Bioscience Evidence 2024, Vol.14, No.2, 39-43 http://bioscipublisher.com/index.php/be 40 substrate consumption observed after the addition of CAL-1 and CAL-2. The properties of compounds 4-6 were confirmed by comparison with synthesized or commercially available standards. The structure of compound 8 was proposed based on MS2 and UV analysis (Supplementary Data Figure 2). The structure of compound 9 was proposed based on MS2, UV analysis, NMR of partially purified 9, and MS2 and complete NMR of the oxidation by-product. Figure 1 Assessing unknown steps in Lycopodium alkaloid biosynthesis Figure 2 Stepwise discovery of early biosynthetic enzymes contributing to scaffold formation Figure 3 demonstrates the roles and studies of newly functionalized CAL enzymes in the biosynthesis of lycopodium alkaloids. Figure 3a proposes the mechanism for the synthesis of compound 9 catalyzed by PtCAL-1a/PtCAL-2a. Figure 3b shows a representative Western Blot of 6xHis-tagged CALs expressed alone or co-expressed with untagged gene constructs in N. benthamiana. As an intracellular protein control, a 6xHis-tagged LcLDC construct is included. The experiment was conducted more than three times, with similar results observed. Figure 3c displays the EICs for 9 (m/z 164.1434) produced by extracellular PtCAL-1a and PtCAL-2a (either alone or co-expressed) with different substrate combinations. Figure 3d shows the dynamics of compound 9 production
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