* 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).