Intended for Biofuel Production

With a lignocellulosic platform for bioethanol production, one obvious target is (as just discussed) the management of energy crop productivity to maximize the capture of solar energy and atmospheric CO2; the chemical composition of the bio­mass is, however, of great practical significance for the industrial bioprocessing of

feedstocks:302,303

1. Developing crop varieties with reduced lignin contents (especially with softwoods)

2. Crops with increased cellulose and, arguably, hemicellulose contents

3. Plants with the increased capability to degrade cellulose, hemicellulose, and lignin — after harvest (i. e., in a controlled manner capable of minimiz­ing biomass pretreatment)

Of these, modifying lignin content has been the most successful — classical genetics suggests that defining quantitative traits and their genetic loci is relatively easy, and (even better) some of these loci are those for increased cellulose biosynthesis.304 As collateral, there is the confidence-building conclusion that lignin contents of commer­cial forest trees have been reduced to improve pulping for the paper industry; the genetic fine-tuning of lignin content, composition, or both is now technically feasible.305

Reductions in plant lignin content have been claimed using both single — and multiple-gene modifications (figure 4.16):

• Down-regulating either of the initial two enzymes of lignin biosynthesis, phenylalanine ammonia lyase and cinnamate 4-hydroxylase (C4H), reduces lignin content and impairs vascular integrity in the structural tissues of

plants.305

• Deletion of the second activity of the bifunctional C4H enzyme, coumarate 3-hydroxylase, results in reduced lignin deposition.306

• Later enzymes in the lignin pathway were considered to be less amena­ble for inhibiting lignification but multiple-gene down-regulation could

be effective.307,308

• Inactivating O-methyltransferase activity with an aspen gene incorporated into a transmissible plasmid in the antisense orientation reduced lignin for­mation in Leucaena leucocephata[45] by 28%, increased monomeric phenolic levels, and increased the cellulose content by 9% but did not visibly affect the plant phenotype.309

Are “lignin-light” plants biologically viable for commercial cultivation? Altered stem lignin biosynthesis in aspen has a large effect on plant growth, reducing total leaf area and resulting in 30% less total carbon per plant; root growth was also

4-OH’Coumaric ||
acid

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I

СН

4’OH’COumarylCoA II

4’OH’Coumarylaldehyde 4-OHcoumaryl alcohol

FIGURE 4.16 Outline of biosynthesis of lignin precursors: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; COMT, caffeate O-methyltransferase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase. (After Hertzberg et al.312)

compromised.310 Vascular impairment can lead to stunted growth.307 On the other hand, aspen wood in reduced-lignin transgenics was mechanically strong because less lignin was compensated for by increased xylem vessel cellulose.308 Smaller plants may be grown, as energy crops, in denser plantations; alternatively, plants with reduced stature may be easier to harvest, and various practical compromises between morphology and use can be imagined — this can be seen as analogous to the introduction of dwarfing rootstocks for fruit trees that greatly reduced plant height and canopy spread and facilitated manual and mechanical harvesting.

Also without obvious effects on plant growth and development was the introduc­tion and heterologous expression in rice of the gene from Acidothermus cellulolyticus encoding a thermostable endo-1,4-P-glucanase; this protein constituted approximately 5% of the total soluble protein in the plant and was used to hydrolyze cellulose in ammonia fiber explosion-pretreated rice and maize.311 More ambitiously, enzymes of polysaccharide depolymerization are being actively targeted by plant biotech compa­nies for new generations of crops intended for biofuels. A large number of genes are under strict developmental stage-specific transcriptional regulation for wood forma­tion in species such as hybrid aspen; at least 200 genes are of unknown function, pos­sibly undefined enzymes and transcription factors, but this implies that heterologous glucanases and other enzymes could be produced during plant senescence to provide lignocellulose processing in plants either before the preparation of substrates for con­ventional ethanol fermentation or in solid-phase bioprocesses (section 4.6.2).312