Structural and biochemical insights of xylose MFS and SWEET transporters in microbial cell factories: challenges to lignocellulosic hydrolysates fermentation.

The production of bioethanol from lignocellulosic biomass requires the efficient conversion of glucose and xylose to ethanol, a process that depends on the ability of microorganisms to internalize these sugars. Although glucose transporters exist in several species, xylose transporters are less common. Several types of transporters have been identified in diverse microorganisms, including members of the Major Facilitator Superfamily (MFS) and Sugars Will Eventually be Exported Transporter (SWEET) families. Considering that Saccharomyces cerevisiae lacks an effective xylose transport system, engineered yeast strains capable of efficiently consuming this sugar are critical for obtaining high ethanol yields. This article reviews the structure-function relationship of sugar transporters from the MFS and SWEET families. It provides information on several tools and approaches used to identify and characterize them to optimize xylose consumption and, consequently, second-generation ethanol production.

XylR regulates genes at xyl cluster, involved in D-xylose catabolism in Herbaspirillum seropedicae Z69.

D-xylose, one of the most abundant sugars in lignocellulosic biomass, is not widely used to produce bioproducts with added value, in part due to the absence of industrial microorganisms able to metabolize it efficiently. Herbaspirillum seropedicae Z69 is a ß-proteobacterium able to accumulate poly-3-hydroxybutyrate, a biodegradable thermoplastic biopolymer, with contents higher than 50%. It metabolizes D-xylose by non-phosphorylative pathways. In the genome of Z69, we found the genes xylFGH (ABC D-xylose transporter), xylB, xylD, and xylC (superior non-phosphorylative pathway), and the transcriptional regulator xylR, forming the xyl cluster. We constructed the knock-out mutant Z69ΔxylR that has a reduced growth in D-xylose and in D-glucose, compared with Z69. In addition, we analyzed the expression of xyl genes by RT-qPCR and promoter fusion. These results suggest that XylR activates the expression of genes at the xyl cluster in the presence of D-xylose. On the other hand, XylR does not regulate the expression of xylA, mhpD (lower non-phosphorylative pathways) and araB (L-arabinose dehydrogenase) genes. The participation of D-glucose in the regulation mechanism of these genes must still be elucidated. These results contribute to the development of new strains adapted to consume lignocellulosic sugars for the production of value-added bioproducts.

Recombinant GH3 ß-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans.

The ß-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified ß-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (ß/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters Vmax, KM, and Kcat/KM for ρ-nitrophenyl-ß-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a Vmax of 212 U mg - 1, KM of 0.0607 mmol L - 1, and Kcat/KM of 4521 mmol L - 1 s - 1 when incubated at pH 6.0 and 65 °C. The KM, Vmax, and Kcat/KM using cellobiose were 2.7 mmol L - 1, 57 U mg - 1, and 27 mmol -1 s - 1, respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L - 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L - 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.

Spathaspora marinasilvae sp. nov., a xylose-fermenting yeast isolated from galleries of passalid beetles and rotting wood in the Amazonian rainforest biome.

Four yeast isolates were obtained from rotting wood and galleries of passalid beetles collected in different sites of the Brazilian Amazonian Rainforest in Brazil. This yeast produces unconjugated allantoid asci each with a single elongated ascospore with curved ends. Sequence analysis of the internal transcribed spacer-5.8 S region and the D1/D2 domains of the large subunit ribosomal RNA (rRNA) gene showed that the isolates represent a novel species of the genus Spathaspora. The novel species is phylogenetically related to a subclade containing Spathaspora arborariae and Spathaspora suhii. Phylogenomic analysis based on 1884 single-copy orthologs for a set of Spathaspora species whose whole genome sequences are available confirmed that the novel species represented by strain UFMG-CM-Y285 is phylogenetically close to Sp. arborariae. The name Spathaspora marinasilvae sp. nov. is proposed to accommodate the novel species. The holotype of Sp. marinasilvae is CBS 13467 T (MycoBank 852799). The novel species was able to accumulate xylitol and produce ethanol from d-xylose, a trait of biotechnological interest common to several species of the genus Spathaspora.

Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.

Effect of pH on metabolic pathway shift in fermentation and electro-fermentation of xylose by Clostridium autoethanogenum.

