EC Number   |
Protein Variants |
Reference |
|---|
  4.2.3.141 | more |
coexpression of gene SsLPPS encoding labd-13-en-8-ol diphosphate synthase (EC 4.2.1.133) and gene SsSS encoding sclareol synthase, recombinant enzyme expression in Physcomitrella patens from vectors pUNI33-SsLPPS-2A-SsSS and pBK3-SsLPPS-2A-SsSS. The transcript levels of SsSS and SsLPPS cannot be directly correlated to the final yields for sclareol of the different transgenic lines, but it is observed that lines with high transcript levels of the genes also have high sclareol levels. The highest yield is 2.84 lg/mg dry weight (2.28 lg/ml) in a liquid culture of wild-type line 2 (WTF2), including sclareol found in the media and in the moss cells. Enzyme expression analysis, overview. Transcriptional changes in the 14-day-old WTF2 shows a similar down-regulation for most genes, but not for HDR, IDI, and HMGS. The HDR, IDI, and HMGS show a higher expression level than in those in the wild-type. Besides sclareol, the three endogenous ent-kaurenoids (ent-kaur-15-ene, ent-kaur-16-ene and ent-16-hydroxy- kaurene) are also observed in the WTF2, but the peaks are significantly lower than in the wild-type line. GC-MS ligand analysis. Of all transgenic lines, the WTF2 line has the most growth impairment and stops growing after 15 days. general, general growth deficiency for transformed lines |
748907 |
| 4.2.3.141 | more |
usage of fed-batch fermentation along with an organic solvent overlay for in situ product removal to develop an industrial-scale platform for sclareol production |
749408 |
| 4.2.3.141 | N431F |
site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively |
746584 |
| 4.2.3.141 | N431I |
site-directed mutagenesis, the mutant exclusively produces isoabienol, resullting from direct deprotonation of the initially generated 13-yl+ intermediate at the neighboring methyl group. There is a below 10% decrease in yield for this mutant relative to wild-type SsSS in the engineered bacterial culture |
746584 |
| 4.2.3.141 | N431Q |
site-directed mutagenesis, the mutant selectively produces epimeric (13S)-sclareol, with SsSS:N431Q exhibiting almost complete reversal of the stereoselective addition of water, i.e., while wild-type SsSS enzyme produces (13R)-sclareol in 80% enantiomeric excess (ee), SsSS:N431Q produced 70% ee of (13S)-sclareol |
746584 |
| 4.2.3.141 | N431Y |
site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively |
746584 |
| 4.2.3.141 | S433C |
site-directed mutagenesis, the mutation has no effect on product outcome |
746584 |
| 4.2.3.141 | T436C |
site-directed mutagenesis, the mutation has no effect on product outcome |
746584 |
