An Unusual Type II Polyketide Synthase System Involved in Cinnamoyl Lipid Biosynthesis

Abstract

As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier‐protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase‐dependent ACP‐tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP‐tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier‐protein‐dependent biosynthetic machineries.

A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis

“…Based on our previous gene deletion experiments, [6a] no candidate gene could be obviously deduced to be involved in the dimerization reaction. Thus, we assumed that this process may occur on the carrier protein.…”

“…Because we were not previously able to successfully reconstitute the entire youssoufene biosynthetic pathway in vitro, [6a] we assumed that this was due to the absence of unidentified protein(s) in the one‐pot assays. Therefore, we performed pull‐down assays for S. coelicolor M1146/pWLI817‐His and its mutants by using the ITCT method developed in our previous study [6a] .…”

“…Because we were not previously able to successfully reconstitute the entire youssoufene biosynthetic pathway in vitro, [6a] we assumed that this was due to the absence of unidentified protein(s) in the one‐pot assays. Therefore, we performed pull‐down assays for S. coelicolor M1146/pWLI817‐His and its mutants by using the ITCT method developed in our previous study [6a] . As shown in Figure S2, SDS‐PAGE and LC–MS analyses suggested that a partner protein with a molecular weight (MW) of ~34,672.85 Da (~34,542.24 Da after N‐terminal methionine cleavage) interacted with YsfA (ACP).…”

“…Previously, we unraveled the assembly line of youssoufenes ( 1 – 5 ) and serpentene ( 6 ), which is an ISO‐dependent acyl carrier protein (ACP)‐tethered elongation process accomplished by three pairs of phylogenetically different ketosynthase/chain length factors (KS/CLF) (Figure 1). YsfB/C is responsible for the first seven cycles of condensation, YsfD/E is involved in the eighth cycle of condensation to yield short‐chain youssoufenes B1 ( 2 ) and B2 ( 3 ), and YsfJ/K catalyzes further elongation to generate long‐chain youssoufenes B3 ( 4 ), B4 ( 5 ) and serpentene ( 6 ) [6a] . Notably, dimeric 1 exhibits increased inhibition against multidrug‐resistant E. faecalis compared to monomeric youssoufene compounds [5] .…”

“…A DH gene ysfR was identified after sequence correction, which interacts with YsfI to form DH heterologous dimer YsfR/I (see Figure S1). B) Proposed biosynthetic pathway of 1 – 6 based on our previous study [6a] . Carbon‐carbon double bonds are indicated in bold.…”