Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands J.G.Daran@TUDelft.
Appl Environ Microbiol. 2020 Jun 2;86(12). doi: 10.1128/AEM.00388-20.
Quantitative physiological studies on commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, -aminobenzoic acid (ABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without -inositol, nicotinic acid, or ABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. Many strains of , a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, -aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.
对常用合成培养基 (SM) 进行定量生理研究，该培养基含有一组水溶性生长因子，根据其在人类营养中的作用，这些因子被称为 B 族维生素。以前的工作表明，在 CEN.PK113-7D 中，通过实验室进化消除了对生物素的需求。在本研究中，当 SM 中缺少肌醇、烟酸、吡哆醇、泛酸、-氨基苯甲酸 (ABA) 或硫胺素时，该实验室菌株的比生长速率表现不佳。随后，在没有单独的 B 族维生素的情况下，该菌株在平行连续传代实验中进行了快速有氧生长的进化。在所有进化系中，在补充有完整 B 族维生素混合物的 SM 中观察到的生长速率至少达到 90%。在没有肌醇、烟酸或 ABA 的 SM 上进行几次传代后，就已经观察到快速生长。在 SM 中缺乏硫胺素、吡哆醇或泛酸盐的情况下，需要超过 300 代的选择性生长才能达到类似的结果。对进化后的单菌落分离物的基因组进行了重新测序，对于每种 B 族维生素，选择了一组与快速维生素独立生长相关的非同义突变。使用基于 CRISPR/Cas9 的基因组编辑将这些突变引入非进化的参考菌株中。对于每种 B 族维生素，引入少量突变就足以在非补充 SM 中实现大幅提高的比生长速率，该速率至少代表在完全补充的完整 SM 中观察到的比生长速率的 87%。在工业生物技术中广泛使用的模式生物 中，许多菌株都携带合成生物素、硫胺素、吡哆醇、-氨基苯甲酸、泛酸、烟酸和肌醇所需的遗传信息。然而，这些 B 族维生素的缺失通常会导致生长不佳。本研究表明，通过适应性实验室进化 (ALE)，对于每种单独的 B 族维生素，都可以实现快速的维生素独立生长。通过全基因组测序和反向工程鉴定负责这些快速生长表型的突变表明，对于每种化合物，只需少数突变就足以在缺乏该化合物的情况下实现快速生长。这些结果是朝着开发在廉价、完全补充矿物质培养基上快速生长的 菌株迈出的重要第一步，这些菌株仅需要与碳源互补，从而降低工业酵母发酵过程中的成本、复杂性和污染风险。
