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. The goals of this study were to investigate whether strategies can be found to eliminate single vitamin requirements and if the found solutions could be combined yielding a vitamin-independent S. cerevisiae strain. Next, the engineered strain was characterized for growth performance in batch and chemostat cultures and compared to a parental strain requiring vitamin supplementation for optimal growth. Finally, transcriptome data of the engineered strain grown in absence of vitamins was analyzed and compared to its isogenic reference grown in presence of vitamins.

酿酒酵母（Saccharomyces cerevisiae）为B族维生素缓生营养缺陷型（bradytroph），即酵母细胞在缺乏此类代谢物的外源供给时，生长速率会显著减慢。解除此类营养需求以实现最优生长表现，对于工业菌株而言是极具价值的表型特征，因为这可降低生产工艺成本，同时减少污染风险。本研究中，针对酿酒酵母在缺乏泛酸、对氨基苯甲酸（para-aminobenzoic acid, pABA）、吡哆醇、肌醇及生物素时的亚最优生长状态，研究人员分别通过单基因或双基因过表达内源基因FMS1、ABZ1/ABZ2、SNZ1/SNO1、INO1，以及费氏赛林达酵母（Cyberlindnera fabianii）来源的BIO1予以纠正。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（thiamine）时的生长情况，结果发现过表达THI4或共过表达THI4与THI5可将生长速率提升至参照菌株CEN.PK113-7D在全维生素补充培养基中最大比生长速率（specific growth rate）的83%。尽管本研究最初目标是将所有已确认的修饰组合至单一菌株，但最终构建的工程菌株IMX2210仅携带了用于纠正生物素、pABA、泛酸及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即可快速生长，最大比生长速率达0.28 ± 0.01 h⁻¹；其二，在稀释率（dilution rate）为0.1 h⁻¹的有氧葡萄糖限制恒化器培养（chemostat cultures）中，该菌株在无维生素条件下的生理参数，与参照菌株CEN.PK113-7D在相同培养条件下且使用完全补充维生素的培养基时的表现高度相似。比较转录组分析（transcriptome analysis）结果显示，两组样本的基因表达差异极为有限，且仅涉及B族维生素生物合成途径（biosynthetic pathways）相关基因，进一步印证了二者的生理相似性。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基础。本研究的核心目标首先是探索能否找到消除单一维生素需求的策略，并将这些解决方案组合以获得不依赖维生素的酿酒酵母菌株；其次对工程菌株的分批培养及恒化器培养生长性能进行表征，并与需要外源维生素补充以实现最优生长的亲本菌株进行对比；最后分析了无维生素培养的工程菌株的转录组数据，并与在有维生素条件下培养的同基因参照菌株进行比较。