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Chapter 28 : Increased Biofuel Production by Metabolic Engineering of

Authors: Leighann Sullivan1, Miles C. Scotcher2, George N. Bennett3
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Affiliations: 1: Department of Biochemistry & Cell Biology, Rice University, Houston, TX 77251-1892; 2: Foodborne Contaminants Research Unit, Western Regional Research Center, USDA Agricultural Research Service, 800 Buchanan St., Albany, CA 94710; 3: Department of Biochemistry & Cell Biology, Rice University, Houston, TX 77251-1892
Content Type: Monograph
Publication Year: 2008

Category: Applied and Industrial Microbiology

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Abstract:

Researched data suggest that membrane-associated proteins in the ethanol-adapted strain are either synthesized in lesser quantities or not properly incorporated into the cell membrane. In this study, 49 different proteins were identified and among them were two highly abundant surface layer proteins, flagellum components, and paralogs of the high-molecular-weight surface layer protein. These identified proteins may represent new virulence factors. The current status for reduction of expression of multiple genes is antisense RNA technology. The introduction of plasmids bearing genes of acid pathways showed only a modest effect on the acid content. This suggests that increasing the levels of certain enzymes may not affect the overall metabolite profile if they are already in adequate amount and the network is limited by other factors. Among the strategies discussed to increase the butanol fraction of solvents in , that is, by manipulating expression levels of genes for acid and solvent production, regulators, heat shock proteins, and sporulation, altering gene expression for cell membrane synthesis would be expected to directly address solvent tolerance as well as production capabilities. The study of the genes encoding the 1,3-PD operon of VPI1718 revealed three genes, dhaB1, dhaB2, and dhaT. Studies detailed in this chapter reflect an increased level of complexity in the process of metabolic engineering and recognize that attempts to improve the desired phenotype must be accompanied by a deeper understanding of the organism as a whole if success in strain engineering is to be achieved.

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
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Increased Biofuel Production by Metabolic Engineering of Clostridium acetobutylicum
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Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
/content/10.1128/9781555815547.ch28.ch28fig01
Figure 1.
Metabolic pathways of . The acid and solvent products are in bold boxes: acetate, butyrate, ethanol, acetone, and butanol. Genes are shown in italics, with those underlined residing on the pSOL1 plasmid. Enzyme names are capitalized: AK, acetate kinase; PTA, phosphotransacetylase; THL, thiolase; CoAT, CoA transferase; AAD, alcohol/alde-hyde dehydrogenase; ADC, acetoacetate decarboxylase; BHBD, β-hydroxylbutyryl dehydrogenase; CRO, crotonase; BCD, butyryl-CoA dehydrogenase; BK, butyrate kinase; PTB, phosphotransbutyrylase; BDHA and BDHB, butanol dehydrogenase isozymes A and B (adapted from , and ).
10.1128/9781555815547/f0362-01_thmb.gif
10.1128/9781555815547/f0362-01.gif
Image of Figure 1.
Figure 1.

Metabolic pathways of . The acid and solvent products are in bold boxes: acetate, butyrate, ethanol, acetone, and butanol. Genes are shown in italics, with those underlined residing on the pSOL1 plasmid. Enzyme names are capitalized: AK, acetate kinase; PTA, phosphotransacetylase; THL, thiolase; CoAT, CoA transferase; AAD, alcohol/alde-hyde dehydrogenase; ADC, acetoacetate decarboxylase; BHBD, β-hydroxylbutyryl dehydrogenase; CRO, crotonase; BCD, butyryl-CoA dehydrogenase; BK, butyrate kinase; PTB, phosphotransbutyrylase; BDHA and BDHB, butanol dehydrogenase isozymes A and B (adapted from , and ).

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
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/content/10.1128/9781555815547.ch28.ch28fig02
Figure 2.
Metabolic engineering stands to gain significantly from advances in complementary biological fields. Omics technologies and computational systems biology can provide large amounts of data about a cellular state and the means by which to analyze it, whereas protein engineering and synthetic biology can provide tool sets for new ways to manipulate a cell to improve the cellular properties. These four fields have a unique set of expertise that could be applied to further metabolic engineering analysis and implementation. Reprinted from ( ) with permission of the publisher.
10.1128/9781555815547/f0363-01_thmb.gif
10.1128/9781555815547/f0363-01.gif
Image of Figure 2.
Figure 2.

Metabolic engineering stands to gain significantly from advances in complementary biological fields. Omics technologies and computational systems biology can provide large amounts of data about a cellular state and the means by which to analyze it, whereas protein engineering and synthetic biology can provide tool sets for new ways to manipulate a cell to improve the cellular properties. These four fields have a unique set of expertise that could be applied to further metabolic engineering analysis and implementation. Reprinted from ( ) with permission of the publisher.

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
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/content/10.1128/9781555815547.ch28.ch28fig03
Figure 3.
Fermentation kinetics of 824(pSOS95del), 824(pCTFB1AS), and 824(pAADB1). (A) Glucose, acid, and solvent profiles. The name of each profile in 824(pSOS95del) (▪), 824(pCTFB1AS) ( ), and 824(pAADB1) ( ) is indicated above each graph. Reprinted from the ( ) with permission of the publisher.
10.1128/9781555815547/f0368-01_thmb.gif
10.1128/9781555815547/f0368-01.gif
Image of Figure 3.
Figure 3.

Fermentation kinetics of 824(pSOS95del), 824(pCTFB1AS), and 824(pAADB1). (A) Glucose, acid, and solvent profiles. The name of each profile in 824(pSOS95del) (▪), 824(pCTFB1AS) ( ), and 824(pAADB1) ( ) is indicated above each graph. Reprinted from the ( ) with permission of the publisher.

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
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Figure 4.
Fermentation profiles of WT (a) and PJC4BK (b) at pH 5.0. Shown are optical density (–X–) and product concentrations of acetate (– –), acetone (– –), butyrate (– –), butanol (– –), and ethanol (– –). The data at pH 5.0 show a more dramatic effect than those published earlier for pH 5.5 cultures by Green et al. ( ). Reprinted from ( ) with permission of the publisher.
10.1128/9781555815547/f0369-01_thmb.gif
10.1128/9781555815547/f0369-01.gif
Image of Figure 4.
Figure 4.

Fermentation profiles of WT (a) and PJC4BK (b) at pH 5.0. Shown are optical density (–X–) and product concentrations of acetate (– –), acetone (– –), butyrate (– –), butanol (– –), and ethanol (– –). The data at pH 5.0 show a more dramatic effect than those published earlier for pH 5.5 cultures by Green et al. ( ). Reprinted from ( ) with permission of the publisher.

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
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Tables

Increased Biofuel Production by Metabolic Engineering of Clostridium acetobutylicum
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Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28
/content/10.1128/9781555815547.ch28.ch28tab01
Table 1.
Proteomic analyses summarized
Generic image for table
Table 1.

Proteomic analyses summarized

Citation: Sullivan L, Scotcher M, Bennett G. 2008. Increased Biofuel Production by Metabolic Engineering of , p 361-376. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch28

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