过表达乙酰辅酶A乙酰基转移酶2基因(RKAcat2)对红冬孢酵母产类胡萝卜素的影响

Effects of RKAcat2 overexpression on carotenoid production in Rhodosporidium kratochvilovae

摘要:

[背景] 乙酰辅酶A乙酰基转移酶（Acetyl Coenzyme A Acyltransferase，Acat）是硫解酶家族的一员，分为I型和II型，而II型作为甲羟戊酸（Mevalonate，MVA）途径的第一个限速酶，其表达水平和催化活性会影响萜类及其衍生物的合成量。[目的] 分析Acat II型基因的过表达对红冬孢酵母产类胡萝卜素的影响。[方法] 从红冬孢酵母YM25235菌株中克隆编码Acat II型的基因RKAcat2，将其回转到红冬孢酵母YM25235菌株中，构建一株RKAcat2基因过表达菌株进行分析。[结果] 与对照菌株相比，RKAcat2基因过表达使YM25235菌株中类胡萝卜素含量提高了50.53%，而菌株中油脂含量降低了22.80%，脂肪酸组成中油酸含量显著下降了17.78%，而且菌株中乙酰辅酶A （Coenzyme A，CoA）的含量也下降了13.64%。[结论] 过表达RKAcat2基因促进更多乙酰CoA进入MVA途径中，从而提高了类胡萝卜素的合成水平，这与部分MVA途径和类胡萝卜素合成途径中基因的转录分析结果一致。研究结果可为进一步通过代谢工程手段提高产油红酵母中类胡萝卜素及其特定组分含量的研究提供参考。

Abstract:

[Background] Acetyl-CoA acetyltransferase (Acat) belongs to thiolase family and it can be classified into two types (I and II). Type II Acat acts as the first rate-limiting enzyme of the mevalonate (MVA) pathway, and its expression level and catalytic activity affect the production of terpenoids and their derivatives. [Objective] To study whether Acat2 overexpression can increase the carotenoid biosynthesis in Rhodosporidium kratochvilovae. [Methods] A gene encoding type II Acat, named RKAcat2, was cloned from R. kratochvilovae strain YM25235 and then introduced into YM25235 for overexpression. [Results] Compared with the control, RKAcat2 overexpression in YM25235 increased the carotenoid content by 50.53% while decreased the lipid content, oleic acid content, and acetyl-CoA level by 22.80%, 17.78%, and 13.64%, respectively. [Conclusion] RKAcat2 overexpression promoted the entry of more acetyl-CoA into MVA pathway. Consequently, the carotenoid biosynthesis in YM25235 was increased, which is consistent with the results of transcriptional analysis of some genes in MVA and carotenoid biosynthesis pathways. This study may provide a reference for further research on improving the contents of carotenoid and its specific compositions in oil-producing red yeast by metabolic engineering.