Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous
Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous
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DOI：
10.4067/s0716-97602008000100011
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发表时间：
2008-01-01
影响因子：
6.7
通讯作者：
CIFUENTES, VÍCTOR
中科院分区：
文献类型：
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作者：
NIKLITSCHEK, MAURICIO;ALCAÍNO, JENNIFER;CIFUENTES, VÍCTOR
获得了控制野生型树状叶黄酵母 ATCC 24230 菌株虾青素生物合成途径的基因（idi、crtE、crtYB、crtI 和 crtS）的基因组和 cDNA 版本的克隆和核苷酸序列。克隆了idi、crtE、crtYB、crtI和crtS基因，分别为10.9、11.5、15.8、5.9和4kb的片段。核苷酸序列数据分析表明，idi、crtE、crtYB、crtI和crtS基因分别具有4、8、4、11和17个内含子和5、9、5、12和18个外显子。此外，通过整合转化，然后进行有丝分裂重组（双重组方法），开发了高效的定点诱变系统。杂合子 idi (idi(+)/idi(-)::hph)、crtE (crtE(+/)crtE(-)::hph)、crtYB (crtYB(+)/crtYB(-)::hph)、crtI (crtI(+)/crtI(-)::hph) 和 crtS构建了 (crtS(+)/crtS(-)::hph) 和纯合突变体 crtYB (crtYB(-)::hph/crtYB(-)::hph)、crtI (crtI(-)::hph/crtI(-)::hph) 和 crtS (crtS(-)::hph/crtS(-)::hph)。所有杂合子突变体均具有苍白表型，并且比野生型菌株产生更少的类胡萝卜素。对野生型、杂合子和纯合子中的crtYB、crtI和crtS基因座的遗传分析提供了ATCC 24230菌株的二倍体构成的证据。此外，还获得了crtYB的截短形式的克隆，其缺乏源自可变剪接mRNA的N端区域的153个氨基酸。它们在携带欧文氏菌胡萝卜素原簇的大肠杆菌中异源表达，导致反式互补，并证明其在该细菌中的功能，保持其八氢番茄红素合酶活性，但不保持番茄红素环化酶活性。
The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtI and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtI and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtI and crtS genes have 4, 8, 4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi(+)/idi(-)::hph), crtE (crtE(+/)crtE(-)::hph), crtYB (crtYB(+)/crtYB(-)::hph), crtI (crtI(+)/crtI(-)::hph) and crtS (crtS(+)/crtS(-)::hph) and homozygote mutants crtYB (crtYB(-)::hph/crtYB(-)::hph), crtI (crtI(-)::hph/crtI(-)::hph) and crtS (crtS(-)::hph/crtS(-)::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtI and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.