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Title: Engineering plant cell walls for second generation biofuel production
Author: Howard, Charis
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2012
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The substrate for second generation biofuels is lignocellulosic material obtained from plant cell walls. In the secondary cell wall, lignin and xylan form a waterproof protective network around cellulose, which is made of chains of glucose. Genetic modification of the cell wall has the potential to improve cellulose accessibility and hydrolysis, therefore decreasing the cost and energy input in biofuel production. Working on tobacco, this study aims to improve understanding of cell wall biosynthesis and organisation in order to increase cellulose content and extractability by genetic modification, and additionally by pretreatment with white rot fungus Phanerochaete chrysosporium. Enzymatic saccharification assays showed differences in soluble sugars released from transgenic tobacco lines down-regulated in both lignin and xylan. Significantly, TOBACCO PEROXIDASE 60 down-regulated line 1074 shows 30% increase in glucose release as compared to the wildtype. Xylan down-regulation by suppression of UDP-GLUCURONATE DECARBOXYLASE, which synthesises the xylan precursor xylose, also caused improvement in saccharification. The amount of glucose released from lignin down-regulated lines suppressed in CINNAMATE-4-HYDROXYLASE and CINNAMOYL-COA REDUCTASE did not increase. The main monosaccharide released from the wildtype lines and all transgenic lines was glucose. Quantitative real time polymerase chain reaction (qRT-PCR) was used to study the effects of suppressing four cell wall biosynthesis genes on the expression of other genes. The results suggest that the lignin biosynthesis pathway is down-regulated at the transcriptional level in lignin modified lines, while the polysaccharide biosynthesis response differs depending on the position of disruption in lignin biosynthesis. The xylan down-regulated line showed suppression of genes involved in both lignin and cell wall carbohydrate synthesis. However with the exception of the suppression of lignin and xylan synthesis genes in the lignin and xylan down-regulated lines respectively, these results were not reflected in cell wall fractionation analysis. At this level, the lignin supressed lines showed no changes in carbohydrate content while the xylan suppressed line had increased levels of lignin. This demonstrates that cell wall synthesis regulation is not wholly dependent on transcriptional regulation, so there must be multiple levels of cell wall regulation. White rot fungus Phanerochaete chrysosporium naturally hydrolyses and metabolises lignin. It is used to pretreat cell wall material to remove lignin before cellulose hydrolysis, but as yet no research has been published the effects it has genetically engineered cell wall material. P. chrysosporium was incubated with cell wall material from the cell wall modified lines. All lines showed improvement in saccharification after pretreatment. The improvement was as much as threefold in one of the wildtype lines and more than twofold in lines down-regulated in CINNAMATE-4-HYDROXYLASE and CINNAMOYL-COA REDUCTASE.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Secondary cell wall ; Bioethanol ; lignolytic fungi