Zhang Yiming, Liu Guodong, Engqvist Martin K M, Krivoruchko Anastasia, Hallström Björn M, Chen Yun, Siewers Verena, Nielsen Jens
Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 412 96, Göteborg, Sweden.
Microb Cell Fact. 2015 Aug 8;14:116. doi: 10.1186/s12934-015-0305-6.
A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol.
In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h(-1), respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH1 (81D) allele restored its growth on glucose at a maximum specific rate of 0.053 h(-1) in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h(-1).
In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.
一株在所有三个丙酮酸脱羧酶（PDC）基因中都存在缺失的酿酒酵母菌株（也称为Pdc阴性酵母）代表了一种用于生产丙酮酸衍生生物化学品的非乙醇生产平台菌株。然而，它不能以葡萄糖作为唯一碳源生长，并且需要向培养基中添加C2化合物，以满足脂肪酸和麦角固醇生物合成中对胞质乙酰辅酶A的需求。
在本研究中，一株Pdc阴性菌株通过连续传代在葡萄糖培养基中进行适应性进化，产生了三个独立进化的菌株，它们能够在以葡萄糖作为唯一碳源的基本培养基中生长，最大比生长速率分别为0.138、0.148、0.141 h⁻¹。通过基因组DNA测序在进化后的Pdc阴性菌株中鉴定出了几个基因变化。在这些基因变化中，发现有4个基因在至少两个进化菌株中携带点突变：编码葡萄糖感应信号转导途径负调控因子的MTH1、编码己糖转运蛋白的HXT2、编码线粒体柠檬酸合酶的CIT1以及编码组蛋白脱乙酰酶的RPD3。通过引入MTH1（81D）等位基因对未进化的Pdc阴性菌株进行逆向工程，使其在含有2%葡萄糖的基本培养基中以0.053 h⁻¹的最大比生长速率在葡萄糖上生长，并且逆向工程菌株中的CIT1缺失进一步将最大比生长速率提高到了0.069 h⁻¹。
在本研究中，通过基因组测序和逆向工程研究了Pdc阴性菌株在葡萄糖上可能的进化机制。MTH1中的非同义突变通过抑制几个己糖转运蛋白基因的表达减轻了葡萄糖阻遏。HXT2和CIT1中的非同义突变可能在存在突变的MTH1等位基因时发挥作用，并且可能与改变的中心碳代谢有关，以确保Pdc阴性菌株中胞质乙酰辅酶A的产生。
