Presentation Information
[3ASBA-07-KL]Exploring synthetic promoter design for gene expression control across different yeast species
○Masahiro Tominaga1,2、Nanami Kawai2、Yoichiro Ito1,2、Akihiko Kondo1,2,3,4、Jun Ishii1,2,3 (1. Engineering Biology Research Center, Kobe University (Japan)、2. Kobe University, Graduate School of Science, Technology and Innovation (Japan)、3. Kobe University, Faculty of Engineering (Japan)、4. Center for Sustainable Resource Science, RIKEN (Japan))
Keywords:
Inducible Promoters,Komagataella phaffii,Saccharomyces cerevisiae,Recombinant Protein Production
Introduction
Gene expression control is essential for enabling microbes to efficiently produce valuable chemicals and biologics as well as for investigating biological functions. To achieve flexible and precise gene expression control, synthetic systems that artificially control gene expression at multiple levels including transcription and translation have been widely developed. In yeasts, synthetic transcription systems can be constructed using inducer-responsive synthetic transcriptional activators (sTAs) and synthetic promoters. Synthetic promoters include sTA-binding sequences fused upstream of the TATA box sequence, followed by a downstream sequence containing transcription start site. The transcription from the synthetic promoter is activated only when the sTA binds to the synthetic promoter in response to inducers. In principle, the promoter activity can be precisely controlled on demand as long as the sTA possessing minimum inducer-independent binding to the promoter. However, even with the well-engineered sTAs with negligible inducer-independent binding to the promoter, sTA-independent promoter activity is often observed, and this complicates the design of yeast synthetic promoters. In this study, we evaluated the effect of different synthetic promoter sequences on gene expression in the methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) and Saccharomyces cerevisiae.
Results
First, we revealed that sTA-independent promoter activity mainly results from regulatory effects of upstream sequences of the promoter, and that this may be substantially mitigated via appropriate insulation. Next, we examined the effects of STA-binding sequence repeats and its distance from the TATA box and found that promoter induction levels were maximized when multiple sTA binding sequences were placed just upstream of the TATA box sequences. We then replaced the sequence downstream of the TATA box with other heterologous TATA box-downstream sequences, including those derived from several fungal species. As a result, several synthetic promoter variants with strong induction levels were identified. Finally, based on these insights, we successfully constructed yeast synthetic promoters capable of inducing reporter gene expression by two to three orders of magnitude. Moreover, these synthetic promoters could be used to efficiently produce various recombinant proteins both at the test-tube and in a jar-fermenter.
Conclusions
We have established a simple design principle to construct strong inducible synthetic promoters in two different yeast species. The set of synthetic promoters enhances the programmability of yeast functions and accelerate engineering biology.
Gene expression control is essential for enabling microbes to efficiently produce valuable chemicals and biologics as well as for investigating biological functions. To achieve flexible and precise gene expression control, synthetic systems that artificially control gene expression at multiple levels including transcription and translation have been widely developed. In yeasts, synthetic transcription systems can be constructed using inducer-responsive synthetic transcriptional activators (sTAs) and synthetic promoters. Synthetic promoters include sTA-binding sequences fused upstream of the TATA box sequence, followed by a downstream sequence containing transcription start site. The transcription from the synthetic promoter is activated only when the sTA binds to the synthetic promoter in response to inducers. In principle, the promoter activity can be precisely controlled on demand as long as the sTA possessing minimum inducer-independent binding to the promoter. However, even with the well-engineered sTAs with negligible inducer-independent binding to the promoter, sTA-independent promoter activity is often observed, and this complicates the design of yeast synthetic promoters. In this study, we evaluated the effect of different synthetic promoter sequences on gene expression in the methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) and Saccharomyces cerevisiae.
Results
First, we revealed that sTA-independent promoter activity mainly results from regulatory effects of upstream sequences of the promoter, and that this may be substantially mitigated via appropriate insulation. Next, we examined the effects of STA-binding sequence repeats and its distance from the TATA box and found that promoter induction levels were maximized when multiple sTA binding sequences were placed just upstream of the TATA box sequences. We then replaced the sequence downstream of the TATA box with other heterologous TATA box-downstream sequences, including those derived from several fungal species. As a result, several synthetic promoter variants with strong induction levels were identified. Finally, based on these insights, we successfully constructed yeast synthetic promoters capable of inducing reporter gene expression by two to three orders of magnitude. Moreover, these synthetic promoters could be used to efficiently produce various recombinant proteins both at the test-tube and in a jar-fermenter.
Conclusions
We have established a simple design principle to construct strong inducible synthetic promoters in two different yeast species. The set of synthetic promoters enhances the programmability of yeast functions and accelerate engineering biology.
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