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

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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Enhanced production of xylanase by Fusarium sp. BVKT R2 and evaluation of its biomass saccharification efficiency

3 Biotech ◽

10.1007/s13205-017-0977-1 ◽

2017 ◽

Vol 7 (5) ◽

Cited By ~ 1

Author(s):

G. Ramanjaneyulu ◽

A. Sridevi ◽

P. Seshapani ◽

A. Ramya ◽

K. Dileep Kumar ◽

...

Keyword(s):

Biomass Saccharification ◽

Enhanced Production ◽

Saccharification Efficiency ◽

Fusarium Sp

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Wood forming tissue-specific expression of PdSuSy and HCHL increases holocellulose content and improves saccharification in Populus

Journal of Forestry Research ◽

10.1007/s11676-020-01220-9 ◽

2020 ◽

Author(s):

Yang Zhang ◽

Hua Xu ◽

Yingzhen Kong ◽

Jiawen Hua ◽

Xianfeng Tang ◽

...

Keyword(s):

Populus Deltoides ◽

Lignin Content ◽

Hybrid Poplar ◽

Structural Complexity ◽

Woody Species ◽

Potential Method ◽

Specific System ◽

Specific Expression ◽

Tissue Specific ◽

Biomass Saccharification

Abstract Development of strategies to deconstruct lignocellulosic biomass in tree species is essential for biofuels and biomaterials production. We applied a wood forming tissue-specific system in a hybrid poplar to express both PdSuSy (a sucrose synthase gene from Populus deltoides × P. euramericana that has not been functionally characterized) and HCHL (the hydroxycinnamoyl-CoA hydratase-lyase gene from Pseudomonas fluorescens, which inhibits lignin polymerization in Arabidopsis). The PdSuSy-HCHL overexpression poplars correspondingly driven by the promoters of Arabidopsis AtCesA7 and AtC4H resulted in a significant increase in cellulose (> 8%), xylan (> 12%) and glucose (> 29%) content, accompanying a reduction in galacturonic acid (> 36%) content, compared to control plants. The saccharification efficiency of these overexpression poplars was dramatically increased by up to 27%, but total lignin content was unaffected. These transgenic poplars showed inhibited growth characteristics, including > 16% reduced plant height, > 10% reduced number of internodes, and > 18% reduced fresh weight after growth of 4 months, possibly due to relatively low expression of HCHL in secondary xylem. Our results demonstrate the structural complexity and interaction of the cell wall polymers in wood tissue and outline a potential method to increase biomass saccharification in woody species.

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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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Wood characteristics and enzymatic saccharification efficiency of field-grown transgenic black cottonwood with altered lignin content and structure

Cellulose ◽

10.1007/s10570-014-0541-7 ◽

2015 ◽

Vol 22 (1) ◽

pp. 683-693 ◽

Cited By ~ 6

Author(s):

Zhouyang Xiang ◽

Suman Kumar Sen ◽

Aparna Roy ◽

Douyong Min ◽

Dhanalekshmi Savithri ◽

...

Keyword(s):

Lignin Content ◽

Enzymatic Saccharification ◽

Black Cottonwood ◽

Saccharification Efficiency

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CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

International Journal of Molecular Sciences ◽

10.3390/ijms22189750 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9750

Author(s):

Hyun-A Jang ◽

Eun-Kyung Bae ◽

Min-Ha Kim ◽

Su-Jin Park ◽

Na-Young Choi ◽

...

Keyword(s):

Lignocellulosic Biomass ◽

Lignin Content ◽

Hybrid Poplar ◽

Lignin Biosynthesis ◽

Biofuel Production ◽

Guide Rnas ◽

Hybrid Poplars ◽

Four Seasons ◽

Promising Target ◽

Saccharification Efficiency

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

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Rational Engineering A Xylanase Hyper-Producing System in Trichoderma Reesei for Efficient Biomass Degradation

10.21203/rs.3.rs-39579/v1 ◽

2020 ◽

Author(s):

Su Yan ◽

Yan Xu ◽

Xiao-Wei Yu

Keyword(s):

Trichoderma Reesei ◽

Xylanase Activity ◽

Biomass Degradation ◽

Over Expression ◽

Rational Engineering ◽

Biomass Saccharification ◽

Saccharification Efficiency ◽

New Perspective ◽

First Time ◽

Rut C30

Abstract Background: The degradation of lignocellulose needs the synergetic work of cellulase and xylanase. Filamentous fungi Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase fermentation with great producing ability. Previous work for biomass saccharification mainly aims to improve the component and production of cellulase, however the low xylanase activity in T. reesei could not meet the needs of a high saccharification efficiency. Results: In this study, for the first time, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous over-expression of the major xylanase XYNⅡ. The expression of key xylanolytic enzymes was significantly upregulated with an increase of 6.78- and 1.98-fold in the xylanase activity and pNPXase (β-xylosidase) activity compared to that of the parent, Rut-C30, respectively. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble lactose or glucose as sole carbon source, which was more efficient and economical than the traditional method of xylan induction. Treated with the crude xylanase cocktails as accessory enzymes, an increase of 39.7% in reducing sugar yield (31.3 mg/mL) in saccharification of alkali pretreated corn stover (5% w/v) was achieved compared to that of the parent. Conclusions: An efficient and economical xylanase hyper-producing platform was developed in T. reesei Rut-C30. The novel platform with outstanding ability for crude xylanase cocktails production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

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Silicification of wood-cell walls

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169870 ◽

1994 ◽

Vol 52 ◽

pp. 428-429

Author(s):

S. E. Keckler ◽

D. M. Dabbs ◽

N. Yao ◽

I. A. Aksay

Keyword(s):

Electron Microscopy ◽

Cell Wall ◽

Cell Walls ◽

Lignin Content ◽

Resin Composite ◽

Middle Lamella ◽

Cellulose Fibers ◽

Complex Structures ◽

Rich Region ◽

Inorganic Precursors

Cellular organic structures such as wood can be used as scaffolds for the synthesis of complex structures of organic/ceramic nanocomposites. The wood cell is a fiber-reinforced resin composite of cellulose fibers in a lignin matrix. A single cell wall, containing several layers of different fiber orientations and lignin content, is separated from its neighboring wall by the middle lamella, a lignin-rich region. In order to achieve total mineralization, deposition on and in the cell wall must be achieved. Geological fossilization of wood occurs as permineralization (filling the void spaces with mineral) and petrifaction (mineralizing the cell wall as the organic component decays) through infiltration of wood with inorganics after growth. Conversely, living plants can incorporate inorganics into their cells and in some cases into the cell walls during growth. In a recent study, we mimicked geological fossilization by infiltrating inorganic precursors into wood cells in order to enhance the properties of wood. In the current work, we use electron microscopy to examine the structure of silica formed in the cell walls after infiltration of tetraethoxysilane (TEOS).

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