Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

Abstract

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50-60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.

Genetic and biotechnological approaches for biofuel crop improvement

“…A related, and possibly favored, approach looked at downregulation of enzymes acting at later stages in monolignol biosynthesis, such as cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD). Targeting CCR and CAD lead to generation of alfalfa lines with up to 60% and 40% improvements in enzymatic saccharification efficiency, respectively, with little to no apparent developmental defects associated [39]. However, extensive metabolomic and transcriptomic analyses have shown that silencing CCR and CAD affects many more biochemical pathways in tobacco and poplar than just lignin biosynthesis [40 ,41 ], suggesting that additional genes can be targeted for more efficient pathway engineering.…”

Salicylic acid mediates the reduced growth of lignin down-regulated plants

“…However, transgenic plants down-regulated in the hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) enzyme, which have a strong reduction in lignin levels, show severe defects in growth (3)(4)(5). Similar, although less severe, effects are observed in plants down-regulated in some, but not all, of the other enzymes of the monolignol pathway (5)(6)(7)(8)(9)(10).…”

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

“…Cinnamoyl-CoA reductase (CCR) catalyzes the first step of the monolignol-specific pathway. It converts the hydroxycinnamoylCoA esters to their corresponding hydroxycinnamaldehydes (mainly feruloyl-CoA to coniferaldehyde), and down-regulation of CCR typically results in reduced lignin content (8)(9)(10)(11)(12)(13). CCR-downregulated poplars are characterized by an orange to wine-red coloration of the xylem that often appears in patches along the stem.…”