Optimising promoters and subcellular localisation for constitutive transgene expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.