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Abstract With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C5) and epi-isozizaene (C15) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing l-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass. Graphical Abstract

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis -muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p - hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated. • Engineered C. glutamicum efficiently uses aromatic monomers from softwood lignin. • Deletion of the reductase FudC eliminates undesired aromatic alcohols as by-products. • C. glutamicum MA-9 converts nine aromatics into biobased cis, cis -muconate. • MA-9 forms cis, cis -muconate from industrially derived softwood-based vanillin. [ABSTRACT FROM AUTHOR]

Azospirillum baldaniorum Sp 245 is a model plant growth-promoting rhizobacterium. The first cross-talk with plants takes place within the roots. Roots cells growth is constrained by the primary cell wall (CW). Also, neighboring CW form the apoplast that should affect cells signaling and biochemical messages. Studies on CW phenolic composition ferulate (FA), diferulates (DFA) and p-coumarate and polyamines (PA) metabolisms of A. baldaniorum Sp 245- inoculated roots and on bacterial PA production in culture media should help to understand more about the mechanisms involved in Azospirillum -root association. For this purpose, CW-bound FA, DFA and p-coumarate contents, putrescine (put) and spermidine contents, diamine and polyamine oxidases activities, and H2O2 content of Cucumis sativus roots from dark grown seedlings inoculated with A. baldaniorum Sp 245 were determined. Also, bacterial PA production under constant agitation or static conditions was evaluated. Results showed lesser contents of all phenolics, and higher FA/DFA ratio in CW of inoculated roots that should be responsible for roots growth promotion. Also, the increased put content, DAO activity, and H2O2 production in the roots should be associated to A. baldaniorum Sp 245 growth promotion in early stages. Finally, the participation of both PA in A. baldaniorum Sp 245 biofilm formation was demonstrated. [ABSTRACT FROM AUTHOR]

With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C5) and epi-isozizaene (C15) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing l-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls.

Abstract Background Arabinoxylan in grass cell walls is acylated to varying extents by ferulate and p-coumarate at the 5-hydroxy position of arabinosyl residues branching off the xylan backbone. Some of these hydroxycinnamate units may then become involved in cell wall radical coupling reactions, resulting in ether and other linkages amongst themselves or to monolignols or oligolignols, thereby crosslinking arabinoxylan chains with each other and/or with lignin polymers. This crosslinking is assumed to increase the strength of the cell wall, and impedes the utilization of grass biomass in natural and industrial processes. A method for quantifying the degree of acylation in various grass tissues is, therefore, essential. We sought to reduce the incidence of hydroxycinnamate ester hydrolysis in our recently introduced method by utilizing more anhydrous conditions. Results The improved methanolysis method minimizes the undesirable ester-cleavage of arabinose from ferulate and p-coumarate esters, and from diferulate dehydrodimers, and produces more methanolysis vs. hydrolysis of xylan-arabinosides, improving the yields of the desired feruloylated and p-coumaroylated methyl arabinosides and their diferulate analogs. Free ferulate and p-coumarate produced by ester-cleavage were reduced by 78% and 68%, respectively, and 21% and 39% more feruloyl and p-coumaroyl methyl arabinosides were detected in the more anhydrous method. The new protocol resulted in an estimated 56% less combined diferulate isomers in which only one acylated arabinosyl unit remained, and 170% more combined diferulate isomers conjugated to two arabinosyl units. Conclusions Overall, the new protocol for mild acidolysis of grass cell walls is both recovering more ferulate- and p-coumarate-arabinose conjugates from the arabinoxylan and cleaving less of them down to free ferulic acid, p-coumaric acid, and dehydrodiferulates with just one arabinosyl ester. This cleaner method, especially when coupled with the orthogonal method for measuring monolignol hydroxycinnamate conjugates that have been incorporated into lignin, provides an enhanced tool to measure the extent of crosslinking in grass arabinoxylan chains, assisting in identification of useful grasses for biomass applications.