Clostridium autoethanogenum can to convert waste gases (CO2, CO, H2) and xylose from hydrolyzed biomass into acetate, lactate, formate, ethanol and 2,3-butanediol, being a candidate for the transformation of waste streams of lignocellulosic biorefineries. Electro-fermentation (EF) modify the pattern of traditional fermentations resulting in improved product yields as has been shown when using Clostridium strains. The aim of this work was to evaluate the influence of pH on microbial growth and product distribution during fermentation and EF of xylose by C. autoethanogenum DSM10061. Fermentation and EF were carried out in a H-type reactor at three controlled pH: 5.0, 5.5 and 5.8, and at a fixed potential of -600 mV (versus Ag/AgCl) in the EF. The experiments showed that maximum biomass concentration increased as the pH increased in fermentation and EF. In accordance with maximum biomass reached, the highest substrate conversion was observed at pH 5.8 for both systems, with 76.80 % in fermentation and 96.18 % in EF. Moreover, the highest concentrations of acetic acid (1.41 ± 0.07 g L-1) and ethanol (1.45 ± 0.15 g L-1) were obtained at the end of cultures in the EF at pH 5.8. The production of lactic and formic acid decreased by the application of the external potential regardless of the pH value, reaching the lowest productivity at pH 5.8. In contrast, the specific productivity of acetic acid and ethanol was lower in both fermentation and EF at the lowest pH. Furthermore, the presence of 0.06 g L-1 of 2,3-butanediol was only detected in EF at pH 5.8. The results revealed that EF modulated microbial metabolism, which can be explained by a possible increased generation of NADP+/NADPH cofactors, which would redirect the metabolic pathway to more reduced products.

An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

Transcriptome profiling brings new insights into the ethanol stress responses of Spathaspora passalidarum.

Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: • Ethanol stress halts Spathaspora passalidarum metabolism and fermentation • Genes encoding nutrient transporters showed downregulation under ethanol stress • Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.

Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic biomass has received considerable attention as an alternative renewable resource toward reducing the demand for fossil energy sources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in a hemicellulosic hydrolysate-based medium. DPY06 exhibited 35% higher volumetric ethanol productivity compared to its parental strain.

Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia.

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

Biochemical characterization of a xylose-tolerant GH43 ß-xylosidase from Geobacillus thermodenitrificans.

Hemicelluloses are the second most abundant polysaccharide in plant biomass, in which xylan is the main constituent. Aiming at the total degradation of xylan and the obtention of fermentable sugars, several enzymes acting synergistically are required, especially ß-xylosidases. In this study, ß-xylosidase from Geobacillus thermodenitrificans (GtXyl) was expressed in E. coli BL21 and characterized. The enzyme GtXyl has been grouped within the family of glycoside hydrolases 43 (GH43). Results showed that GtXyl obtained the highest activity at pH 5.0 and temperature of 60 °C. In the additive's tests, the enzyme remained stable in the presence of metal ions and EDTA, and showed high tolerance to xylose, with a relative activity of 55.4% at 400 mM. The enzyme also presented bifunctional activity of ß-xylosidase and α-l-arabinofuranosidase, with the highest activity on the substrate p-nitrophenyl-ß-d-xylopyranoside. The specific activity on p-nitrophenyl-ß-d-xylopyranoside was 18.33 U mg-1 and catalytic efficiency of 20.21 mM-1 s-1, which is comparable to other ß-xylosidases reported in the literature. Putting together, the GtXyl enzyme presented interesting biochemical characteristics that are desirable for the application in the enzymatic hydrolysis of plant biomass, such as activity at higher temperatures, high thermostability and stability to metal ions.

Xylose Metabolization by a Saccharomyces cerevisiae Strain Isolated in Colombia.

Saccharomyces cerevisiae (S. cerevisiae) is the most widely used yeast in biotechnology in the world because its well-known metabolism and physiology as well as its recognized ability to ferment sugars such as hexoses. However, it does not metabolize pentoses such as arabinose and xylose, which are present in lignocellulosic biomass. Lignocellulose is a widely available raw material, with xylose content of approximately 35% of total sugars. This xylose fraction could be used to obtain high added-value chemical products such as xylitol. One of these yeasts isolated from a Colombian locality, designated as 202-3, showed interesting properties. 202-3 was identified through different approaches as a strain of S. cerevisiae, with an interesting consumption of xylose metabolizing into xylitol, in addition with excellent ability as a hexose fermenter with high ethanol yields and shows resistance to inhibitors present in lignocellulosic hydrolysates. The xylose metabolization by the 202-3 strain and their kinetics parameters had not been previously reported for any other natural strain of S. cerevisiae. These results suggest the great potential of natural strains for obtaining high value-added chemical products using sugars available in lignocellulosic biomass. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01054-z.

Glycosyltransferases Expression Changes in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 Grown on Different Carbon Sources.

