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Enzyme and Biosynthetic Pathway Engineering for Controlling Intracellular Localization and Chemical Biosynthesis

  • Author(s): Lin, Jyun-Liang
  • Advisor(s): Wheeldon, Ian R.
  • et al.
Abstract

Chemical biosynthesis with enzymes and microorganisms holds great promise for renewable production of chemicals, fuels, and therapeutics. To achieve high productivity, identification of active enzymes and engineering of efficient metabolic or synthetic pathways are essential for massive production. Enzyme co-localization orchestrated by synthetic DNA and protein scaffolds have been shown improvement of pathway yields in vitro and in vivo. However, an investigation of the effect of DNA scaffold to enzyme activity of enzyme-DNA nanostructure and development of tools for enzyme co-localization on intracellular membranes are limited.

Using a model system of horseradish peroxide and a multi-valent DNA scaffold, we demonstrated that DNA-conjugated horseradish peroxidase (HRP) activity can be enhanced by tuning the binding between substrates and DNA. The concept extracted from this work can be extended to rational designing of enzyme activity toward given substrates.

Biological production of esters can be accomplished by the enzymatic reaction catalyzed by alcohol-O-acetyltransferase (AATase) with condensation of acyl-CoA and alcohol. Ethyl acetate synthesized by Atf1, an AATase, in S. cerevisiae relies on the availability of acetyl-CoA metabolized by aldehyde dehydrogenase (Ald6) and acetyl-CoA synthetase (Acs1) from acetaldehyde and ethanol produced by alcohol dehydrogenase (Adh). Both Ald6 and Acs1 have been shown to localize to the cytosol or mitochondria, whereas Atf1 is found on the endoplasmic reticulum (ER) and lipid droplets (LDs). To engineering enzyme co-localization of Ald6, Acs1, and Atf1, we first elucidated molecular transport of Atf1 to the ER and LDs, and essential domains required for membrane association. We then developed an Oleosin-Cohesin-Dockerin based synthetic protein scaffold that functionally localizes Ald6 and Acs1 to Atf1 on LDs. Such intracellular organization of the engineered pathway has improved the yield of ethyl acetate production. In addition, we also developed a spectrophotometric-based high throughput screening assay for determination of AATase activity and discovered a previously unexplored Atf-Sl from tomato with high activity toward a diverse set of alcohols and acyl-CoAs. This work not only broaden our understanding of LD biology, but also expand our capabilities to control intracellular localization of enzymes for efficient chemical conversion and to rapidly investigate enzyme activity of AATase.

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