The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae yeast strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. In contrast to the fermentation of hexose sugar-rich substrates, such as corn syrup or sugar cane bagasse, these hydrolysates contain mixtures of the hexose sugar D-glucose and the pentose sugars D-xylose and L-arabinose. While S. cerevisiae performs excellently in fermenting hexose sugars to ethanol, efficient utilization of pentose sugars required extensive metabolic and evolutionary engineering.@en

Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K _m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10^-3 and 1.8 g L^-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L^-1 l-arabinose and 20 g L^-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

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Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.

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D-Glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three ‘specialist’ Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose-non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved ‘generalist’ strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.@en

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.@en

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h−1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex ‘transporter landscapes’, consisting of sugar transporters with complementary kinetic and regulatory properties.@en

Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca²⁺/Mn²⁺ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn²⁺ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn²⁺ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.

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The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.@en