Assay for Cyclase Activity by complement viability
3.2.5 The construction of novel gene disruption strain TKW14 for analysis of inactive mutated cyclase inactive mutated cyclase
In order to unambiguously determine the functional role of inactive mutations, a novel cyclase-deficient strain, TKW14, derived from CBY57[pZS11] was generated.
Accordingly, the pZS11 plasmid bearing the wild-type ERG7 was removed and the viability of yeast cells was mediated via the exogenous addition of the ergosterol. As it has been suggested that mutations in the heme biosynthetic pathway could allow the uptake of sterol from the media under aerobic conditions, efforts were thus made to incorporate a hem1 gene disruption into the CBY57[pZS11] strain.122, 123 Using homologous recombination technology, we have successfully exchanged the HEM1 gene with a drug Geneticin (G418) resistance gene in the CBY57[pZS11] strain. In order to confirm the accurate genetic recombination in the HEM1 gene, the genomic DNA of yeast transformants was extracted and verified via PCR based amplification with the HEM1 gene terminus sequence primers. Moreover, the restriction enzyme mapping with NcoI, a unique restriction site for the Geneticin resistance gene (G418R) but not for HEM1 gene, was further used for genetic verification (Figure 3.3). After the successful genetic knockout of the HEM1 gene in the CBY57[pZS11] strain, the pZS11 plasmid was removed from the yeast cell. Based on the similar principle of plasmid shuffle, the presence of 5’-FOA in the selection plates would completely eliminate yeast containing the pZS11 plasmid. The viable yeast colonies which bear the genomic hem1/erg7 double knockout, eliminate the pZS11 plasmid, and maintain cell viability via the uptake of exogenous ergosterol from the medium were renamed as the TKW14 strain. The genotype of novel haploid yeast TWK14 is (MATa or MATα ERG7∆::LEU2 ade2-101 lys2-801 his3-∆200 leu2-∆1 trp1-∆63 ura3-52
HEM1∆::kanR).
(a)
(b)
Figure 3.3 (a) The homology recombination strategy for exchanging the HEM1 gene with a drug geneticin resistance gene (G418R) in the CBY57[pZS11] strain. (b) The PCR amplicons (1.5 kb) of the genomic DNA with specific primers (left), and the result of restriction enzyme mapping with NcoI, a unique mapping site for Geneticin resistance gene (G418R), with the expected sizes of 1 kb and 500 bp (right). Lane 1~Lane 9 indicated the individual colonies from selection plates, Lane 10 showed the host CBY57 strain, as a negative control.
HEM1
Homologous recombination in
Selection agaist G418
PCR and restriction enzyme mapping verifaction
MATa or MATα ERG7 ::LEU2 ade2-101 lys2-801△ his3- 200 leu2- 1 trp1- 63 ura3-52 HEM1 ::kanR△ △ △ △ CBY57 chromosome
CBY57 chromosome
3.2.6 Characterizing the mutant products in the novel gene disruption strain
The Tyr510Lys and Try510Trp mutated plasmids were each transformed into the novel TKW14 strain, respectively. As expected, the Tyr510Lys and Try510Trp substitutions failed to maintain the cell viability in the absence of the exogenous ergosterol. This result further supported the inactive characteristic from the previously mentioned plasmids shuffle experiments. Both mutants were then grown in the liquid media and harvested. Preliminary NSL extract showed that both mutations were less efficient than the native ERG7 and gave fewer amounts of NSL per liter. The chromatographic results with no lanosterol-positioned products isolation also demonstrated the inactive activity of these two mutations. In addition, two non-lanosterol products, which migrate between oxidosqualene and lanosterol, were detected from TLC and GC-MS analysis. After co-injection with the authentic samples, one of the triterpene alcohols was determined to be achilleol A (Figure 3.4).However, attempt to separate the other NSL always presented difficulties due to its indistinguishable characteristic with the monocyclic achilleol A. Thus, the detailed analyses of the partially purified mixture with the ratio of 1:0.88 were carried out via different GC-MS, 1H-NMR, and 13C-NMR spectra (Appendix 3.4). A multiplet signal at 5.216 ppm on 1H-NMR suggested that the second NSL product might contain a trisubstituted olefin distinct from that in the side chain. In addition, the chemical shifts for the region of methyl groups on 1H-NMR showed that δ = 0.691 and 1.001 ppm are the signals of achilleol A and theδ = 0.806 and 0.946 ppm belonged to the other NSL. Similarly, the δ = 108.379 and 147.212 ppm from 13C-NMR spectrum are the unique chemical shifts of achilleol A, whereas theδ = 118.257 and 137.083 ppm are the characteristic peaks of the second NSL. Fortunately, a natural monocyclic triterpene, camelliol C, which was extracted from sasanqua oil (Camellia sasanqua) exhibited a identical and consistent spectroscopic result with ours in GC-MS,
1H-NMR, and 13C-NMR analyses.124 This result confirmed that the identity of the second compound is camelliol C, which was identified from the ERG7 mutations for the first time. 1H-NMR and GC-FID analysis of the non-lanosterol-positioned products showed that Tyr510Lys and Tyr510Trp mutants produced achilleol A and camelliol C in the ratio of 86:14 and 94:4, respectively. Interestingly, the achilleol A and camelliol C were also produced from the other inactive mutants of Trp443Ala and Lys448Ala. The analysis in the new TKW14 strain showed that these two mutants produced different ratio of monocyclic triterpene products from that of Tyr510 mutants. These results indicated that both Trp443Ala and Lys448Ala mutations might compromise the lanosterol production, halt the cyclization reaction at the monocyclic stage, and result in the accumulation of achilleol A and camelliol C as its end product.
The ratio of product from the S. cerevisiae ERG7 single-point mutants are listed in Table 3.2. The chemical structure and postulated mechanism of the relevant oxidosqualene cyclization/rearrangement are shown in Scheme 3.3.
Figure 3.4 GC analyses of the nonsaponifiable lipid, derived from the yeast ERG7Y510K mutant. Two non-lanosterol-positioned products, which migrate between oxidosqualene and lanosterol, were detected. The blue color line indicates the authentic achilleol A from ERG7Y510A mutant. The red color line indicates the partially purified NSL from ERG7Y510K mutant. The co-injection of the authentic samples is shown in the green color line.
achilleol A
camelliol C
Table 3.2 Product ratio from the various S. cerevisiae ERG7 single-point mutants