热带假丝酵母ctpxa1基因缺失菌的构建及对长链二元酸积累的影响

Effect of ctpxa1 gene deletion in Candida tropicalis on long chain dicarboxylic acid accumulation

摘要:

Pxa1p为酿酒酵母Saccharomyces cerevisiae过氧化物酶体上的膜蛋白，与Pxa2p组成二聚体，参与转运长链脂肪酸进入过氧化物酶体过程。热带假丝酵母能够发酵烷烃和脂肪酸生产长链二元酸，而过氧化物酶体中发生的β-氧化会消耗产生的长链二元酸造成产率降低。本研究以热带假丝酵母Candida tropicalis 1798为宿主菌，通过基于PCR片段的同源单交换法，快速构建ctpxa1基因敲除菌株C. tropicalis 1798-pxa1。利用半定量RT-PCR技术，检测ctpxa1基因在C. tropicalis 1798、C. tropicalis 1798-pxa1的表达量，灰度值比值为2.03，表明ctpxa1在C. tropicalis 1798-pxa1中的表达被弱化。经144 h发酵，C. tropicalis 1798-pxa1比C. tropicalis 1798的十二碳二元酸产量明显提升，其产出浓度为10.3 g/L，比野生型菌株C. tropicalis 1798提高了94.3%。

Abstract:

Candida tropicalis uses alkanes and fatty acids to produce long chain dicarboxylic acids. However, the yield can be affected by β-oxidation in peroxisomes. Pxa1p was a membrane protein of Saccharomyces cerevisiae peroxisomes. Pxa1p and Pxa2p form a dimer that is involved in transporting of long chain fatty acids into peroxisomes, but the similar transporting system of Candida tropicalis has not yet been reported. In this study, a ctpxa1 gene deletion strain named C. tropicalis 1798-pxa1 was constructed by homologous single exchange method using PCR fragment. The expression of ctpxa1 gene in C. tropicalis 1798, C. tropicalis 1798-pxa1 was detected by semi-quantitative RT-PCR, and the ratio of gray value was 2.03, implying that the expression of ctpxa1 in C. tropicalis 1798-pxa1 was weakened. After 144 h fermentation, the dodecanedioic acid production of C. tropicalis 1798-pxa1 was increased 94.3% than the former strain, the maximum yield was 10.3 g/L.