The biomass of plants consists predominately of cell walls, a sophisticated composite material composed of various polymer networks including numerous polysaccharides and the polyphenol lignin. In order to utilize this renewable, highly abundant resource for the production of commodity chemicals such as biofuels, major hurdles have to be surpassed to reach economical viability. Recently, major advances in the basic understanding of the synthesis of the various wall polymers and its regulation has enabled strategies to alter the qualitative composition of wall materials. Such emerging strategies include a reduction/alteration of the lignin network to enhance polysaccharide accessibility, reduction of polymer derived processing inhibitors, and increases in polysaccharides with a high hexose/pentose ratio.

The development of second-generation biofuels - those that do not rely on grain crops as inputs - will require a diverse set of feedstocks that can be grown sustainably and processed cost-effectively. Here we review the outlook and challenges for meeting hoped-for production targets for such biofuels in the United States.

The development of alternative transportation fuels that can meet future demands while reducing global warming is critical to the national, environmental, and economic security of the United States. Currently, biofuels are produced largely from starch, but there is a large, untapped resource (more than a billion tons per year) of plant biomass that could be utilized as a renewable, domestic source of carbon-neutral, liquid fuels. However, significant roadblocks hamper the development of cost-effective and energy-efficient processes to convert lignocellulose biomass into fuels. Lignin is a very strong phenolic polymer, which embeds cellulose and hemicellulose, and its recalcitrance to chemical and biological degradations inhibits the conversion of cell wall polysaccharides (cellulose and hemicellulose) into fermentable sugars. Unfortunately, lignin provides such compressive resistance to plant cells that it cannot simply be genetically removed without incurring deleterious consequences on plant productivity. Alternative strategies to significantly reduce lignin recalcitrance would be modifying its composition and deposition. We are currently developing an alternative strategy, which is focusing on the partial replacement of the ?hard bonds? (e.g. ether, carbon bonds) in the lignin polymer with ?easily cleavable? ones (e.g., amide or ester bonds). For this propose, we are rerouting part of the lignin biosynthesis towards the synthesis of phenylpropanoid-derived molecules such as hydroxycinnamic acid amides and esters in order to partially replace conventional lignin monomers in the cell wall. Biosynthetic pathways and preliminary data for de novo synthesis in Arabidopsis of selected phenylpropanoid-derived compounds are presented.

The process of plant cell wall biosynthesis involves a complex series of biochemical processes involving many hundreds of proteins. Determining the function of a specific gene through functional genomics techniques have proved problematic due to genetic redundancies and undetectable changes. Common techniques have used mutant collections in forward genetic screens or reverse genetics to directly target and disrupt genes of interest. Since such techniques are heavily reliant on some phenotypic discrimination to asses gene function, the absence of a measureable difference often results in little useful information. Furthermore the complete absence of many genes produced by such techniques results in a lethal phenotype as the gene is absolutely required for normal function of the plant. A more subtle approach utilizes genetic differences in naturally occurring variants to provide important information about gene function and genetic diversity. In collaboration with the Joint Genome Institute we have sequenced two Arabidopsis accessions (Bay-0 and Sha-0) that have previously been shown to have measureable differences in Ara-Rha ratios in cell wall extracts. This genetic information and the utilization of recombinant inbred lines (RIL?s) will be used to map QTL?s identified in these and other Arabidopsis accessions to identify loci that contribute to functional differences in plant cell walls.

Genetic variation in different accessions of Arabidopsis thaliana has occurred through thousands of years of geographic spread and adaptation. As a result of this diversity, disparate accessions have phenotypic differences that can be used to identify genes that contribute to the biosynthesis and composition of cell walls. Forward and reverse genetic screens to identify single mutants often prove difficult for detection of genetic differences that may lead to more subtle phenotypes. Exploiting the inherent genetic variation in Arabidopsis accessions through quantitative trait analysis will allow for the detection of variation in cell wall biosynthesis and composition. We selected two methods for screening parental accessions: measuring monosaccharide composition by HPAEC and structural changes by Near Infrared (NIR) spectroscopy. From these analyses, we selected Ri-0 as the most different accession from the reference accession Col-0. Recombinant inbred lines (RILs) derived from Col-0 and Ri-0 parents were used to determine quantitative trait loci (QTL) that contribute to the differences observed in monosaccharide and pectin content and NIR spectroscopy. Two putative candidate genes encoding enzymes involved in nucleotide sugar conversion have been identified. We are currently determining differences between the parental versions of the genes and how any differences contribute to the observed phenotypes. Additionally, we have re-sequenced two Arabidopsis accessions, Bay-0 and Shahdara, in collaboration with the Joint Genome Institute. This effort has resulted in a collection of SNPs between these accessions and the reference Arabidopsis accession, Col-0. Re-seqeuencing efforts are being expanded to other accessions, beginning with Ri-0. Information obtained from resequencing will aid in QTL analysis and be of service to the general Arabidopsis community.

Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive 'hypersensitive response' (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.

Background

Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli.

Results

We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4'-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3',4'-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F.

Conclusion

We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

Abstract Background Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli. Results We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4′-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F. Conclusion We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

Muconic acid (MA) is a dicarboxylic acid used for the production of industrially relevant chemicals such as adipic acid, terephthalic acid, and caprolactam. Because the synthesis of these polymer precursors generates toxic intermediates by utilizing petroleum-derived chemicals and corrosive catalysts, the development of alternative strategies for the bio-based production of MA has garnered significant interest. Plants produce organic carbon skeletons by harvesting carbon dioxide and energy from the sun, and therefore represent advantageous hosts for engineered metabolic pathways towards the manufacturing of chemicals. In this work, we engineered Arabidopsis to demonstrate that plants can serve as green factories for the bio-manufacturing of MA. In particular, dual expression of plastid-targeted bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase (CatA) resulted in the conversion of the endogenous salicylic acid (SA) pool into MA via catechol. Sequential increase of SA derived from the shikimate pathway was achieved by expressing plastid-targeted versions of bacterial salicylate synthase (Irp9) and feedback-resistant 3-deoxy-D-arabino-heptulosonate synthase (AroG). Introducing this SA over-producing strategy into engineered plants that co-express NahG and CatA resulted in a 50-fold increase in MA titers. Considering that MA was easily recovered from senesced plant biomass after harvest, we envision the phytoproduction of MA as a beneficial option to add value to bioenergy crops.