Modularised Engineering of Regulatory Circuits to Expand Artificial Control Mechanisms for Metabolic Engineering in Yeast

Precise control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. However, current regulatory mechanisms for transcriptional control do not deliver sufficiently precise control for many applications – including industrial applications – because they are not induced at the right time during culture growth. This is particularly problematic for the more complex regulatory control mechanisms in Eukaryotes. Predictable design of transcriptional control circuits remains a significant challenge because the common transcriptional tools are not well characterised for application in metabolic engineering. Here, we employ a modularised engineering strategy to deploy novel synthetic circuits for conditional control of heterologous pathways in Saccharomyces cerevisiae. To better understand operational parameters, each genetic part was characterised under industry-relevant conditions. A novel tetracycline-mediated circuit was developed to deliver stringent pathway repression to minimise metabolic burden during strain development and maintenance. This was combined with a novel temperature induction circuit to deliver an economical induction system suitable for industrial applications. This delivered a 44 % increase in production of the terpenoid nerolidol, to 2.54 g L^-1 in flask cultivation. Using these positive/negative transcriptional regulatory circuits to control heterologous pathways expands global strategies to control metabolisms in industrial microorganisms, and provides a regulatory control mechanism which may be more suitable for industry applications.