The nonlinear nature of metabolism and the use of metabolic intermediates for numerous cellular reactions makes the balancing of flux through these pathways paramount to meeting the needs of growing cells and an efficient production pathway. Pathway balancing is often hard wired into the organism via the use of promoters of different strength. In eukaryotes, such as yeast and fungi, creating promoters of different strength typically requires the use of different endogenous promoters, each having the potential different regulatory behaviors in different media. An alternative strategy is to create a library of random promoter mutants; however, this often results in mostly weaker promoters and has the potential to generate undesired regulatory differences. Here, we have developed two new methods for pathway balancing that largely preserves the regulatory features of a common parent promoter. The first uses TATA-box promoter libraries to identify promoters across a range of strengths, including those stronger than strongest natural promoters. A select number of these promoters were then used to systematically vary the strength of expression of each gene in a four gene pathway to find optimal combinations of gene expression for the production of deoxyviolecin. The optimal concentration produced significantly higher titers than the unbalanced pathway. The second tool we developed addresses the need to create promoter libraries in the TATA-box region of endogenous genomically encoded pathways. We will describe the engineering of robust Cas9-guided base-editors, with specific tuning of the mutagenesis window to target a narrow range near the TATA-box. Together, these tools can provide more effective means of balancing endogenous and heterologous pathways in Yarrowia lipolytica.

TATA-box engineering for heterologous and endogenous pathway balancing in Yarrowia lipolytica

ACS Spring 2022

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