Our lab previously demonstrated galactose (GAL) regulon of Saccharomyces cerevisiae can serve as an excellent global regulatory infrastructure to aid the growth of this yeast on a non-native substrate, xylose. By engineering the sensor protein, Gal3p, to respond xylose, we created a semi-synthetic regulon where this pentose could activate its own metabolic genes and thousands of endogenous yeast genes required for rapid growth. Using minimal engineering approach, the semi-synthetic regulon exhibited higher growth rate compared to traditionally metabolically engineered S. cerevisiae. In this talk we will discuss our latest efforts to extend semi-synthetic regulon for utilization of another abundant pentose, arabinose. We found that the xylose-responsive Gal3p variant (called Syn4.1) exhibited strong interaction with arabinose as well. We incorporated the arabinose isomerase pathway genes - araA, araB, and araD from Lactobacillus plantarum under the control of GAL promoters (ARA-REG) or under constitutive promoter (ARA-CONS) as control. ARA-REG strain exhibited approximately 2.5-fold higher growth rate and 3.5-fold higher endpoint OD₆₀₀compared to the ARA-CONS strain when grown in YP-arabinose suggesting that a regulon-based strategy may be a general strategy to yield superior for engineering non-native sugar utilization when compared to the conventional strategy that employs only constitutive heterologous gene expression. We improved the growth rate further by 2-fold using mutagenic gene-promoter shuffling and directed evolution strategy on the three arabinose catabolic genes. Gene expression analysis indicated requirement of high expression of AraA for optimum growth. Using transcriptomic analysis, we compared genes that are differentially expressed between pentose (xylose and arabinose) and hexose (galactose) to identify factors that specifically promote growth on pentoses and elucidate how information from metabolic processes percolates and permeates through the yeast regulatory network.

Nikhil Nair

Tufts University

Appearances