棉粕发酵菌株M-15的筛选及全基因组测序分析

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

摘要:

【背景】 棉粕中的游离棉酚致使其释放营养价值及蛋白饲料应用受到限制，微生物发酵是棉粕脱酚的有效策略。【目的】 筛选具有棉酚降解活性的发酵菌株，初步探索菌株降解棉酚的机制。【方法】 以棉酚降解率为检测指标筛选棉酚高效降解菌株，采用形态学观察、生理生化鉴定和基于16S rRNA基因序列的系统发育分析进行菌株鉴定。同时，分析筛选到的菌株代谢棉酚可能的中间产物及对多环芳烃类物质代谢关键中间产物的利用能力，并通过菌株全基因组测序发掘菌株中相关代谢酶对应基因的存在。【结果】 从河北省保定市近郊某土壤样品中筛选出一株具有较高棉酚降解活性的菌株，命名为M-15。该菌株发酵棉粕，可将其中的游离棉酚含量由882.07 mg/kg降至32.38 mg/kg，降解率高达96.33%；菌株M-15被鉴定为枯草芽孢杆菌(Bacillus subtilis)。此外，菌株M-15降解棉酚的培养液中检测到关键中间产物有水杨酸、邻苯二酚及龙胆酸，结合该菌株对多环芳烃类物质代谢关键中间产物利用能力，推测菌株M-15发酵棉粕降解棉酚可能的代谢途径由棉酚的联萘结构分解形成萘类化合物，再经水杨酸途径形成邻苯二酚或龙胆酸，通过邻苯二酚和龙胆酸分别进行间位开环，最后由三羧酸循环途径代谢。【结论】 本研究筛选得到高效降解棉酚的菌株M-15，初步推定其应该通过水杨酸途径降解棉酚，为微生物发酵棉粕脱毒技术的开发与应用奠定了一定的理论基础。

关键词:棉粕;枯草芽孢杆菌;固态发酵;棉酚;脱毒

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.

Key words:cottonseed meal;Bacillus subtilis;solid-state fermentation;gossypol;detoxification