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.
The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a ...worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.
Corallopyronins (COR) are α-pyrone antibiotics from myxobacteria representing highly promising lead structures for the development of antibacterial therapeutic agents. Their ability to inhibit RNA ...polymerase through interaction with the “switch region”, a novel target, distant from binding sites of previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising as antibiotic candidates. Corallopyronin A is currently also investigated as a lead compound for the treatment of lymphatic filariasis because of its superb activity against the nematode symbiont Wolbachia. As total synthesis is not a valid production option biotechnological optimization of compound supply is of utmost importance to further develop this highly potent compound class.
Here we describe decisive improvements of the previously reported heterologous COR production and engineering platform yielding production of ~100 mg/L COR A. Furthermore, we provide a revised model of COR biosynthesis shedding light on the function of several biosynthetic proteins, including an unusual ECH-like enzyme providing dehydration functionality in trans and an uncharacterized protein conferring COR self-resistance in the myxobacterial heterologous host Myxococcus xanthus DK1622. We also report two new COR derivatives, COR D and oxyCOR A discovered in genetically engineered strains.
•Total corallopyronin yield increased threefold by promoter exchange.•Revision of corallopyronin biosynthesis.•Isolation and structure elucidation of two novel corallopyronin derivatives.•Identification of a novel self-resistance determinant for myxobacterial RNAP inhibitors.
In this article, we briefly review the potential of myxobacteria as 'natural product factories' by highlighting results from the recently sequenced myxobacterial model strain Myxococcus xanthus. We ...will focus on the production of polyketides, non-ribosomally-made peptides, and their hybrids, and discuss the evaluation of biosynthetic potential using genome-based methods, as well as biosynthetic process engineering.
Myxopyronins (MXN) and corallopyronins (COR) are structurally related α-pyrone antibiotics from myxobacteria that represent a highly promising compound class for the development of broad-spectrum ...antibacterial therapeutic agents. Their ability to inhibit RNA polymerase through interaction with the “switch region”, a novel target, distant from previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising candidates for further research. To improve compound supply for further investigation of MXN, COR and novel derivatives of these antibacterial agents, establishment of an efficient and versatile microbial production platform for myxobacterial α-pyrone antibiotics is highly desirable. Here we describe design, construction and expression of a heterologous production and engineering platforms for MXN and COR to facilitate rational structure design and yield improvement approaches in the myxobacterial host strain Myxococcus xanthus DK1622. Optimization of the cultivation medium yielded significantly higher production titers of MXN A at around 41-fold increase and COR A at around 25-fold increase, compared to the standard CTT medium.
•myxopyronin gene cluster cloned, modified and expressed in heterologous host.•myxopyronin/corallopyronin hybrid gene cluster expressed in heterologous host.•Production of respective metabolites by heterologous producer confirmed.•Production medium optimized for both heterologous producers.•Production yield of both metabolites significantly increased.
Myxopyronins and corallopyronins are structurally related α-pyrone antibiotics from myxobacteria. They are thought to represent a highly promising compound class for the development of broad-spectrum ...antibacterial therapeutic agents, because of their ability to inhibit RNA polymerase through interaction with the "switch region", a recently identified novel drug target. Here we describe the identification and characterization of the myxopyronin biosynthetic pathway from Myxococcus fulvus Mx f50. A detailed comparison with the recently identified corallopyronin biosynthetic pathway revealed the genetic and biochemical basis, thus explaining the observed structural differences between the two natural product families. Directed mutagenesis procedures for M. fulvus Mx f50 were developed to enable functional studies and pathway modifications. Our work provided new insights into myxopyronin biosynthesis and led to the production of a novel and unexpected myxopyronin derivative.
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The ...current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
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
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.
Polyunsaturated fatty acids (PUFAs), particularly the omega-3 long-chain PUFAs (LC-PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are well known for their beneficial health ...effects. The obvious limitation of the present EPA/DHA key source, fish oil, demands for alternative and sustainable PUFA resources and several biotechnological approaches addressing this problem are currently under development. Different marine microorganisms are known to produce PUFAs de novounder strictly anaerobic conditions employing polyketide synthase (PKS)-like enzymes known as PUFA synthases. Here, we report for the first time the characterization of such PUFA synthases from terrestrial origin. Two distinct types of PUFA biosynthetic gene clusters were discovered, originating from linoleic acid producing myxobacteria of the genus Sorangiumas well as from species of the recently discovered myxobacterial genus Aetherobacter, that turned out to be prolific producers of EPA and DHA. The identified biosynthetic pathways differ significantly from the marine systems in terms of gene organization, catalytic domain arrangement, and sequence identity of the encoded PUFA synthases. Notably, a unique domain, which most likely acts as 1-acylglycerol-3-phosphate O-acyltransferase, was identified in these myxobacterial PUFA synthases. As the native producer strains grow slowly, are difficult to handle, and genetic modification has proven difficult, synthetic biotechnology approaches were applied to establish a heterologous production platform in the myxobacterial model strain Myxococcus xanthus.
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