3.1 Identification of a CYP catalyzing HLDOA
Previously, from the G. lucidum genome, we narrowed down to 82
candidate genes which may be responsible for GA biosynthesis (Wang et
al., 2018). In order to extend the biosynthetic step from HLDOA to a
further GA, here we re-screened those genes by using the previously
developed yeast chassis. Both plasmids pRS426-CYPx (Ura3 selection
marker) and pRS425-CYP5150L8-iGLCPR (Leu selection marker) were
co-transformed into S. cerevisiae YL-T3 to yield various
engineered strains (Fig. 1a, Table S3). Considering that the
biosynthetic genes of secondary metabolites may be arranged in cluster,
we first examined whether the CYP genes located in the same gene cluster
as CYP5150L8, i.e., CYP5150L6, CYP5150L9, CYP5150K5, CYP5150J5 and
CYP5150J10, could catalyze HLDOA. The results showed that they could not
catalyze, as no new peak appeared (Fig. 1b), suggesting that the GA
biosynthetic genes might not be simply clustered together.
From the fermentation samples of the CYP5139G1 and CYP5150L8 (L8)
co-overexpressed strain, generation of a new peak in the HPLC
chromatogram was found (Fig. 1c). The peak with retention time (Rt) of
17.5 min could not be detected in either CYP5150L8 or CYP5139G1 single
gene expressed strain, not to mention in the control with void plasmid.
By LC-MS analysis, compound A was detected with m/z at 455.3513
and 437.3410 (Fig. 1d), which was consistent with the molecular weight
of
[C30H48O4-H2O+H]+455.3525 and
[C30H48O4-2H2O+H]+437.3420, respectively. Compared to HLDOA
(C30H48O3), it was
supposed that hydroxylation of HLDOA to compound A might have
occurred, and cyp5139G1 was considered responsible for the hydroxylation
of HLDOA.