Supplementary Data for Schniete et al 2019.Differential transcription in expanded gene families of central carbon metabolism of Streptomyces coelicolor A3(2)

<b>Background: </b>Streptomycete<i> </i>bacteria are prolific producer of specialised metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by HGT or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalisation or sub-functionalisation. To test whether expanded gene families in <i>Streptomyces </i>exhibit differential expression an RNA-Seq approach was used to examine cultures of wild-type <i>Streptomyces coelicolor </i>grown with either glucose or tween as the sole carbon source.<b>Results: </b>RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose, in addition, expression of specialised metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. <b>Conclusions: </b>Expression of genes encoding the same enzymatic function exhibited transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning function under different physiological conditions. These<b> </b>approaches can inform metabolic engineering of industrial <i>Streptomyces </i>strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters. . <br>Supplementary Figure 1 Growth profiles of liquid Streptomyces coelicolor M145 cultures grown in glucose or tween with sampling point indicator (green arrow) at mid log phase. Mean of three biological replicates is shown as ln of cell dry weight (CDW) over time. Each experimental condition was carried out in three biological replicates, points are the mean of three independent experiments and error bars represent the standard deviation of the data. Supplementary Fig. 2 Overview of differential gene expression by functional categories of genes Supplementary Figure 3. Verification of fold change data from RNA-Seq and qPCR obtained from three biological replicates with standard deviation of five genes, one constitutively expressed gene (hrdB), two from glycolysis (pyk1, pyk2) and two from gluconeogenesis (ppdk1, ppdk2). Experimental details are in the Materials and Methods section of the associated manuscript. Pyk= pyruvate kinase; PPDK= pyruvate phosphate dikinase; hrdB = housekeeping sigma factor Supplementary Figure 4 Standard curves of each gene for the quantification of transcripts in qPCR experiments A purified PCR product from a PCR for each gene from genomic DNA served as template (Supp. Table 8), this template was diluted to get in total seven different standards ranging from 101-107 molecules. The standard curves were prepared in triplicate and were used to calculate the concentration in the unknown samples and were then compared to the results obtained in the RNA Sequencing. . Pyk= pyruvate kinase; PPDK= pyruvate phosphate dikinase; hrdB = housekeeping sigma factor Supplementary Table Legends Supplementary Table 1 Differential gene expression (DE) and expression category on tween versus glucose in central carbon metabolism showing all genes annotated for the function Legend: green = up, red = down, yellow highlighted genes = expanded gene in Streptomyces, '-' symbol indicates no significant change in expression detected, expression type meanings: I) same direction of change or no change in all genes II) different direction of change in all genesSupplementary Table 2 Specialised metabolite gene clusters with individual genes showing differential expression on Tween and Glucose given SCO number, gene function, fold change and p-valueSupplementary Table 3 Genes involved in fatty acid metabolism and EM-CoA pathway showing differential expression on Tween and Glucose given SCO number, gene function, fold change and p-valueSupplementary Table 4 Regulatory genes showing differential expression on Tween and Glucose given SCO number, gene function, fold change and p-valueSupplementary Table 5 List of all genes showing differential expression on Tween and Glucose given SCO number, gene function, fold change and p-valueSupplementary Table 6 List of all genes showing differential expression on Tween and Glucose given SCO number, normalised fold change (+1 highest increase in expression to -1 highest decrease in expression), fold change and p-valueSupplementary Table 7 GO enrichment analysis (geneontology.org,https://www.ncbi.nlm.nih.gov/pubmed/23868073)Supplementary Table 8 Raw data output from analysis of RNA-Seq data from CLC Genomics Workbench 7.5 Supplementary Table 9 Primer utilised for qPCR specifying for which gene, direction, sequence,melting temperature, amplicon size

