 g/L xylose with incubation at 30 °C for 48 h. Four isolates tested, namely SLI (1), SL6, SL7, and R5, can produce xylitol in media containing xylose carbon sources. The concentration of xylitol produced was determined using high-pressure liquid chromatography (HPLC), and the results ranged from 5.0 to 6.0 g/L. Five isolates tested, namely SLI (1), SL6, SL3, R5, and OPT4B, can produce ethanol. The ethanol content produced was determined using gas chromatography (GC), with concentrations ranging from 0.85 to 1.34 g/L. Three isolates, namely SL1(1), R5, and SL6, were able to produce xylitol and ethanol from xylose as carbon sources and were also able to grow on liquid hydrolyzate from pretreated oil palm trunk waste with the subcritical water method. The three isolates were further analyzed using the 18S rDNA sequence to identify the species and confirm their phylogenetic position. Identification based on DNA sequence analysis revealed that isolates SL1(1) and R5 were Pichia kudriavzevii, while isolate SL6 was Candida xylopsoci. The yeast strains isolated from this study could potentially be used for the bioconversion process of lignocellulosic biomass waste to produce value-added derivative products.Graphical

Engineering Thermoanaerobacterium aotearoense SCUT27 with the deficiency of a hypothetic protein regulated by ArgR1864 for enhanced ethanol production from lignocellulosic hydrolysates

Arginine repressor (ArgR1864) is a multifunctional regulator in Thermoanaerobacterium aotearoense SCUT27 (SCUT27). For better understanding its function in SCUT27, the transcriptome of SCUT27/ΔargR1864 under xylose was further investigated. 2014 (V518_2014) denoted as a hypothetic protein gene was mined, which played important roles on xylose metabolism and carbon metabolic flux distribution in SCUT27. In 2014 deletion mutant, no lactic acid could be produced along with enhanced ethanol formation. Same changes were also observed in SCUT27/ΔargR1864. ArgR1864 is the direct regulator of 2014 identified by electrophoretic mobility shift assays (EMSA). When 2014 was complemented in SCUT27/ΔargR1864, the fermentation traits of SCUT27/ΔargR1864/2014 were returned to wild-type levels, suggesting that the improved xylose utilization and ethanol formation of SCUT27/ΔargR1864 were attributed to the low expression of 2014. When cultured under sorghum stalk, corn cob and wheat straw hydrolysates, SCUT27/Δ2014 also showed the excellent capability for sugar utilization and ethanol production with the consumption rate of xylose and glucose increased by 20%–37.93 and 64.29%–190.91% separately, the ethanol production improved by 326.61%–547.89% and ethanol yield enhanced by 216.67%–337.50%. Thus, 2014, as a hypothetic protein gene, could be used as the new genetic reference to predict functional genes to engineer the microorganism for biofuel production from lignocellulosic hydrolysates.

Co-Fermentation of Glucose-Xylose Mixtures from Agroindustrial Residues by Ethanologenic Escherichia coli: A Study on the Lack of Carbon Catabolite Repression in Strain MS04.

The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.

The role of hexose transporter-like sensor hxs1 and transcription activator involved in carbohydrate sensing azf1 in xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha

BackgroundFuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied.ResultsWe isolated an insertional mutant of O.polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O.polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O.polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20–40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains.ConclusionsThis paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.

Adaptation on xylose improves glucose–xylose co-utilization and ethanol production in a carbon catabolite repression (CCR) compromised ethanologenic strain

