Evidence for rapid uptake of d-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system

Abstractd-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of d-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in d-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including d-glucuronic acid, which was also transported. The characteristics of d-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates

Genomic saturation mutagenesis for isolation and characterization of mutants in non-selectable polygenic traits

Isolation of mutants in populations of yeast and other microorganisms, has been a valuable tool in experimental genetics for decades. The main disadvantages of classical mutant hunts are the inability to isolate mutants in non-selectable phenotypes and the difficulty of obtaining multiple mutations affecting a single phenotype, i.e. the isolation of mutants in polygenic traits. Most traits of organisms, however, are polygenic and non-selectable. This includes many, if not most, commercially-important traits of industrial yeast strains. The advent of powerful technologies for polygenic linkage analysis of complex traits now allows efficient identification of multiple mutations responsible for a complex trait among many thousands of irrelevant mutations. In this study, we have optimized a methodology for introducing hundreds of mutations into a single haploid S288c strain using multiple rounds of EMS mutagenesis, while maintaining genetic proficiency. Two mutants with about 900 mutations were screened for multiple non-selectable phenotypes. One mutant showed strongly reduced ethyl acetate production in semi-anaerobic fermentations, while in the other increased isoamyl acetate production was observed. Since there are still one or more unknown enzymes responsible for ethyl acetate production, we have mapped the quantitative loci (QTLs) underlying this trait using pooled-segregant whole-genome sequence analysis. Further dissection of the QTLs identified induced non-synonymous single nucleotide polymorphisms in both CEM1 and PMA1 as being causative for low ethyl acetate production. The CEM1 gene encodes a beta-ketoacyl synthase that is involved in mitochondrial fatty acid synthesis. PMA1 is an essential gene that encodes an H+-ATPase and the cytosolic pH is primarily regulated by Pma1. We have also identified the TPS1 allele of S288c, present in the background of the mutant strain, to be causative in a QTL with a strong link to low ethyl acetate production. TPS1 encodes trehalose-6-P synthase that is involved in the synthesis of trehalose, a well-known storage carbohydrate and a stress protectant. Trehalose-6-phosphate has also been implicated in the regulation of the influx of sugars into glycolysis. Our results demonstrate that mutant strains can be obtained in a straightforward way that have a multitude of mutations randomly spread all over the genome. A collection of such saturated mutants could be used for phenotypic screens, in order to identify the genetic basis of a wide range of monogenic and polygenic phenotypes for which no selectable system can be devised. A major advantage of this method is that only a limited number of mutant strains has to be screened in order to identify with great probability at least one strain that is affected in the trait of interest. Moreover, the genes underlying the defect in the trait of interest can then be identified in a relatively straightforward manner. This method also allows identification of causative alleles of traits, which are actually absent in the multiple mutant strain because of one or more other mutations. Because of the fact that the different mutations segregate in the descendants after crossing with a wild type strain, they become visible in the segregants and can effectively be detected. The number of mutations that can be accumulated in a single yeast strain is therefore limited by the ability of the multiple mutants to cope with these mutations rather than by the possibility of observing the trait of interest in the mutant strain. In this way our method eliminates the most important limitations of the classical mutagenesis and selection methods. It is able to generate mutants that are deficient in many polygenic and non-selectable phenotypes. Moreover, the underlying causative alleles of these phenotypes can be identified in a relatively easy and straightforward manner.nrpages: 238status: publishe

Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait

Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to identify causative alleles underlying many non-selectable, polygenic traits in small collections of haploid strains with multiple induced mutations

Docking Motif-Guided Mapping of the Interactome of Protein Phosphatase-1

The ubiquitous protein Ser/Thr phosphatase-1 (PP1) interacts with dozens of regulatory proteins that are structurally unrelated. However, most of them share a short, degenerate "RVxF"-type docking motif. Using a broad in silico screening based on a stringent definition of the RVxF motif, in combination with a multistep biochemical validation procedure, we have identified 78 novel mammalian PP1 interactors. A global analysis of the validated RVxF-based PP1 interactome not only provided insights into the conserved features of the RVxF motif but also led to the discovery of additional common PP1 binding elements, described as the "SILK" and "MyPhoNE" motifs. In addition to the doubling of the known mammalian PP1 interactome, our data contribute to the design of PP1 interaction networks. Notably, an interaction network linking PP1 interactors discloses a pleiotropic role of PP1 in cell polarity.status: publishe