Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli . Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs , which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms ( Saccharomyces cerevisiae ) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe(177)Ser) and, similarly, Tyr(195)His, and Lys(218)Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.In both functional genomics and metabolic engineering, strategies for fine-tuning gene expression are required to study the control exerted by target genes on phenotypes or metabolic fluxes of interest. Traditionally, gene expression varies among three conditions: (i) the wild type, (ii) the gene knockout, and (iii) strong overexpression of the target gene. However, several examples where the optimization of target phenotypes was achieved by moderate rather than strong multicopy expression of a target gene have been given (12,13,30). These examples highlight the need for tools that enable the fine-tuning and precise control of gene expression in Saccharomyces cerevisiae.Several strategies have been explored to obtain a graded target gene expression. One strategy is to clone native promoters of various strengths to drive the expression of a desired gene (34). However, this method is not robust, since endogenous promoters may be subject to different regulation modalities, even despite their designation as "constitutive." Moreover, a native promoter of a given desired strength may be unavailable or difficult to identify. A second strategy uses vectors of different copy numbers to adjust gene expression levels (12,13,30). This approach is limited by the availability of plasmids of any given desired copy number. Other problems with this technique are the metabolic burden associated with maintenance of high-copy number plasmids and the cell-cell heterogeneity in expression caused by copy number variance between single cells of a culture. A third strategy for modulating gene expression has been to titrate an inducible promoter sy...

Summary There is huge variability among yeasts with regard to their efficiency in utilizing glycerol as the sole source of carbon and energy. Certain species show growth rates with glycerol comparable to those reached with glucose as carbon source; others are virtually unable to utilize glycerol, especially in synthetic medium. Most of our current knowledge regarding glycerol uptake and catabolic pathways has been gained from studying laboratory strains of the model yeast Saccharomyces cerevisiae. The growth of these strains on glycerol is dependent on the presence of medium supplements such as amino acids and nucleobases. In contrast, there is only fragmentary knowledge about S. cerevisiae isolates able to grow in synthetic glycerol medium without such supplements as well as about growth of non‐Saccharomyces yeast species on glycerol. Thus, more research is required to understand why certain strains and species show superior growth performance on glycerol compared with common S. cerevisiae laboratory strains. This mini‐review summarizes what is known so far about the gene products and pathways involved in glycerol metabolism and transport in yeast and fungi as well as the regulation of these processes.

BackgroundGlycerol has attracted attention as a carbon source for microbial production processes due to the large amounts of crude glycerol waste resulting from biodiesel production. The current knowledge about the genetics and physiology of glycerol uptake and catabolism in the versatile industrial biotechnology production host Saccharomyces cerevisiae has been mainly based on auxotrophic laboratory strains, and carried out in the presence of growth-supporting supplements such as amino acids and nucleic bases. The latter may have resulted in ambiguous conclusions concerning glycerol growth in this species. The purpose of this study was to re-evaluate growth of S. cerevisiae in synthetic glycerol medium without the addition of supplements.ResultsInitial experiments showed that prototrophic versions of the laboratory strains CEN.PK, W303, and S288c did not exhibit any growth in synthetic glycerol medium without supporting supplements. However, a screening of 52 S. cerevisiae isolates for growth in the same medium revealed a high intraspecies diversity. Within this group significant variation with respect to the lag phase and maximum specific growth rate was observed. A haploid segregant of one good glycerol grower (CBS 6412-13A) was selected for detailed analysis. Single deletions of the genes encoding for the glycerol/H+ symporter (STL1), the glycerol kinase (GUT1), and the mitochondrial FAD+-dependent glycerol 3-phosphate dehydrogenase (GUT2) abolished glycerol growth in this strain, implying that it uses the same glycerol utilization pathway as previously identified in auxotrophic laboratory strains. Segregant analysis of a cross between CBS 6412-13A and CEN.PK113-1A revealed that the glycerol growth phenotype is a quantitative trait. Genetic linkage and reciprocal hemizygosity analysis demonstrated that GUT1 CBS 6412-13A is one of the multiple genetic loci contributing to the glycerol growth phenotype.ConclusionThe S. cerevisiae intraspecies diversity with regard to glycerol growth is a valuable starting point to identify the genetic and molecular basis of this phenotype. This knowledge can be applied for further rational strain improvement with the goal of using glycerol as a carbon source in industrial biotechnology processes based on S. cerevisiae as a production organism.

Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the ratecontrolling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost of reduced glycerol yield have most often been based on deletion of either one or two isogenes (GPD1 and GPD2). While single deletions of GPD1 or GPD2 reduced glycerol formation only slightly, the gpd1⌬ gpd2⌬ double deletion strain produced zero glycerol but showed an osmosensitive phenotype and abolished anaerobic growth. Our current approach has sought to generate "intermediate" phenotypes by reducing both isoenzyme activities without abolishing them. To this end, the GPD1 promoter was replaced in a gpd2⌬ background by two lower-strength TEF1 promoter mutants. In the same manner, the activity of the GPD2 promoter was reduced in a gpd1⌬ background. The resulting strains were crossed to obtain different combinations of residual GPD1 and GPD2 expression levels. Among our engineered strains we identified four candidates showing improved ethanol yields compared to the wild type. In contrast to a gpd1⌬ gpd2⌬ double-deletion strain, these strains were able to completely ferment the sugars under quasi-anaerobic conditions in both minimal medium and during simultaneous saccharification and fermentation (SSF) of liquefied wheat mash (wheat liquefact). This result implies that our strains can tolerate the ethanol concentration at the end of the wheat liquefact SSF (up to 90 g liter ؊1 ). Moreover, a few of these strains showed no significant reduction in osmotic stress tolerance compared to the wild type.

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