Engineered Lignin in Poplar Biomass Facilitates Cu-Catalyzed Alkaline-Oxidative Pretreatment

Abstract

Both untransformed poplar and genetically modified “zip-lignin” poplar, in which additional ester bonds were introduced into the lignin backbone, were subjected to mild alkaline and copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment. Our hypothesis was that the lignin in zip-lignin poplar would be removed more easily than lignin in untransformed poplar during this alkaline pretreatment, resulting in higher sugar yields following enzymatic hydrolysis. We observed improved glucose and xylose hydrolysis yields for zip-lignin poplar compared to untransformed poplar following both alkaline-only pretreatment (56% glucose yield for untransformed poplar compared to 67% for zip-lignin poplar) and Cu-AHP pretreatment (77% glucose yield for untransformed poplar compared to 85% for zip-lignin poplar). Compositional analysis, glycome profiling, and microscopy all supported the notion that the ester linkages increase delignification and improve sugar yields. Essentially no differences were noted in the molecular weight distributions of solubilized lignins between the zip-lignin poplar and the control line. Significantly, when zip-lignin poplar was utilized as the feedstock, hydrogen peroxide, catalyst, and enzyme loadings could all be substantially reduced while maintaining high sugar yields.

Lignin Engineering in Forest Trees

“…Poplars have also been engineered to contain ester linkages in the lignin polymer backbone. Coniferyl ferulate esters were introduced into the polymer via expression of a FERULOYL-CoA:MONOLIGNOL TRANSFERASE ( FMT ) gene derived from Angelica sinensis (Wilkerson et al, 2014), leading to an improved saccharification efficiency under various pretreatment conditions (Wilkerson et al, 2014; Kim et al, 2017; Bhalla et al, 2018), and an improved kraft pulping efficiency as compared to wild type (Zhou et al, 2017). Monolignol p -coumarate esters have also been engineered in poplar, via expression of a rice p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE ( PMT ) gene, resulting in a higher frequency of resistant interunit bonds and a higher frequency of G and S terminal units with free phenolic groups (Smith et al, 2015; Sibout et al, 2016).…”

Improved analysis of arabinoxylan-bound hydroxycinnamate conjugates in grass cell walls

“…Transferase genes have now been identified for this monolignol acylation via the activated intermediate p -coumaroyl-CoA [ 4 – 6 ]; the equivalent alternative viewpoint is that p CA (via p -coumaroyl-CoA) is esterified by monolignols. More recently, plants have been engineered to produce analogous monolignol ferulate (FA) conjugates [ 3 , 7 , 8 ], that also participate in lignification and, because ferulate is more compatible with the monolignols in its radical coupling reactions [ 2 , 9 ], biosynthesize lignins with readily cleavable ester bonds in the lignin backbone, allowing more facile delignification of such materials [ 8 , 10 – 12 ]. The DFRC (derivatization by reductive cleavage) method, which cleaves lignin β-ethers while leaving γ-esters intact, was extended to provide unambiguous evidence for the existence of such conjugates in lignin, and to assess their (relative) levels in the polymer [ 7 , 13 – 17 ]; DFRC releases both p CA and FA conjugates (and in fact also benzoate, vanillate, and other conjugates) that are involved in β-ether units in lignin that can be cleaved by the method [ 13 , 18 – 20 ].…”

Opportunities and barriers for biofuel and bioenergy production from poplar

“…Expressing the AsFMT gene in poplar using tissue‐specific promoters was shown to introduce novel ester bonds into the lignin in transgenic poplar ( P. alba × grandidentata ) effectively manifesting in natural “zip‐lignin” (Wilkerson et al, 2014). Compared with untransformed poplar, the “zip‐lignin” poplar showed higher glucose and xylose yields following both alkaline‐only pretreatment (Wilkerson et al, 2014), copper‐catalyzed alkaline hydrogen peroxide pretreatment (Bhalla et al, 2018), and ILs pretreatment (Kim et al, 2017).…”