Characterization of an anti-pyruvate decarboxylase nanobody and its application for the arrest and redistribution of flux of ethanol fermentation

Abstract

Chemical production is dominated by the use of petroleum feedstocks, making the chemical industry one of the largest industrial emitters of greenhouse gases. Bioprocesses offer a more sustainable alternative to current production methods by replacing petroleum with renewable feedstocks, operating at lower temperatures, and reducing or eliminating the need for environmental pollutants such as heavy metals and organic solvents. However, a major challenge in the engineering of microorganisms as biocatalysts is the tradeoff between production of chemicals of interest and microbial growth. Splitting bioprocesses into two stages allows for the decoupling of growth and production. The successful implementation of two-stage processing requires a mechanism with which to switch between stages. Most synthetic tools available for the regulation of gene expression operate at the level of transcription. However, these tools introduce an inherent lag in the switching response time, as growth-promoting proteins already accumulated within cells are unaffected by transcriptional repression. While tools exist that operate at the protein level directly (post-translationally), these tools are relatively scarce in number, and require the introduction of foreign tags to target proteins, limiting their generalizability. Here, I present a heavy-chain-only antibody fragment (nanobody) that targets the key metabolic enzyme pyruvate decarboxylase (Pdc1p) in the baker’s yeast Saccharomyces cerevisiae. In chapter 2, I characterize the nanobody in vitro, measuring its specificity, affinity, and inhibition mechanism, and also discuss corroborating data from my collaborators. In chapter 3, I characterize the nanobody’s ability to inhibit cellular growth. In chapter 4, I layer transcriptional and nanobody-mediated post-translational control of pyruvate decarboxylase to rewire carbon flux from ethanol production to production of a chemical of interest. Finally, in Chapter 5, I outline possible future research directions and provide concluding remarks.