Metabolic engineering ofClostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

1993 ◽  
Vol 42 (9) ◽  
pp. 1053-1060 ◽  
Author(s):  
Lee D. Mermelstein ◽  
Eleftherios T. Papoutsakis ◽  
Daniel J. Petersen ◽  
George N. Bennett
Keyword(s):  
Metabolic Engineering ◽  
Enzyme Activities ◽  
Solvent Production ◽  
Ofclostridium Acetobutylicum

scholarly journals Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Nicolaus A. Herman ◽  
Jeffrey Li ◽  
Ripika Bedi ◽  
Barbara Turchi ◽  
Xiaoji Liu ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Efficiency ◽  
Heterologous Gene Expression ◽  
Transformation Method ◽  
Type I ◽  
Exchange Method ◽  
Content Type ◽  
Solvent Production ◽  
Rational Engineering ◽  
Transformation Methods

ABSTRACT While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 106 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

2017 ◽  
Vol 39 ◽  
pp. 38-48 ◽  
Author(s):  
Zhiqiang Wen ◽  
Nigel P. Minton ◽  
Ying Zhang ◽  
Qi Li ◽  
Jinle Liu ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Solvent Production

Metabolic engineering of d-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid

2012 ◽  
Vol 14 (5) ◽  
pp. 569-578 ◽  
Author(s):  
Han Xiao ◽  
Zhilin Li ◽  
Yu Jiang ◽  
Yunliu Yang ◽  
Weihong Jiang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Clostridium Beijerinckii ◽  
Solvent Production

scholarly journals Examination of Physiological and Molecular Factors Involved in Enhanced Solvent Production by Clostridium beijerinckii BA101

1999 ◽  
Vol 65 (5) ◽  
pp. 2269-2271 ◽  
Author(s):  
Chih-Kuang Chen ◽  
Hans P. Blaschek
Keyword(s):  
Mrna Expression ◽  
Growth Phase ◽  
Exponential Growth ◽  
Enzyme Activities ◽  
Clostridium Beijerinckii ◽  
Exponential Growth Phase ◽  
Solvent Production ◽  
Acetoacetate Decarboxylase ◽  
Specific Activities ◽  
Mrna Expression Levels

ABSTRACT The specific activities and the mRNA expression levels associated with coenzyme A transferase, acetoacetate decarboxylase, and butyraldehyde dehydrogenase were elevated in hyper-solvent-producingClostridium beijerinckii BA101 during the exponential growth phase. The increase in expression of the sol operon and associated enzyme activities may be responsible for enhanced solvent production by C. beijerinckii BA101.

High time resolution enzyme cytochemistry of rod outer segment

1990 ◽  
Vol 48 (3) ◽  
pp. 878-879
Author(s):  
Takuma Saito ◽  
Toshihiro Takizawa
Keyword(s):  
Enzyme Activities ◽  
Outer Segment ◽  
Rapid Change ◽  
High Time Resolution ◽  
Visual Cell ◽  
Enzyme Cytochemistry ◽  
Activation Effect ◽  
Rod Outer Segment ◽  
Enzymatic Cascades ◽  
Rapid Drop

Cells and tissues live on a number of dynamic metabolic pathways, which are made up of sequential enzymatic cascades.Recent biochemical and physiological studies of vision research showed the importance of cGMP metabolism in the rod outer segment of visual cell, indicat ing that the photon activated rhodopsin exerts activation effect on the GTP binding protein, transducin, and this act ivated transducin further activates phosphodiesterase (PDEase) to result in a rapid drop in cGMP concentration in the cytoplasm of rod outer segment. This rapid drop of cGMP concentration exerts to close the ion channel on the plasma membrane and to stop of inward current brings hyperpolarization and evokes an action potential.These sequential change of enzyme activities, known as cGMP cascade, proceeds quite rapidly within msec order. Such a rapid change of enzyme activities, such as PDEase in rod outer segment, was not a matter of conventional histochemical invest igations.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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