Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7

Abstract

Background: Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes.

Results: Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc.

Conclusions: This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.

Keywords: Ethanol productivity; Ethanol titers; Ethanol tolerance; Global transcription machinery engineering (gTME); Oxidative tolerance; RNA polymerase II; Subunit Rpb7; Transcriptional engineering; VHG fermentation.

Fig. 1

Multifunction of Rpb7 in gene expression. Rpb7 usually fulfills its function by forming sub-complex with Rpb4, but the major role of Rpb4 is to augment the interaction of Rpb7 with Pol II [42]. a In the transcription initiation complex, the Rpb4/7 sub-complex is situated closed to the nascent-transcript-exit groove and adjacent to Rpb1 C-terminal domain (CTD) linker region [10], and it is also located near general transcription factor TFIIB and can physically interact with TFIIF [43]. b The role of Rpb4/7 in post-transcription regulation, including mRNA export, translation, and mRNA decay [19]

Fig. 2

Ethanol tolerance. M1 in a 0% ethanol; b 8% (v/v) ethanol; c 10% (v/v) ethanol. All cells were grown in YPAD medium at 30 °C, 225 rpm. d Spot assay. Tenfold serial dilutions of the culture (5 μL) were spotted on YPAD agar with or without 10% (v/v) ethanol. The spotted agar plates were incubated at 30 °C for 2 days

Fig. 3

Cross-tolerance towards different inhibitors. M1 in a 3.5 mM H₂O₂; b 80 mM acetic acid; c 1.5 M NaCl. d Intracellular ROS level in M1 when cells reached early log phase (OD₆₀₀ = 1). ROS level is positively correlated to the fluorescence intensity of probe H₂DCFDA. All experiments were performed in triplicates

Fig. 4

Cross-tolerance towards inhibitors from lignocellulose hydrolysates. M1 in a 196 mM levulinic acid; b 1.16 g/L furfural; c 17.5 mM HMF; d 1 mM ferulic acid; e 13.1 mM vanillin; f 12 mM p-coumaric acid

Fig. 5

Fermentation characteristics during laboratory VHG fermentation. a ethanol concentration (solid line) and specific productivity (dashed line); b glucose concentration (solid line) and OD600 (dashed line); c acetic acid profile; d glycerol profile. Cells were cultured in biological replicates in 300 g/L glucose with a high inoculum of initial cell density of OD600 = 15 (~6 g DCW/L). Metabolites were analyzed by HPLC. *Specific productivity is expressed by ethanol productivity per viable cell following the equation below [29]: $\frac{{\text{EtOH}}_t-{\text{EtOH}}_{t-1}}{\left(\frac{{\text{DCW}}_{\text{viable},t}+{\text{DCW}}_{\text{viable},t-1}}{2}\right)(t-t_{-1})}\left[\text{g}·{\text{g DCW}}^{-1}·{\text{h}}^{-1}\right]$

Fig. 6

Fermentation characteristics. Ethanol production (solid) and glucose consumption (dashed) of M1 in CEN.PK and BY strains. CEN.PK2-1C strains containing mutated and native operon of p41K-RPB7 were denoted as CEN-M1 and CEN-P41K-RPB7, respectively

Fig. 7

Gene expression level changes in ethanol synthesis pathway. Up-regulated genes (red arrows) from M1 in ethanol synthesis pathway