Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon

Summary To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML‐FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML‐FA production are as follows: (1) The presence of an active FERULOYL‐CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16‐fold increase in lignin‐bound ML‐FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon . On its own, knocking out the native p ‐ COUMAROYL‐CoA MONOLIGNOL TRANSFERASE (BdPMT ) pathway that competes for monolignols and the p ‐coumaroyl‐CoA intermediate did not change ML‐FA incorporation, nor did partial loss of CINNAMOYL‐CoA REDUCTASE1 ( CCR1 ) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt‐1 mutant resulted in a 32‐fold increase in ML‐FA incorporation into lignin over the wild‐type level.