BackgroundSugar hydrolysates from lignocellulosic biomass are majorly composed of glucose and xylose that can be fermented to biofuels. Bacteria, despite having the natural ability to consume xylose are unable to consume it in presence of glucose due to a carbon catabolite repression (CCR) mechanism. This leads to overall reduced productivity as well as incomplete xylose utilization due to ethanol build-up from glucose utilization. In our effort to develop a strain for simultaneous fermentation of glucose and xylose into ethanol, we deleted ptsG in ethanologenic E. coli SSK42 to make it deficient in CCR and performed adaptive laboratory evolution to achieve accelerated growth rate, sugar consumption and ethanol production. Finally, we performed proteomics study to identify changes that might have been responsible for the observed improved phenotype of the evolved strain.ResultsThe parental strain of SSK42, i.e., wild-type E. coli B, did not co-utilize glucose and xylose as expected. After deleting the ptsG gene encoding the EIIBCGlc subunit of PTS system, glucose consumption is severely affected in wild-type E. coli B. However, the ethanologenic, SSK42 strain, which was evolved in our earlier study on both glucose and xylose, didn’t show such a drastic effect of EIIBCGlc deletion, instead consumed glucose first, followed by xylose without delay for switching from one sugar to another. To improve growth on xylose and co-utilization capabilities, the ptsG deleted SSK42 was evolved on xylose. The strain evolved for 78 generations, strain SCD78, displayed significant co-utilization of glucose and xylose sugars. At the bioreactor level, the strain SCD78 produced 3-times the ethanol titer of the parent strain with significant glucose–xylose co-utilization. The rate of glucose and xylose consumption also increased 3.4-fold and 3-fold, respectively. Proteome data indicates significant upregulation of TCA cycle proteins, respiration-related proteins, and some transporters, which may have a role in increasing the total sugar consumption and co-utilization of sugars.ConclusionThrough adaptive evolution, we have obtained a strain that has a significant glucose–xylose co-utilization phenotype with 3-fold higher total sugar consumption rate and ethanol production rate compared to the unevolved strain. This study also points out that adaptation on xylose is enough to impart glucose–xylose co-utilization property in CCR compromised ethanologenic strain SSK42.

Genomic insights to facilitate the construction of a high-xylose-utilization Enterococcus faecalis OPS2 for 2,3-BDO production

Sustainable bioconversion of plant biomass sugars for 2,3-butanediol (2,3-BDO) production is a carbon-neutral practice, however, its economic feasibility has been hindered by inefficient utilization of xylose. In this study, a novel Enterococcus faecalis strain OPS1 was isolated from palm oil-polluted soil, which assimilates xylose for 2,3-BDO production. While this wild-type strain OPS1 was suppressed with an increase in xylose concentration (60 g/L), strain OPS2, constructed using an adaptive laboratory evolution, achieved a desirable 2,3-BDO production with high-xylose (>100 g/L) tolerance. Whole-genome sequencing analysis reconstructed the metabolic pathways facilitating xylose utilization and 2,3-BDO production in these two novel strains that evolved within from a lactic acid-producing genus Enterococcus. A rpiB gene encoded for ribose-5-phosphate isomerase B enzyme of xylose metabolism in these two strains was identified to be horizontally acquired from a different family Enterobacteriaceae. The detection of a bacteriocin gene UviB and an antitoxin gene hicB in strain OPS1 and OPS2 which are absent in the E. faecalis type strain indicates their beneficial probiotic and antitoxin features. A mutation in uge (UDP-xylose 4-epimerase) was identified between strain OPS1 and OPS2 which may cater to the high-xylose stress. The evolved E. faecalis OPS2 showed a 3-fold increase in xylose consumption rate (2.19 g/L/h) with 0.67 g/L/h 2,3-BDO productivity than OPS1. For the first time, a maximum of 65.3 g/L 2,3-BDO was successfully produced via fed batch xylose fermentation. These results provide the genomic insights to the xylose regulatory metabolism in Enterococci and pave the way for industrial implication as a microbial biocatalyst in a 2,3-BDO biorefinery.

Cell Immobilization Using Alginate-Based Beads as a Protective Technique against Stressful Conditions of Hydrolysates for 2G Ethanol Production.

