Metabolic Engineering for Aromatic Amino Acids in Cyanobacteria

Jeremiah Vue, Purdue University

Abstract

Phenylalanine and tryptophan are valuable chemicals in the beverage, pharmaceutical and animal feed industries. Currently, engineered heterotrophic microorganisms produce phenylalanine and tryptophan from carbon sources, such as sugars. As an alternative production strategy, we are investigating photosynthetic cyanobacteria to produce aromatic amino acids from CO2. We are combining directed and evolutionary approaches. Initially, we performed chemical mutagenesis on the cyanobacteria strains Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 and selected colonies for resistance to tryptophan (Trp) or phenylalanine (Phe) analogs. The resistant colonies were subsequently screened for overproduction of the aromatic amino acids. The mutants resistant to phenylalanine analogs had significant increases of all three aromatic amino acids, but mutant strains resistant to tryptophan analogs only had increased tryptophan. The aromatic amino acids were detected both intracellularly and in the media. After one round of mutagenesis, the best Synechocystis strain resistant to Phe had the intracellular levels of Phe increase 38-fold compared to wild type, and the best Trp analog resistant strain increased Trp 130-fold. To determine the mutations leading to the phenotype, we used next generation sequencing to identify single nucleotide polymorphisms in genes of the shikimate and aromatic amino acid pathway enzymes using next-generation sequencing (NGS).

Degree

M.S.Ch.E.

Advisors

Morgan, Purdue University.

Subject Area

Chemical engineering

COinS