Natalia Bolotina

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid.

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L(-1) (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)(-1). Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.

A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae

Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His(6) tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.

Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His6 tag fusion.The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the ‘Berlin Protein Structure Factory’. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).

Shifting the Fermentative/Oxidative Balance in Saccharomyces cerevisiae by Transcriptional Deregulation of Snf1 via Overexpression of the Upstream Activating Kinase Sak1p

ABSTRACT With the aim to reduce fermentation by-products and to promote respiratory metabolism by shifting the fermentative/oxidative balance, we evaluated the constitutive overexpression of the SAK1 and HAP4 genes in Saccharomyces cerevisiae. Sak1p is one of three kinases responsible for the phosphorylation, and thereby the activation, of the Snf1p complex, while Hap4p is the activator subunit of the Hap2/3/4/5 transcriptional complex. We compared the physiology of a SAK1-overexpressing strain with that of a strain overexpressing the HAP4 gene in wild-type and sdh2 deletion (respiratory-deficient) backgrounds. Both SAK1 and HAP4 overexpressions led to the upregulation of glucose-repressed genes and to reduced by-product formation rates (ethanol and glycerol). SAK1 overexpression had a greater impact on growth rates than did HAP4 overexpression. Elevated transcript levels of SAK1, but not HAP4, resulted in increased biomass yields in batch cultures grown on glucose (aerobic and excess glucose) as well as on nonfermentable carbon sources. SAK1 overexpression, but not the combined overexpression of SAK1 and HAP4 or the overexpression of HAP4 alone, restored growth on ethanol in an sdh2 deletion strain. In glucose-grown shake flask cultures, the sdh2 deletion strain with SAK1 and HAP4 overexpression produced succinic acid at a titer of 8.5 g liter−1 and a yield of 0.26 mol (mol glucose)− 1 within 216 h. We here report for the first time that a constitutively high level of expression of SAK1 alleviates glucose repression and shifts the fermentative/oxidative balance under both glucose-repressed and -derepressed conditions.