* Average TPM values of duplicates.
Figure 1. Physiological comparison between E3 and NRRL23338. Spores on XM agar plates over time (a), biomass formation (b), residual glucose concentration (c), and erythromycin titer (d). Error bars show the standard deviation from three independent experiments.
Figure 2. COG annotation of CDSs with nonsynonymous or intergenic SNP variations.
Figure 3. Functional enrichment analysis of DEGs in exponential phase based on KEGG (a) and GO (b) database. Y axis represents KEGG or GO terms, X axis shows the rich factor, which refers to the ratio of the number of DEGs belonging to the KEGG or GO term (sample number) compared to the number of all genes categorized in the KEGG or GO term (background number). The greater the rich factor is, the greater the enrichment degree is. The size of the node indicates the number of DEGs of the KEGG or GO term. The color of the node corresponds to different FDR (False Discovery Rate, corrected P-value) value.
Figure 4. Reporter metabolites in the exponential phase (a) and in the stationary phase (b) identified by Piano. Nodes represent metabolites around which the most significant changes in expression occurred. The number in the center of the node corresponds to the metabolites name in the table below (only top 10 metabolites were listed). The color of nodes indicates the P-value of significance. The size represents the number of genes around the metabolite according to the GSMM (Licona-Cassani et al., 2012; Zhuang, Huang, & Chu, 2018). The postfix, i.e. [c], of reporter metabolites means “intracellular”. QH2, ubiquinol; Q, ubiquinone; XMP, xanthosine 5’-phosphate; IMP, inosine monophosphate.
Figure 5. Hierarchical clustering of genes in erythromycin biosynthesis gene cluster and genes related to polyketide sugar unit biosynthesis. Red, up-regulation in E3. Blue, down-regulation in E3.
Figure 6. Schematic map of putative mechamisms by which E3 enhances its erythromycin production. Pathways shown on red background were stimulated in E3, while a green background indicates the repression and grey indicates altered activity. KDPG, 2-keto-3-deoxy-6-phospho-gluconate.
Figure 7. Metabolic engineering around the node of 2-oxoglutarate (α-ketoglutarate) in E3. Identification of the key node, α-ketoglutarate (a). # indicates that SNP occurred in intergenic region of the gene. * indicates that nonsynonymous mutations occurred inside the CDS. The inset on the up-right showed the transcriptional change of genes around α-ketoglutarate node. Erythromycin titers of engineered strains and E3 in shake flasks (b). Strains were cultivated in 300 mL shake flasks with the minimal liquid medium. Data was collected from six independent replicates. Boundaries of the boxes indicate the 1st and the 3rd quartile of the sample populations and horizontal lines represent the median values. The squares inside the boxes represent the average values. Whiskers indicate the highest and lowest values of the results, whereas the solid rhombus indicate abnormal outliers.
Figure 8. Comparison of physiological process parameters between industrial strain E3 and mutant strain E3::sucBA. Dry cell weight (DCW) (a), consumed glucose (b), CO2 emission rate (CER) (c), erythromycin titer (d).