Identification of traits to improve co-assimilation of glucose and xylose by adaptive evolution of Spathaspora passalidarum and Scheffersomyces stipitis yeasts.

Lignocellulosic biomass is a renewable raw material for producing several high-value-added chemicals and fuels. In general, xylose and glucose are the major sugars in biomass hydrolysates, and their efficient utilization by microorganisms is critical for an economical production process. Yeasts capable of co-consuming mixed sugars might lead to higher yields and productivities in industrial fermentation processes. Herein, we performed adaptive evolution assays with two xylose-fermenting yeasts, Spathaspora passalidarum and Scheffersomyces stipitis, to obtain derived clones with improved capabilities of glucose and xylose co-consumption. Adapted strains were obtained after successive growth selection using xylose and the non-metabolized glucose analog 2-deoxy-D-glucose as a selective pressure. The co-fermentation capacity of evolved and parental strains was evaluated on xylose-glucose mixtures. Our results revealed an improved co-assimilation capability by the evolved strains; however, xylose and glucose consumption were observed at slower rates than the parental yeasts. Genome resequencing of the evolved strains revealed genes affected by non-synonymous variants that might be involved with the co-consumption phenotype, including the HXT2.4 gene that encodes a putative glucose transporter in Sp. passalidarum. Expression of this mutant HXT2.4 in Saccharomyces cerevisiae improved the cells' co-assimilation of glucose and xylose. Therefore, our results demonstrated the successful improvement of co-fermentation through evolutionary engineering and the identification of potential targets for further genetic engineering of different yeast strains. KEY POINTS: • Laboratory evolution assay was used to obtain improved sugar co-consumption of non-Saccharomyces strains. • Evolved Sp. passalidarum and Sc. stipitis were able to more efficiently co-ferment glucose and xylose. • A mutant Hxt2.4 permease, which co-transports xylose and glucose, was identified.

Recent progress in the microbial production of xylonic acid.

Interest in the production of renewable chemicals from biomass has increased in the past years. Among these chemicals, carboxylic acids represent a significant part of the most desirable bio-based products. Xylonic acid is a five-carbon sugar-acid obtained from xylose oxidation that can be used in several industrial applications, including food, pharmaceutical, and construction industries. So far, the production of xylonic acid has not yet been available at an industrial scale; however, several microbial bio-based production processes are under development. This review summarizes the recent advances in pathway characterization, genetic engineering, and fermentative strategies to improve xylonic acid production by microorganisms from xylose or lignocellulosic hydrolysates. In addition, the strengths of the available microbial strains and processes and the major requirements for achieving biotechnological production of xylonic acid at a commercial scale are discussed. Efficient native and engineered microbial strains have been reported. Xylonic acid titers as high as 586 and 171 g L-1 were obtained from bacterial and yeast strains, respectively, in a laboratory medium. Furthermore, relevant academic and industrial players associated with xylonic acid production will be presented.

Xylitol production on sugarcane biomass hydrolysate by newly identified Candida tropicalis JA2 strain.

Xylitol is a building block for a variety of chemical commodities, besides being widely used as a sugar substitute in the food and pharmaceutical industries. The aim of this work was to develop a microbial process for xylitol production using sugarcane bagasse hydrolysate as substrate. In this context, 218 non-Saccharomyces yeast strains were screened by growth on steam-exploded sugarcane bagasse hydrolysate containing a high concentration of acetic acid (8.0 g/L). Seven new Candida tropicalis strains were selected and identified, and their ability to produce xylitol on hydrolysate at low pH (4.6) under aerobic conditions was evaluated. The most efficient strain, designated C. tropicalis JA2, was capable of producing xylitol with a yield of 0.47 g/g of consumed xylose. To improve xylitol production by C. tropicalis JA2, a series of experimental procedures were employed to optimize pH and temperature conditions, as well as nutrient source, and initial xylose and inoculum concentrations. C. tropicalis JA2 was able to produce 109.5 g/L of xylitol with a yield of 0.86 g/g of consumed xylose, and with a productivity of 2.81 g·L·h, on sugarcane bagasse hydrolysate containing 8.0 g/L acetic acid and177 g/L xylose, supplemented with 2.0 g/L yeast nitrogen base and 4.0 g/L urea. Thus, it was possible to identify a new C. tropicalis strain and to optimize the xylitol production process using sugarcane bagasse hydrolysate as a substrate. The xylitol yield on biomass hydrolysate containing a high concentration of acetic acidobtained in here is among the best reported in the literature.

