(17g) Keynote: Overproduction of Essential Aromatic Amino Acids in Synechocystis sp. PCC 6803

Phenylalanine and tryptophan are used as supplements in the animal feed and in pharmaceutical industries. Currently, engineered heterotrophic microorganisms are used to produce phenylalanine and tryptophan from carbon sources such as glucose. As an alternative, photoautotrophic cyanobacteria can fix carbon dioxide to produce aromatic amino acids via the shikimate pathway. This approach converts the two step process of obtaining the carbon source such as molasses/glucose from plants and its subsequent conversion to aromatic amino acids to a single step. However, the native shikimate pathway is subject to extensive regulation primarily by feedback inhibition. 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase has been shown to be feedback inhibited by phenylalanine and anthranilate synthase (AS) by tryptophan, respectively, in several cyanobacteria strains. Therefore, we used a strategy combining random mutagenesis and rational metabolic engineering to develop cyanobacterial strains engineered to improve the productivity and efficiency of converting carbon dioxide to phenylalanine or tryptophan. We selected the well characterized Synechocystis sp. PCC 6803 as a model cyanobacteria because it is easy to transform via homologous recombination.

To improve the overall flux to the aromatic amino acids, the first step was to express the feedback resistant enzyme DAHP synthase. Next we expressed the feedback resistant AS from E.coli. The resulting cyanobacteria strains accumulated phenylalanine and tryptophan titers 6 fold and 180 fold as much as the empty vector controls, respectively. To further improve on the production of aromatic amino acids by our strain expressing a combination of feedback resistant DAHPS and AS, the wild type strain was modified by chemical mutagenesis followed by heterologous expression of feedback resistant enzymes from E.coli. Random mutagenesis was performed using methyl methanesulfonate and resistant strains were screened for overproduction of aromatic amino acids using phenylalanine or tryptophan analogues. The best phenylalanine and tryptophan overproducers were selected and had their genomes sequenced. Previous work in cyanobacterial amino acid overproduction did not determine the mutations of the deregulated aromatic amino acid biosynthesis pathway enzymes. However, in this work, single nucleotide polymorphisms (SNPs) were identified in the shikimate pathway from next generation sequencing data of overproducing mutant strains that were responsible for enhanced aromatic amino acid production. The phenylalanine accumulating strains had a mutant DAHPS, while the tryptophan accumulating strain had a mutant chorismate mutase (CM). The top Trp accumulating strain was subjected to further rational engineering by expressing feedback insensitive DAHPS and AS individually as well as together. The resulting engineered strain expressing both feedback resistant DAHPS and AS combined with a mutated CM showed a significant 580 fold increase in tryptophan titers.

In this work, we have identified novel mutations responsible for a feedback resistant DAHPS and CM in Synechocystis sp. PCC 6803. The combination of rational and random mutagenesis approaches proved to be a superior strategy than either approach alone. This method could be used to guide the development of cyanobacteria strains that are more suited for production of chemicals on an industrial scale.