3.4 Improving the DHLDOA biosynthesis in yeast with a dual tunable plasmid system
To explore whether the production of DHLDOA can be improved via regulating the CYP5150L8 and CYP5139G1 gene expression levels, here, two plasmids pRS425-Hyg-CYP5150L8-iGLCPR and pRS426-KanMX-CYP5139G1, which could be regulated by Hyg and G418, respectively, were co-transformed into S. cerevisiae YL-T3 to generate the strain HygL8-G418G1. By testing different combinations of Hyg (0, 100, 200, 300 mg/L) and G418 (0, 100, 300, 500, 700 mg/L) concentrations, we observed that DHLDOA had the highest titer when the Hyg and G418 concentration was at 100 mg/L and 300 mg/L, respectively (Fig. 6b, Fig. S1). To better understand this phenomenon, we further analyzed the production of HLDOA and DHLDOA, copy number of plasmids as well as transcription levels of CYP5150L8 and CYP5139G1 in the HygL8-G418G1 strain (Fig. 6). For the HygL8-G418G1 strain, the control without addition of antibiotics (H0G0) was set; fermentations with Hyg concentration at 100 mg/L while G418 concentration at 0, 100, 300, and 500 mg/L (H100G0, H100G100, H100G300, H100G500, respectively) were done; and those with G418 concentration at 300 mg/L while Hyg at 0, 100, and 200 mg/L (H0G300, H100G300, H200G300, respectively) were also conducted. It was found that in the control (H0G0), the production of HLDOA and DHLDOA was relatively low (4.62 mg/L and 0.27 mg/L, respectively). When 100 mg Hyg was added without G418 (H100G0), the DHLDOA titer decreased to 0.19 mg/L, although the accumulation of HLDOA increased to 14.43 mg/L (Fig. 6b). By comparing the copy number change of pRS425-Hyg-CYP5150L8-iGLCPR and pRS426-KanMX-CYP5139G1 plasmids between strains at H0G0 and H100G0, it was seen that with the addition of Hyg, the copy number of pRS425-Hyg-CYP5150L8-iGLCPR increased by 28 times and the transcription level of CYP5150L8 increased by 13.6 times; in contrast, the copy number of the plasmid pRS426-KanMX-CYP5139G1 and the transcription level of CYP5139G1 were low due to no addition of G418 (H0G0, H100G0) (Fig. 6c, 6d).
Under 100 mg/L of Hyg, with a higher addition of G418 (H100G0, H100G100, H100G300, H100G500), both the copy number of the plasmid pRS426-KanMX-CYP5139G1 and the transcription level of CYP5139G1 were increased by about 7~10 times compared to H0G0. However, interestingly, compared with H100G0, although Hyg concentration was the same, with more addition of G418 (H100G100, H100G300, H100G500), the copy number of the plasmid pRS425-Hyg-CYP5150L8-iGLCPR decreased significantly (Fig. 6c). The results implied that the copy number of the two plasmids regulated by Hyg and G418 was not completely independent, which coincided with previous reports [32, 34]. A similar trend was seen in the transcription levels of CYP5150L8 and CYP5139G1 genes located in pRS425-Hyg-CYP5150L8-iGLCPR and pRS426-KanMX-CYP5139G1, respectively (Fig. 6d).
Under 300 mg/L of G418, the copy number of pRS426-KanMX-CYP5139G1 plasmid and the transcription level of CYP5130G1 were significantly increased (approximately 6-10 times) compared to that of H0G0. In addition, with the increase of Hyg (H0G300, H100G300, H200G300), the copy number of pRS425-Hyg-CYP5150L8-iGLCPR and the transcription level of CYP5150L8 also increased (Fig. 6c, 6d). Although the CYP5139G1 transcription level at H0G300 was 5.5 fold that of H0G0, due to the low copy number of pRS425-Hyg-CYP5150L8-iGLCPR plasmid and low transcription level of CYP5150L8, the production of HLDOA was low (3.1 mg/L), and the final DHLDOA titer was also very limited (0.48 mg/L). In the case of more pRS425-Hyg-CYP5150L8-iGLCPR plasmids and higher CYP5150L8 transcription levels at H200G300 vs. H100G300, the production of HLDOA and DHLDOA was 17.5 mg/L and 0.84 mg/L, respectively, which was almost 90% and 40% of the HLDOA and DHLDOA produced at H100G300, respectively, i.e., 17.5 vs. 21.8 mg/L; and 0.84 vs. 2.2 mg/L (Fig. 6). It seems that excessive expression of CYP5150L8 not only reduced the production of HLDOA, but also greatly reduced the production of DHLDOA. All the above results indicated that by manipulating antibiotics concentrations, both the plasmid copy number and the target gene transcription level could be adjusted; and both the gene expression level and their balanced expression were important. A suitable enhancement of CYP5150L8 and CYP5139G1 expression levels could improve the DHLDOA accumulation.