图1 基于增变基因的酿酒酵母基因组连续进化［酿酒酵母中存在一些维持遗传稳定性的基因，其敲除或抑制会引发不同的突变类型，提高基因组突变率，即增变基因（mutator genes）。利用CRISPRi系统抑制增变基因的表达水平，构建4个基因增变器（mutators），可诱导酿酒酵母基因组发生不同类型的突变］

Fig. 1 Continuous genome evolution of S. cerevisiae through mutator genes(There are mutator genes in S. cerevisiae to maintain its genetic stability, and their knockdown or suppression can increase the genome mutation rate, and also result in different types of mutations. Using the CRISPRi system to inhibit the expression of mutator genes, four mutators were constructed to induce different types of mutations in the genome of S. cerevisiae.)

图2 以mVenus为报告基因检测不同gRNA对增变基因表达的抑制效果（显著性差异：*表示p < 0.05；**表示p < 0.01；***表示p < 0.001）

Fig. 2 Inhibition efficiency of different gRNAs on the expression of mutator genes with mVenus as a reporter(*, ** and *** for the significance p < 0.05, 0.01 and 0.001, respectively)

图3 基因增变器诱导酿酒酵母基因组产生的突变率和突变类型（a）基因增变器诱导酿酒酵母基因组产生不同的相对突变率；（b）基因增变器均诱导酿酒酵母基因组产生不同的突变类型，4个基因增变器均产生了点突变，其中TM和RM诱导产生短片段缺失和插入，RM和CM还诱导产生了中长片段插入缺失等其他突变类型（显著性差异：*表示p < 0.05；**表示p < 0.01）

Fig. 3 Mutation rates and types in the genome of S. cerevisiae induced by mutators(a) Relative mutation rates of the genome of S. cerevisiae induced by mutators (* for significance p < 0.05); (b) Mutation type of the genome of S. cerevisiae induced by mutators. All 4 mutators induced SNPs, TM and RM induced small Indels, and RM and CM also induced other mutation types. * and ** for significance p < 0.05 and 0.01, respectively.

图4 基因增变器提高酿酒酵母异丁醇耐受性（a）当培养基中不存在异丁醇时，进化组和对照组之间的生长趋势无显著差异；（b）当培养基中异丁醇浓度达到4%时，yTM的生长速率显著高于其他菌株；（c）当异丁醇浓度达到4.5%时，相比对照y426菌株，各进化组都具有显著的生长优势，其中提升效果最好的增变酵母为yTM

Fig. 4 Evaluation of the mutators for improved isobutanol tolerance(a) No significant difference in the growth profiles of the mutated and control strains when isobutanol was not present in the medium; (b) When the concentration of isobutanol in the medium reached 4%, the growth rate of yTM was significantly higher than that of other strains; (c) When isobutanol concentration reached 4.5%, all mutated strains showed significant growth advantages over the control, with yTM as the best strain.

图5 基因增变器提高酿酒酵母β-胡萝卜素产量（a）～（e）基因增变器诱导酿酒酵母产生颜色深浅不一的菌落；（f）通过HPLC测定进化菌株的β-胡萝卜素产量（显著性差异：**表示p < 0.01；***表示p < 0.001）

Fig. 5 Evaluation of the mutators in increasing β-carotene biosynthesis（a）～（e） Mutation resulted in the formation of colonies with different color densities; (f) Production of β-carotene in the mutated and control strains determined by HPLC. ** and *** for significance p < 0.01 and 0.001, respectively.

图6 通过基因增变器提高酿酒酵母对木糖的利用率［不同基因增变器诱导的酿酒酵母在以木糖为唯一碳源的液体培养基中的生长曲线（a）和木糖消耗曲线（b）；（c）混合增变器MTRC和多重增变器MTRC*2诱导的酿酒酵母的生长曲线（空心图例）和木糖消耗曲线（实心图例）；（d）混合增变器MTRC诱导酿酒酵母在以木糖为唯一碳源的液体培养基中连续传代培养后菌液中的gRNA丰度分布］

Fig. 6 Evaluation of the mutators in improving xylose utilization[Profiles for growth (a) and xylose consumption (b) of the mutated and control strains with xylose as the sole carbon source. (c) Profiles for growth (hollow symbols) and xylose consumption (solid symbol) of the single (MTRC) and double (MTRC*2) mutated strains. (d) Distribution of gRNA abundance when the MTRC mutated strain was continuously subcultured in the liquid medium containing xylose.]

附图1 荧光酶标仪分析MSH2p-mVenus的CRISPRi结果Supplementary Fig. S1　Repression of MSH2p-mVenus by CRISPRi analyzed by a microplate reader

附图2 产β-胡萝卜素的酿酒酵母进化菌株的遗传稳定性（eCarTM菌株丢质粒前所携带的质粒为p426-Sg25，eCarCM菌株丢质粒前所携带的质粒为p426-Sg41，为验证菌株获得目的性状后能够保持遗传性状的稳定性，将获得目的性状的进化菌株去除其gRNA质粒，然后再对其性状的稳定性进行测试。以产β-胡萝卜素的进化菌株为例，具体方法如下：①在增变菌株yCarMM、yCarTM、yCarRM、yCarCM和对照菌株yCar426生长的SED-URA/G418 固体培养基上挑取颜色最深的菌落，每类菌种各挑3个单菌落，接种于SED完全液体培养基中，连续传代培养2次，同时通过测序确认所挑取的单菌落的gRNA种类；②取5 μL菌液在SED完全固体培养基上划线培养，培养2～3天后，从划线的固体培养基上随机挑取16个单菌落，每个菌落都同时在SED-URA/G418和 SED完全固体培养基上接种，筛选出只能够在SED完全固体培养基上生长的菌落，即丢弃质粒的进化菌株；③将去除质粒的进化菌株继续在SED完全液体培养基中连续传代培养3～5次后，取菌液涂布至SED完全固体培养基，通过其菌落颜色的均一性判断性状遗传的稳定性。TM和CM的进化菌株经过传代后，其菌落颜色保持均一，证明其遗传稳定性。）Supplementary Fig. S2　Genetic stability of the evolved β-carotene producing strains