Synthetic biology tools for scalable production of biorenewable 3-hydroxypropionic acid

Abstract

The ability to rewire microbial metabolism for the production of bio-based chemicals is critical for achieving a sustainable future. However, engineering cellular metabolism for bioproduction can lead to significant metabolic burdens, challenging the cost-efficient production of many relevant chemicals at an industrial scale. For instance, efforts to achieve commercial bio-based production of the building block chemical 3-hydroxypropionic acid (3HP) have been unsuccessful, primarily due to unbalanced biosynthetic pathway designs that can overload cells and also due to a direct competition between production and endogenous pathways required for cellular growth. In this proposal, we will apply modern synthetic biology and high-throughput technologies to overcome these challenges and identify an optimal engineered design for 3HP production in the budding yeast Saccharomyces cerevisiae. To do so, we will investigate two design strategies that can reduce the metabolic burden caused by 3HP biosynthesis. First, we will balance the expression levels of each enzyme in the pathway by systematically testing combinatorial combinations of promoters with varying strengths. Second, we will reduce the competition between growth and the 3HP production pathway by developing sophisticated genetic circuits that will enable cells to autonomously separate fermentation into two distinct phases, a minimum-productivity biomass propagation phase and a maximum-productivity production phase. The stability of engineered strains during scale up will be assessed experimentally and modelled mathematically. Whole-genome sequencing of non-producing escaper mutants will reveal common routes that lead to loss of production and will inform new rounds of strain engineering aimed at preventing the emergence of such mutations. Finally, once the best strain design is identified, we will conduct a genome-wide search for genes conferring 3HP tolerance in wild acid-tolerant yeast strains. These genes will be transferred to our engineered strain, thus generating a final top producer and highly tolerant strain. This integrated strategy has the potential to minimize the metabolic burden caused by 3HP production, improving strain performance and stability and generating a final top producer amenable for scale-up testing. (AU)