Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.
TL;DR: It is demonstrated that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH- dependent pathway and transhydrogenase over-expressing strain.
About: This article is published in Metabolic Engineering.The article was published on 2011-05-01. It has received 277 citations till now. The article focuses on the topics: Isobutanol & Alcohol dehydrogenase.
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TL;DR: The general strategies of systems metabolic engineering are discussed and examples of its application are offered and insights are offered as to when and how each of the different strategies should be used.
Abstract: Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of chemicals, fuels and materials from renewable resources. Metabolic engineering is a key enabling technology for transforming microorganisms into efficient cell factories for these compounds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology and evolutionary engineering at the systems level, offers a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.
668 citations
TL;DR: This Review discusses how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill.
Abstract: Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.
473 citations
TL;DR: It is found that it is possible to confer antimicrobial activity to Au NPs through precise control of their size down to NC dimension (typically less than 2 nm), which would allow them to better interact with bacteria.
Abstract: Bulk gold (Au) is known to be chemically inactive. However, when the size of Au nanoparticles (Au NPs) decreases to close to 1 nm or sub-nanometer dimensions, these ultrasmall Au nanoclusters (Au NCs) begin to possess interesting physical and chemical properties and likewise spawn different applications when working with bulk Au or even Au NPs. In this study, we found that it is possible to confer antimicrobial activity to Au NPs through precise control of their size down to NC dimension (typically less than 2 nm). Au NCs could kill both Gram-positive and Gram-negative bacteria. This wide-spectrum antimicrobial activity is attributed to the ultrasmall size of Au NCs, which would allow them to better interact with bacteria. The interaction between ultrasmall Au NCs and bacteria could induce a metabolic imbalance in bacterial cells after the internalization of Au NCs, leading to an increase of intracellular reactive oxygen species production that kills bacteria consequently.
428 citations
TL;DR: In this article, the Ehrlich pathway was shown to increase the production of isobutanol in Saccharomyces cerevisiae, compared with overexpression of the enzymes involved in the same pathways in the cytoplasm.
Abstract: Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains revealed that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves greater local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion and reducing the loss of intermediates to competing pathways.
409 citations
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
14,389 citations
"Engineered ketol-acid reductoisomer..." refers methods in this paper
...Strain 1993 was constructed by integrating PLlacO1::Ll_kivd1::Ec_ilvD_coEc::FRT into the ilvC locus of JCL260 (Atsumi et al., 2008a) using SOE (Horton et al., 1989), gene integration (Datsenko and Wanner, 2000), and P1 transduction....
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..., 1989), gene integration (Datsenko and Wanner, 2000), and P1 transduction....
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TL;DR: The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
8,474 citations
"Engineered ketol-acid reductoisomer..." refers methods in this paper
...The AdhA recombination library was constructed by SOE PCR introducing beneficial mutations found in the error-prone library, while allowing for wild-type sequence at all targeted sites....
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...After successful assembly PCR, homologous recombination, as described previously (Chao et al., 2006), was used to create the yeast libraries using the recombination PCR product as insert and pGV1662 as backbone....
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...The clean assembly product served as template for a second round of SOE PCR introducing mutations at positions 76 and 78 with a mixture of equimolar concentrations of primers....
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...Point mutations, site-saturation mutagenesis, and recombination libraries were made by splicing by overlap extension PCR (Kunkel et al., 1987)....
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...All primers are listed in Table B, Appendix A. Error-prone PCR was performed according to a published protocol (Bloom et al., 2006)....
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5,207 citations
TL;DR: Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase.
3,339 citations
"Engineered ketol-acid reductoisomer..." refers methods in this paper
...The AdhA recombination library was constructed by SOE PCR introducing beneficial mutations found in the error-prone library, while allowing for wild-type sequence at all targeted sites....
[...]
...The clean assembly product served as template for a second round of SOE PCR introducing mutations at positions 76 and 78 with a mixture of equimolar concentrations of primers....
[...]
...Strain 1993 was constructed by integrating PLlacO1::Ll_kivd1::Ec_ilvD_coEc::FRT into the ilvC locus of JCL260 (Atsumi et al., 2008a) using SOE (Horton et al., 1989), gene integration (Datsenko and Wanner, 2000), and P1 transduction....
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..., 2008a) using SOE (Horton et al., 1989), gene integration (Datsenko and Wanner, 2000), and P1 transduction....
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...The IlvC recombination library was constructed using SOE PCR to introduce beneficial mutations identified at the five targeted sites, while allowing for wild-type sequence as well....
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