## 2 Results
### Identification of the Differentially Expressed Regulator Genes  
Given the lack of regulatory-related genes in the _oka_ gene cluster [21], the control of Okaramine B synthesis may be derived from regulatory factors elsewhere in the genome. In accordance with the genome annotation data, 259 regulatory genes were identified among the DEGs. Investigation of the transcription patterns of these regulatory genes, combined with the patterns of the _oka_ gene clusters under the two fermentation processes, suggested that five regulatory genes, including genes 1.1084, 1.213, 2.485, 4.1080, and 4.1188, could play a role in the regulation of Okaramine B biosynthesis (Figure 7b). The transcription trend of gene 1.1084 exhibited a similar pattern to that of the Okaramine B biosynthesis gene cluster during submerged fermentation (e.g., 3.7-fold on day two, 5.8-fold on day four, and 4.1-fold on day eight, respectively; Figure 7b, Table S5). Although the level of up-regulation was statistically significant during semi-solid fermentation, it was lower than that observed during submerged fermentation (e.g., 2.4-fold on day two, 1.8-fold on day four, and 1.5-fold on day eight, respectively; Figure 7b, Table S5). The annotation results derived from the NCBI NR database indicated that the protein Reg2, encoded by gene 1.1084, shares 87.7% as a identity with the transcriptional regulatory protein pro-1 found in _P. brasilianum_ (accession no. OOQS4656.1). Conserved domain analysis revealed that Reg2 belongs to the Zn(II)2 Cys6 transcription factors family (domain architecture ID 10049156), which includes a GAL4-like Zn2 Cys6 binuclear cluster DNA-binding domain (GAL4; accession no. cd00067) and a fungal-specific transcription factor domain (Fungal_trans_2; accession no. pfam11951). The transcriptional pattern of gene 4.1080 during submerged fermentation was similar to that of gene 1.1084. However, in the latter stages of the semi-solid fermentation process, gene 4.1080 did not exhibit a decrease in transcriptional up-regulation as Okaramine B production stabilized. Instead, it maintained a consistently high level of up-regulation (3.8-fold on day eight) (Figure 7b, Table S5). Protein Reg21, encoded by gene 4.1080, shares 76.9% aa identity with the condiophore development regulator BrIA in _P. rubens_ (accession no. B6 GVZ2.1) [28]. It was hypothesized that unlike gene 4.1080, gene 1.1084 might be associated with the regulation of Okaramine B biosynthesis during submerged fermentation. Gene 1.213 exhibited significant transcriptional activity during the initial stages of both liquid (2.3-fold to 3.7-fold between day two and four) and semi-solid fermentation conditions (3.2-fold to 2.3-fold between day two and four; Figure 7b, Table S5). Protein Reg19, encoded by gene 1.213, shares 91.5% aa identity with the Zn(II)2 Cys6 transcription factor in _P. brasilianum_ (accession no. OOQ91732.1). The transcription factor belongs to another group of Zn(II)2 Cys6 transcription factors (domain architecture ID 10048070) and contains a domain of fungal transcription factor regulatory middle homology region (fungal_TF_MHR; accession no. cd12148) and a GAL4 domain. It is postulated that Reg19 may play a role in promoting the initiation of Okaramine B biosynthesis. The expression levels of genes 2.485 and 4.1188 were delayed in relation to Okaramine B biosynthesis during both liquid and semi-solid fermentation conditions (Figure 7b, Table S5). Notably, gene 2.485 displayed down-regulation in transcription at the onset of liquid fermentation, but later transitioned to a significant up-regulation on the fourth day, coinciding with a substantial increase in Okaramine B production. Protein conserved domain and function analysis indicated that the protein Reg5, encoded by gene 2.485, shares 66.9% aa identity with the transcription factor stel1 in _P. subrbescens_ (accession no. OKP12827.1) and an HMG-box_ROX1-like domain (accession no. cd01389).