The development of biorefineries brings the necessity of an efficient consumption of all sugars released from biomasses, including xylose. In addition, the presence of inhibitors in biomass hydrolysates is one of the main challenges in bioprocess feasibility. In this study, the application of Ca-alginate hybrid gels in the immobilization of xylose-consuming recombinant yeast was explored with the aim of improving the tolerance of inhibitors. The recombinant yeast Saccharomyces cerevisiae GSE16-T18SI.1 (T18) was immobilized in Ca-alginate and Ca-alginate–chitosan hybrid beads, and its performance on xylose fermentation was evaluated in terms of tolerance to different acetic acid concentrations (0–12 g/L) and repeated batches of crude sugarcane bagasse hemicellulose hydrolysate. The use of the hybrid gel improved yeast performance in the presence of 12 g/L of acetic acid, achieving 1.13 g/L/h of productivity and reaching 75% of the theoretical ethanol yield, with an improvement of 32% in the xylose consumption rate (1:1 Vbeads/Vmedium, 35 °C, 150 rpm and pH 5.2). The use of hybrid alginate–chitosan gel also led to better yeast performance at crude hydrolysate, yielding one more batch than the pure-alginate beads. These results demonstrate the potential of a hybrid gel as an approach that could increase 2G ethanol productivity and allow cell recycling for a longer period.

Production of Xylitol from Corn Biomass using Candida sp. As Microbial Agent

Xylitol, C5H12O5, is a white and odorless crystalline powder of sweetening agents that included as low-calorie sweetener. It could be used as a healthy ingredient for food and pharmaceutical. Natural sources of xylitol are fruit and vegetable, even in minute quantities. At industrial scale, xylitol is produced through hydrolysis and hydrogenation process of lignocellulosic materials. The aim of this research was to study the production of xylitol from corn biomass using Candida sp. as a microbial agent. The research was conducted using different species of Candida sp. (C. guilliermondii and C. tropicalis) and supplement media growth (with or without glucose). Xylitol concentration was examined after fermentation for 3 and 5 days. The results showed that corncob is a promising material to use in producing xylitol from lignocellulosic biomass. The longer fermentation time, the higher xylitol concentration, ranged from 0.049 to 0.088 g/L. However, compare to another microbe species, the treatment using Candida tropicalis showed that long fermentation resulted in a lower xylitol concentration. The addition of glucose as co-substrate increased xylose consumption rate and xylitol productivity. These results provide useful information to develop further study about xylitol production using agricultural biomass.

Physiological comparisons among Spathaspora passalidarum, Spathaspora arborariae, and Scheffersomyces stipitis reveal the bottlenecks for their use in the production of second-generation ethanol.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44g/g) and productivity (1.02g/L.h) were obtained using Sp. passalidarum grown in 100g/L xylose at 32°C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells.

Conversion of xylose to bioproducts on bifunctional supported platinum-group metals catalysts

Nb2O5-supported platinum-group metals catalysts were investigated in the one-pot aqueous-phase isomerization-dehydration-hydrogenation cascade conversion of xylose. The interplay between Nb2O5 acid sites and noble metal particles was discussed with a particular focus on catalyst stability. Pt/Nb2O5 was the best-performing catalyst for xylose upgrading regarding xylose consumption rates and high selectivity to furfuryl alcohol. It was chemically stable as no metal leaching to the aqueous phase was observed. Deposition of carbonaceous materials was identified as the main cause of deactivation and it disturbed the Brønsted acid sites in a more significant way. The impact of choosing an organic co-solvent was also demonstrated. Catalytic activity and distribution of bioproducts were determined by the organic solvent:water mixture. While polarity influenced activity, intermolecular interactions were suggested to tune selectivity.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

BackgroundEfficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.ResultsHeterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.ConclusionsThis study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

