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3-Amino-4-Hydroxy Benzoic Acid Production Via 3,4-AcAHBA in Escherichia coli

Shozui, F., Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc.
Yokoyama, K., Ajinomoto Co., Inc.
Khrushcheva, A., Ajinomoto-Genetika Research Institute
Slivinskaya, E., Ajinomoto-Genetika Research Institute

The bio-based production of chemicals is presently of great importance in the field of biotechnology. Production of aromatic compounds from renewable bio resources is recognized as one of the key aspects of biorefinery research. 3-Amino-4-hydroxy benzoic acid (3,4-AHBA) is a precursor of bioplastics as it increases the thermostability of the polymer when used as a monomer. Most aromatic compounds are produced via the shikimate pathway; however, in Streptomyces griseus, 3,4-AHBA is produced through a two-step catalyzed reaction from L-aspartate-4-semialdehyde (ASA) and dihydroxyacetone phosphate (DHAP). 3,4-AHBA was found to be a metabolic intermediate in grixazone biosynthesis in S. griseus and it is synthesized by the activities of the enzymes GriI and GriH. GriI catalyzes an aldol condensation reaction between ASA and DHAP, while GriH converts the resulting C7 metabolite into 3,4-AHBA.

However, there are two problems with biological production of 3,4-AHBA in E. coli. The first problem is its rapid conversion to other chemical compounds. 3,4-AHBA is non-enzymatically converted to 2-aminophenoxazin-3-one-8-carboxylic acid (APOC) under aerobic/anaerobic condition, while enzymatically, it is converted to 3-acetylamino-4-hydroxybenzoic acid (3,4-AcAHBA). Approximately 39% of the exogenously added initial amount of 3,4-AHBA is converted into 3,4-AcAHBA in the presence of E. coli cells. The second problem is an inhibitory effect of 3,4-AHBA on the growth of E. coli cells. Addition of 60 mM exogenous 3,4-AHBA led to significant growth retardation of MG1655 cells.

In contrast to the instability of 3,4-AHBA in culture medium, exogenously added 3,4-AcAHBA maintains a constant concentration in culture medium during aerobic/anaerobic cultivation conditions. Furthermore, 3,4-AcAHBA is non-toxic for E. coli cells. Taking these observations into account, biotechnological production of 3,4-AcAHBA and its chemical deacetylation were considered to be feasible processes.

In S. griseus, N-acetyltransferase (NAT) encoded by natA is solely responsible for the N-acetylation of 3,4-AHBA. Homology analysis showed that an S. griseus NAT-like protein, NhoA, is conserved in E. coli. NhoA functions as an arylamine acetyltransferase that catalyzes the acetyl-CoA dependent N-acetylation of a variety of arylamine substrates. Our study demonstrated that the disruption of nhoA led to a loss of function and consequently the loss of 3,4-AcAHBA production, thus indicating that NhoA is solely responsible for the N-acetylation of 3,4-AHBA in E. coli.

For the synthesis of 3,4-AHBA, NspI and NspH from Streptomyces murayamaensis (the GriIH homologs) were expressed in E. coli. The E. coli MG1655 strain harboring the nspIH operon expression plasmid produced around 2.7 %(mol/mol) of 3,4-AHBA from glucose and by-produced AHBA derivatives. By combining genetic modification to improve ASA supply in the host strain and expression of nspI, nspH and nhoA, 3,4-AcAHBA production increased up to 13.3 %(mol/mol) without any by-production of AHBA derivative compounds. These results suggest a technological advantage of 3,4-AcAHBA fermentation.