Fig. 6
From: Complete microbial synthesis of crocetin and crocins from glycerol in Escherichia coli
Construction of crocin-3/4 pathway and crocin-3/4 production in batch bioreactor fermentations. (A) HPLC and UV/Vis analysis of in vitro activity of three second step UGT candidates (GjUGT1, GT1-316, and NtUGT) for determining the glycosylation activity on crocin-1/2. In vitro reaction was performed with combinations of the first reaction of two first-step UGTs (GjUGT1 and NtUGT), which converted crocetin into crocin-1/2, and the second reaction of three second-step UGT (SpUGT, NsUGT, and CaUGT3): (upper) crocin-3/4 standard (upper-middle) empty + SpUGT, empty + NsUGT, empty + CaUGT3, empty + empty (a control); (lower-middle) NtUGT + SpUGT, NtUGT + NsUGT, NtUGT + CaUGT3, NtUGT + empty (a control); (lower) GjUGT1 + SpUGT, GjUGT1 + NsUGT, GjUGT1 + CaUGT3, GjUGT1 + empty (a control). Peak 1 corresponds to crocin-4; peak 2, crocin-2; peak 3, crocetin. (B) UV/VIS spectrum corresponds to each HPLC chromatogram peak (A). (C) HPLC analysis of culture medium (upper) and Z1pCA7942(P)pNC cell extract. (D) LC-MS analysis of crocin-4 and crocin-3. (E) Cell growth and glycerol consumption in bioreactor batch fermentations of the Z1pCA7942(P)pNC (upper). The time-course production of crocin-1/2/3/4 in bioreactor batch fermentations of the Z1pCA7942(P)pNC (lower). Bioreactor fermentations were performed in biological triplicate, and error bars represent mean ± SD