Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.
Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.The baker's yeast Saccharomyces cerevisiae has long served as a model for studying glucose repression, the multi-layer process by which glucose is consumed before all other carbon sources 1,2 . A wide variety of interconnected mechanisms contribute to yeast's ability to sense, respond, and optimize internal metabolism to preferentially consume glucose 3,4 . Transcriptional repressors such as Mig1, Cat8, and the Ssn6/Tup1 complex prevent transcription of glucose-repressed genes, such as those involved in gluconeogenesis and metabolism of alternative carbon sources [5][6][7][8] . The activities of these repressors are then mediated by kinases and phosphatases such as Snf1 and Glc7/Reg1, respectively 6,9,10 . Beyond these intracellular sensing mechanisms, membrane sensors such as Snf3 and Rgt2 allow yeast to sense extracellular sugar concentrations and internalize signals 11 . In sum, the S. cerevisiae glucose repression pathway is a complex network of signals and regulations comprising significant amounts of research and a continuously growing base of knowledge.Recently, a new layer of glucose repression of galactose consumption has been reported to be linked to the kinetic properties of sugar transporters 12 . Because sugars compete for cellular uptake, relative transport efficiency between two sugars will depend on extracellular sugar concentrations as well as transporter affinities (K m values) for each sugar. Consequently, it was reported that the extracellular sugar concentrations coupled with transporter substrate affinity determine the intracellular sugar concentrations 12 . As GAL ge...
eThe haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13⌬), we investigated the response of S. cerevisiae to pho13⌬ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13⌬ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13⌬ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13⌬ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13⌬ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.
Genomic integration of expression cassettes containing heterologous genes into yeast with traditional methods inevitably deposits undesirable genetic elements into yeast chromosomes, such as plasmid-borne multiple cloning sites, antibiotic resistance genes, Escherichia coli origins, and yeast auxotrophic markers. Specifically, drug resistance genes for selecting transformants could hamper further industrial usage of the resulting strains because of public health concerns. While we constructed an efficient and rapid xylose-fermenting Saccharomyces cerevisiae, the engineered strain (SR8) might not be readily used for a large-scale fermentation because the SR8 strain contained multiple copies of drug resistance genes. We utilized the Cas9/CRISPR-based technique to refactor an efficient xylose-fermenting yeast strain without depositing any undesirable genetic elements in resulting strains. In order to integrate genes (XYL1, XYL2, and XYL3) coding for xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis, and delete both PHO13 and ALD6, a double-strand break formation by Cas9 and its repair by homologous recombination were exploited. Specifically, plasmids containing guide RNAs targeting PHO13 and ALD6 were sequentially co-transformed with linearized DNA fragments containing XYL1, XYL2, and XYL3 into S. cerevisiae expressing Cas9. As a result, two copies of XYL1, XYL2, and XYL3 were integrated into the loci of PHO13 and ALD6 for achieving overexpression of heterologous genes and knockout of endogenous genes simultaneously. With further prototrophic complementation, we were able to construct an engineered strain exhibiting comparable xylose fermentation capabilities with SR8 within 3 weeks. We report a detailed procedure for refactoring xylose-fermenting yeast using any host strains. The refactored strains using our procedure could be readily used for large-scale fermentations since they have no antibiotic resistant markers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.