Characterizing yeast promoters used in Kluyveromyces marxianus

Fermentation at higher temperatures can potentially reduce the cooling cost in large-scale fermentation and reduce the contamination risk. Thus, the thermotolerant yeast, Kluyveromyces marxianus, which can grow and ferment at elevated temperatures, is a promising biotechnological tool for future applications. However, the promoters used in K. marxianus are not well characterized, especially at elevated temperatures, which is important in efficient metabolic pathway construction. In this study, six constitutive promoters (PTDH3, PPGK, and PADH1 from both Saccharomyces cerevisiae and K. marxianus) were evaluated in K. marxianus through the heterologous expression of the KlLAC4, GUSA, and SH BLE genes at various temperatures, with various carbon sources and oxygen conditions. The expression was evaluated at the transcription and protein level using real-time PCR and protein activity determination to eliminate the effect of heterologous protein stability. While the transcription of all the promoters decreased at higher temperatures, the order of their promoting strength at various temperatures with glucose as the carbon source was PKmPGK > PKmTDH3 > PScPGK > PScTDH3 > PKmADH1 > PScADH1. When glycerol or xylose was supplied as the carbon source at 42 °C, the order of promoter strength was PKmPGK > PScPGK > PKmADH1 > PScADH1 > PScTDH3 > PKmTDH3. The promoter activity of PTDH3 decreased significantly, while the promoter activity of both of the PADH1 promoters increased. Oxygen conditions had non-significant effect. The results of this study provide important information for fine-tuned pathway construction for the metabolic engineering of K. marxianus.