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1.
J Ind Microbiol Biotechnol ; 42(3): 349-60, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25416472

ABSTRACT

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.


Subject(s)
Biotechnology/methods , Green Chemistry Technology , Butylene Glycols/metabolism , Carbon Isotopes , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Metabolic Engineering , Metabolic Networks and Pathways/genetics , Systems Biology
2.
Appl Microbiol Biotechnol ; 74(1): 113-24, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17103163

ABSTRACT

A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different beta-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes beta-1,4-linked mannan substrates, while the second catalytic site hydrolyzes beta-1,4-linked xylan and beta-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.


Subject(s)
Glycoside Hydrolases , Multienzyme Complexes , Rumen/microbiology , Animals , Base Sequence , Cattle , Gene Library , Glucans/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Industrial Microbiology , Mannans/metabolism , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutagenesis, Site-Directed , Sequence Analysis, DNA , Xylans/metabolism
3.
Appl Environ Microbiol ; 70(6): 3609-17, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15184164

ABSTRACT

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.


Subject(s)
Bacteria/enzymology , Digestive System/microbiology , Endo-1,4-beta Xylanases/genetics , Endo-1,4-beta Xylanases/metabolism , Fungi/enzymology , Isoptera/microbiology , Moths/microbiology , Amino Acid Sequence , Animals , Bacteria/genetics , DNA, Bacterial/analysis , DNA, Bacterial/isolation & purification , DNA, Fungal/analysis , DNA, Fungal/isolation & purification , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/classification , Fungi/genetics , Gene Library , Molecular Sequence Data , Phylogeny , Sequence Alignment
4.
Curr Opin Chem Biol ; 8(2): 141-9, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15062774

ABSTRACT

The use of enzymes in industrial catalysis continues to grow because of the considerable advantages of natural catalytic systems. The need for enantiomerically pure fine chemicals and the movement away from chemically burdened technologies will drive the acceptance of enzyme-assisted processes. New technologies for enzyme discovery and optimization have enabled the application of enzymes in harsh industrial conditions and in processes demanding stringent selectivity. These discovery and laboratory evolution methods entail genomic approaches that by their nature engender screening of extremely large numbers of gene types and variants. By extension, the fitness of an individual high-throughput screen requires an intelligent, process-targeted assay amenable to a chosen screening platform.


Subject(s)
Biotechnology/trends , Enzymes/genetics , Catalysis , Enzyme Stability , Enzymes/isolation & purification , Enzymes/metabolism , Gene Expression , Gene Library , Genomics , Mutagenesis , Substrate Specificity
5.
Protein Sci ; 13(2): 494-503, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14718652

ABSTRACT

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).


Subject(s)
Directed Molecular Evolution/methods , Endo-1,4-beta Xylanases/genetics , Endo-1,4-beta Xylanases/metabolism , Amino Acid Substitution , Endo-1,4-beta Xylanases/chemistry , Enzyme Stability , Genetic Variation/genetics , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Peptide Mapping , Sequence Homology, Amino Acid , Transition Temperature
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