Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs

Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases.

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica. Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains

Synthetic biology approaches and combinatorial biosynthesis towards heterologous lipopeptide production.

Synthetic biology techniques coupled with heterologous secondary metabolite production offer opportunities for the discovery and optimisation of natural products. Here we developed a new assembly strategy based on type IIS endonucleases and elaborate synthetic DNA platforms, which could be used to seamlessly assemble and engineer biosynthetic gene clusters (BGCs). By applying this versatile tool, we designed and assembled more than thirty different artificial myxochromide BGCs, each around 30 kb in size, and established heterologous expression platforms using a derivative of Myxococcus xanthus DK1622 as a host. In addition to the five native types of myxochromides (A, B, C, D and S), novel lipopeptide structures were produced by combinatorial exchange of nonribosomal peptide synthetase (NRPS) encoding genes from different myxochromide BGCs. Inspired by the evolutionary diversification of the native myxochromide megasynthetases, the ancestral A-type NRPS was engineered by inactivation, deletion, or duplication of catalytic domains and successfully converted into functional B-, C- and D-type megasynthetases. The constructional design approach applied in this study enables combinatorial engineering of complex synthetic BGCs and has great potential for the exploitation of other natural product biosynthetic pathways

Modular Construction of a Functional Artificial Epothilone Polyketide Pathway

Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from <i>Sorangium cellulosum</i> for expression in the high GC host <i>Myxococcus xanthus</i>. The codon composition was adapted to a modified codon table for <i>M. xanthus</i>, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in <i>M. xanthus</i> at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy