耐L-山梨糖伴生菌的比较基因组学研究

Comparative Genomics Study of L-Sorbose-Tolerant Helper Strain

摘要: 维生素C工业化生产以“二步发酵法”为核心技术，其第二步发酵依赖由产酮古洛糖酸菌(Ketogulonicigenium vulgare)与伴生菌组成的混合发酵体系完成从L-山梨糖向2-酮基-L-古龙酸(2-KLG，维生素C前体)的转化。提高L-山梨糖浓度对提高2-KLG转化效率有积极的作用。前期研究结果显示，高浓度L-山梨糖对伴生菌具有强烈的抑制作用，进一步影响2-KLG的转化。筛选获得耐高浓度L-山梨糖伴生菌并研究其耐受机理对实现高浓度底物转化意义重大。本研究以筛选获得的伴生菌，植物内生芽孢杆菌(Priestia endophytica)的出发株Y0及耐L-山梨糖突变株Y31为研究对象，利用比较基因组学技术，研究Y31与Y0在基因水平上差异，为进一步分子改造奠定基础。结果显示，Y31与Y0相比，形态上无显著差异，但可耐受15%的L-山梨糖。Y0的基因组总长度为4,442,862 bp，编码基因数量为5572个，Y31的基因组总长度为4,437,048 bp，编码基因数量为5556个。通过比较基因组学分析发现，Y31与Y0相比，在染色体的局部变异(主要集中在3,026,400 bp~3,034,400 bp的区间内)，涉及多个基因的变化。同时，与Y0相比，Y31发生了质粒P7的丢失，其涉及多个功能未知蛋白。推测突变区域和P7丢失可能是Y31表现出高L-山梨糖耐受性的原因。

Abstract: The industrial production of vitamin C is based on the “two-step fermentation process”, in which the second fermentation step relies on a co-culture system composed of Ketogulonicigenium vulgare and helper strain to convert L-sorbose into 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. Increasing the concentration of L-sorbose has a positive effect on improving the conversion efficiency of 2-KLG. However, previous studies have shown that high concentrations of L-sorbose strongly inhibit the growth and activity of helper strain, thereby adversely affecting the conversion of 2-KLG. Therefore, the screening of helper strain tolerant to high L-sorbose concentrations and the elucidation of their tolerance mechanisms are of great significance for achieving efficient conversion under high-substrate fermentation of vitamin C. In this study, the helper strain, Priestia endophytica, including the original strain Y0 and an L-sorbose-tolerant mutant strain Y31 obtained through screening, was used as the research object to investigate the genetic differences between the Y31 and Y0 by comparative genomics approaches. The results showed that, compared with the Y0, the mutant strain Y31 exhibited no significant morphological differences but was able to tolerate up to 15% (w/v) L-sorbose. The genome length of Y0 was 4,442,862 bp, encoding 5572 predicted genes, whereas Y31 possessed a genome of 4,437,048 bp with 5556 predicted genes. Comparative genomic analysis revealed localized chromosomal variations in Y31 relative to the parental strain Y0, primarily concentrated within the region from 3,026,400 bp to 3,034,400 bp. In addition, strain Y31 was found to have lost plasmid P7 compared with Y0. This plasmid harbors multiple genes encoding proteins with unknown functions. It is therefore inferred that the identified chromosomal mutation region and the loss of plasmid P7 may be responsible for the enhanced tolerance to high L-sorbose concentrations observed in the Y31.