Connecting moss lipid droplets to patchoulol biosynthesis
Our new paper about connecting moss lipid droplets to patchoulol biosynthesis has finally been published in PlosOne.
Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. In this paper, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants
Figure: Microscopy images of WT protonema cells grown in for 10 days (i) and WT gametophytes grown for 6 weeks (ii). Stained with BODIPY (green) and overlaid with chloroplast autofluorescence (red), showing the presence of cytosolic LDs.
Figure: Representative confocal Z-stack images of LDs in gametophyte leaves of different P. patens lines. Green colour shows the BODIPY stained LDs and the red shows the chloroplast autofluorescence. Images were collected at the same laser intensity and magnification (scale = 10 μm).
Figure: Log (x+) normalized heatmap of the different P. patens lines and the wildtype P. patens with a 256-fold up (red) or down (blue) regulation of gene expression in one or more P. patens lines compared to the wild type. For some this also show that there is little to no change in one or more P. patens lines, but a 256-fold change in just one P. patens line.
We observed 26% less growth in plant height when patchoulol is biosynthesized in P. patens, just by over-expressing PTS. This finding was in line with similarly published works, however, in the LD targeted PTS line, ZmUbi:AtLDAP1-PTS we observed 2-fold increased growth in plant height compared to WT. This shows that we can biosynthesize terpenoids in P. patens, increase growth, and potentially device a new extraction method, which is very useful for future industrial production.
All the LD protein-PTS attached lines retained more patchoulol in the cell, over none-attached ZmUbi:PTS and ZmUbi:PpOle1-LP4/2A-PTS lines. The size and number of LDs in patchoulol biosynthesis lines were increased; the size was increased with up to 188% and about 25% more patchoulol was retained inside the cell, though the production was significantly reduced. Thus, there is a potential to use the LDs for storage of small molecules during production in bioreactors. However, it requires further work to be established for the production of any small molecule.
The paper is freely available here: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243620