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

Abstract 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.

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Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

Frontiers in Plant Science ◽

10.3389/fpls.2021.762067 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yogesh Kumar Ahlawat ◽

Akula Nookaraju ◽

Anne E. Harman-Ware ◽

Crissa Doeppke ◽

Ajaya K. Biswal ◽

...

Keyword(s):

Lignin Content ◽

Woody Biomass ◽

Bioenergy Crops ◽

Transgenic Lines ◽

Saccharification Efficiency ◽

Transgenic Poplars ◽

Specific Alteration ◽

Guaiacyl Lignin ◽

Transcription Factor Expression ◽

The Impact

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

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LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar

Biotechnology for Biofuels ◽

10.1186/s13068-020-01843-4 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Shifei Qin ◽

Chunfen Fan ◽

Xiaohong Li ◽

Yi Li ◽

Jian Hu ◽

...

Keyword(s):

Cell Wall ◽

Genetic Manipulation ◽

Lignin Content ◽

Biomass Conversion ◽

Enzymatic Saccharification ◽

Lignin Biosynthesis ◽

Populus Tomentosa ◽

Wall Structure ◽

Biomass Saccharification

Abstract Background The recalcitrance of lignocellulosic biomass provided technical and economic challenges in the current biomass conversion processes. Lignin is considered as a crucial recalcitrance component in biomass utilization. An in-depth understanding of lignin biosynthesis can provide clues to overcoming the recalcitrance. Laccases are believed to play a role in the oxidation of lignin monomers, leading to the formation of higher-order lignin. In plants, functions of only a few laccases have been evaluated, so little is known about the effect of laccases on cell wall structure and biomass saccharification. Results In this study, we screened a gain-of-function mutant with a significant increase in lignin content from Arabidopsis mutant lines overexpressing a full-length poplar cDNA library. Further analysis confirmed that a Chinese white poplar (Populus tomentosa) laccase gene PtoLAC14 was inserted into the mutant, and PtoLAC14 could functionally complement the Arabidopsis lac4 mutant. Overexpression of PtoLAC14 promoted the lignification of poplar and reduced the proportion of syringyl/guaiacyl. In contrast, the CRISPR/Cas9-generated mutation of PtLAC14 results in increased the syringyl/guaiacyl ratios, which led to integrated enhancement on biomass enzymatic saccharification. Notably, the recombinant PtoLAC14 protein showed higher oxidized efficiency to coniferyl alcohol (precursor of guaiacyl unit) in vitro. Conclusions This study shows that PtoLAC14 plays an important role in the oxidation of guaiacyl deposition on cell wall. The reduced recalcitrance of the PtoLAC14-KO lines suggests that PtoLAC14 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of lignocellulose.

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Forages for feedstocks of biorefineries in temperate environments: review of lignin research in bioenergy crops and some insight into Miscanthus studies

Crop and Pasture Science ◽

10.1071/cp14039 ◽

2014 ◽

Vol 65 (11) ◽

pp. 1199 ◽

Cited By ~ 3

Author(s):

Maria S. Dwiyanti ◽

J. Ryan Stewart ◽

Toshihiko Yamada

Keyword(s):

