Our goal is to make a new material — LIGGREEN. We mimic the lignin of Picea abies and determine to use one type of monolignol for polymerization. LIGGREEN is biodegradable, plastically, and more transparently than natural lignin.
The structure of Picea abies (Norway spruce) contains up to 94% of coniferyl alcohol (Figure1). In general, the lignin structure of a tree with high coniferyl alcohol content is relatively easily biodegraded and structurally tough, so it is also called soft lignin. Picea abies is also the mainstream of European tree species research. The lignin structure and related research on Picea abies are comprehensive, so we chose Picea abies as our tree species reference.
Figure1: Typical H:G:S Ratio for Lignin from Biomass(source)
In the production of LIGGREEN, we start from the production of enzymes. We found three enzymes:Px16, Px18 and Lac1 (Shigeto J et al.2016, Zhao Q et al. 2013), that help coniferyl alcohol form bonds. In this regard, we want to produce dehydrogenase polymer (DHP) lignin (Ferrer JL et al. 2008). As mentioned, bonds between coniferyl alcohol are promoted by peroxidase and laccase. Peroxidase and laccase of different tree species will produce different bonds,while in Picea abies, the enzymes present are mainly Lac1, Px16 and Px18.
Figure3: Project outline
We use commercial vector pGAPZ A from Invitrogen, which containing ZeocinTM resistance. However, ZeocinTM is expensive and licensed under patent. In order to keep the cost down, we have designed another selection—histidine deficiency.
Figure2: The design and construction of histidine deficiency.
In the production of LIGGREEN, two enzymes extracted from plants are used for monolignol polymerization: peroxidase and laccase. Laccase reacts at specific point on coniferyl alcohol according to enzyme specificity, generating three binding structures—β-β, β-O-4, β-5 (Vanholme R et al. 2010); Peroxidase mainly cause polymerization between dimers, polymerizing β-O-4 and β-5 bonds.
Figure4: Dimerization of two dehydrogenated coniferyl alcohol monomers, resonance forms of dehydrogenated coniferyl alcohol.(Vanholme R. et al. 2010)
Based on the above theory, we want to synthesize LIGGREEN using Coniferyl alcohol, Lac1, Px16 and Px18. We produce these enzymes by synthetic biology. Using P. pastoris to produce enzymes, P. pastoris can provide the N-Link glycosylation modification required by our enzymes (Spadiut O et al. 2013), and achieve high protein expression and exogenous gene regulation (α-factor).
Figure5: Design of our entire production line.
Jun S.,Yuji T. (2016). Diverse functions and reactions of class III peroxidases. New Phytologist (2016) 209: 1395–1402. doi: 10.1111/nph.13738
Ferrer JL, Austin MB, Stewart C Jr, Noel JP (2008). Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol Biochem (2008) 46(3):356-70. doi: 10.1016/j.plaphy.2007.12.009
Shigeto J, Tsutsumi Y. (2016) Diverse functions and reactions of class III peroxidases. New Phytol.(2016) 209(4):1395-402. doi: 10.1111/nph.13738
Zhao Q, Nakashima J, Chen F, Yin Y, Fu C, Yun J, Shao H, Wang X, Wang ZY, Dixon RA. (2013) Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis. Plant Cell (2013) 25(10):3976-87. doi: 10.1105/tpc.113.117770
Spadiut O, Herwig C, (2013) Production and purification of the multifunctional enzyme horseradish peroxidase. Pharm Bioprocess. 1(3): 283–295. doi: 10.4155/pbp.13.23
Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W. (2010) Lignin biosynthesis and structure. Plant Physiol. (2010) 153(3):895-905. doi: 10.1104/pp.110.155119