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Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

ABSTRACT: Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

SUBMITTER: Chen Y

PROVIDER: S-EPMC6016917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundLignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2.ResultsWith all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth.ConclusionsLaboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.

Project description:BackgroundSaccharomyces cerevisiae, a key organism used for the manufacture of renewable fuels and chemicals, has been engineered to utilize non-native sugars derived from plant cell walls, such as cellobiose and xylose. However, the rates and efficiencies of these non-native sugar fermentations pale in comparison with those of glucose. Systems biology methods, used to understand biological networks, hold promise for rational microbial strain development in metabolic engineering. Here, we present a systematic strategy for optimizing non-native sugar fermentation by recombinant S. cerevisiae, using cellobiose as a model.ResultsDifferences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induces mitochondrial activation and reduces amino acid biosynthesis under fermentation conditions. Furthermore, glucose-sensing and signaling pathways and their target genes, including the cAMP-dependent protein kinase A pathway controlling the majority of glucose-induced changes, the Snf3-Rgt2-Rgt1 pathway regulating hexose transport, and the Snf1-Mig1 glucose repression pathway, were at most only partially activated under cellobiose conditions. To separate correlations from causative effects, the expression levels of 19 transcription factors perturbed under cellobiose conditions were modulated, and the three strongest promoters under cellobiose conditions were applied to fine-tune expression of the heterologous cellobiose-utilizing pathway. Of the changes in these 19 transcription factors, only overexpression of SUT1 or deletion of HAP4 consistently improved cellobiose fermentation. SUT1 overexpression and HAP4 deletion were not synergistic, suggesting that SUT1 and HAP4 may regulate overlapping genes important for improved cellobiose fermentation. Transcription factor modulation coupled with rational tuning of the cellobiose consumption pathway significantly improved cellobiose fermentation.ConclusionsWe used systems-level input to reveal the regulatory mechanisms underlying suboptimal metabolism of the non-glucose sugar cellobiose. By identifying key transcription factors that cause suboptimal cellobiose fermentation in engineered S. cerevisiae, and by fine-tuning the expression of a heterologous cellobiose consumption pathway, we were able to greatly improve cellobiose fermentation by engineered S. cerevisiae. Our results demonstrate a powerful strategy for applying systems biology methods to rapidly identify metabolic engineering targets and overcome bottlenecks in performance of engineered strains.

Project description:BackgroundConsiderable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.ResultsGenes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.ConclusionThe results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

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Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

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