Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae

Abstract

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Keywords: intron-mediated enhancement; promoter-proximal introns; recombinant amylase; starch-based biofuels.

Figure 1.

SC^−URA starch plates showing clear zones produced by recombinant yeast strains expressing the (A) ateA, (B) temA and (C) temG_Opt amylases, as well as (D) recombinant strains expressing an α-amylase and glucoamylase. Strain Y294[ENO1] was a negative control (no amylase gene).

Figure 2.

Extracellular amylase activity displayed by S. cerevisiae strains expressing the (A) ateA, (B) temA and (C) temG_Opt genes under the control of different native and intron-engineered promoters when aerobically cultivated in 2 × SC^−URA at 30°C. The solid line represents the negative control, and the different broken lines represent the intron-less, RPS25A_i and COX4_i-containing strains. Strains with the ENO1_p, TEF1_p, HXT7_p and TDH3_p promoters are represented by green, blue, red and orange graphs, respectively. Error bars represent standard deviations from the mean of three replicates.

Figure 3.

(A) Total extracellular amylase activity on soluble corn starch using supernatant from the recombinant and control S. cerevisiae strains cultivated aerobically in 2 × SC^−URA at 30°C for 48 hours. (B) SDS-PAGE analysis of the protein species in the supernatant after 72 hrs. MM represents the molecular weight marker (kDa). (C) Glucose, (D) ethanol, (E) glycerol and (F) maltose concentration during the fermentation of raw starch for the recombinant and control S. cerevisiae strains. Error bars represent the standard deviations from the mean of three replicates.