The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (PGAL10) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. PGAL10 in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.
anaerobic alcoholic fermentation
Bioethanol production from renewable feedstocks has been rapidly increasing in the world. It is produced from a complicated process including pretreatment of the feedstocks, saccharification of the constituent polysaccharides, and subsequent fermentation of the released hexose and pentose sugars. It would greatly enhance the cost-effectiveness of bioethanol production to combine the production of enzyme for hydrolysis of polysaccharide, its hydrolysis and fermentation in one step, called consolidated bioprocessing (CBP; Lynd et al., 2002). Microorganisms suitable for the CBP should have excellent ethanol-forming capacity and can produce recombinant protein at high yield during the anaerobic alcoholic fermentation.
The yeast Saccharomyces cerevisiae has been used in bioethanol industry from renewable feedstocks due to its capability of producing ethanol at close to theoretical yield (0.51 gethanol per gglucose), high osmotolerance and ethanol tolerance, natural robustness in industrial processes, ease of genetic manipulation, and generally regarded as safe status (Den Haan et al., 2007). Also, S. cerevisiae has been widely used as a host strain to produce a variety of recombinant proteins. Saccharomyces cerevisiae-derived gene expression has been widely accomplished using the GAL system, whose expression is tightly repressed during growth on glucose and induced by growth on galactose up to 1000-fold (Hopper et al., 1978). The GAL regulatory system has been well-studied: Gal4 activates genes involved in galactose metabolism in response to galactose in the medium, and in the absence of galactose, induction is prevented by the regulatory protein Gal80 (Lohr et al., 1995). However, to date, recombinant protein production using the GAL system has been performed only in an aerobic environment, and limited information is available concerning the production of recombinant protein under anaerobic condition.
The present study investigated the production of recombinant protein in S. cerevisiae using the GAL promoter under an anaerobic alcoholic fermentation. The reporter was Candida antarctica lipase B, one of the most frequently used enzymes in industrial chemical reactions. Gene expression with GAL10 promoter (PGAL10) was compared with ones under the control of promoters known to work well under anaerobic glucose-excess condition (Ishida et al., 2005; Matsushika & Sawayama, 2008).
Firstly, efficiency of PGAL10-driven gene expression at the anaerobic alcoholic fermentation was examined using a variant (CALB14) of lipase B from C. antarctica as a reporter (Kim et al., 2007). Wild-type S. cerevisiae 2805 (MATα pep4::HIS3 prb1-δ can1 his3 ura3-52) was transformed with pGAL-MFαopt-CALB14opt for the secreotory production of CALB14 under a control of PGAL10 (Whang et al., 2009), and then, the transformants were cultivated under aerobic and anaerobic conditions using galactose as the sole carbon source. The lipase activity of culture supernatants was determined by measuring the release of p-nitrophenol by the action of an enzyme on p-nitrophenyl palmitate (Kim et al., 2007). During anaerobic culture, lipase activity, which is a product of CALB14 expression, and lipase activity per biomass were considerably lower compared to aerobic culture (Supporting Information, Table S1).
To explore the possible ways to improve gene expression using PGAL10 at anaerobic condition, use of a Δgal80 mutant where the GAL genes can be constitutively transcribed independent of galactose (Štagoj et al., 2005) was tested as a host strain. When the Δgal80 mutant transformed with pGAL-MFαopt-CALB14opt was grown anaerobically with fermentation of glucose, secreted lipase activity in the mutant was 6.2-fold higher as compared to anaerobic culture of wild-type strain (Table S1). It was noted that this enhanced CALB14 products by Δgal80 mutation were also observed in the other host strain, S. cerevisiae BY4741 (MATα his3Δ0 leu2Δ0 met15Δ0 ura3Δ0) (Table S1). The CALB14 transcript levels measured by quantitative real-time PCR method (Livak & Schmitten, 2001) were significantly higher in the mutant as compared to wild-type strain (Fig. S1). These results indicate that use of Δgal80 mutant as a host strain could enhance transcription of PGAL10 anaerobically. Thus, the Δgal80 mutant offers efficient PGAL-driven heterologous gene expression with the pronounced transcriptional enhancement of the GAL promoter not only under the aerobic condition observed in our previous study (Whang et al., 2009), but also under an anaerobic alcoholic fermentation condition.
