Abstract
For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers. A common cell factory Saccharomyces cerevisiae is a Crabtree-positive yeast and is typically fermented at industrial scale under glucose-limited conditions to avoid the formation of ethanol. In this work, we aimed to identify and characterize promoters that depend on glucose concentration for use as dynamic control elements. Through analysis of mRNA data of S. cerevisiae grown in chemostats under glucose excess or limitation, we identified 34 candidate promoters that strongly responded to glucose presence or absence. These promoters were characterized in small-scale batch and fed-batch cultivations using a quickly maturing rapidly degrading green fluorescent protein yEGFP3-Cln2PEST as a reporter. Expressing 3-hydroxypropionic acid (3HP) pathway from a set of selected regulated promoters allowed for suppression of 3HP production during glucose-excess phase of a batch cultivation with subsequent activation in glucose-limiting conditions. Regulating the 3HP pathway by the ICL1 promoter resulted in 70% improvement of 3HP titer in comparison to PGK1 promoter.
Highlights
Budding yeast, Saccharomyces cerevisiae, is widely used for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals (Nielsen and Jewett, 2008; Hong and Nielsen, 2012)
We performed an analysis of this dataset in order to directly compare gene expression levels in the glucose limiting condition to the average of expression levels observed while not glucose limited which we describe as “glucose excess” conditions
Four archetypes of gene expression were identified for their known roles and are represented in Figure 2: (1) TEF1 archetype with constitutive expression in both conditions, (2) HXT1 archetype with high expression in glucose-excess and low expression in glucoselimiting conditions, (3) ADH2, and (4) MAL12 archetypes with low expression in glucose-excess and high expression in glucose limiting conditions
Summary
Saccharomyces cerevisiae, is widely used for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals (Nielsen and Jewett, 2008; Hong and Nielsen, 2012). Serial cultivation steps aim at building up biomass while for the final-stage cultivation it is desirable to re-direct the metabolic fluxes such that the product of interest is formed at high yield and rate (Borodina and Nielsen, 2014) (Figure 1). Constraints are very different between the seed train, which prioritizes strain stability, efficient biomass production, process convenience and robustness, and the main cultivation step, often referred to as the “production phase,” where maximal yield and product formation rate are ultimate goals. The slope of any rectilinear section of the LOS plot will be the specific growth rate μ We extend this past what is described by Poulsen et al (2003) by taking the derivative of Equation (2) with respect to time and further simplifying with the chain rule to get:. This expression for μ is not dependent on the magnitude of b and is independent of background
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