## 2 Results
### Differential Expression of Okarantine Biosynthesis Gene Cluster  
Homologs of the _oka_ gene cluster have been identified in the genome of _P. daleae_ NBP-49626. The amino acid (aa) identities of the proteins encoded by these homologs (OkaA to OkaG) were 96.84%, 98.67%, 97.44%, 98.49%, 99.00%, 98.95%, and 99.24%, respectively.  
The _oka_ gene cluster exhibited a notable gradual increase in up-regulation from the second to the third day of submerged fermentation (average 4.3-fold up-regulated of these seven genes), and this level of up-regulation of gene transcripts was sustained until the eighth day (average 5.5-fold up-regulated, Figure 7a, Table S5), mirroring the trend observed in the yield enhancement of Okarantine B (Figure 2). Contrariwise, in semi-solid fermentation, the production of Okarantine B increased from the first to the second day (Figure 2), and the levels of _oka_ gene cluster transcripts significantly increased by an average of 3.8-fold from the second day onwards but decreased relative to the later stages (average 1.6-fold on day eight; Figure 7a, Table S5).  
Figure 6: KEGG pathway enrichment analysis of differentially expressed genes (\(\geq\)2-fold change) at various periods during the fermentation processes. (**a–c**) Days 2, 3, and 4 vs. day 1 in submerged fermentation. (**d–f**) Days 2, 3, and 4 vs. day 1 in semi-solid fermentation. GeneRatio = DEGs number/total gene number identified from transcriptome of a certain process.  
During the biosynthesis of Okaramine B, the protein products encoded by the _oka_ gene clusters exhibit their roles sequentially, with OkaA, OkaC, OkaB, OkaD, OkaE, OkaG, and OkaF [21,27]. More detailed analyses revealed that the differences in the patterns of the two transcript levels suggested two distinct synthesis strategies. In contrast to submerged fermentation, in which the transcript levels of all seven genes increased with fungal growth, the transcript levels of genes involved in the initial steps of Okaramine B synthesis were significantly higher on the third day of semi-solid fermentation, resulting in a greater peak of Okaramine B production. During semi-solid fermentation, the expression levels of the _okaA_, _okaC_, _okaB_, and _okaD_ genes involved in the initial synthesis of Okaramine B decreased from the third to the fourth day (e.g., 3.1-fold on day two and 1.4-fold on day four of gene _okaA_, respectively), although the production of Okaramine B was still at its highest  
Figure 7: Expression patterns of key genes involved in Okaramine B biosynthesis at various periods during the fermentation processes. (**a**) The _oka_ gene cluster of Okaramine B biosynthesis. (**b**) Putative regulator genes. (**c**) Genes involved in tryptophan metabolic pathway. (**d**) Putative transporter genes. The blue to red color scale values represent fold changes.  
point (Figure 2). In contrast, the _okaE_ and _okaF_ genes responsible for the subsequent steps showed a slower increase and then a decrease in expression levels (e.g., 4.0-fold on day two and 2.7-fold on day four of gene _okaE_, respectively). Notably, the _okaG_ gene remained consistently up-regulated at a high level (e.g., 6.4-fold on day two and 5.0-fold on day eight, respectively; Figure 7a, Table S5).