To compare the relative strengths of PGAL10 to the promoters (PADH1,PPGK, and PPDC1) known to work well under anaerobic glucose-excess condition (Ishida et al., 2005; Matsushika & Sawayama, 2008), these promoters were amplified by PCR using genomic DNA of S. cerevisiae 2805 with synthetic primers (for PADH1, forward: 5′-GAGCTCTGTAGCCCTAGACTTGAT-3′, reverse: 5′-GAATTCTGTATATGAGATAGTTGA-3′; for PPGK, forward: 5′-GAGCTCGGGCCAGAAAAAGGAAGT-3′, reverse: 5′-GAATTCTGTTTTATATTTGTTGTA-3′; for PPDC1, forward: 5′-ATATATGGATCCGCGTTTATTTACCTATCTC-3′, reverse: 5′-ATATAT GAATTCTTTGATTGATTTGACTGTGTTA-3′) in which the nucleotides in italics are the added sequences required for the addition of the SacI (BamHI for PPDC1) and EcoRI sites at the 5′ and 3′ ends, respectively. PGAL10 in pGAL-MFαopt-CALBopt was replaced with PADH1, PPGK, and PPDC1, respectively.
For a precisely controlled environment, a bench-top stirred-tank fermentor was used for comparison of the strength of PGAL10 to those of PADH1, PPGK, and PPDC1 at anaerobic alcoholic fermentation. Four 5-L fed-batch cultures were performed with the transformants harboring different promoter-containing expression plasmid. Each seed culture was inoculated into 5-L jar fermentors (KoBiotech, Korea) with initial medium containing (g L−1): 100 of glucose, 10 of yeast extract, 20 of Bacto peptone. 100 g of glucose was supplied when initial glucose is nearly depleted, and subsequently, anaerobic glucose-excess conditions were maintained. Agitation was fixed at 200 rpm, and no aeration was supplied. In all four fermentations, cell growth and ethanol production displayed similar patterns, and ethanol yields were close to the theoretical maximum (Table 1, Fig. S2). The amount of lipase activity for PADH1 was maximal, similar with that for PGAL10, and was 4- and 50-fold higher than those for PPDC1 and PPGK, respectively (Table 1).
↵* Ethanol yields were calculated through dividing produced ethanol by consumed glucose.
↵† Relative CalB activities correspond to the normalized lipase activities divided by lipase activity obtained with PGAL10.
Ostergaard et al. (2000), (2001) reported that it is possible to control the flux through the galactose utilization pathway by reducing the levels of negative regulators or increasing the levels of positive activators of gene expression. They also demonstrated that specific growth rates of the negative regulators–deficient mutant are unaffected compared with the wild-type strain. These observations were consistent with our result in which disruption of gal80 gene gave no effect on ethanol yield and cell growth.
In conclusion, the current work provides valuable information on suitable promoters, which are highly efficient in anaerobic alcoholic fermentation of S. cerevisiae. To our knowledge, this is the first report on the different behavior of PGAL under aerobic and anaerobic condition and that the PGAL activity can be restored using Δgal80 mutant at the anaerobic alcoholic fermentation. Furthermore, the PGAL-driven gene expression in this mutant can be constitutively transcribed independent of galactose. Therefore, GAL promoter-driven gene expression with Δgal80 mutant may be effectively used in the field of metabolic engineering and recombinant protein production in the anaerobic alcoholic fermentation of S. cerevisiae.
Additional Supporting Information may be found in the online version of this article:
Fig. S1. The abundances of CALB14 mRNA determined by quantitative PCR.
Fig. S2. Five litre fed-batch cultures with the Δgal80 mutant harbouring each promoter [PGAL10 (A) or PADH1 (B) or PPDC (C) or PPGK (D)]-containing expression plasmid at anaerobic alcoholic fermentation.
Table S1. Expression of CalB14p by S. cerevisiae under an aerobic or anaerobic condition.
This study was supported by KRIBB research initiative program and the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2010-C1AAA001-0029084).