Panicum Virgatum ◽

Target Genes ◽

Lignin Content ◽

Genetic Regulation ◽

Warm Season ◽

Lignin Biosynthesis ◽

Biofuel Production ◽

Bioenergy Crops ◽

Cost Efficient ◽

Insight Into

Rhizomatous and perennial warm-season C4 grasses such as Miscanthus spp. and switchgrass (Panicum virgatum) are potential bioenergy crops for temperate regions. However, lignin in Miscanthus and switchgrass inhibits the cellulose digestion process during bioethanol production. One of the targets for improvement of forages from feedstocks to bioenergy crops is to develop a cost-efficient biorefinery process through lignin content manipulation. Numerous reports have shown that RNAi suppression of lignin-biosynthesis pathway genes can increase biomass fermentable sugar yields for biofuel production. These studies have also reported that RNAi suppression of cell-wall lignin biosynthesis can decrease biomass yield and resistance to biotic stress in the transgenic plants. Transcriptome and metabolome approaches can be used to clarify the networks and pathways of lignin biosynthesis to facilitate the identification of appropriate target genes for transformation. However, whole-genome sequencing of the forage species, which provides much-needed genomic information, is limited. Germplasm of natural, low-lignin mutants also plays a role in identification of genetic regulation of lignin content and this would be useful breeding material. Molecular markers have been developed and utilised to accelerate identification of quantitative trait loci/genes for traits relating to the biorefinery process. All of these studies will serve as basic information for supporting genetic improvement through classical breeding or genetic transformation, and offer the opportunity to develop cultivars which have enhanced biomass and are cost-efficient for biorefinery process.

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Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Plant Biotechnology Journal ◽

10.1111/pbi.12310 ◽

2015 ◽

Vol 13 (9) ◽

pp. 1241-1250 ◽

Cited By ~ 42

Author(s):

Aymerick Eudes ◽

Noppadon Sathitsuksanoh ◽

Edward E. K. Baidoo ◽

Anthe George ◽

Yan Liang ◽

...

Keyword(s):

Lignin Content ◽

Biomass Saccharification ◽

Saccharification Efficiency

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Distinct Miscanthus lignocellulose improves fungus secreting cellulases and xylanases for consistently enhanced biomass saccharification of diverse bioenergy crops

Renewable Energy ◽

10.1016/j.renene.2021.04.107 ◽

2021 ◽

Author(s):

Peng Liu ◽

Ao Li ◽

Youmei Wang ◽

Qiuming Cai ◽

Haizhong Yu ◽

...

Keyword(s):

Bioenergy Crops ◽

Biomass Saccharification

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Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials

Biotechnology for Biofuels ◽

10.1186/s13068-021-01892-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Anne E. Harman-Ware ◽

Samuel Sparks ◽

Bennett Addison ◽

Udaya C. Kalluri

Keyword(s):

Organic Carbon ◽

Soil Organic Carbon ◽

Biomass Conversion ◽

Terrestrial Ecosystems ◽

Bioenergy Crops ◽

System Management ◽

Performance Improvements ◽

Future Technologies ◽

Analysis Models ◽

Analytical Approaches

AbstractSuberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

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Exploiting xylan as sugar donor for the synthesis of an antiproliferative xyloside using an enzyme cascade

Microbial Cell Factories ◽

10.1186/s12934-019-1223-9 ◽

2019 ◽

Vol 18 (1) ◽

Author(s):

Manuel Nieto-Domínguez ◽

José Alberto Martínez-Fernández ◽

Beatriz Fernández de Toro ◽

Juan A. Méndez-Líter ◽

Francisco Javier Cañada ◽

...

Keyword(s):

