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Redesign of metabolic pathways for photosynthetic production of n-butanol using cyanobacteria

  • Author(s): Lan, Ethan I.
  • Advisor(s): Liao, James C
  • et al.
Abstract

As world population increases and fossil fuel-based resource depletes, production of renewable chemical and fuel using microorganisms is an attractive approach to solving both energy and environmental problems. n-Butanol has received increasing attention because it is both a potential fuel substitute and an important chemical feedstock, and has been produced by microorganisms from sugars or biomass. However, direct conversion of CO2 into n-butanol has never been accomplished.

In this work, we constructed an n-butanol biosynthesis pathway in cyanobacteria and demonstrated the first production of n-butanol from CO2. However, the production requires anoxic treatment in dark and the inhibition of photosystem II in light. This difficulty turns out to be originated in both the lack of driving force and the oxygen sensitivity of the key CoA-acylating aldehyde dehydrogenase. We overcame this difficulty of n-butanol production in cyanobacteria by redesigning the pathway with ATP expenditure as a driving force and expressing an oxygen tolerant enzyme for converting butyryl-CoA to butyraldehyde. The final synthetic pathway was constructed using enzymes from five different organisms and accomplished direct photosynthetic n-butanol production up to 400 mg/L. These results demonstrate both the feasibility of direct n-butanol production from CO2 and the importance of redesigning pathways according to the characteristics of host metabolism.

With the above success, the next bottleneck resides in the malonyl-CoA synthesis, which is highly regulated across all organisms. To solve this problem, we redesigned malonyl-CoA biosynthesis by constructing pathways that are independent of acetyl-CoA carboxylase, which is used universally for malonyl-CoA synthesis. We designed several novel pathways, and experimentally demonstrated one route for malonyl-CoA biosynthesis in vitro from oxaloacetate in four enzymatic steps. This result presents a novel method for engineering malonyl-CoA availability, which is crucial for the production of fatty acids, polyketides, and flavonoids.

Together, this work presents a collection of metabolic engineering design principles which enhances our understanding of fundamental evolution of natural pathways and our ability to design new metabolic pathways for the green production of desirable compounds.

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