Key Laboratory of Metabolic Engineering and Biosynthesis Technology, Ministry of Industry and Information Technology, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing, 210094, China.
Department of Chemistry, Bengbu Medical University, Bengbu, 233030, China.
World J Microbiol Biotechnol. 2024 Nov 28;40(12):395. doi: 10.1007/s11274-024-04205-6.
Rhodotorula toruloides, an oleaginous yeast known for its high lipid productivity, produces lipids with low very-long-chain fatty acid (VLCFA) content. Meanwhile, the roles of enzymes, particularly the condensing enzymes, involved in VLCFA biosynthesis in R. toruloides remained unclear. In this study, two elongases, RtELO1 and RtELO2, were identified from R. toruloides U13N3 and their tertiary structure and catalytic mechanism were investigated using molecular dynamic methods. Both enzymes exhibited typical ELO-like characteristics, with active sites located within cavities formed by seven transmembrane helixes. RtELO2 displayed higher binding affinity to acyl-CoAs compared to RtELO1, and at least seven amino acid residues, including two crucial histidines in the "HXXHH" box, were identified as important for the condensation reaction. To enhance VLCFA production, an internal ribosome entry site (IRES)-mediated bicistronic strategy was developed to integrate multiple genes into the R. toruloides genome. The efficiency of IRES-mediated translation initiation reached 85.4% of cap-dependent upstream translation, based on EGFP fluorescent intensity. Using this strategy, four genes encoding enzymes involved in the VLCFA biosynthesis cycle (Rtelo2, RtKCR, RtHCD, and RtECR) were introduced into the U13N3 genome in various combinations. The results indicated that the expression of a single elongase had a modest effect on VLCFA production, but the simultaneous expression of multiple genes resulted in cumulative effects. Notably, the transformant harboring four genes exhibited a remarkable 436.8% increase in C22 and C24 VLCFA yield compared to the original strain.
罗伦隐球酵母是一种产油酵母，以其高产脂特性而闻名，其脂质中含有较低含量的超长链脂肪酸 (VLCFA)。然而，罗伦隐球酵母中参与 VLCFA 生物合成的酶，特别是缩合酶的作用尚不清楚。在本研究中，从罗伦隐球酵母 U13N3 中鉴定出两种延伸酶，RtELO1 和 RtELO2，并使用分子动力学方法研究了它们的三级结构和催化机制。这两种酶均表现出典型的 ELO 样特征，活性位点位于由七个跨膜螺旋形成的腔体内。与 RtELO1 相比，RtELO2 对酰基辅酶 A 具有更高的结合亲和力，并且确定了至少七个氨基酸残基，包括“HXXHH”盒中的两个关键组氨酸，对缩合反应很重要。为了提高 VLCFA 的产量，采用内部核糖体进入位点 (IRES) 介导的双顺反子策略将多个基因整合到罗伦隐球酵母的基因组中。基于 EGFP 荧光强度，IRES 介导的翻译起始效率达到帽依赖性上游翻译的 85.4%。使用该策略，将参与 VLCFA 生物合成循环的四个基因（Rtelo2、RtKCR、RtHCD 和 RtECR）编码酶以不同组合引入 U13N3 基因组中。结果表明，单个延伸酶的表达对 VLCFA 的产量仅有适度的影响，但多个基因的同时表达则会产生累积效应。值得注意的是，与原始菌株相比，同时表达这四个基因的转化体的 C22 和 C24 VLCFA 产量显著增加了 436.8%。