**背景：** 链霉菌属（Streptomycete）细菌是次级代谢产物的高产菌株，其中诸多产物具有临床相关的生物活性。其基因组的一个显著特征是：编码同一酶功能的基因家族发生了扩张。通过水平基因转移（HGT）或基因复制实现扩张的基因，可产生多种演化结局：基因可能丢失，或经历新功能化（neo-functionalisation）、亚功能化（sub-functionalisation）过程。为探究链霉菌属中扩张的基因家族是否存在差异表达，本研究采用RNA测序（RNA-Seq）技术，分析了以葡萄糖或吐温（tween）为唯一碳源培养的野生型天蓝色链霉菌（*Streptomyces coelicolor*）的样本。

**结果：** RNA测序分析显示，相较于葡萄糖培养条件，扩张基因家族中三分之二的基因在吐温培养下出现转录差异；此外，次级代谢产物基因簇（放线菌红素、异瑞替烷、天蓝色链霉菌素以及一个隐秘的非核糖体肽合成酶（NRPS）基因簇）的表达也受碳源种类影响。

**结论：** 编码同一酶功能的基因，在不同碳源培养条件下呈现转录差异。这种转录分化使得细菌可在不同生理条件下实现功能分配。本研究方法可为工业链霉菌菌株的代谢工程改造提供参考，也有助于开发培养条件以激活所谓的“沉默”生物合成基因簇。

补充图1 以葡萄糖或吐温为碳源培养的液态天蓝色链霉菌M145生长曲线，绿色箭头标注了对数中期的采样节点。结果以三次生物学重复的细胞干重（CDW）的自然对数随时间变化呈现。每组实验均设置三次生物学重复，数据点为三次独立实验的平均值，误差棒表示数据的标准差。
补充图2 基于基因功能分类的差异基因表达概况
补充图3 基于三次生物学重复的RNA测序与实时定量PCR（qPCR）结果的倍数变化验证，共涉及5个基因：1个组成型表达基因（hrdB）、2个糖酵解相关基因（pyk1、pyk2）以及2个糖异生相关基因（ppdk1、ppdk2）。实验细节见相关论文的材料与方法部分。注：Pyk=丙酮酸激酶；PPDK=丙酮酸磷酸双激酶；hrdB=管家σ因子
补充图4 实时定量PCR（qPCR）实验中用于转录本定量的各基因标准曲线。以基因组DNA为模板通过PCR扩增得到的纯化PCR产物作为标准品（补充表8），将其稀释为10¹~10⁷分子数共7个梯度浓度。标准曲线均设置三次重复，用于计算未知样本的转录本浓度，并与RNA测序结果进行对比。注：Pyk=丙酮酸激酶；PPDK=丙酮酸磷酸双激酶；hrdB=管家σ因子

补充表图例
补充表1 中心碳代谢中以吐温vs葡萄糖为条件的差异表达（DE）基因与表达分类，列出所有注释了对应功能的基因。图例：绿色=上调，红色=下调，黄色高亮基因=链霉菌中的扩张基因；‘-’表示未检测到显著表达变化。表达类型含义：I）所有基因变化方向一致或无变化；II）所有基因变化方向不同。
补充表2 存在单个基因差异表达的次级代谢产物基因簇（以吐温与葡萄糖为培养条件），列出SCO编号、基因功能、倍数变化与p值。
补充表3 参与脂肪酸代谢与EM-CoA通路的差异表达基因（以吐温与葡萄糖为培养条件），列出SCO编号、基因功能、倍数变化与p值。
补充表4 存在差异表达的调控基因（以吐温与葡萄糖为培养条件），列出SCO编号、基因功能、倍数变化与p值。
补充表5 所有以吐温与葡萄糖为培养条件的差异表达基因列表，列出SCO编号、基因功能、倍数变化与p值。
补充表6 所有以吐温与葡萄糖为培养条件的差异表达基因列表，列出SCO编号、标准化倍数变化（+1为表达上调最高，-1为表达下调最高）、倍数变化与p值。
补充表7 GO富集分析（数据来源：geneontology.org，https://www.ncbi.nlm.nih.gov/pubmed/23868073）
补充表8 来自CLC Genomics Workbench 7.5的RNA测序数据分析原始输出结果。
补充表9 实时定量PCR（qPCR）实验所用引物，标注了对应基因、引物方向、序列、解链温度与扩增子大小。