Chemical Synthesis ◽

Biomass Conversion ◽

Value Added ◽

Cost Effective ◽

Raw Material ◽

Birchwood Xylan ◽

Enzyme Cascade ◽

Antiproliferative Agent ◽

Xylanolytic Enzymes ◽

Value Added Products

Abstract Background Currently, industrial societies are seeking for green alternatives to conventional chemical synthesis. This demand has merged with the efforts to convert lignocellulosic biomass into value-added products. In this context, xylan, as one of main components of lignocellulose, has emerged as a raw material with high potential for advancing towards a sustainable economy. Results In this study, the recombinant endoxylanase rXynM from the ascomycete Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris and used as one of the catalysts of an enzyme cascade developed to synthesize the antiproliferative 2-(6-hydroxynaphthyl) β-d-xylopyranoside, by transglycosylation of 2,6-dihydroxynaphthalene. The approach combines the use of two fungal xylanolytic enzymes, rXynM and the β-xylosidase rBxTW1 from the same fungus, with the cost-effective substrate xylan. The reaction conditions for the cascade were optimized by a Central Composite Design. Maximal productions of 0.59 and 0.38 g/L were reached using beechwood xylan and birchwood xylan, respectively. For comparison, xylans from other sources were tested in the same reaction, suggesting that a specific optimization is required for each xylan variety. The results obtained using this enzyme cascade and xylan were similar or better to those previously reported for a single catalyst and xylobiose, an expensive sugar donor. Conclusions Beechwood and birchwood xylan, two polysaccharides easily available from biomass, were used in a novel enzyme cascade to synthetize an antiproliferative agent. The approach represents a green alternative to the conventional chemical synthesis of 2-(6-hydroxynaphthyl) β-d-xylopyranoside using a cost-effective substrate. The work highlights the role of xylan as a raw material for producing value-added products and the potential of fungal xylanolytic enzymes in the biomass conversion.

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Rapid Assessment of Lignin Content and Structure in Switchgrass (Panicum virgatum L.) Grown Under Different Environmental Conditions

BioEnergy Research ◽

10.1007/s12155-009-9054-x ◽

2009 ◽

Vol 2 (4) ◽

pp. 246-256 ◽

Cited By ~ 63

Author(s):

David G. J. Mann ◽

Nicole Labbé ◽

Robert W. Sykes ◽

Kristen Gracom ◽

Lindsey Kline ◽

...

Keyword(s):

Environmental Conditions ◽

Panicum Virgatum ◽

Lignin Content ◽

Rapid Assessment ◽

Panicum Virgatum L

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Silica Production across Candidate Lignocellulosic Biorefinery Feedstocks

Agronomy ◽

10.3390/agronomy10010082 ◽

2020 ◽

Vol 10 (1) ◽

pp. 82

Author(s):

Yifeng Xu ◽

Nick Porter ◽

Jamie L. Foster ◽

James P. Muir ◽

Paul Schwab ◽

...

Keyword(s):

Pearl Millet ◽

Panicum Virgatum ◽

Pennisetum Glaucum ◽

Value Added ◽

Neutral Detergent Fiber ◽

Yield Potential ◽

High Yield ◽

Energy Cane ◽

Lignocellulosic Feedstocks ◽

Sunn Hemp

Biofuels produced from non-food lignocellulosic feedstocks have the potential to replace a significant percentage of fossil fuels via high yield potential and suitability for cultivation on marginal lands. Commercialization of dedicated lignocellulosic crops into single biofuels, however, is hampered by conversion technology costs and decreasing oil prices. Integrated biorefinery approaches, where value-added chemicals are produced in conjunction with biofuels, offer significant potential towards overcoming this economic disadvantage. In this study, candidate lignocellulosic feedstocks were evaluated for their potential biomass and silica yields. Feedstock entries included pearl millet-napiergrass (“PMN”; Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), napiergrass (P. purpureum Schumach.), annual sorghum (Sorghum bicolor [L.] Moench), pearl millet (P. glaucum [L.] R. Br.), perennial sorghum (Sorghum spp.), switchgrass (Panicum virgatum L.), sunn hemp (Crotalaria juncea L.), giant miscanthus (Miscanthus × giganteus J.M. Greef and Deuter), and energy cane (Saccharum spp.). Replicated plots were planted at three locations and characterized for biomass yield, chemical composition including hemicellulose, cellulose, acid detergent lignin (ADL), neutral detergent fiber (NDF), crude protein (CP), and silica concentration. The PMN, napiergrass, energy cane, and sunn hemp had the highest biomass yields. They were superior candidates for ethanol production due to high cellulose and hemicellulose content. They also had high silica yield except for sunn hemp. Silica yield among feedstock entries ranged from 41 to 3249 kg ha−1. Based on high bioethanol and biosilica yield potential, PMN, napiergrass, and energy cane are the most promising biorefinery feedstock candidates for improving biofuel profitability.

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