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Chapter 28 : Increased Biofuel Production by Metabolic Engineering of Clostridium acetobutylicum
Category: Applied and Industrial Microbiology
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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 Clostridium acetobutylicum, 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 Clostridium butyricum 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.
Metabolic pathways of C. acetobutylicum. 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 Bahl and Durre, 2001 , and Mitchell, 1998 ).
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 Trends in Biotechnology ( Tyo et al., 2007 ) with permission of the publisher.
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 Journal of Bacteriology ( Tummala et al., 2003a ) with permission of the publisher.
Fermentation profiles of WT (a) and PJC4BK (b) at pH 5.0. Shown are optical density A 600 (–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. ( 1996 ). Reprinted from Biotechnology and Bioengineering ( Harris et al., 2000 ) with permission of the publisher.
Proteomic analyses summarized a