Formation Mechanism Of Gossypol Enantiomers And Creation Of New Materials With Low Toxicity For Cottonseed

Nature Plants published on line a research paper entitled Dirigent gene editing of gossypol antioxidants for toxicity replicated cotton seeds written by Chen Xiaoya, a research group of the Center for Excellence and Innovation in Molecular Plant Science, Chinese Academy of Sciences. This study identified and characterized the key proteins in cotton that control the biosynthesis of levo and dextro gossypol. By removing levo gossypol through gene editing technology, low or non-toxic cottonseed was obtained and had no significant impact on cotton insect resistance. This work is the first to realize the specific selection of enantiomers of compounds through gene editing, which opens up a new way for the asymmetric synthesis of complex natural products, and is of great significance for crop genetic improvement and molecular design breeding.

Gossypol is an important terpenoid phytoalexin in cotton, which widely exists in cotton plants of Malvaceae family. It is formed by oxidative coupling of two molecules of hemigossypol and has axial chirality. The biological activities of the two enantiomers formed are quite different (Figure A). Among them, L-gossypol has reproductive toxicity to humans and non ruminant animals, which limits the use of rich oil and protein in cottonseed. Although cotton varieties without gossypol have been bred, they are more vulnerable to diseases and pests. In contrast, d-Gossypol does not affect mammalian sperm production, but it has similar anti insect and antibacterial activities with L-Gossypol. Therefore, cottonseed that selectively inhibits the synthesis of L-gossypol and retains D-gossypol can reduce the reproductive toxicity of cottonseed while retaining its resistance to pests.

Through bioinformatics analysis, the team found that two leading protein (DIR) genes GhDIR5 and GhDIR6 were co expressed with known gossypol synthesis pathway genes. Unlike enzyme proteins, GhDIR5 and GhDIR6 are secreted into cotton glands and located outside the cell. GhDIR5 and many DIR genes are arranged in tandem in the same string of DIR gene clusters (A04), while GhDIR6 is isolated on another chromosome (D05) and spatially separated. The enzyme activity experiment shows that GhDIR6 can control the biosynthesis of dextral gossypol, while GhDIR5 controls the biosynthesis of levogypol (Figure A). Research shows that GhDIR5 can be knocked out by CRISPR-Cas9 mediated gene editing technology, which can prevent the formation of L-gossypol in cottonseed (Figure B), while the level of D-gossypol and other terpenoids with similar structures has not changed significantly, so cotton insect resistance can be retained. Compared with wild type cottonseed, the toxicity of gene edited cottonseed with reduced levogypol content to mouse spermatocytes and other cell lines was significantly reduced. In addition, molecular simulation and mutation analysis revealed three key amino acid residues of the levo and dextro gossypol control proteins, which play a key role in enantioselectivity. At the same time, combined with evolutionary analysis, it is speculated that the DIR gene responsible for levo gossypol was selected from the evolution and diffusion of cotton plants 5 million to 13 million years ago, which is exactly the time of the rodent outbreak, It is speculated that the prosperity of rodents accelerated the emergence of levogypol and its control protein with anti reproductive toxicity.

To sum up, this study identified the key proteins that control the chirality of gossypol axis in cotton, and selectively removed levogypol harmful to mammals through gene editing, which not only can maintain the ability of cotton pests and diseases, but also opens a new way for the safe use of cottonseed protein and cottonseed oil. This study has important reference value for the selective manipulation of the formation of axial chiral isomers using synthetic biology. In addition, GhDIR5 can be used for industrial production of L-gossypol, providing a new technical route for the preparation of antisperm, antiviral and anti-tumor drug precursors.

The research work was supported by the national key research and development plan, the CAS strategic leading science and technology project, the National Natural Science Foundation of China, etc. Scientific researchers from Kunming Institute of Botany, Chinese Academy of Sciences and Yazhouwan Seed Laboratory, Hainan Province participated in the research.

Gene editing GhDIR5 reduces the reproductive toxicity of cottonseed. (A) Gossypol is formed by oxidative coupling of two hemigossypol molecules. GhDIR5 and GhDIR6 control the production of left and right gossypol respectively. (B) The proportion of L-gossypol in cottonseed of GhDIR5 gene knockout cotton lines was significantly reduced.