Oxidative Decarboxylation of Short-Chain Fatty Acids to 1-Alkenes.

The enzymatic oxidative decarboxylation of linear short-chain fatty acids (C4:0-C9:0) employing the P450 monooxygenase OleT, O2 as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C3 to C8  alkenes with product titers of up to 0.93 g L(-1) and TTNs of >2000. Key to this process was the construction of an efficient electron-transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite.

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin.

This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway.

In vitro evolution of a fungal laccase in high concentrations of organic cosolvents.

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae.

Directed evolution of ribosomal protein S1 for enhanced translational efficiency of high GC Rhodopseudomonas palustris DNA in Escherichia coli.

The expression of foreign DNA in Escherichia coli is important in biotechnological applications. However, the translation of genes from GC-rich organisms is inefficient in E. coli.

Screening mutant libraries of fungal laccases in the presence of organic solvents.

Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents).

A synthetic gene-metabolic oscillator.

Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed oscillators have been demonstrated, using components that are not part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour.

Design of artificial cell-cell communication using gene and metabolic networks.

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable.

A perspective of metabolic engineering strategies: moving up the systems hierarchy.

Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation.

Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants widely distributed in terrestrial and aquatic environments. In the present work, 2 colorimetric assays for laccase-catalyzed degradation of PAHs were developed based on studies of the oxidation of 12 aromatic hydrocarbons by fungal laccases from Trametes versicolor and Myceliophthora thermophila. Using a sodium borohydride water-soluble solution, the authors could reduce the single product of laccase-catalyzed anthracene biooxidation into the orange-colored 9,10-anthrahydroquinone, which is quantifiable spectrophotometrically.

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution.

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold.

Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases.

The enzymatic oxidation of uridine 5'-diphospho-alpha-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-alpha-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-epsilon-amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-alpha-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-N-acetyl-alpha-D-galactosamine (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases beta3Gal-T5, beta4Gal-T1, and beta4Gal-T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin-p-aminophenyl N-acetyl-beta-D-glucosaminide (BSA-(GlcNAc)17) and ovalbumin.

Directed enzyme evolution.

Laboratory evolutionists continue to generate better enzymes for industrial and research applications. Exciting developments include new biocatalysts for enantioselective carbon-carbon bond formation and fatty acid production in plants. Creative contributions to the repertoire of evolutionary methods will ensure further growth in applications and expand the scope and complexity of biological design problems that can be addressed.

A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity.

Epidemiological studies have documented a reduced prevalence of Alzheimer's disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Abeta42 peptide (the 42-residue isoform of the amyloid-beta peptide) produced from a variety of cultured cells by as much as 80%.

Enzymatic synthesis of nucleotide sugars.

The present review gives a survey on the biosynthetic pathways of nucleotide sugars which are important for the in vitro synthesis of mammalian glycoconjugates. With respect to the use of these enzymes in glycotechnology the availability as recombinant enzymes from different sources, the large-scale synthesis of nucleotide sugars and their in situ regeneration in combination with glycosyltransferases are summarized and evaluated.