Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity

Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process.