Mechanism and evolutionary analysis of Yarrowia lipolytica CA20 capable of producing erythritol with a high yield based on comparative genomics.

Combined mutagenesis is widely applied for the breeding of robust Yarrowia lipolytica used in the production of erythritol. However, the changes of genome after mutagenesis remains unclear. This study aimed to unravel the mechanism involved in the improved erythritol synthesis of CA20 and the evolutionary relationship between different Y. lipolytica by comparative genomics analysis. The results showed that the genome size of Y. lipolytica CA20 was 20,420,510 bp, with a GC content of 48.97%. There were 6330 CDS and 649 ncRNA (non-coding RNA) in CA20 genome. Average nucleotide identity (ANI) analysis showed that CA20 genome possessed high similarity (ANI > 99.50%) with other Y. lipolytica strains, while phylogenetic analysis displayed that CA20 was classified together with Y. lipolytica IBT 446 and Y. lipolytica H222. CA20 shared 5342 core orthologous genes with the 8 strains while harbored 65 specific genes that mainly participated in the substrate and protein transport processes. CA20 contained 166 genes coding for carbohydrate-active enzymes (CAZymes), which was more than that found in other strains (108－137). Notably, 4, 2, and 13 different enzymes belonging to glycoside hydrolases (GHs), glycosyltransferases (GTs), and carbohydrate esterases (CEs), respectively, were only found in CA20. The enzymes involved in the metabolic pathway of erythritol were highly conserved in Y. lipolytica, except for transaldolase (TAL1). In addition, the titer and productivity of erythritol by CA20 were 190.97 g/L and 1.33 g/L/h, respectively, which were significantly higher than that of WT5 wherein 128.61 g/L and 0.92 g/L/h were obtained (P< 0.001). Five frameshift mutation genes and 15 genes harboring nonsynonymous mutation were found in CA20 compared with that of WT5. Most of these genes were involved in the cell division, cell wall synthesis, protein synthesis, and protein homeostasis maintenance. These findings suggested that the genome of Y. lipolytica is conserved during evolution, and the variance of living environment is one important factor leading to genome divergence. The varied number of CAZymes existed in Y. lipolytica is one factor that contributes to the performance difference. The increased synthesis of erythritol by Y. lipolytica CA20 is correlated with the improvement of the stability of cell structure and internal environment. The results of this study provide a basis for the directional breeding of robust strains used in erythritol production.解脂亚罗酵母(Yarrowia lipolytica)是赤藓糖醇生产中使用的主要菌种，复合诱变是选育优良菌株的常用方法。然而，诱变处理所引起的基因组变化尚待探索。本研究对前期获得的高产菌株CA20和原始菌株WT5进行基因组测序，并与已发布的8株解脂亚罗酵母进行比较基因组分析，旨在探究菌株CA20高产赤藓糖醇的机理以及不同菌株的基因组进化关系。结果显示，菌株CA20基因组大小为20,420,510 bp，GC碱基含量为48.97%，编码6330个CDS和649个ncRNA，菌株CA20和其他8株菌高度同源，平均核苷酸同源性(average nucleotide identity，ANI)> 99.50%，其中与菌株IBT 446和H222具有更近的亲缘关系。比较基因组分析显示，CA20和其他8株菌共有5342个核心基因，而CA20特有的65个基因主要参与物质跨膜运输和蛋白质转运过程。CA20基因组中含有166个碳水化合物活性酶(carbohydrate-active enzymes，CAZymes)基因，远多于其他菌株(108~137个)，包括特有的4个糖苷水解酶类(glycoside hydrolases，GHs)、2个糖基转移酶类(glycosyltransferases，GTs)和13个碳水化合物酯酶类(carbohydrate esterases，CEs)。除转醛酶TAL1外，赤藓糖醇代谢途径有关酶在不同菌株中高度保守。此外，菌株CA20的赤藓糖醇产量和产率为190.97 g/L和1.33 g/L/h，显著高于WT5的128.61 g/L和0.92 g/L/h (P<0.001)。相比于WT5，CA20中5个基因发生移码变异，15个基因存在非同义突变位点，这些基因主要参与细胞分裂、细胞壁合成、蛋白质合成及稳态维持等过程。以上结果表明，解脂亚罗酵母基因组在进化过程中保守；生存环境不同是导致菌株间基因组差异的重要因素；基因组中CAZymes数量的差异是不同菌株间性能差异的原因之一；菌株CA20高产赤藓糖醇与其细胞结构及内环境稳定性的提升有关。本研究结果为赤藓糖醇高产菌株的定向选育提供基础。.