At the beginning of the project, when we explored the solution of late spring cold (LSC), we found that exogenous spraying abscisic acid (ABA) was the most common and effective methods. Chemical synthesis of ABA might produce amounts of by-products and toxic compounds, while it’s difficult to purify, so biosynthesis seemed to be a better solution. Through literature review, we acquired that Prof. Hong Tan of Chengdu Institute of Biology, Chinese Academy of Sciences has been committed to ABA synthesis by Botrytis cinerea and obtained excellent research achievements. Therefore, we visited Prof. Tan, hoping to bring some inspiration to our project design.
On July 21, 2020, six members of SCU-China went to Chengdu Institute of Biology, Chinese Academy of Sciences for an interview with Professor Tan Hong. During the interview, we asked some questions about ABA fermentation and the treatment of late spring cold, and Prof. Tan provided positive answers. For the fermentation microorganism Botrytis cinerea, Prof. Tan made a few points: First, this mold is a plant pathogen, may cause a large range of plant pollution if without thoroughly purified; Second, although natural Botrytis cinerea has ABA synthesis pathway, but its synthesis efficiency is very low, also there is a problem of unthoroughly research on the strain. Their research group can only improve the strain through metabolic engineering, but not in synthetic biology; Finally, the purification method of abscisic acid from Botrytis cinerea is also very difficult and expensive.
Fig 1. Group photo
The chemical synthesis of ABA can be produced on a large scale, but due to its special chemical structure, the production process usually produces many toxic wastes and biologically inactive analogs (due to chiral problems). Allowing farmers to directly spray ABA crude products may cause soil contamination that does more harm than good. The difficulty in separating and purifying the crude product results in high cost of pure ABA, which makes it unsuitable for farmers. Professor Hong Tan's research team at the Chengdu Institute of Biology, Chinese Academy of Sciences (CAS) has realized the biosynthesis of ABA in Botrytis cinerea. During our communication with Prof. Tan, we learned that the biosynthesis of ABA requires complex fermentation steps. Although ABA can be freely transported out of the eukaryotic cell membrane, it is difficult to purify as same as chemical synthesis. At the same time, B. cinerea is a plant pathogenic bacteria, which may lead to widespread plant fungal diseases if biosafety is not met. Prof. Tan also mentioned that the ABA synthesis pathway in B. cinerea has been well studied.
Fig 2-3. Lab visit
After we proposed the idea of ABA synthesis based on Saccharomyces cerevisiae, Professor supported this proposal quickly. She pointed out that since Saccharomyces cerevisiae is much more thoroughly studied than B. cinerea, synthetic biology methods should push producing more efficient engineered microorganisms. Because the four enzyme genes in the BcABA cluster are not well understood yet, modeling may help us improve our understanding of the enzyme structure, and also increase ABA yields, she said. For aspect to ABA treatment of late spring cold, she showed that ABA can indeed significantly improve plant stress resistance, but we need to ensure that the concentration of ABA fermentation is sufficient for agricultural application. We adopted all of Prof. Tan's suggestions in the subsequent project design.
After the interview, Professor Tan showed us around the fermentation laboratory and briefly introduced the fermentation technology used in the research institute. At last, we took a group photo in front of the institute, and we expressed our gratitude to Professor Tan Hong again.
Fig 4-5. Discussion with Prof. Tan
Based on the interview, we learned about the drawbacks of Botrytis cinerea in biosynthesis and decided to conduct ABA biosynthesis by engineering Saccharomyces cerevisiae. Similarly, Tan's suggestion also made us realize the necessity of assisting the subject design by modeling.