Background: The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results: We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions: Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls. [ABSTRACT FROM AUTHOR]

Background: Arabinoxylan in grass cell walls is acylated to varying extents by ferulate and p-coumarate at the 5-hydroxy position of arabinosyl residues branching off the xylan backbone. Some of these hydroxycinnamate units may then become involved in cell wall radical coupling reactions, resulting in ether and other linkages amongst themselves or to monolignols or oligolignols, thereby crosslinking arabinoxylan chains with each other and/or with lignin polymers. This crosslinking is assumed to increase the strength of the cell wall, and impedes the utilization of grass biomass in natural and industrial processes. A method for quantifying the degree of acylation in various grass tissues is, therefore, essential. We sought to reduce the incidence of hydroxycinnamate ester hydrolysis in our recently introduced method by utilizing more anhydrous conditions. Results: The improved methanolysis method minimizes the undesirable ester-cleavage of arabinose from ferulate and p-coumarate esters, and from diferulate dehydrodimers, and produces more methanolysis vs. hydrolysis of xylan-arabinosides, improving the yields of the desired feruloylated and p-coumaroylated methyl arabinosides and their diferulate analogs. Free ferulate and p-coumarate produced by ester-cleavage were reduced by 78% and 68%, respectively, and 21% and 39% more feruloyl and p-coumaroyl methyl arabinosides were detected in the more anhydrous method. The new protocol resulted in an estimated 56% less combined diferulate isomers in which only one acylated arabinosyl unit remained, and 170% more combined diferulate isomers conjugated to two arabinosyl units. Conclusions: Overall, the new protocol for mild acidolysis of grass cell walls is both recovering more ferulate- and p-coumarate-arabinose conjugates from the arabinoxylan and cleaving less of them down to free ferulic acid, p-coumaric acid, and dehydrodiferulates with just one arabinosyl ester. This cleaner method, especially when coupled with the orthogonal method for measuring monolignol hydroxycinnamate conjugates that have been incorporated into lignin, provides an enhanced tool to measure the extent of crosslinking in grass arabinoxylan chains, assisting in identification of useful grasses for biomass applications. [ABSTRACT FROM AUTHOR]

Summary: Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular‐breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin‐enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9‐mediated genome editing. The OsMYB108‐knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two‐dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ‐p‐coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants. Significance Statement: Comprehensive understanding of secondary cell wall formation in grasses, as major biomass feedstocks, is important for their molecular‐breeding applications toward improved biorefinery processes. We demonstrated that CRISPR/Cas9‐mediated mutagenesis of the transcriptional repressor gene OsMYB108 preferentially enriched grass‐specific lignin units, i.e. p‐coumaroylated units and tricin units, in rice (Oryza sativa), providing insight into the transcriptional regulatory network in secondary cell wall formation specific to grass species. [ABSTRACT FROM AUTHOR]

Summary: The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice. Significance Statement: CRISPR/Cas9‐mediated gene knockout of rice coniferaldehyde 5‐hydroxylase (OsCAld5H1) resulted in lignins enriched in guaiacyl (G) units. However, the mutants still produced considerable numbers of syringyl (S) units. The enrichment of G units was limited to the non‐γ‐p‐coumaroylated lignin units, and the grass‐specific γ‐p‐coumaroylated lignin units were apparently unchanged, suggesting that OsCAld5H1 is mainly involved in the biosynthesis of non‐γ‐p‐coumaroylated monolignols common in both eudicots and grasses, but not in that of grass‐specific γ‐p‐coumaroylated monolignols. [ABSTRACT FROM AUTHOR]

Summary: p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification. [ABSTRACT FROM AUTHOR]

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase. [ABSTRACT FROM AUTHOR]

CouR from Rhodopseudomonas palustris is a member of the MarR transcriptional regulator family. It regulates the expression of CouA and CouB, enzymes that are involved in the degradation of p-coumarate. In vivo, CouR binds to a DNA fragment containing the couAB promoter and suppresses the expression of CouA and CouB, while binding of p-coumaroyl-CoA attenuates its affinity towards DNA and activates the expression of CouA and CouB. Here, the crystallization and X-ray diffraction analyses of CouR alone and in complex with p-coumaroyl-CoA are reported. Apo and ligand-complexed CouR crystals diffracted to 2.5 and 3.3 Å resolution, respectively. The crystals of apo CouR belonged to space group P22121, with unit-cell parameters a = 62.78, b = 76.15, c = 87.38 Å, whereas the crystals of the CouR-ligand complex belonged to space group P212121, with unit-cell parameters a = 61.37, b = 69.82, c = 70.32 Å. The crystals were predicted to contain two CouR molecules or CouR-ligand complexes per asymmetric unit. [ABSTRACT FROM AUTHOR]

