Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield

Abstract

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

Lignin engineering in forest trees: From gene discovery to field trials

“…(2020) P. deltoides × P. euramericana LTF1 vessel-specific suppression ↓16% ↑S/G n.d. n.d. ↓ Gui et al. (2020) P. tremula × P. alba ClDCS and ClCURS2 overexpression ↑23% ↓S/G WT n.d. ↓ De Meester et al. (2022a) P. alba × Populus grandidentata MdCHS3 overexpression ↓10% WT S/G ↑ n.d. WT Mahon et al.…”

“…Second, curcumin is a soluble metabolite in Curcuma longa (turmeric) that is not known to be part of the natural lignin of tree species. By overexpression of two genes from turmeric, DIKETIDE-CoA SYNTHASE ( DCS ) and CURCUMIN SYNTHASE ( CURS ), curcumin has been successfully biosynthesized and integrated into the cell walls of Arabidopsis and poplar ( Oyarce et al., 2019 ; De Meester et al., 2022a ). However, in contrast to transgenic Arabidopsis plants, transgenic poplars have 23% more lignin, an altered lignin composition that resembles stress lignin, no increase in saccharification efficiency after alkaline pretreatment, and a yield penalty.…”

“…Indeed, the very same transgenic line can behave significantly differently depending on whether it is grown in the greenhouse or the field. For example, the reduction in lignin in 4CL -, CCoAOMT -, CCR -, or CAD -downregulated poplars was greater when the trees were grown in the greenhouse than when they were grown in the field ( Wang et al., 2012 ; Stout et al., 2014 ; Van Acker et al., 2014 , 2017 ; Xiang et al., 2017 ; De Meester et al., 2022a ). Moreover, although CCR -, CAD -, and vessel-specific 4CL -downregulated poplars grew normally in the greenhouse, the same lines displayed a yield penalty when grown in the field ( Leplé et al., 2007 ; Van Acker et al., 2017 ; Cao et al., 2020 ; De Meester et al., 2022a ).…”

“…For example, 4CL -downregulated lines grown in the greenhouse had severely reduced lignin amounts (up to 47% less lignin than the WT control), whereas the same transgenic lines grown on a mountain site field had mildly reduced lignin amounts (up to 8%) after 2 years and lignin amounts similar to that of the WT after 3 years of growth ( Xiang et al., 2017 ). Similarly, greenhouse-grown CAD -downregulated poplars had 10% less lignin ( Van Acker et al., 2017 ), whereas the same transgenic lines grown in the field displayed 10% less lignin after 1 year and had lignin amounts equal to that of the WT after 3 years of growth ( De Meester et al., 2022a ). The lignin content in 1-year-old greenhouse-grown 4CL - and CCoAOMT -downregulated poplars was reduced by 16%–42%, whereas that in 5-year-old field-grown clonal ramets was reduced by only 6%–10% ( Wang et al., 2012 ).…”

“…The lignin content in 1-year-old greenhouse-grown 4CL - and CCoAOMT -downregulated poplars was reduced by 16%–42%, whereas that in 5-year-old field-grown clonal ramets was reduced by only 6%–10% ( Wang et al., 2012 ). This trend might be explained by the fact that greenhouse-grown trees continuously grow and develop new xylem because of the long-daylight regime typically used in greenhouses, but trees grown in the field in a temperate climate zone cease growth in late summer every year and may continue to lignify their cell walls—aided by the good neighbor cells—until they enter dormancy ( Van Acker et al., 2014 ; De Meester et al., 2022a ). Alternatively, these observations might be explained by the enhanced exposure to (multiple cycles of) environmental stresses in the field, such as drought, leading to enhanced lignin biosynthesis ( Jubany-Marí et al., 2009 ; Lee et al., 2012 ; Cesarino, 2019 ; Liu et al., 2021b ).…”