Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering

Abstract

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

“…All of these observations suggested the need for identifying effective gene targets through methods that directly select resistant yeast strains with traceable genetic perturbations. Successful application of inverse metabolic engineering in the present study and some previous works (34,(61)(62)(63)(64) demonstrated the effectiveness of this approach in discovering novel gene perturbation targets for improving the desirable target phenotypes.…”

“…The genomic DNA of the S. cerevisiae S288C strain was used to construct the genomic library as previously described (34). Briefly, genomic DNA fragments (2 to 5 kb) were generated by sonication and ligated into a multicopy plasmid, pRS424 (a yeast episomal plasmid), with TRP1 as an auxotrophic selection marker (35,36).…”

Recovery of Phenotypes Obtained by Adaptive Evolution through Inverse Metabolic Engineering

“…In our previous study we inferred that the identified mutations in the Ras/PKA signaling pathway may have triggered the upregulation of PGM2 and reserve carbohydrates metabolism because not only were the mutations identified in all of the evolved mutants but also on the promoter region of PGM2, and genes involved in reserve carbohydrate metabolism contained STER elements involved in Ras activation (27). The upregulation of PGM2 in the evolved strains could increase galactose utilization as proven in earlier studies (3,13,17,21). In the present study, the mutations RAS2 Lys77 (RAU) and RAS2 Tyr112 (RBU) showed a certain relation to the upregulation of PGM2 but not to any changes in reserve carbohydrate metabolism.…”

Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries

“…The modification targets to improve galactose utilization have been well elucidated in S. cerevisiae, which include engineering of the regulatory network (inactivation of repressors and up-regulation of activator) and overexpression of the final enzyme, phosphoglucomutase (PGM2) in the Leloir pathway responsible for galactose catabolism [75][76][77]. All these targets were directly associated with the galactose metabolic pathway, but using a cDNA library, another target that is not part of galactose metabolism was also found [71]. In this study, three beneficial over-expression targets, SEC3, tTUP1, and SNR84 were identified.…”