Andreas Raab

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.

Oxidative versus reductive succinic acid production in the yeast saccharomyces cerevisiae

Bio-based succinic acid is receiving increasing attention, as it could provide a cost-effective, ecologicallysustainable alternative to the current petrochemical production process, thus promising a significantly higher market potential. The yeastSaccharomyces cerevisiae is a robust and well-established industrial production organism exhibiting an extraordinarily high acid- and osmotolerance. These features in conjunction with the sophisticated toolbox for genetic engineering make it particularly suitable for succinic acid production. The high tolerance towards acidity is a major advantage over previously established bacterial succinic acid production hosts, since it makes the use of neutralisation salts dispensable and thus enormously facilitates the downstream process. By constructing yeast strains capable of producing significant amounts of succinic acid, we have recently established S. cerevisiae as a promising host for succinic acid production. Our metabolic engineering strategy relied on the implementation of an oxidative production route using the glyoxylate cycle. We here discuss theoretical and practical aspects of oxidative and reductive succinic acid production routes in S. cerevisiae.

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.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon‐limited fed‐batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory‐scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo‐polygalacturonase (EPG) was characterized in microwell plates with an enzyme‐based fed‐batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200–400 U (gx h)−1 were determined. As a reference, bioreactor experiments using the change‐stat cultivation technique were performed. The growth‐dependent product formation was analyzed over dilution rates of D = 0.01–0.35 with smooth change of D at a rate of 0.003 h−2. EPG production was found to be comparable with a qp of 400 U (gx h)−1 at D = 0.27 h−1. The presented results indicate that parallel miniaturized fed‐batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.