Chemistry Reference
In-Depth Information
clusters were identified from the nanchangmycin producer
S. nanchangensis
,and
the nanchangmycin gene cluster was identified through gene disruption (18). The
organization and assignment of function of each ORF is carried out by sequence
comparison. Cloning and overexpression of individual enzymes in the gene clus-
ter is an important step in confirming the function and mechanism of a particular
ORF. For example, sequence comparisons of both the monensin and the nan-
changmycin clusters revealed no TE function required for the release of the
polyketide from the PKS. However, cloning, overexpression, and characteriza-
tion of MonCII (14) and NanE (20) resulted in the reassignment from an epoxide
cyclase to Type I TE.
The preparation of mutants is an important tool to investigate the role of
individual enzymes, and it is used to ascertain which genes in the cluster are
essential for the biosynthesis of the polyether and whether modified/truncated
products are formed. The effected gene can then be supplied
in trans
on a plasmid
to see whether activity can be restored. Chemists are required to analyze the
fermentation mixtures for minor and truncated metabolites. In this manner, the
triene lactones
22
and
23
were isolated from a 7L culture of a mutant strain of
S.
cinnamonensis
lacking
MonCI
, and their structure was determined by NMR (15).
HPLC-MS is an essential technique for the separation and identification of
these trace compounds, but unless sufficient quantities can be isolated for struc-
ture determination by NMR, chemists are also required to synthesize standards
for comparison. Characterization of the gene cluster requires chemists to synthe-
size intermediates (often with stable isotopes at specific places) to be used as
substrates for purified enzymes or to confirm the structure of products formed
from modified genes.
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2006;45:4406-4413.
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501-530.
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on biohydrogenation in continuous flow-through ruminal fermenters. J. Dairy Sci.
1997;80:921-928.
