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. Many microorganisms produce vitamins naturally, but their corresponding metabolic pathways are tightly regulated since vitamins are needed only in catalytic amounts. Metabolic engineering is accelerating the development of microbial cell factories for vitamins that could compete with chemical methods that have been optimized over decades, but scientific hurdles remain. Additional technological and regulatory issues need to be overcome for innovative bioprocesses to reach the market. Here, we review the current state of development and challenges for fermentative processes for the B vitamin group.

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. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence-function relationships of small-molecule transport systems in bacteria.

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. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

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. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.

BackgroundMany 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. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF.ResultsIn accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class.ConclusionsWe identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.

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. In reviewing this field, we find that its potential for new biological discovery is dramatically increasing. Functional metagenomic mining using genetic circuits has led to the discovery of novel bioactivity such as amidases, NF-κB modulators, naphthalene degrading enzymes, cellulases, lipases and transporters. Using these genetic circuits as a template, improvements are made by designing biosensors, such as in vitro-evolved riboswitches and computationally redesigned transcription factors. Thus, powered by the rapidly expanding repertoire of biosensors and streamlined processes for automated genetic circuit design, a greater variety of complex selection circuits can be built, with resulting impacts on drug discovery and industrial biotechnology.

Synopsis Mycophenolic acid (MPA) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMP dehydrogenase (IMPDH). The MPA biosynthetic cluster of P. brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA-resistance. Surprisingly, all members of subgenus Penicillium contain genes encoding IMPDHs of both the A and B type, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred prior to and independent of the acquisition of the MPA biosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant while the IMPDHs from a nonproducer are MPA-sensitive. Resistance comes with a catalytic cost: while P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its value of kcat/Km is 0.5% of “normal”. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in Pencillium subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.

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