Corn stover was subjected to dilute sulfuric acid pretreatment to assess the impact of pretreatment conditions on lignin extractability, properties, and utility as a phenol replacement in wood phenol-formaldehyde (PF) adhesives. It was identified that both formic acid and NaOH could extract and recover 60-70% of the lignin remaining after pretreatment and enzymatic hydrolysis under the mildest pretreatment conditions while simultaneously achieving reasonable enzymatic hydrolysis yields (>60%). The availability of reaction sites for the incorporation of lignins into the PF polymer matrix (i.e., unsubstituted phenolic hydroxyl groups) was shown to be strongly impacted by the pretreatment time and the recovery. Finally, a lignin-based wood adhesive was formulated by replacing 100% of the phenol with formic-acid-extracted lignin, which exhibited a dry shear strength exceeding a conventional PF adhesive. These findings suggest that both pretreatment and lignin extraction conditions can be tailored to yield lignins with properties targeted for this co-product application.

Grasses, such as Zea mays L. (maize), contain relatively high levels of p -coumarates ( pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p -coumaroyl CoA:hydroxycinnamyl alcohol transferase ( pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. [ABSTRACT FROM AUTHOR]

Hydroxycinnamates incorporate into lignins by various mechanisms. The polysaccharide esters of ferulate, in particular, and the range of dehydrodiferulates and higher oligomers in grasses, participate in free-radical (cross-)coupling reactions during lignification to become integrally bound into the lignin polymer, resulting in extensive cross-linking between lignins and polysaccharides. Monolignol-hydroxycinnamate (primarily monolignol- p-coumarate) conjugates are primary building blocks for lignins, again in grasses (but analogously with monolignol acetates and p-hydroxybenzoates in other plants); radical coupling reactions of the monolignol moiety of the conjugate result in lignins with pendant p-coumarate units acylating a variety of lignin structures. Recent evidence suggests that even the hydroxycinnamic acids themselves can be monomers in lignification in wild-type and transgenic plants, undergoing radical cross-coupling reactions to incorporate into the polymer with interesting consequences. The compatibility of ferulate, in particular, with lignification suggests that plants able to utilize monolignol-ferulate conjugates in their primary monomer supply will be particularly well suited for subsequent chemical delignification, potentially improving processes for biomass conversion to biofuels, and for chemical pulping. [ABSTRACT FROM AUTHOR]

Abstract: p-Coumaric acid is one of the predominant phenolic acids acylating the cell walls of grasses; p-coumarates are mainly esterified by lignins and arabinoxylans. Here we describe the production and characterisation of two monoclonal antibodies against p-coumarates. The 5-O-pCou-Ara(1→4)Xyl was chemically synthesized and conjugated to a carrier protein. Two interesting antibodies were obtained, hereinafter named INRA-COU1 and INRA-COU2. The specificity of these monoclonal antibodies has been evaluated using competitive-inhibition assays with different oligosaccharides and phenolic compounds. INRA-COU1, recognized free p-coumaric acid or p-coumarate esters. INRA-COU1 did not react with any of the other hydroxycinnamic acids and related compounds found in plants. INRA-COU2, only recognizes esterified p-coumarate. These antibodies were used to study the localization of p-coumarates in the cell walls of grasses. Immunocytochemical analyses indicated noticeable amounts of p-coumarate in the cell walls of the aleurone layer of wheat grain, in the epiderm of cereal straw, and in the exoderm of wheat root. The use of these antibodies will contribute to a better understanding of the organisation and developmental dynamics of cell walls in Graminaceae. [Copyright &y& Elsevier]

Grass lignins are differentiated from other lignin types by containing relatively large amounts of p-coumaric acid ( pCA) acylating the C-9 position of lignin subunits. In the case of a mature corn ( Zea mays L.) stems, pCA constitutes 15–18% of a dioxane soluble enzyme lignin. The major portion of the pCA is specifically attached to syringyl residues. Studies with isolated corn wall peroxidases show that pCA readily undergoes radical coupling in the presence of hydrogen peroxide, whereas sinapyl alcohol radical coupling proceeds more slowly. Analysis of corn wall peroxidases did not reveal specific enzymes that would lead to the preferred incorporation of sinapyl alcohol as seen in other plants. The addition of ethyl ferulate, methyl p-coumarate, or sinapyl p-coumarate conjugates to a reaction mixture containing peroxidase, sinapyl alcohol, and hydrogen peroxide stimulated the rate of sinapyl alcohol radical coupling by 10–20-fold. Based on spectral analysis it appears that pCA and ferulate radicals form rapidly, but the radical is readily transferred to sinapyl alcohol. The newly formed sinapyl alcohol radicals undergo coupling and cross-coupling reactions. However, sinapyl alcohol radicals do not cross-couple with pCA radicals. As long as hydrogen peroxide is limiting pCA remains uncoupled. Ferulates have similar reaction patterns in terms of radical transfer though they appear to cross-couple in the reaction mixture more readily then pCA. The role of pCA may be to internally provide a radical transfer mechanism for optimizing radical coupling of sinapyl alcohol into the growing lignin polymer. Attachment of some pCA to sinapyl alcohol ensures localization of the radical transfer mechanism in areas where sinapyl alcohol is being incorporated into lignin. [ABSTRACT FROM AUTHOR]

