Abstract
BackgroundSaccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve production efficiency under industrially relevant conditions, coordinated expression of multiple genes by manipulating promoter strengths is an efficient approach. It is known that gene expression is highly dependent on the practically used environmental conditions and is subject to dynamic changes. Therefore, investigating promoter activities of S. cerevisiae under different culture conditions in different time points, especially under stressful conditions is of great importance.ResultsIn this study, the activities of various promoters in S. cerevisiae under stressful conditions and in the presence of xylose were characterized using yeast enhanced green fluorescent protein (yEGFP) as a reporter. The stresses include toxic levels of acetic acid and furfural, and high temperature, which are related to fermentation of lignocellulosic hydrolysates. In addition to investigating eight native promoters, the synthetic hybrid promoter P3xC-TEF1 was also evaluated. The results revealed that PTDH3 and the synthetic promoter P3xC-TEF1 showed the highest strengths under almost all the conditions. Importantly, these two promoters also exhibited high stabilities throughout the cultivation. However, the strengths of PADH1 and PPGK1, which are generally regarded as ‘constitutive’ promoters, decreased significantly under certain conditions, suggesting that cautions should be taken to use such constitutive promoters to drive gene expression under stressful conditions. Interestingly, PHSP12 and PHSP26 were able to response to both high temperature and acetic acid stress. Moreover, PHSP12 also led to moderate yEGFP expression when xylose was used as the sole carbon source, indicating that this promoter could be used for inducing proper gene expression for xylose utilization.ConclusionThe results here revealed dynamic changes of promoter activities in S. cerevisiae throughout batch fermentation in the presence of inhibitors as well as using xylose. These results provide insights in selection of promoters to construct S. cerevisiae strains for efficient bioproduction under practical conditions. Our results also encouraged applications of synthetic promoters with high stability for yeast strain development.
Highlights
Saccharomyces cerevisiae is widely studied for production of biofuels and biochemicals
The promoter activities of PTDH3, promoters of translational elongation factor EF-1 alpha (PTEF1), PHSP12, PTPS1 and P3xC-TEF1 were investigated in the presence or absence of antibiotics (Additional file 1: Figure S1), indicating that the promoter activities revealed by centromericplasmid reporter system are relatively stable
The mRNA level of yeast enhanced green fluorescent protein (yEGFP) under the control of different promoters at log-phase was well correlated with promoter strength determined by quantification of yEGFP fluorescence (Additional file 1: Figure S2), which is consistent with the previous report [22], suggesting that the promoter strengths reflected by fluorescence intensities of yEGFP were convincible
Summary
Saccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve overall performance of the strains, it is critical to ensure that efficient pathways with balanced gene expression levels are achieved. The optimal gene expression levels are variable and dependent on different environmental conditions [1,2,3]. Successful examples have been reported to improve production efficiency by fine-tuning gene expression through manipulating multiple promoter strengths [7,8,9,10,11,12]. With different combinations of promoters to control the expression of pathway genes, combinatorial method has been successful in developing efficient strains [8, 13, 14]
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