[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-357] Development of Cyanobacteria with High Production of 3-Hydroxybenzoic Acid by Metabolic Engineering

○Shodai Yoshioka¹, Kenya Tanaka^1,2,3, Tomohisa Hasunuma^1,2,4,5 (1. Grad. Sch. Sci. Technol. Innov., Kobe Univ. (Japan), 2. EGBRC, Kobe Univ. (Japan), 3. Grad. Sch. Eng. Sci., Univ Osaka (Japan), 4. CSRS, RIKENS (Japan), 5. Fac. Eng., Kobe Univ. (Japan))

Keywords:

Synechococcus sp. PCC 7002,Shikimate pathway,3-Hydroxybenzoic Acid,Metabolic Engineering

3-Hydroxybenzoic acid (3-HBA) is an aromatic compound possessing antibacterial, anti-inflammatory, and antioxidant properties, and has been utilized as an intermediate for pharmaceuticals and a monomer for fully aromatic copolyesters. Conventionally, it has been produced through multi-step organic synthesis; however, this requires harsh reaction conditions such as high temperature and high pressure, generating substantial waste and by-products. Consequently, poor yields and high environmental impacts have been inevitable challenges. Conversely, microbial production using the actinomycete Streptomyces hygroscopicus-derived chorismatase Hyg5 introduced into Escherichia coli has enabled the selective synthesis of 3-HBA from chorismate. However, the use of E. coli requires sugars that compete with food supplies as a carbon source, and the production of these sugars necessitates pretreatment and saccharification of plant biomass, leading to significant cost and environmental burdens. Therefore, we developed a 3-HBA-producing strain using a cyanobacterium, a photosynthetic microorganism easily amenable to genetic modification, as a host, and enhanced its production through a metabolic engineering approach. This aims to achieve carbon-negative 3-HBA production utilizing carbon dioxide as the carbon source and light as the energy source.In this study, we adopted Synechococcus sp. PCC 7002 as the host, which is a well-studied cyanobacterium with abundant genetic engineering insights and a particularly high growth rate. To construct the 3-HBA-producing strain, Hyg5 from S. hygroscopicus was introduced into PCC 7002. Cultivation tests of this strain confirmed the production of 3-HBA. Next, to achieve higher 3-HBA production, we focused on enhancing the precursor supply. To increase the supply of the precursor chorismate, we overexpressed AroG^fbr, an E. coli-derived 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase that is relieved of phenylalanine feedback inhibition. Furthermore, to recycle pyruvate, which is generated as a by-product when 3-HBA is cleaved from chorismate, we introduced PpsA, a phosphoenolpyruvate (PEP) synthase derived from PCC 7002. It is known that the overexpression of AroG^fbr inhibits growth, and it is common strategy to use an inducible promoter activated by the addition of an inducer for its expression. However, since the use of inducers such as isopropyl-β-D-1-thiogalactopyranoside (IPTG) directly leads to increased production costs, this study employed the sbtA promoter, derived from the PCC 7002 bicarbonate transporter gene. This promoter is strongly induced under low CO₂ conditions without requiring an inducer and was used to drive the expression of AroG^fbr and PpsA. By implementing these approaches, cultivation tests revealed that the optimized strain achieved a 3-HBA production approximately three times higher than that of the initially constructed strain.