CAJ | 학술논문

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力，但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株，通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalisXZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase，PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase，乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP)，并转化宿主菌株C. tropicalisXZX，筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子 XZX02。在此基础上，将转化子 XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上，筛选得到 URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm，并转化C. tropicalisXZX03菌株，获得转化子C. tropicalisXZX04。经PCR和DNA测序确认转化子C. tropicalisXZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术，利用该技术成功敲除了细胞的 PDC 基因，为进一步利用代谢工程改造热带假丝酵母奠定了基础。
열대가사효모(Candida tropicalis)재발효공업중구유중요적응용잠력，단이배체유전결구화교저적유전전화효솔한제료기대사공정육충기술적응용。건립가고적유전전화기술병고효적산제목적기인시대사공정개조열대가사효모적중요전제。문장이C. tropicalis ATCC 20336위출발균주，통과화학유변사선획득료뇨밀정결함형돌변주C. tropicalisXZX(ura3/ura3)。이병동산탈최매(Pyruvate decarboxylase，PDC)기인작위파기인구건료량단포함동원비병재선택성표기C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase，유청산핵감-5-린산탈최매)기인량측동향삽입원우사문씨균(Salmonella typhimurium)적hisG서렬적기인고제합PDC1-hisG-URA3-hisG- PDC1(PHUHP)，병전화숙주균주C. tropicalisXZX，사선획득PHUHP편단정학정합도염색체적PDC기인위점적전화자 XZX02。재차기출상，장전화자 XZX02도포우5-FOA(5-불유청산)선택배양기상，사선득도 URA3기인종PHUHP편단중주실적영양결함형균주XZX03。진일보구건료제2개PDC등위기인적산제표체합PDCm- URA3-PDCm，병전화C. tropicalisXZX03균주，획득전화자C. tropicalisXZX04。경PCR화DNA측서학인전화자C. tropicalisXZX04세포염색체상적량개PDC등위기인피성공고제。문장건립료일충영양결함형표기가중복사용적열대가사효모유전전화기술，이용해기술성공고제료세포적 PDC 기인，위진일보이용대사공정개조열대가사효모전정료기출。
Candida tropicalis, a diploid asporogenic yeast, is frequently utilized in industrial applications and research studies. However, the low efficiency of genetic transformation limits the strain improvement by metabolic engineering. A reliable transformation and efficient deletion of target gene are prerequisite for molecular improve-ment ofC. tropicalis. In this study, an efficient approach for genetic transformation ofC. tropicalis was devel-oped based on the URA3 gene as a reusable selection marker and both ofPDC allele genes encoding pyruvate decar-boxylase were successfully deleted by this approach. Firstly, an auxotrophic mutant strain ofC. tropicalis XZX which is defective in orotidine-5′-phosphate decarboxylase (URA3) was isolated by chemical mutagenesis combined with nystatin enrichment selection and 5-fluoro-orotic acid (5-FOA) resistance selection using C. tropicalisATCC 20336 as the parent strain. Then, the firstPDC deletion cassettePDC1-hisG-URA3-hisG-PDC1(PHUHP) which contains a 1.6 kb URA3 marker gene, two copies of 1.1 kbSalmonellahisG fragments and homologous arms of target gene was con-structed and transformed intoC. tropicalis XZX cells. Transformants with a single copy ofPDC deleted were isolated and identified by PCR and DNA sequencing, which was designated asC.tropicalis XZX02. TheC.tropicalis XZX02 cells were spread on the minimal medium containing 5-FOA to generate mutant C. tropicalis XZX03 in whichURA3 marker gene was excised from PHUHP fragment integrated into thePDC gene site. The secondPDC gene dele-tion cassettePDCm-URA3-PDCm(MUM) was constructed and transformed intoC. tropicalis XZX03 to generate C.tropicalis XZX04 in which both ofPDC allele genes were deleted. All strains were confirmed by PCR and DNA sequencing. This efficient genetic transformation approach laid a foundation for further metabolic engineering ofC. tropicalis.