Production of industrial aromatic chemicals from renewable resources could provide a competitive alternative to traditional chemical synthesis routes. This review describes the engineering of microorganisms for the production of p-hydroxycinnamic acid (pHCA) and p-hydroxystyrene (pHS) from glucose. The initial process concept was demonstrated using a tyrosine-producing Escherichia coli strain that overexpressed both fungal phenylalanine/tyrosine ammonia lyase (PAL) and bacterial pHCA decarboxylase (pdc) genes. Further development of this bioprocess resulted in uncoupling the pHCA and pHS production steps to mitigate their toxicity to the production host. The final process consists of a fermentation step to convert glucose to tyrosine using a tyrosine-overproducing E. coli strain~ This step is followed by a single biotransformation reaction to deaminate tyrosine to pHCA through immobilized E. coli cells that overexpress the Rhodotorula glutinis PAL gene. Finally, chemical decarboxylation of pHCA produces pHS. This multifaceted approach, which integrates biology, chemistry, and engineering, has allowed development of an economical process at scales suitable for industrial applications. [ABSTRACT FROM AUTHOR]

Grass cell walls are atypical because their xylans are acylated with ferulate and lignins are acylated with p-coumarate. To probe the role and interactions of these p-hydroxycinnamates during lignification, feruloylated primary cell walls isolated from maize cell suspensions were lignified with coniferyl and sinapyl alcohols and with varying levels of p-coumarate esters. Ferulate xylan esters enhanced the formation of wall-bound syringyl lignin more than methyl p-coumarate, however, maximal concentrations of syringyl lignin were only one-third that of guaiacyl lignin. Including sinapyl p-coumarate, the presumed precursor of p-coumaroylated lignins, with monolignols unexpectedly accelerated peroxidase inactivation, interfered with ferulate copolymerization into lignin, and had minimal or adverse effects on cell wall lignification. Free phenolic groups of p-coumarate esters in isolated maize lignin and pith cell walls did not undergo oxidative coupling with each other or with added monolignols. Thus, the extensive formation of syringyl-rich lignins and the functional role of extensive lignin acylation by p-coumarate in grasses remains a mystery. [ABSTRACT FROM AUTHOR]

Abstract: Livestock performance can be improved by increasing the digestibility of feeds, one of the key elements of nutritional quality. Digestibility of feeds can be improved by breeding forage crops with modified cell-wall structure, increasing the potential availability of energy from the cell wall to rumen microbes and livestock. The objectives of this research were to identify interrelationships among lignin and phenolic components of the fibre fraction of three perennial grasses and to determine their influence on in vitro fibre digestibility. Differences in etherified and esterified ferulate and esterified p-coumarate among clones of three perennial grasses were generally repeatable across harvests. The concentration of neutral detergent fibre (NDF) and Klason lignin within the NDF fraction were the factors most limiting to 24-h in vitro digestibility, with NDF being the most important. Klason lignin and etherified ferulate were the factors most limiting to 96-h in vitro digestibility for all three species. Due to its positive correlation with NDF, selection for low etherified ferulate should be avoided in smooth bromegrass (Bromus inermis Leyss) and cockfsoot (Dactylis glomerata L.). It should be possible to select and breed for low concentrations of lignin to improve digestibility without decreasing NDF in these two species. However, in reed canarygrass (Phalaris arundinacea L.), both lignin and etherified ferulate were positively correlated with NDF, indicating that selection for increased digestibility should be based directly on some measure of in vitro digestibility to avoid the fitness problems associated with reduced NDF. [Copyright &y& Elsevier]

