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首页> 外文期刊>Molecular and Cellular Biology >Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.
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Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.

机译：通过顺序基因破坏​​测定热带念珠菌酰基辅酶A氧化酶同工酶的功能。

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A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.
机译：最近开发的转化系统已用于促进热带假丝酵母染色体POX4和POX5基因的顺序破坏，这些基因编码酰基辅酶A（酰基辅酶A）氧化酶的不同同工酶，可催化β氧化途径中的第一个反应。通过选择自发突变或通过在URA 3编码序列内定向缺失来恢复对转化菌株的尿嘧啶需求，可以重复生成基于URA3的转化系统，从而允许在单个热带梭菌中连续破坏基因。这些基因破坏通过显示每个基因包含两个拷贝，揭示了这种利用烷烃和脂肪酸的酵母的二倍体性质。比较其中两个POX4或两个POX5基因均被破坏的突变体，发现这两个同工酶受到差异调节，显示出独特的底物谱和动力学特性。 POX4在葡萄糖的生长过程中组成性表达，并由十二烷或月桂酸甲酯强烈诱导，其程度比POX5更大，后者主要由十二烷诱导。与POX5编码的窄谱长链氧化酶相比，POX4编码的同工酶显示了较宽的底物谱。在所有POX4和POX5基因均被破坏的菌株中，由于缺乏可检测的酰基CoA氧化酶活性，这证实了所有功能性酰基CoA氧化酶基因均已失活。该菌株不能利用烷烃或脂肪酸进行生长，表明β-氧化途径已被功能性阻断。

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