Leuconostoc mesenteroides strains are common contributors in fermented foods producing a wide variety of polysaccharides from sucrose through glycosyltransferases (GTFs). These polymers have been proposed as protective barriers against acidity, dehydration, heat, and oxidative stress. Despite its presence in many traditional fermented products and their association with food functional properties, regulation of GTFs expression in Ln. mesenteroides is still poorly understood. The strain Ln. mesenteroides ATCC 8293 contains three glucansucrases genes not found in operons, and three fructansucrases genes arranged in two operons, levLX and levC-scrB, a Glycoside-hydrolase. We described the first differential gene expression analysis of this strain when cultivated in different carbon sources. We observed that while GTFs are expressed in the presence of most sugars, they are down-regulated in xylose. We ruled out the regulatory effect of CcpA over GTFs and did not find regulatory elements with a direct effect on glucansucrases in the condition assayed. Our findings suggest that only operon levLX is repressed in xylose by LexA and that both fructansucrases operons can be regulated by the VicK/VicR system and PerR. It is essential to further explore the effect of environmental conditions in Ln. mesenteroides bacteria to better understand GTFs regulation and polymer function.

Spathaspora brunopereirae sp. nov. and Spathaspora domphillipsii sp. nov., two d-xylose-fermenting ascosporogenous yeasts from Amazonian Forest biomes.

Four isolates of Spathaspora species were recovered from rotting wood collected in two Brazilian Amazonian biomes. The isolates produced unconjugated allantoid asci with a single elongated ascospore with curved ends. Sequence analysis of the ITS-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent two different novel Spathaspora species, phylogenetically related to Sp. boniae. Two isolates were obtained from rotting wood collected in two different sites of the Amazonian forest in the state of Pará. The name Spathaspora brunopereirae sp. nov. is proposed to accommodate these isolates. The holotype of Spathaspora brunopereirae sp. nov. is CBS 16119T (MycoBank MB846672). The other two isolates were obtained from a region of transition between the Amazonian forest and the Cerrado ecosystem in the state of Tocantins. The name Spathaspora domphillipsii sp. nov. is proposed for this novel species. The holotype of Spathaspora domphillipsii sp. nov. is CBS 14229T (MycoBank MB846697). Both species are able to convert d-xylose into ethanol and xylitol, a trait with biotechnological applications.

Wide distribution of D-xylose dehydrogenase in yeasts reveals a new element in the D-xylose metabolism for bioethanol production.

D-xylose utilization by yeasts is an essential feature for improving second-generation ethanol production. However, industrial yeast strains are incapable of consuming D-xylose. Previous analyzes of D-xylose-consuming or fermenting yeast species reveal that the genomic features associated with this phenotype are complex and still not fully understood. Here we present a previously neglected yeast enzyme related to D-xylose metabolism, D-xylose dehydrogenase (XylDH), which is found in at least 105 yeast genomes. By analyzing the XylDH gene family, we brought evidence of gene evolution marked by purifying selection on codons and positive selection evidence in D-xylose-consuming and fermenting species, suggesting the importance of XylDH for D-xylose-related phenotypes in yeasts. Furthermore, although we found no putative metabolic pathway for XylDH in yeast genomes, namely the absence of three bacterial known pathways for this enzyme, we also provide its expression profile on D-xylose media following D-xylose reductase for two yeasts with publicly available transcriptomes. Based on these results, we suggest that XylDH plays an important role in D-xylose usage by yeasts, likely being involved in a cofactor regeneration system by reducing cofactor imbalance in the D-xylose reductase pathway.

Xylose consumption and ethanol production by Pichia guilliermondii and Candida oleophila in the presence of furans, phenolic compounds, and organic acids commonly produced during the pre-treatment of plant biomass.

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

Microbial lipid production from soybean hulls using Lipomyces starkeyi LPB53 in a circular economy.

Soybean hulls are lignocellulosic residuesgeneratedinthe industrial processing of soybean, representing about 5 % of the mass of the whole bean. This by-product isan importantsource of polymers suchas cellulose(34 %) and hemicellulose (11 %),which could bevalorizedvia biotechnology to improvethe economic returnof the oilseed chain. In the present work,soybean hulls were evaluated as a carbon sourcefor biolipid productionbyLipomycesstarkeyi LPB 53. Initially the hulls were treated physicochemically and enzymatically to obtain fermentable sugars. Subsequently, biomass growth was evaluated using different nitrogen sources andthe lipid production was optimized, reaching a maximum cell biomass concentration of 26.5 g/L with 42.5 % of lipids. Around 65 % of the xylose content was consumed.The obtained oil wasmajorlycomposed of oleic, palmitic, palmitoleic, linoleic and stearic fatty acids in a proportion of 54 %, 32 %, 4 %, 3 % and 2 %, respectively.

Prospecting and engineering yeasts for ethanol production under inhibitory conditions: an experimental design analysis.

The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.

Improving 3-hydroxypropionic acid production in E. coli by in silico prediction of new metabolic targets.

3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the ß-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose.

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.