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.