Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations.

BACKGROUND: The selection of bioengineering platform strains and engineering strategies to improve the stress resistance of Saccharomyces cerevisiae remains a pressing need in bio-based chemical production. Thus, a systematic effort to exploit genotypic and phenotypic diversity to boost yeast's industrial value is still urgently needed. RESULTS: We analyzed 5,400 growth curves obtained from 36 S. cerevisiae strains and comprehensively profiled their resistances against 13 industrially relevant stresses. We observed that bioethanol and brewing strains exhibit higher resistance against acidic conditions; however, plant isolates tend to have a wider range of resistance, which may be associated with their metabolome and fluxome signatures in the tricarboxylic acid cycle and fatty acid metabolism. By deep genomic sequencing, we found that industrial strains have more genomic duplications especially affecting transcription factors, showing that they result from disparate evolutionary paths in comparison with the environmental strains, which have more indels, gene deletions, and strain-specific genes. Genome-wide association studies coupled with protein-protein interaction networks uncovered novel genetic determinants of stress resistances. CONCLUSIONS: These resistance-related engineering targets and strain rankings provide a valuable source for engineering significantly improved industrial platform strains.

Close to the Edge: Growth Restrained by the NAD(P)H/ATP Formation Flux Ratio.

Most fermentative microorganisms grow well-under anaerobic conditions managing a balanced redox and appropriate energy metabolism, but a few species do exist in which cells have to cope with inadequate energy recovery or capture and/or redox balancing. Two cases of these species, i.e., the metabolically engineered Saccharomyces cerevisiae enabling it to ferment xylose and Lactobacillus reuteri fermenting glucose via the phosphoketolase pathway, are here used to introduce a quantification parameter to capture what limits the growth rate of these microorganisms under anaerobic conditions. This dimensionless parameter, the cofactor formation flux ratio (RJ ), is the ratio between the redox formation flux (JNADH+NADPH), and the energy carrier formation flux (JATP), which are mainly connected to the central carbon pathways. Data from metabolic flux analyses performed in previous and present studies were used to estimate the RJ -values. Even though both microorganisms possess different central pathways, a similar relationship between RJ and the specific growth rate (µ) was found. Furthermore, for both microorganisms external electron acceptors moderately reduced the RJ -value, thereby raising the µ accordingly. Based on the emerging profile of this relationship an interpretation is presented suggesting that this quantitative analysis can be applied beyond the two microbial species experimentally investigated in the current study to provide data for future targeted strain development strategies.

Physiology of Saccharomyces cerevisiae strains isolated from Brazilian biomes: new insights into biodiversity and industrial applications.

Fourteen indigenous Saccharomyces cerevisiae strains isolated from the barks of three tree species located in the Atlantic Rain Forest and Cerrado biomes in Brazil were genetically and physiologically compared to laboratory strains and to strains from the Brazilian fuel ethanol industry. Although no clear correlation could be found either between phenotype and isolation spot or between phenotype and genomic lineage, a set of indigenous strains with superior industrially relevant traits over commonly known industrial and laboratory strains was identified: strain UFMG-CM-Y257 has a very high specific growth rate on sucrose (0.57 ± 0.02 h-1), high ethanol yield (1.65 ± 0.02 mol ethanol mol hexose equivalent-1), high ethanol productivity (0.19 ± 0.00 mol L-1 h-1), high tolerance to acetic acid (10 g L-1) and to high temperature (40°C). Strain UFMG-CM-Y260 displayed high ethanol yield (1.67 ± 0.13 mol ethanol mol hexose equivalent-1), high tolerance to ethanol and to low pH, a trait which is important for non-aseptic industrial processes. Strain UFMG-CM-Y267 showed high tolerance to acetic acid and to high temperature (40°C), which is of particular interest to second generation industrial processes.

Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae.

BACKGROUND: Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. RESULTS: Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. CONCLUSIONS: Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.

Engineering yeast hexokinase 2 for improved tolerance toward xylose-induced inactivation.

Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks.

Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose.

BACKGROUND: The concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production. RESULTS: Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool. CONCLUSIONS: The low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae.

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.