Fusion of Pyruvate Decarboxylase and Alcohol Dehydrogenase Increases Ethanol Production in <i>Escherichia coli</i>

Lewicka, Aleksandra Jolanta; Lyczakowski, Jan J.; Blackhurst, Gavin; Pashkuleva, Christiana; Rothschild-Mancinelli, Kyle; Tautvaišas, Dainius; Thornton, Harry; Villanueva, Hugo; Xiao, Weike; Slikas, Justinas; Horsfall, Louise; Elfick, Alistair; French, Christopher E.

doi:10.1021/sb500020g

Cited by 23 publications

(19 citation statements)

References 4 publications

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“…Fusion proteins can lead to increased effectiveness due to substrate channelling even if the measured activities of the two individual partners decreases. This was reported earlier from our lab [49] as well as is shown in this study. Presently assays to measure quantitative cellulose degradation in vivo are not available.…”

Section: Discussionsupporting

confidence: 92%

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

Duedu

French

2016

Enzyme and Microbial Technology

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Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields.

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Section: Discussionsupporting

confidence: 92%

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

Duedu

French

2016

Enzyme and Microbial Technology

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“…Previously we and others have shown that individual shell proteins, which form the hexameric tiles of the BMC casing, generate long filamentous macromolecular structures when overproduced in the host bacterial cells 30,38 . These structures are particularly apparent with…”

Section: Discussionmentioning

confidence: 99%

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

et al. 2017

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We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

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“…Genes from Z. mobilis contribute to the standard biology of the International Genetically Engineered Machine registry, especially with unique characteristics such as pdc (ZMO1360, EC: 4.1.1.1), adhB (ZMO1596, EC:1.1.1.1), extracellular sucrase gene sacC (ZMO0375, EC:3.2.1.26), and glf (ZMO0366, glucose facilitated diffusion protein). For example, the ethanol production module of the fusion enzymes, Pdc and AdhB from Z. mobilis (Part:BBa_K1122673), could increase ethanol yields and productivity in E. coli and lactic acid bacteria (Nichols et al ., ; Chen et al ., ; Flynn et al ., ; Lewicka et al ., ).…”

Section: Classical Genetics Tools and Emerging Technologymentioning

confidence: 97%

Zymomonas mobilis as a model system for production of biofuels and biochemicals

Yang

Fei

Zhang

et al. 2016

Microbial Biotechnology

172

110

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Summary Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. This review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.

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Fusion of Pyruvate Decarboxylase and Alcohol Dehydrogenase Increases Ethanol Production in Escherichia coli

Cited by 23 publications

References 4 publications

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

Zymomonas mobilis as a model system for production of biofuels and biochemicals

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