(276f) Functional Characterization of Saccharomyces Cerevisiae Promoters and Application in Pathway Engineering

Synthetic biology is considered as an emerging research field that will bring new opportunities to biotechnology. Among the microbial platforms in synthetic biology, Saccharomyces cerevisiae is one of the most widely used organisms due to its well-characterized physiology and genetics, fast cell growth rate, and availability of abundant genetic tools.

    Most metabolic engineering and synthetic biology studies have focused on selecting the appropriate enzymes, because enzyme homologs cloned from a variety of organisms could have different behavior in terms of expression and activity in a heterologous host. However, selection of proper promoters is also important especially for yield and productivity optimization. This is not only because the protein transcription level is determined by the promoter strength, but also because promoters could be regulated and behave differently under different growth conditions. The constitutive promoters have been reported mostly come from housekeeping genes, including glycolysis pathway genes and elongation factor. Here we report the cloning of eighteen constitutive promoters employing eGFP as a reporter from the S. cerevisiae genome and the comparison of their strengths. Five of them come from glycolysis pathway genes, the rest thirteen come from amino-acyl tRNA synthetase genes. To our knowledge, this is the first time report about the promoters that come from amino-acyl tRNA synthetase genes. By analyzing the sequence of the promoters, we found some transcription factors could affect the strengths of promoters. In the batch fermentation, the strengths of promoters that come from different pathways behave in different patterns. We chose the promoters with relatively high strength and cloned the xylan degradation pathway and xylose reductase genes under these promoters, and transformed into S. cerevisiae. The resulting strain synthesized xylitol directly from beechwood xylan, and therefore can be utilized as a consolidated bioprocessing (CBP) organism for biomass utilization and conversion. During the fermentation we found the promoter not only affected the expression of gene, but also the growth of the cell.