Expression of a bacterial 3-dehydroshikimate dehydratase (QsuB) reduces lignin and improves biomass saccharification efficiency in switchgrass (Panicum virgatum L.).

BACKGROUND: Lignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading. RESULTS: The 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass (Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene (pShOMT) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process. CONCLUSION: This study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.

Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.

A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction.

BACKGROUND: Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established. RESULTS: We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 (GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation. CONCLUSIONS: This screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.

Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility.

Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet). In this study, we demonstrate in Arabidopsis stems that targeted expression of AdoMet hydrolase (AdoMetase, E.C. 3.3.1.2) in secondary cell wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H) units and a reduction of dimethylated syringyl (S) units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild-type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.