Synthetic-biology approach for plant lignocellulose engineering

Abstract

Plant biomass is an abundant, renewable resource that offers multiple advantages for the production of green chemicals and recombinant proteins. However, the adoption of plant-based systems by industry is hindered because mammalian and other cell cultures are well-established and better characterized in an industrial setting, and thus it is difficult for plant-based processes to gain a foothold in the marketplace. Therefore, additional benefits of plant-based systems may be essential to tip the balance in favor of sustainable plant-derived products. A crucial factor in biomass valorization is to design mid- to high-value co-products that can be derived cost-effectively from the residual lignocellulose (LC). However, the utility of LC remains limited because LCs are, in general, too recalcitrant for industries to utilize their components (lignin, cellulose, and hemicelluloses). To overcome this issue, in planta engineering to reduce LC recalcitrance has been ongoing in recent decades, with essential input from synthetic biology owing to the complexity of LC pathways and the massive number of genes involved. In this review, we describe recent advances in LC manipulation and eight strategies for redesigning the pathways for lignin and structural glycans to reduce LC recalcitrance while mitigating against the growth penalty associated with yield loss.

Keywords: biomass; cell wall; hemicellulose; lignin; lignocellulose engineering.

Figure 1. Schematic diagram of lignin biosynthesis pathway in plant cell. The magenta arrows and letters indicate unconventional steps and units of lignin after some genetic manipulations, respectively (F5H overexpression in the COMT mutant for 5-Hydroxy G units or down regulations of the methionine and folate cycles for C units, see also Figure 2). The shikimic pathway in plastids, phenylpropanoid pathway in cytosol and endoplasmic reticulum (ER) are described. Lignin units are H (hydroxyphenyl), G (guaiacyl) and S (syringyl). ADT, arogenate dehydratase; C3H, p-coumarate 3-hydroxylase; C3′H, p-coumaroyl-shikimate 3′-hydroxylase; C4H, cinnamate 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CM, chorismate mutase, COMT, caffeate/5-hydroxyferulate O-methyltransferase; CS, chorismate synthase; CSE, caffeoyl shikimate esterase; C units, catechyl units; DAHP, 3-deoxy-d-arabino-heptulosonate 7-phosphate; DAHPS, 3-deoxy-d-arabino-2-heptulosonate 7-phosphate synthase; DHQ, 3-dehydroquinate; 3DHS, 3-dehydroshikimate; DHQS, 3-dehydroquinate synthase; DHQ/SDH, 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase; E4P, d-erythrose 4-phosphate; EPSP, 5-enolpyruvyl shikimate-3-phosphate, EPSPS, 5-enolpyruvylshikimate 3-phosphate synthase; F5H, ferulate 5-hydroxylase or coniferyl aldehyde 5-hydroxylase; HCT, p-hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase; LAC, laccase; PAL, phenylalanine ammonia-lyase; PAT, prephenate aminotransferase; PEP, phosphoenol pyruvate; PER, peroxidase; PTAL, a bifunctional phenylalanine and tyrosine ammonia-lyase from grass; TAL, tyrosine ammonia-lyase; TyrA, tyrosine arogenate dehydrogenase; S3P, shikimate-3-phosphate; SK, shikimate kinase.

Figure 2. Interconnection between the lignin biosynthetic pathway and the methionine and folate cycles (Adapted from Liu and Eudes 2022 and Eudes et al. 2016b). Enzymes are: CCoAOMT, caffeoyl-CoA O-methyltransferase; COMT, caffeate/5-hydroxyferulate O-methyltransferase; CY-β-L, cystathionine β-lyase; CYS, cystathionine synthase; FPGS, folylpolyglutamate synthase encoded by maize brown midrib4 (bm4) gene or sorghum brown midrib19 (bmr19) gene; HSK, homoserine kinase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase encoded by maize brown midrib2 (bm2); SAHH, S-adenosylhomocysteine hydrolase; SAMase, S-adenosylmethionine hydrolase from coliphage T3 in red; SAMS, SAM synthase in blue, SHMT, serine hydroxymethyl transferase; ThrS, threonine synthase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate. Red and Blue characters are genes which up regulated genes and down regulated, respectively.

Figure 3. Manipulation of the lignin biosynthetic pathway based on studies from transgenic plants involving of heterologous expression of bacterial, plant and newly designed enzymes. Canonical monolignols and alternative monomers are biosynthesized from the shikimic pathway and phenylpropanoid pathway. Black character and arrows indicate metabolites and enzymes in the pathway for canonical monolignols biosynthesis. Magenta character and arrows indicates metabolites and enzyme for alternative monomer biosynthesis. Abbreviations are as follows, E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate; TAL, tyrosine ammonia-lyase; LAC, laccase; PER, peroxidase (See also Figure 1). Enzyme in black indicate the conventional steps in lignin pathway. Enzymes in magenta indicate the up-regulated genes. CALDH, coniferyl aldehyde dehydrogenase; CouA, hydroxycinnamoyl-CoA hydratase/lyase, DCS:CURS2, diketide-CoA synthase and curcumin synthase2; F6′H:COSY, feruloyl-CoA 6′-hydroxylase 1 and coumarin synthase; FMT, feruloyl-CoA monolignol transferase; HCHL, hydroxycinnamoyl-coa hydratase-lyase; MOMT4 and MOMT9, monolignol O-methyl transferase; pHBMT, p-hydroxybenzoyl-CoA monolignol transferase; PMT, p-coumaroyl-CoA monolignol transferase; QsuB, 3-dehydroshikimate dehydratase; UbiC, chorismate pyruvate lyase. The metabolic inhibition of HCT by QsuB-synthesized 3,4-dihydroxybenzoate (DHB) is also indicated.

Figure 4. Engineering strategies for 2-pyrone-4,6-dicarboxylic acid and p-hydroxybenzoate overproductions in plants. Schematic diagram of the shikimate, aromatic amino acid metabolic networks in plants (Adapted from Yokoyama et al. 2022) and engineering approach for PDC and pHBA. Arrows represent a single enzymatic step (See also Figure 1). Enzymes in magenta indicate introduction of foreign genes. AroGL175Q, 3-deoxy-d-arabino-2-heptulosonate 7-phosphate synthase with L175Q mutation; QsuB, 3-dehydroshikimate dehydratase; PmdA, 3,4-dihydroxybenzoate (DHB) 4,5-dioxygenase α-subunit; PmdB, DHB 4,5-dioxygenase β-subunit; PmdC, 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase.