Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds
/Camelina sativa/(camelina) is emerging as an alternative oilseed crop
due to its short growing cycle, low input requirements, adaptability to
less favorable growing environments and a seed oil profile suitable for
biofuel and industrial applications.
Camelina meal and oil are also registered for use in animal and fish
feeds; however, like meals derived from most cereals and oilseeds, it is
deficient in certain essential amino acids, such as lysine.
In higher plants, the reaction catalyzed by dihydrodipicolinate synthase
(DHDPS) is the first committed step in the biosynthesis of lysine and is
subject to regulation by lysine through feedback inhibition. Here, we
report enhancement of lysine content in/C. sativa/seed via expression of
a feedback inhibition-insensitive form of DHDPS from/Corynebacterium
Two genes encoding/C. sativa/DHDPS were identified and the endogenous
enzyme is partially insensitive to lysine inhibition. Site-directed
mutagenesis was used to examine the impact of alterations, alone and in
combination, present in lysine-desensitized DHDPS isoforms
from/Arabidopsis thaliana/DHDPS (W53R),/Nicotiana tabacum/(N80I) and/Zea
mays/(E84K) on/C. sativa/DHDPS lysine sensitivity. When introduced
alone, each of the alterations decreased sensitivity to lysine; however,
enzyme specific activity was also affected.
There was evidence of molecular or structural interplay between residues
within the/C. sativa/DHDPS allosteric site as coupling of the W53R
mutation with the N80V mutation decreased lysine sensitivity of the
latter, but not to the level with the W53R mutation alone. Furthermore,
the activity and lysine sensitivity of the triple mutant
(W53R/N80V/E84T) was similar to the W53R mutation alone or the/C.
glutamicum/DHDPS. The most active and most lysine-insensitive/C.
sativa/DHDPS variant (W53R) was not inhibited by free lysine up to 1¬†mM,
comparable to the/C. glutamicums/enzyme. Seed lysine content increased
13.6 -22.6% in CgDHDPS transgenic lines and 7.6‚??13.2% in the mCsDHDPS
lines. The high lysine-accumulating lines from this work may be used to
produce superior quality animal feed with improved essential amino acid
Engineering a feedback inhibition-insensitive plant dihydrodipicolinate
synthase to increase lysine content in Camelina sativa seeds |