ENGINEERING GRASSES WITH REDUCED CROSS-LINKING OF CELL WALLS: IMPACT FOR ANIMAL AND BIOFUEL INDUSTRIES
Marcia M de O. Buanafina
August, 2008

Cell walls are complex structures that serve several important functions in the life of plants. They provide shape and strength to cells, glue cells together, give rigidity to the whole plant, and function as a physical barrier to pathogen attack. Moreover, as a major sink for photosynthates, they factor heavily in the nutrition of farm animals, providing the major source of carbon and energy. In a more industrial vein, the need for new forms of renewable and eco-friendly energy has gained wide recognition in recent years, partly due to the economic and political hazards of U.S. dependence on foreign oil and partly due to the growing realization that burning fossil fuels contributes to global warming¹.

Recalcitrance of grass cell wall
Plant cell walls—particularly of grasses—are expected to supply most of the biomass for the production of renewable biofuels in the U.S. by 2030¹. The potential use of grasses as feedstock material stems from their high yield, low cost, suitability for marginal land, and minimal environmental impact.

However, grass cell walls are characterized by a large quantity of esterified ferulates. Ferulic acid (FA), the most abundant hydroxycinnamic acid (HCA) identified in grass cell wall, is attached to the cell wall via an ester linkage to the arabinose side chain of arabinoxylans (AX)² (Fig. 1). HCAs can be oxidatively coupled to form a variety of dehydrodiferulate dimers, cross-linking hemicellulose polysaccharide chains³ (Fig.2). Feruloylation of AX is important because it directly cross-links xylans and because ferulate esters become incorporated into lignin through ether and C-C bonds, acting as nucleating sites for the formation of lignin. The arabinoxylans in grasses serve a major structural role by binding to cellulose microfibrils. Feruloylation is also responsible for the linkage of lignin to the xylan/cellulose network via lignin-ferulate-xylan complexes⁴. FA ester-ether bridges between lignin and arabinoxylan are ascribed a significant role for limiting cell wall degradability of grasses⁵.

Manipulating ferulate cross-linking
Ferulic acids esterified to arabinoxylans in grasses are expected to interfere with cell wall digestion by hindering the binding of xylanase to its substrate, which is essential for hydrolysis. Removing labile phenolics by chemical treatment with alkali increases biodegradability and the nutritional value of low-quality feed. Therefore reducing the level of cross-linking of cell wall carbohydrates would be expected to improve the rate and extent of digestibility in grasses and their bioconversion to ethanol.

The availability of a ferulic acid esterase gene (faeA) from Aspergillus niger⁶, together with advances in genetic transformation techniques, provide the tools that we have combined into a new strategy to produce grasses able to efficiently synthesize ferulic acid esterase (FAEA). Since the recombinant enzyme can cleave the 1→5 ester bond between ferulic acid and arabinose, releasing ferulic acid and diferulic dimers from grass cell walls, we expect that the targeted expression of FAEA in planta would result in plants with reduced levels of ferulate cross-linking. This in turn would be highly desirable for improvement of biomass for energy production or animal feed.

To test this strategy, faeA was initially transformed into Lolium (ryegrass) ⁷, driven by the rice actin promoter, with the recombinant enzyme targeted to the vacuole. FAEA expression was confirmed.

Considering the importance of Festuca arundinacea (tall fescue) as a forage crop, which forms the basis for beef and milk production worldwide, and also as a potential feedstock crop for biofuel production, FAEA was more recently introduced into this species⁸ to extend the utility of our approach. Festuca, with a much lower digestibility and a different cell wall composition compared to Lolium, is expected to present a different, more recalcitrant substrate to FAEA. Consistent with our prior results in Lolium, vacuole-targeted FAEA was successfully expressed in Festuca, resulting in the release of p-coumarate, monomeric, and dimeric ferulates from cell walls upon cell death.

In contrast to other studies in which the specificity of Aspergillus FAEA to hydrolyze ester bonds between HCA and AX has been studied by analyzing its action on substrates in vitro, we evaluated FAEA action in planta by producing grass that constitutively expresses FAEA. Expression in planta results in the release of a broader range of esterified ferulates compared to FAEA action in vitro. Additionally, we showed that the release of ferulates and diferulates is enhanced several fold with the addition of exogenous endo-1,4-β-xylanase. Endo-1,4-β-xylanase should open the cell wall hemicellulose complex, which in turn should allow more efficient FAEA access to ferulate esters bound to AXs, compared with untreated walls.

This particular approach, targeting FAEA expression to the vacuole, was taken to test the potential use of vacuole-stored FAEA that is released on cell death; no effect on cell wall structure was expected until the cells died or were otherwise disrupted. However, most FAEA-expressing plants show reduced levels of cell wall esterified HCAs before cell death. This indicates that the enzyme may leak into the ER/Golgi membrane system where it reduces the feruloylation of arabinoxylan during its formation. FAEA-expressing plants are stable when vegetatively propagated via tillering as well as by meristem culture, over three generations.

In our study, line T7 in particular showed high FAEA expression, the highest released level of cell wall ferulates upon xylanase (XYN) digestion, substantially reduced amounts of esterified cell wall p-coumarate, monomeric, and dimeric ferulates, and a significant increase in in vitro dry matter digestibility (IVDMD) and initial rate of digestion, compared to controls, as summarized in Table 1.

Our results indicate that FAEA expression in Festuca can indeed result in plants with enhanced digestibility, measured as end point digestibility (IVDMD), as well as an improved initial rate of fermentation, an important parameter in ruminal digestion of forages (see Fig. 3).

Safety concerns / alternative strategies
The potential risk of transgene flow into wild species or conventional crops could be overcome if genetically engineered grasses expressing FAEA were harvested before becoming sexually mature, eliminating pollen and seed spread. Additionally Festuca needs a strong vernalization to flower, and the line we engineered has a very low fertility and thus must be propagated vegetatively, representing no risk in terms of transgene flow.

Implications for agbiotechnology and future prospects
Our results demonstrate the potential for further manipulation of cell wall cross-linking by targeting FAEA expression directly to the cell wall or alternatively to the ER/Golgi system (to reduce feruloylation of arabinoxylans).

During the last 15 years, more than 40 FAEAs have been purified, and they show great diversity of physicochemical characteristics, catalytic properties, and substrate specificity. In addition, feruloylated arabinoxylan fragments generated by hydrolysis with different xylanases differ in length and structure. Finding the best synergism between a cell wall hydrolase such as FAEA and a main-chain cleaving enzyme such as xylanase and their co-expression in planta is a potential strategy that can help achieve an optimum level of cell wall hydrolysis. These potential approaches could prove even more efficient for altering cross-linking of cell walls and consequently have a greater effect on cell wall degradability.

We conclude that, in the context of forage improvement, the generation of genetically engineered plants expressing FAEA is an effective strategy for improving wall digestibility, as demonstrated in Festuca and Lolium. The effectiveness of this strategy also reinforces the importance and potential of genetic engineering for plant improvement. We anticipate applications of this strategy in other grass species, where phenolic cross-linking is a limiting factor for cell wall degradability, as well as in a large number of biotechnological processes and industries in order to improve: (1) biomass processing for biofuels; (2) pulp bleaching applications; (3) bread quality; (4) production of flavorants for food industry; and (5) quality of animal feedstock. This effort could be translated as:

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Marcia M de O. Buanafina
Department of Biology
Penn State University, State College, PA
mmb26@psu.edu