Physiological and comparative genomic analysis of new isolated yeasts Spathaspora sp. JA1 and Meyerozyma caribbica JA9 reveal insights into xylitol production.

Xylitol is a five-carbon polyol of economic interest that can be produced by microbial xylose reduction from renewable resources. The current study sought to investigate the potential of two yeast strains, isolated from Brazilian Cerrado biome, in the production of xylitol as well as the genomic characteristics that may impact this process. Xylose conversion capacity by the new isolates Spathaspora sp. JA1 and Meyerozyma caribbica JA9 was evaluated and compared with control strains on xylose and sugarcane biomass hydrolysate. Among the evaluated strains, Spathaspora sp. JA1 was the strongest xylitol producer, reaching product yield and productivity as high as 0.74 g/g and 0.20 g/(L.h) on xylose, and 0.58 g/g and 0.44 g/(L.h) on non-detoxified hydrolysate. Genome sequences of Spathaspora sp. JA1 and M. caribbica JA9 were obtained and annotated. Comparative genomic analysis revealed that the predicted xylose metabolic pathway is conserved among the xylitol-producing yeasts Spathaspora sp. JA1, M. caribbica JA9 and Meyerozyma guilliermondii, but not in Spathaspora passalidarum, an efficient ethanol-producing yeast. Xylitol-producing yeasts showed strictly NADPH-dependent xylose reductase and NAD+-dependent xylitol-dehydrogenase activities. This imbalance of cofactors favors the high xylitol yield shown by Spathaspora sp. JA1, which is similar to the most efficient xylitol producers described so far.

Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid.

BACKGROUND: Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. RESULTS: We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export. CONCLUSIONS: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Optimization of ethylene glycol production from (D)-xylose via a synthetic pathway implemented in Escherichia coli.

BACKGROUND: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. RESULTS: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (D)-xylose isomerase, (D)-xylulose-1-kinase, (D)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (D)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. CONCLUSIONS: We have demonstrated the feasibility to produce EG from (D)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.

Applications of the Methylotrophic Yeast Komagataella phaffii in the Context of Modern Biotechnology.

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.

Construction of a synthetic pathway for the production of 1,3-propanediol from glucose.

In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.

Molecular analysis of maltotriose active transport and fermentation by Saccharomyces cerevisiae reveals a determinant role for the AGT1 permease.

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K(m), 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

The synthetic xylulose-1 phosphate pathway increases production of glycolic acid from xylose-rich sugar mixtures.

BACKGROUND: Glycolic acid (GA) is a two-carbon hydroxyacid with applications in the cosmetic, textile, and medical industry. Microbial GA production from all sugars can be achieved by engineering the natural glyoxylate shunt. The synthetic (d)-xylulose-1 phosphate (X1P) pathway provides a complementary route to produce GA from (d)-xylose. The simultaneous operation of the X1P and glyoxylate pathways increases the theoretical GA yield from xylose by 20 %, which may strongly improve GA production from hemicellulosic hydrolysates. RESULTS: We herein describe the construction of an E. coli strain that produces GA via the glyoxylate pathway at a yield of 0.31 , 0.29 , and 0.37 g/g from glucose, xylose, or a mixture of glucose and xylose (mass ratio: 33:66 %), respectively. When the X1P pathway operates in addition to the glyoxylate pathway, the GA yields on the three substrates are, respectively, 0.39 , 0.43 , and 0.47 g/g. Upon constitutive expression of the sugar permease GalP, the GA yield of the strain which simultaneously operates the glyoxylate and X1P pathways further increases to 0.63 g/g when growing on the glucose/xylose mixture. Under these conditions, the GA yield on the xylose fraction of the sugar mixture reaches 0.75 g/g, which is the highest yield reported to date. CONCLUSIONS: These results demonstrate that the synthetic X1P pathway has a very strong potential to improve GA production from xylose-rich hemicellulosic hydrolysates.

Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals.

A synthetic pathway for (d)-xylose assimilation was stoichiometrically evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose to (d)-xylulose, phosphorylation of (d)-xylulose to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses showed that this pathway provides access to ethylene glycol with a theoretical molar yield of 1. Alternatively, both glycolaldehyde and DHAP can be converted to glycolic acid with a theoretical yield that is 20% higher than for the exclusive production of this acid via the glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and X1P aldolase activities, provided by human ketohexokinase-C and human aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase mutant on xylose. This strain produced ethylene glycol as the major metabolic endproduct. Metabolic engineering provided strains that assimilated the entire C2 fraction into the central metabolism or that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake flasks.