[3S05Aa] 21. Open Topics in Biotechnology - Part 1

2:45 PM - 3:00 PM JST(5:45 AM - 6:00 AM UTC)

[3Open-05] CRISPR/Cas9-based genome engineering for indole-3-acetic acid production in Escherichia coli K12

○Pattarawan Intasian¹, Supachanee Vichit, Chalermroj Sutthaphirom¹, Valalak Jintasakul¹, Chonticha Rueanpeng¹, Sirus Kongjaroon, Pimchai Chaiyen¹ (1. Vidyasirimedhi Institute of Science and Technology (Thailand))

Keywords:

Indole-3-acetic acid,Bioproduction,CRISPR/Cas9

Metabolic engineering and synthetic biology contribute to development of green methodology for production of valuable biochemicals, biofuels and bio-based materials. Creation of genome editing tools to allow manipulation of microorganisms is important for cell functions as microbial cell factories. In this work, we have developed CRISPR/Cas systems for integrating genes into the E. coli K12 genome. Neutral integration sites in E. coli K12 genome which allow gene integration without creating negative effects were identified. The neutral integration sites were used for integrating genes relevant for engineering the strain to produce indole-3-acetic acid (IAA), one of the most important plant hormones. We have overcome the issues of protein insolubility and stability of enzymes in the IAA producing pathway to improve IAA production. The thermotolerant tryptophan-2-monooxygenase, indole acetamide hydrolase and chaperone systems were employed for IAA production. Our current system can produce IAA at >8 g/L, which is the highest IAA production from E. coli reported to date. This advancement could be beneficial for scaling up the system in the future to obtain high production of IAA for real applications.