Engineering class-B vitamin biosynthesis in Saccharomyces cerevisiae

Saccharomyces cerevisiae is bradytroph for class B vitamins, it means that yeast cells exhibit slower growth in the absence of an external source of these metabolites. Alleviating these nutritional requirements for optimal growth performance would represent a valuable phenotypic characteristic for industrial strains since this would result in cheaper processes that would also be less susceptible to contaminations. In the present study, suboptimal growth of S. cerevisiae in absence of either pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and biotin were corrected by single or double gene overexpression of native FMS1, ABZ1/ABZ2, SNZ1/SNO1, INO1 and the Cyberlindnera fabianii BIO1, respectively. Several strategies were attempted to improve growth of S. cerevisiae CEN.PK113-7D in absence of thiamine, revealing that overexpression of THI4 and THI4/THI5 was able to improve growth up to 83% of the maximum specific growth rate of the reference CEN.PK113-7D in medium including all vitamins. Although the initial aim of this study was to combine all identified mutations in a single strain, the engineered strain IMX2210 only harboured genes to correct biotin, pABA, pantothenate and inositol bradotrophies. Firstly, this strain was fast-growing at a maximum specific growth rate of 0.28 ± 0.01 h-1 in medium devoid of all vitamins. Secondly, this strain exhibited physiological variables in aerobic glucose limited chemostat cultures at a dilution rate of 0.1 h-1 in absence of vitamins similar to that of the reference strain CEN.PK113-7D grown in the same conditions but in a fully supplemented complete medium. These physiological similarities were further emphasized by the limited differences observed in comparative transcriptome analysis from the chemostat culture grown cells that were essentially affecting genes of the class B vitamins biosynthetic pathways. This work paves the way towards construction of the first fast growing vitamin-independent S. cerevisiae strain.酿酒酵母（Saccharomyces cerevisiae）为B族维生素缓生营养缺陷型菌株，即当其缺乏此类代谢物的外源来源时，酵母细胞的生长速率会显著降低。解除此类营养需求以实现最优生长性能，对于工业生产菌株而言是极具价值的表型特征，因为这可简化生产工艺并降低污染风险。在本研究中，研究人员分别通过单或双基因过表达酿酒酵母内源基因FMS1、ABZ1/ABZ2、SNZ1/SNO1、INO1以及费比恩类林德纳酵母（Cyberlindnera fabianii）来源的BIO1，修复了酿酒酵母在缺乏泛酸、对氨基苯甲酸（pABA）、吡哆醇、肌醇与生物素时的亚优生长情况。此外，研究团队尝试了多种策略以改善酿酒酵母CEN.PK113-7D在缺乏硫胺素时的生长表现，结果发现过表达THI4或THI4与THI5联合过表达，可将其生长速率提升至含全维生素培养基中参考菌株CEN.PK113-7D最大比生长速率的83%。尽管本研究最初目标是将所有已鉴定的突变整合至单一菌株中，但工程菌株IMX2210仅携带了用于修复生物素、pABA、泛酸与肌醇缓生营养缺陷的基因。其一，该菌株在不含所有维生素的培养基中可快速生长，最大比生长速率达0.28 ± 0.01 h⁻¹；其二，在稀释率为0.1 h⁻¹的有氧葡萄糖限制恒化器培养中，该菌株的生理参数与在完全补充维生素的同款培养基中培养的参考菌株CEN.PK113-7D高度相似。通过对恒化器培养细胞开展比较转录组分析，进一步验证了上述生理相似性：二者仅在B族维生素生物合成通路相关基因上存在少量表达差异。本研究为构建首个快速生长且不依赖维生素的酿酒酵母菌株奠定了重要基础。