Part:BBa_K2753010
pSB1C3-TDH3 promoter-G8H-tENO2
The complete genetic circuit encoding for Catharanthus roseus 8-hydroxylase (G8H) codon-optimised for use in S. cerevisiae, responsible for the hydroxylation of geraniol by geraniol to form 8-hydroxygeraniol. Expression of this circuit is regulated by a strong yeast constitutive promoter pTDH3 and terminator tENO2.
Characterisation
In our study, we aim at achieving the conversion of geraniol to nepetalactol, which requires heterologous expression of G8H, GOR, and ISY[1].
Figure. 1 Heterologous biocatalytic route in S. cerevisiae and relevant plasmids design. (A) Three cytochrome P-450, G8H, GOR, and ISY, are needed for converting geraniol to nepetalactol.
From literature we have comprehended that fact that the conversion was unlikely unless promiscuous endogenous enzymes were knocked out[2][3], so we began expression vector construction and gene deletion at the same time.
At first we used E. coli as the cloning host for constructing the vector, but to our surprise, no matter what assembly method we used — Gibson or biobrick assembly, the E. coli colony grown always missed the G8H component. After careful debug, we found that pTDH3 contains wild-type E. coli promoters which have trigger the expression of G8H in E. coli, resulting in cytotoxicity which forced E. coli to cleave G8H. So later we changed out method by transforming the ligated DNA directly into yeast and using colony PCR to determine if the construction was successful.
Figure. 2 The results of the conversion of geraniol to nepetalactol. (A) S. cerevisiae expression vector design. G8H expression is under the regulation of a strong constitutive promoter pTDH3 and terminator tADH1, but later we abandoned this design and changed pTDH3 to a galactose-inducible promoter pGAL. pTEF1 and tENO1 controls the expression of GOR. pRP18B and tTDH3 controls the expression of ISY. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful. (C) Gas chromatography results of geraniol consumption and conversion to nepetalactol. (1) is geraniol. (2),(3),and (4) are isomers of nepetalactol. (5) is an unknown products of metabolism. (D) The standard curve plotting to quantify nepetalactol yield
When we have eventually acquired the expression vector, the gene oye2 was knocked out. Therefore we started the characterization of using engineered yeast to convert geraniol to nepetalatcol. Having transferred the vector into both BY4741 and △oye2 BY4741, we shared the yeast at 30℃ and fed them with 40μl 200mM geraniol every two hours for 24h, then used gas chromatography for biocatalytic activity analysis. From the GC result, BY4741 has not produced nepetalactol, but we verified that geraniol was consumed by geraniol and nepetalactol was produced by △oye2 BY4741. However, an unknown metabolite was also generated and in a larger quantity than nepetalactol. Our assumption was that this peak was a shunt product reported. And if oye3 and adh7 were deleted in addition to oye2, it would be very likely that the nepetalactol yield increased and the shunt product decreased. At this moment, the nepetalactol yield we have obtained is 1.267mg/L.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 956
Illegal BglII site found at 1034
Illegal BglII site found at 1442 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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