Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB.

Metabolic engineering of Escherichia coli for high-level production of free lipoic acid.

L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms.

Improved biotin, thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR.

Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin.

Wiring cell growth to product formation.

Microbial cell factories offer new and sustainable production routes for high-value chemicals. However, identification of high producers within a library of clones remains a challenge. When product formation is coupled to growth, millions of metabolic variants can be effectively interrogated by growth selection, dramatically increasing the throughput of strain evaluation.

Microbial cell factories for the sustainable manufacturing of B vitamins.

Vitamins are essential compounds in human and animal diets. Their demand is increasing globally in food, feed, cosmetics, chemical and pharmaceutical industries. Most current production methods are unsustainable because they use non-renewable sources and often generate hazardous waste.

The evolving interface between synthetic biology and functional metagenomics.

Nature is a diverse and rich source of bioactive pathways or novel building blocks for synthetic biology. In this Perspective, we describe the emerging research field in which metagenomes are functionally interrogated using synthetic biology. This approach substantially expands the set of identified biological activities and building blocks.

Directed Evolution of Membrane Transport Using Synthetic Selections.

Understanding and engineering solute transporters is important for metabolic engineering and the development of therapeutics. However, limited available experimental data on membrane transporters makes sequence-function relationships complex to predict. Here we apply ligand-responsive biosensor systems that enable selective growth of E.

Functional mining of transporters using synthetic selections.

Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer.

Molecular Buffers Permit Sensitivity Tuning and Inversion of Riboswitch Signals.

Predictable integration of foreign biological signals and parts remains a key challenge in the systematic engineering of synthetic cellular actuations, and general methods to improve signal transduction and sensitivity are needed. To address this problem we modeled and built a molecular signal buffer network in Saccharomyces cerevisiae inspired by chemical pH buffer systems. The molecular buffer system context-insulates a riboswitch enabling synthetic control of colony formation and modular signal manipulations.

deFUME: Dynamic exploration of functional metagenomic sequencing data.

Background: Functional metagenomic selections represent a powerful technique that is widely applied for identification of novel genes from complex metagenomic sources. However, whereas hundreds to thousands of clones can be easily generated and sequenced over a few days of experiments, analyzing the data is time consuming and constitutes a major bottleneck for experimental researchers in the field.

Findings: Here we present the deFUME web server, an easy-to-use web-based interface for processing, annotation and visualization of functional metagenomics sequencing data, tailored to meet the requirements of non-bioinformaticians.

Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides.

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously.

Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes.

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi.

Background: Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides.

Adaptive evolution of drug targets in producer and non-producer organisms.

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance.

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications.