Studies on the biosynthetic enzymes of abscisic acid toward its
fermentation production
Atsushi MINAMI
Department of Chemistry, Faculty of Science, Hokkaido University
Research aims
Abscisic acid (ABA, 1) is a sesquiterpene phytohormone that regulates physiological
functions in plants as well as in a wide range of organisms from bacteria to human cells. For
example, 1 mediates adaptive responses toward abiotic stresses such as drought stress andsalt stress in plants, as well as temperature-induced oxygene consumption and water
filtration in sponges. In a previous study, we successfully elucidated the biosynthetic
pathway of 1 by through the heterologous expression of four ABA biosynthetic genes(bcABA1-4) in Aspergillus oryzae (Fig 1). 1, 2 Efficient production of 1 (68 mg/kg) in thetransformant revealed that cyclization of farnesyl diphosphate (FPP) by the action ofBcABA3 can be a bottleneck in the metabolic pathway, strongly suggesting that improved
catalytic activity of BcABA3 would enable ABA production with a more efficient yield.
Considering this hypothesis, in this study, we performed 1) re-analysis of the metabolic
profile in the transformant and 2) a kinetics study of BcABA3.

Figure 1. Proposed biosynthetic pathway of ABA in fungi
Methods1) Re-examination of the metabolic profile in the transformant
The previously decribed transformant harboring four bcABA genes was constructedusing traditional methods via non-homologous end joining. In this case, the bcABA genes
were integrated into random positions in the chromosome, necessitating the search forpositive clones producing ABA. Further, the production yield as well as the metabolic
profiles of each positive clone were different.
To obtain a reliable result especially focusing on the metabolic profile in thetransformant, I decided to re-construct the transformant using an established hot spot
knock-in method. In this method, the introduced genes are highly expressed in thetransformant because they locate at recently identified hot spot loci, which guarantees theirefficient expression. The metabolites produced by the new transformant were analyzed by
UPLC-MS.
2) Kinetic study of BcABA3
BcABA3 catalyzes the cyclization of FPP to afford a cyclization product 2 via twoneutral intermediates, β-farnesene and allofarnesene (Figure 2). To avoid complicateddiscussion, we quantified 2 to determine the optimal pH, temperature, metal ion requirement,and kinetic parameters of BcABA3.
Results
1) Re-examination of the metabolic profile in the transformant
Applying the established method, we constructed the expected transformant with 70%
efficiency. Metabolite analysis revealed that the transformant only produced 228 mg/kg ofABA. The fact that no intermediates such as 2–5 were observed in the metabolites indicatedthat, as expected, cyclization is a bottleneck in the ABA biosynthetic pathway.
2) Kinetic study of BcABA3The highest BcABA3 activity with FPP was observed with 50 mM HEPES buffer, pH
8.0, at 30°C. The Mg2+ cation (10 mM) was effective for BcABA3 activity. Theserequirements are almost the same as those of the traditional sesquiterpene cyclase. The
kinetic parameters are as follows: Km = 3.2 mM and kcat = 6.7 × 10-2 s-1.
Conclusion
In this study, we constructed an ABA-producing transformant by harnessing the fourbcABA genes in A. oryzae. Efficient production of ABA revealed that BcABA3 catalyzed
cyclization is a limiting step in ABA biosynthesis. Kinetic study of BcABA3 revealed theKm and kcat values under the optimal conditions. Thus, fine-tuning of the BcABA3 function
based on these kinetic studies will allow efficient production of ABA in A. oryzae.
Finally, I sincerely appreciate Noda Institute for Scientific Research for kindly
providing financial support.

References
1）Takino, J., Kozaki, T., Sato, Y., Liu, C., Ozaki, T., Minami, A., and Oikawa, H. (2018) Unveilingbiosynthesis of the phytohormone abscisic acid in fungi: Unprecedented mechanism of corescaffold formation catalyzed by an unusual sesquiterpene synthase. J. Am. Chem. Soc. 140:
12392-12395.
2）Takino, J., Kozaki, T., Ozaki, T., Liu, C., Minami, A., and Oikawa, H. (2019) Elucidation of
biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi. Biosci.
Biotechnol. Biochem. 83: 1642-1649.