Regarding the expression analysis, in all the transformants analysed the levels of CzPSY mRNA were higher than those of the endogenous PSY, since exogenous CzPSY was constitutively expressed under the control of the strong promoters rbcS2 and hsp70A.
After a few transformants were selected to see the level of expression, a single transformed P. pastoris colony presenting the highest expressed enzyme level was inoculated into 25 ml of BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% (w/v) yeast nitrogen base, 4 μg/ml D-biotin and 1% (v/v) glycerol).
Western blot analysis of bacterial cells and culture supernatants confirmed that B. subtilis inhA-transformant expressed InhA at the level comparable with B. anthracis Ames 35 (not shown).
The transformant indicated a significant rise of PME and the level of methanol in the transformant increased by 28.7% compared to the vector control transformant.
Compared with the control transformants not expressing RNF4, the levels of di-SUMO2 were reduced to approximately 55%, and this effect was enhanced if the chain was composed of three or four SUMOs (45% and 43% of control levels respectively).
However, the level of sorbitol was not detectable in the transformant.
In M184 and the three transformants, guaA was expressed at a level 20- to 50-fold higher than the level of expression in R6.
Transformants T1 and T10 showed the highest CzPSY levels, which reached 140-fold the level of the endogenous PSY.
As shown in Table 3, all of the transformants produced significant levels of EPA, confirming the identity of the P. ramorum and P. sojae Δ-17 desaturases.
The CzPSY transcript level reached 140-fold the level of the endogenous PSY of the wild type in the transformants T1 and T10, however the violaxanthin and lutein contents in these transformants were only 2.0- and 2.2-fold higher than the parental strain, respectively, in T10 transformant, and 7% and 21% higher, respectively, in T1.
