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

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.