(462c) An Integrated Computational and Experimental Study for the Overproduction of Fatty Acids in Escherichia Coli

Sridhar Ranganathan¹,
Anupam Chowdhury², Ali R. Zomorrodi², Ting Wei Tee ³,
Jong Moon Yoon ³⁺, Yanfen Fu ³,
Jacqueline V. Shanks ³, and Costas D. Maranas²

¹ Huck Institutes of Life
Sciences, Pennsylvania State University, University Park, PA

² Department of Chemical
Engineering, Pennsylvania State University, University Park, PA

³
Department of Chemical and Biological Engineering, Iowa State University, Ames,
IA

Fatty acids of varying chain
lengths (C₆ – C₁₆) naturally synthesized in many
organisms are promising starting points for the catalytic production of
industrial chemicals and diesel-like biofuels. However, bio-production of fatty
acids from plants and other microbial production hosts relies heavily on
manipulating tightly regulated fatty acid biosynthetic pathways. In addition,
precursors for fatty acids are used along other central metabolic pathways for
the production of amino acids and biomass, which further complicates the
engineering of microbial hosts for higher yields. In this study, we demonstrate
an iterative metabolic engineering effort that integrates computationally
driven predictions and metabolic flux analysis techniques to meet this
challenge. The OptForce procedure, our recently developed computational strain
design algorithm, was used for suggesting and prioritizing genetic
manipulations that overproduce fatty acids of different chain lengths from C₆
to C₁₆ starting with wild-type E. coli. We identified some
chain-specific genetic interventions alluding to the possibility of fine-tuning
overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP
thioesterase were upregulated
and fadD
was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty
acid/g glucose (~ 39% maximum theoretical yield) of C_14-16 fatty
acid in minimal M9 medium. OptForce suggested additional modifications distal from
the fatty acid synthesis pathways that led to additional improvements in yield.
Our study reinforces the advantage of integrating computational and experimental
tools for the design and engineering of microbial strains to overproduce
value-added chemicals or biofuels.