Sugiyama H, Ohkuma M, Masuda Y, Park S M, Ohta A, Takagi M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Yeast. 1995 Jan;11(1):43-52. doi: 10.1002/yea.320110106.
An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.
为了建立适合疏水性化合物生物转化的系统，对一种同化烷烃的酵母——麦芽糖假丝酵母进行了研究。然而，为此目的测试的异源基因在几种情况下功能表达均未成功。另一方面，据报道，通用的亮氨酸密码子CUG在圆柱假丝酵母中被读作丝氨酸。在一些与麦芽糖假丝酵母系统发育密切相关的假丝酵母中，体外实验也表明存在相同的密码子使用改变情况。在本研究中，我们表明异源基因功能表达失败是由于在麦芽糖假丝酵母中密码子CUG被读作丝氨酸。这一结论是基于以下实验结果得出的：（1）当含有CTG密码子的麦芽糖假丝酵母细胞色素P450基因在麦芽糖假丝酵母中表达时，发现相应的氨基酸是丝氨酸，而不是亮氨酸；（2）可以从麦芽糖假丝酵母基因组中分离出一个与白色念珠菌tRNASerCAG基因结构几乎相同的tRNA基因；（3）含有一个CTG密码子的酿酒酵母URA3基因本身不能互补麦芽糖假丝酵母的ura3突变，但当CTG密码子改为另一个亮氨酸密码子CTC时，突变基因可以互补ura3突变。最后一个结果是通过将基因中的CTG密码子改为另一个密码子，在密码子使用改变的假丝酵母物种中成功实现异源基因功能表达的首个例子。
