3.1 Sequence screening
In the sequence mining, the prokaryote-derived SuSy templatesAn SuSy and Mr SuSy were used for sequence collection and
the sequences without conservative residues G302, G303, H438, R580,
L581, K585, and E675,[22] and residues Q648, N654,
and E683 that contribute to UDPG binding were removed (the residue
number refers to At SuSy1 in the multiple sequence
alignment).[11, 24] As a result, only a few
sequences from lower eukaryote sources like green algae remained
together with a large number of the putative plant SuSys. Mc SuSy
from M. conductrix and Cb SuSy1 and Cb SuSy2 fromC. braunii were selected and synthesized after codon optimization
for heterologous expression in E. coli . Enzyme activities of the
crude extracts containing Mc SuSy and Cb SuSy2 were around
15.5 and 5.5 mU/mg total protein, respectively. However, it is difficult
to detect the SuSy activity of Cb SuSy1. As well, the obvious band
corresponding to Mc SuSy (Fig. S1) was found in the soluble
fraction prepared from the induced cells, indicating the better soluble
expression of recombinant Mc SuSy than the other two enzymes.
Therefore, Mc SuSy was chosen for further study of enzymatic
properties.