Alex Huang, Cathy Coutu, Myrtle Harrington, Kevin Rozwadowski & Dwayne D. Hegedus

Transgenic Research (2021) Published: 20 November 2021

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 glutamicums (CgDHDPS). 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 profile.

See: https://link.springer.com/article/10.1007/s11248-021-00291-6

Fig. 1:

Conservation between C. sativa and other plant and microbial DHDPS enzymes. Amino acid sequence alignment of DHDPSs from A. thaliana (AtDHDPS2, Q9LZX6, 365 aa), C. sativa CsDHDPS B4 (Csa16g004020, 364 aa) and CsDHDPS B6 (Csa05g092770, 365 aa), N. tabacum (NtDHDPS, NP_001313049, 359 aa), Z. mays (ZmDHDPS, NP_001105425.1, 380 aa), Vitis vinifera (VvDHDPS, PDB 3TUU, 346 aa), C. glutamicum (CgDHDPS, X53993, 301 aa) and E. coli (EcDHDPS, WP_061350668, 292 aa). The predicated chloroplast transit peptides at the N-terminus in the plant enzymes are in bold text. Conserved amino acids involved in catalysis within the catalytic site (C) and those involved in lysine binding in the allosteric (L) sites are shown. The sites of the three mutations introduced into CsDHDPS B6 allosteric site (L and highlighted in yellow) are designated as mA (W53R), mB (N80V) and mC (E84T) according the numbering associated with the E. coli DHDPS. Amino acids found in all enzymes (white letters on black background) or amino acids with conserved properties (black letters on grey background) are shown. Alignment performed using the Vector NTI Suite (Life Technologies)