The mechanisms of iron (Fe) absorption and translocation in plants have received much study because they are the key processes in the supply of Fe to plants. The objective of this research was to study the effectiveness of phytosiderophore (PS) in the absorption and translocation of 59 Fe in Fe-deficient barley ( Hordeum vulgare L. cv. ‘Minorimugi’) plants in the presence of plant-borne, synthetic, or microbial chelators. Plants grown under Fe-deficient conditions in a phytotron at pH 5.5 for 7–18 d were fed with Fe 3+ (10 μ M labeled with 59 Fe) in the presence of 10 μ M of different chelators with or without 10 μ M PS for 4 h starting at 2 p.m. (6 h after the onset of light period). The absorption and translocation of 59 Fe in plants treated with PS and Fe 3 + were increased relative to plants fed solely with Fe 3 + (control). There was no effect found on absorption and translocation of 59 Fe in plants treated with EDTA or p -coumarate relative to the control, but a differential increase was observed in 59 Fe absorption and translocation in plants treated with EDTA or p -coumarate in the presence of PS. In comparison with the control, a decrease in 59 Fe absorption and translocation was observed in plants treated with HEDTA or EDDHA or FOB, but this decrease was avoided in plants treated with HEDTA or EDDHA or FOB in the presence of PS. The enhancement of 59 Fe absorption and translocation in plants treated with citrate, and the highest 59 Fe absorption and translocation in plants treated with citrate and PS, indicated that citrate had an additive effect on Fe absorption and translocation in plants. Our results showed that PS effectively played a role in Fe absorption and translocation in plants in the presence of other chelators. Plants treated with any chelators had lower extracellular 59 Fe in the roots compared with the control. [ABSTRACT FROM AUTHOR]

Prior work has identified that lignins recovered from dilute acid-pretreated corn stover exhibit superior performance in phenol–formaldehyde resins used in wood adhesive applications when compared to diverse process-modified lignins derived from other sources. This improved performance is hypothesized to be due to the higher content of unsubstituted phenolic groups specifically p- coumarate lignin esters. In this work, a diverse set of corn stover samples are employed that exhibit diversity in p -coumarate content and total lignin content to explore the relationship between dilute acid pretreatment conditions, p -coumarate ester hydrolysis, xylan solubilization, and the resulting glucose enzymatic hydrolysis yields. The goal of this study is to identify pretreatment conditions that preserve a significant fraction of the p -coumarate esters while simultaneously achieving high enzymatic hydrolysis yields. Kinetic parameters for p -coumarate ester hydrolysis were quantified and pretreatment-biomass combinations were identified that result in glucose hydrolysis yields of more than 90% while retaining nearly 50 mg p -coumarate/g lignin. [ABSTRACT FROM AUTHOR]

The hydroxycinnamates (HCAs) ferulate and p -coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacterium Sagittula stellata E-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis of S. stellata revealed the presence of two feruloyl-CoA ( fcs ) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism, fcs-1 and fcs-2 were disrupted using insertional mutagenesis, yielding both single and double fcs mutants. Growth on p -coumarate was abolished in the fcs double mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where the fcs-1 mutant showed impaired growth (extended lag and ∼60% of wild-type rate) on p -coumarate, and the fcs-2 mutant showed impaired growth (extended lag and ∼20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encoded fcs homologs, with fcs-1 primarily dedicated to p -coumarate utilization and fcs-2 playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs in S. stellata E-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria. IMPORTANCE Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors., (Copyright © 2018 American Society for Microbiology.)

The arabinosyl side chains of grass arabinoxylans are partially acylated by p-coumarate ( pCA) and ferulate (FA). These aromatic side chains can cross-couple wall polymers resulting in modulation of cell wall physical properties. The determination of p-coumaroylated and feruloylated arabinose units has been the target of analytical efforts with trifluoroacetic acid hydrolysis the standard method to release feruloylated and p-coumaroylated arabinose units from arabinoxylans. Herein, we report on a more robust method to measure these acylated units. Acidolysis of extractive-free grass samples in a dioxane/methanol/aqueous 2 M HCl mixture provided the methyl 5- O- p-coumaroyl- and 5- O-feruloyl-l-arabinofuranoside anomers ( pCA-MeAra and FA-MeAra). These conjugates were readily analyzed by liquid chromatography combined with both UV and MS detection. The method revealed the variability of the relative acylation of arabinose units by pCA or FA in grass cell walls. This methodology will permit delineation of hydroxycinnamate acylation patterns in arabinoxylans.

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice ( O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs ( AtMYB55 , AtMYB61 , and AtMYB63 ) in transgenic rice T 1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61 -overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p -coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum , switchgrass, Miscanthus and Erianthus .