BackgroundAs the second most abundant polysaccharide in nature, hemicellulose can be degraded to xylose as the feedstock for bioconversion to fuels and chemicals. To enhance xylose conversion, the engineered Saccharomyces cerevisiae with xylose metabolic pathway is usually adapted with xylose as the carbon source in the laboratory. However, the mechanism under the adaptation phenomena of the engineered strain is still unclear.ResultsIn this study, xylose-utilizing S. cerevisiae was constructed and used for the adaptation study. It was found that xylose consumption rate increased 1.24-fold in the second incubation of the yYST12 strain in synthetic complete-xylose medium compared with the first incubation. The study figured out that it was observed at the single-cell level that the stagnation time for xylose utilization was reduced after adaptation with xylose medium in the microfluidic device. Such transient memory of xylose metabolism after adaptation with xylose medium, named “xylose consumption memory”, was observed in the strains with both xylose isomerase pathway and xylose reductase and xylitol dehydrogenase pathways. In further, the proteomic acetylation of the strains before and after adaptation was investigated, and it was revealed that H4K5 was one of the most differential acetylation sites related to xylose consumption memory of engineered S. cerevisiae. We tested 8 genes encoding acetylase or deacetylase, and it was found that the knockout of the GCN5 and HPA2 encoding acetylases enhanced the xylose consumption memory.ConclusionsThe behavior of xylose consumption memory in engineered S. cerevisiae can be successfully induced with xylose in the adaptation. H4K5Ac and two genes of GCN5 and HPA2 are related to xylose consumption memory of engineered S. cerevisiae during adaptation. This study provides valuable insights into the xylose adaptation of engineered S. cerevisiae.

Exploring metal ion metabolisms to improve xylose fermentation in Saccharomycescerevisiae.

SummaryThe development of high‐performance xylose‐fermenting yeast is essential to achieve feasible conversion of biomass‐derived sugars in lignocellulose‐based biorefineries. However, engineered C5‐strains of Saccharomyces cerevisiae still present low xylose consumption rates under anaerobic conditions. Here, we explore alternative metabolisms involved in metal homeostasis, which positively affect C5 fermentation and analyse the non‐obvious regulatory network connection of both metabolisms using time‐course transcriptome analysis. Our results indicated the vacuolar Fe2+/Mn2+ transporter CCC1, and the protein involved in heavy metal ion homeostasis BSD2, as promising new targets for rational metabolic engineering strategies, enhancing xylose consumption in nine and 2.3‐fold compared with control. Notably, intracellular metal concentration levels were affected differently by mutations and the results were compared with positive controls isu1Δ, a Fe‐S cluster scaffold protein, and ssk2Δ, a component of HOG pathway. Temporal expression profiles indicate a metabolic remodelling in response to xylose, demonstrating changes in the main sugar sensing signalling pathways.

Metabolic and Transcriptional Analysis of Recombinant Saccharomyces Cerevisiae for Xylose Fermentation: A Feasible and Efficient Approach

Lignocellulose is an abundant xylose-containing biomass found in agricultural wastes, and has arisen as a suitable alternative to fossil fuels for the production of bioethanol. Although Saccharomyces cerevisiae has been thoroughly used for the production of bioethanol, its potential to utilize lignocellulose remains poorly understood. In this work, xylose-metabolic genes of Pichia stipitis and Candida tropicalis, under the control of different promoters, were introduced into S. cerevisiae. RNA-seq analysis was use to examine the response of S. cerevisiae metabolism to the introduction of xylose-metabolic genes. The use of the PGK1 promoter to drive xylitol dehydrogenase (XDH) expression, instead of the TEF1 promoter, improved xylose utilization in "XR-pXDH" strain by overexpressing xylose reductase (XR) and XDH form C. tropicalis, enhancing the production of xylitol (13.66 ± 0.54 g/L after 6 days fermentation). Overexpression of xylulokinase and XR/XDH from P. stipitis remarkably decreased xylitol accumulation (1.13 ± 0.06 g/L and 0.89 ± 0.04 g/L xylitol, respectively) and increased ethanol production (196.14 % and 148.50 % increases during the xylose utilization stage, respectively), in comparison with the results of XR-pXDH. This result may be produced due to the enhanced xylose transport, Embden-Meyerhof and pentose phosphate pathways, as well as alleviated oxidative stress. The low xylose consumption rate in these recombinant as well as alleviated strains comparing with P. stipitis and C. tropicalis may be explained by the insufficient supplementation of NADPH and NAD +. The results obtained in this work provide new insights on the potential utilization of xylose using bioengineered S. cerevisiae strains.

Understanding the differences in 2G ethanol fermentative scales through omics data integration.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.