RNA sequencing reveals metabolic and regulatory changes leading to more robust fermentation performance during short-term adaptation of Saccharomyces cerevisiae to lignocellulosic inhibitors.

BACKGROUND: The limited tolerance of Saccharomyces cerevisiae to inhibitors is a major challenge in second-generation bioethanol production, and our understanding of the molecular mechanisms providing tolerance to inhibitor-rich lignocellulosic hydrolysates is incomplete. Short-term adaptation of the yeast in the presence of dilute hydrolysate can improve its robustness and productivity during subsequent fermentation. RESULTS: We utilized RNA sequencing to investigate differential gene expression in the industrial yeast strain CR01 during short-term adaptation, mimicking industrial conditions for cell propagation. In this first transcriptomic study of short-term adaption of S. cerevisiae to lignocellulosic hydrolysate, we found that cultures respond by fine-tuned up- and down-regulation of a subset of general stress response genes. Furthermore, time-resolved RNA sequencing allowed for identification of genes that were differentially expressed at 2 or more sampling points, revealing the importance of oxidative stress response, thiamin and biotin biosynthesis. furan-aldehyde reductases and specific drug:H+ antiporters, as well as the down-regulation of certain transporter genes. CONCLUSIONS: These findings provide a better understanding of the molecular mechanisms governing short-term adaptation of S. cerevisiae to lignocellulosic hydrolysate, and suggest new genetic targets for improving fermentation robustness.

Nutrient-supplemented propagation of Saccharomyces cerevisiae improves its lignocellulose fermentation ability.

Propagation conditions have been shown to be of considerable importance for the fermentation ability of Saccharomyces cerevisiae. The limited tolerance of yeast to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. We have investigated the hypothesis that the addition of nutrients during propagation leads to yeast cultures with improved ability to subsequently ferment lignocellulosic materials. This hypothesis was tested with and without short-term adaptation to wheat straw or corn stover hydrolysates during propagation of the yeast. The study was performed using the industrial xylose-fermenting S. cerevisiae strain CR01. Adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate from 0.04 g g-1 to 0.19 g g-1 and in corn stover hydrolysate from 0.02 g g-1 to 0.08 g g-1. The combination of short-term adaptation and supplementation with the vitamin mixture during propagation led to ethanol yields of 0.43 g g-1 in wheat straw hydrolysate fermentation and 0.41 g g-1 in corn stover hydrolysate fermentation. These ethanol yields were improved compared to ethanol yields from cultures that were solely short-term adapted (0.37 and 0.33 g g-1). Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation.

Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae.

The limited tolerance of Saccharomyces cerevisiae to the inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The aim of this study was to investigate the short-term adaptation effects in yeast strains with different genetic backgrounds. Fed-batch propagation cultures were supplemented with 40% wheat straw hydrolysate during the feed phase to adapt two different pentose-fermenting strains, CR01 and KE6-12. The harvested cells were used to inoculate fermentation media containing 80% or 90% wheat straw hydrolysate. The specific ethanol productivity during fermentation was up to 3.6 times higher for CR01 and 1.6 times higher for KE6-12 following adaptation. The influence of physiological parameters such as viability, storage carbohydrate content, and metabolite yields following short-term adaptation demonstrated that short-term adaptation was strain dependent.

Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae.

BACKGROUND: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. RESULTS: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. CONCLUSIONS: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.

Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

BACKGROUND: Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. RESULTS: Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. CONCLUSIONS: The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.

Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase.

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald(-) strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald(-) A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.