Exploiting Spermidine N-Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast

Abstract

Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products.

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory

“…To this aim, a precursor-directed synthesis strategy was employed. A yeast strain was first transformed with At4Cl5 allowing CoA ester production from hydroxycinnamates added to the external medium as required for BAHD acyltransferases activities ( Perrin et al, 2021 ). Then CiSHT1 and CiSHT2 alone or combined were introduced in the yeast chassis.…”

“…N -dihydrocoumaroyl, N′ , N″ -dicoumaroyl spermidine and N -dihydrocoumaroyl, N′ , N″ -dicoumaroyl spermine, Supplemental Figures S6 and S7 ). The yeast enoyl reductase TSC13 (temperature sensitive CSG2 suppressor protein 13) is known to reduce coumaroyl-CoA into dihydrocoumaroyl-CoA which is further used, in our case, as a substrate by CiSHTs and competed with coumaroyl-CoA ( Perrin et al, 2021 ). This heterologous system confirms our findings, that is both enzymes are needed for tetracoumaroyl spermine accumulation in vivo and provide an efficient tool for the bioproduction of phenolamides derived either from spermidine or from spermine.…”

“…The engineering of a strain secreting the final products in the culture media should be considered. It was recently shown that yeast could be a suitable host platform for the production and excretion of spermidine-derived phenolamides ( Perrin et al, 2021 ). Finally, the catalytic versatility of the enzymatic system characterized in this study may be used to synthetize related bioactive compounds such as the kukoamines widely studied, among other, that possess numerous pharmacological effects, such as antihypertensive, anti-inflammatory, anti-analgesic, anti-sepsis, autoimmune-enhancing, and neuroprotective activities ( Chantrapromma and Ganem, 1981 ; Wang et al, 2016 ; Li et al, 2017 ).…”

“…Finally, the catalytic versatility of the enzymatic system characterized in this study may be used to synthetize related bioactive compounds such as the kukoamines widely studied, among other, that possess numerous pharmacological effects, such as antihypertensive, anti-inflammatory, anti-analgesic, anti-sepsis, autoimmune-enhancing, and neuroprotective activities ( Chantrapromma and Ganem, 1981 ; Wang et al, 2016 ; Li et al, 2017 ). Yeast expressing SHTs from different species was already exploited to produce spermidine acylated with dihydrocinnamic acids ( Perrin et al, 2021 ). Our work showed that engineered yeast can also produce spermine acylated with dihydrocinnamic acids.…”

“…Both chicory enzymes were shown to be involved in tetracoumaroyl spermine synthesis ( Delporte et al, 2018 ). The only enzymes characterized so far that have the capability to acylate secondary amino groups to synthesize fully substituted polyamines are SHTs ( Pichersky et al, 2006 ; Grienenberger et al, 2009 ; Elejalde-Palmett et al, 2015 ; Peng et al, 2016 , 2019 ; Delporte et al, 2018 ; Perrin et al, 2021 ). The accumulation of fully substituted spermidine derivatives was shown to be restricted to the pollen coat of Arabidopsis and apple tree and suggested as a marker of the pollen exine of Eudicotyledons ( Grienenberger et al, 2009 ; Elejalde-Palmett et al, 2015 ).…”