背景:根孢霉是一种植物病原真菌,可产生一种称为水色色素的色素。这种真菌的perylenequinone在光敏剂的生产中起着重要作用,光敏剂是光动力疗法的必要组成部分。我们使用酿酒酵母应用合成生物学生产静脉色素。
结果:基因Cppks1,它编码一种非还原性聚酮合成酶(NR-PKS),负责在C.phlei中生物合成水,克隆到酵母附加型载体中并用于转化酿酒酵母。此外,将编码构巢曲霉的磷酸肽酰转移酶(PPTase)的基因克隆到酵母整合载体中,并将其引入酿酒酵母中,用于酶促活化Cppks1的蛋白质产物。在缺乏亮氨酸/尿嘧啶的选择性培养基上筛选共转化的酵母,并通过菌落PCR确认酵母中整合和附加型重组质粒的存在。Cppks1表达的附加型载体在培养过程中非常不稳定,以至于大多数细胞在非选择性培养基中迅速失去其附加型载体。在选择性培养基中也观察到较小程度的这种损失。该数据强烈表明Cppks1基因的存在对转化的酵母细胞的生长产生显著的有害影响,并且需要选择压力来维持表达Cppks1的载体。在20°C和25°C下长时间培养后,选择性培养基上的共转化体显示出色素沉着的独特变化,但不是在30°C此外,薄层层析(TLC)显示,在来自共转化子的细胞的提取物中存在与纯化的水色素相对应的斑点。液相色谱(LC/MS/MS)证实新表达的色素确实是水色色素。
结论:我们的结果表明,通过多基因表达进行代谢工程改造是可能的,并且能够在酿酒酵母中产生真菌色素黄花。这一结果增加了我们对真菌PKS基因特征的理解,表现出复杂的结构和多样的生物活性。
BACKGROUND: Cladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a photosensitizer that is a necessary component of photodynamic therapy. We applied synthetic biology to produce phleichrome using Saccharomyces cerevisiae.
RESULTS: The gene Cppks1, which encodes a non-reducing polyketide synthase (NR-PKS) responsible for the biosynthesis of phleichrome in C. phlei, was cloned into a yeast episomal vector and used to transform S. cerevisiae. In addition, a gene encoding a phosphopantetheinyl transferase (PPTase) of Aspergillus nidulans was cloned into a yeast integrative vector and also introduced into S. cerevisiae for the enzymatic activation of the protein product of Cppks1. Co-transformed yeasts were screened on a leucine/uracil-deficient selective medium and the presence of both integrative as well as episomal recombinant plasmids in the yeast were confirmed by colony PCR. The episomal vector for Cppks1 expression was so dramatically unstable during cultivation that most cells lost their episomal vector rapidly in nonselective media. This loss was also observed to a less degree in selective media. This data strongly suggests that the presence of the Cppks1 gene exerts a significant detrimental effect on the growth of transformed yeast cells and that selection pressure is required to maintain the Cppks1-expressing vector. The co-transformants on the selective medium showed the distinctive changes in pigmentation after a period of prolonged cultivation at 20 °C and 25 °C, but not at 30 °C. Furthermore, thin layer chromatography (TLC) revealed the presence of a spot corresponding with the purified phleichrome in the extract from the cells of the co-transformants. Liquid chromatography (LC/MS/MS) verified that the newly expressed pigment was indeed phleichrome.
CONCLUSIONS: Our results indicate that metabolic engineering by multiple gene expression is possible and capable of producing fungal pigment phleichrome in S. cerevisiae. This result adds to our understanding of the characteristics of fungal PKS genes, which exhibit complex structures and diverse biological activities.