[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-370] Increased fumarate titer by relieving arginine feedback inhibition in cyanobacteria

○Haruka Higuchi¹, Satoko Ohneda¹, Ryosuke Mitsui², Tomokazu Shirai², Takashi Osanai¹ (1. Meiji Univ. (Japan), 2. RIKEN (Japan))

Keywords:

Arginine biosynthetic pathway,Biorefinery,Cyanobacteria,Fumarate,Metabolic engineering

[Purpose]
Cyanobacterial cell factories are widely studied as a biorefinery host to produce useful chemicals such as fumarate directly from CO₂. Fumarate is an organic acid widely used as a raw material for bioplastics and as a sour flavoring agent. In cyanobacterium Synechocystis sp. PCC 6803, fumarate is excreted under photoautotrophic conditions with deletion of the fumarase gene (fumC, slr0018) in the tricarboxylic acid (TCA) cycle (Du et al., 2019). Fumarate is synthesized through arginine biosynthetic pathway, and the activity of key enzyme, N-acetylglutamate kinase (NAGK, encoded by argB, slr1898) is decreased by arginine feedback inhibition. Arginine feedback inhibition on NAGK is regulated by a signal transduction protein, named P_Ⅱ (glnB, ssl0707) (Heinrich et al., 2004). The P_Ⅱ(I86N) variant, a single amino acid substitution in glnB, Ile86 to Asp86 constitutively binds NAGK and alleviates arginine feedback inhibition on NAGK, enhancing its enzymatic activity (Fokina et al., 2010). We here increased fumarate production through arginine biosynthetic pathway, by activating NAGK via the P_Ⅱ(I86N) overexpression.
[Method]
We constructed ΔfumC/glnBox strain and ΔfumC/glnB(I86N)ox strain, which were glnB or glnB(I86N) overexpression in the ΔfumC background, respectively. These strains were cultivated at a 50-mL scale in modified BG-11 medium, aerated with 1% (v/v) CO₂ in air, and maintained at 30℃ under continuous white light (300 µmol photons m^-2 s^-1). Excreted organic acids were quantified using high-performance liquid chromatography (HPLC). Intracellular polymers were observed using transmission electron microscopy (TEM) and intracellular metabolites were quantified using liquid chromatography-mass spectrometry (LC-MS).
[Results]
The ΔfumC/glnBox strain showed no change in fumarate production compared to the ΔfumC strain. However, the ΔfumC/glnB(I86N)ox strain excreted approximately two-fold more fumarate (up to 3 g/L) throughout the cultivation. Furthermore, glnB(I86N) overexpression increased cyanophycin polymers, and intracellular metabolite levels of arginine biosynthetic pathway.
[Consideration]
Our results indicate that the constitutive activation of NAGK via the P_Ⅱ(I86N) variant successfully relieved feedback inhibition, thereby increased fumarate production. These results revealed that NAGK acts as a rate-limiting enzyme in the fumarate production under photoautotrophic conditions. The enlargement of cyanophycin granules and the coordinated increase in related intracellular metabolites of arginine biosynthetic pathway suggests that metabolic flux through the arginine pathway was widely enhanced via introduction of glnB(I86N).
[Conclusion]
Genetic engineering of the P_Ⅱ protein to constitutively has activated NAGK and increased fumarate production, reaching the highest titer to date in cyanobacteria. This study demonstrates that utilizing P_Ⅱ-mediated metabolic control acts as an innovative strategy to improve fumarate production in cyanobacteria.