Screening and whole genome sequencing of strain M-15 for cottonseed meal fermentation

Abstract:

[Background] Free gossypol in cottonseed meal limits the release of nutrients and the application of cottonseed meal as protein feed, and microbial fermentation is an effective approach for the removal of gossypol from cottonseed meal. [Objective] To screen a strain capable of degrading gossypol and decipher the mechanism of gossypol degradation by the strain. [Methods] The strain capable of degrading gossypol was screened with the degradation rate of gossypol as the indicator and identified by morphological observation, physiological and biochemical tests, and phylogenetic analysis based on 16S rRNA sequences. The possible intermediates of gossypol metabolism by the selected strain were analyzed and the utilization capability of the strain for the key intermediates of polycyclic aromatic hydrocarbons was determined. Furthermore, whole genome sequencing of the strain was carried out to mine the genes encoding the enzymes involved in the degradation of gossypol. [Results] A strain capable of degrading gossypol was isolated from a soil sample in the outskirts of Baoding and named M-15. After fermentation by strain M-15, the content of free gossypol in cottonseed meal decreased from 882.07 mg/kg to 32.38 mg/kg, which indicated a degradation rate as high as 96.33%. The strain was identified as Bacillus subtilis. Salicylic acid, catechol, and gentianic acid were the major intermediates in the degradation of gossypol by strain M-15. According to the utilization capacity of the intermediates in the metabolism of polycyclic aromatic hydrocarbons, the possible degradation pathway of gossypol in the fermentation of cottonseed meal by strain M-15 was deduced as follows. Naphthalenes were produced from gossypol with the binaphthalene structure and then formed catechol or gentianic acid via the salicylic acid pathway. Catechol and gentianic acid underwent ring opening at the meta position, and the products entered the tricarboxylic acid cycle to complete the degradation. [Conclusion] The strain M-15 highly effective in degrading gossypol was screened out. It may degrade gossypol via the salicylic acid pathway. The findings provided a theoretical basis for the development and application of microbial fermentation for detoxification of cottonseed meal.