Targeted cloning of a large gene cluster from Lecanicillium genome by Cre/loxP based method.

Functionally related genes often form a large gene cluster on fungal genomes. To analyze, by heterologous expression system, overall pathway in which a series of related genes are involved, the whole gene cluster should be introduced intact into the host strain. However, the construction of a genomic library based on cosmid or bacterial artificial chromosome, and screening of a clone harboring the target region are time consuming and usually require additional cloning of missing regions. The available PCR-based methods are convenient, but are likely to cause unexpected errors during long-range PCR. Therefore, in this study we developed a method for targeted cloning of a large gene cluster based on Cre/loxP-mediated recombination. loxP sequences were integrated at both edges of the targeted region, and the region was excised and cloned as a circular fosmid by in vitro Cre recombination. To facilitate the Cre/loxP-based method, a competent host-vector system was developed, including a double auxotrophic Lecanicillium PTk3 (ΔpyrG trp1-ku80-) strain and two vectors for introducing the loxP sequences, pUTlox and pCCPlox. A targeted region longer than 45 kb in length was successfully cloned by the Cre/loxP-based method.

Identification of a gene cluster responsible for the biosynthesis of cyclic lipopeptide verlamelin.

Only limited studies are available on the molecular-level biosynthesis of cyclic lipopeptides (cyclic and hybrid molecules consisting of peptide and fatty acid moieties) in filamentous fungi. Here, we identified and characterized biosynthetic genes of the cyclic lipopeptides, known as verlamelins. Only four genes, coding for non-ribosomal peptide synthetase (NRPS), fatty acid hydroxylase, thioesterase, and AMP-dependent ligase, were found to be involved in verlamelin biosynthesis by the analysis of corresponding gene knockouts. Surprisingly, no gene(s) coding for fatty acid synthase or polyketide synthase was present in the cluster, while verlamelin A/B contained a 5-hydroxytetradecanoic acid moiety. Precursor feeding experiment indicated that both fatty acid hydroxylase and thioesterase are involved to supply 5-hydroxytetradecanoic acid. The results suggested that 5-hydroxytetradecanoic acid was supplied from primary metabolism via fatty acid hydroxylase and loaded onto NRPS. Elongation of the peptide and final cyclization were accomplished by NRPS. The knowledge obtained through this study should provide new insight into fungal lipopeptide biosynthesis.

Cyclic lipodepsipeptides verlamelin A and B, isolated from entomopathogenic fungus Lecanicillium sp.

Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val). This is the first study that precisely analyzed the structure of verlamelin.

Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species.

Lack of genetic tools in entomopathogenic fungi, especially those for targeted homologous recombination, hindered the advance in this field. To facilitate the genetic study, we constructed a transformation system in entomopathogenic fungus Lecanicillium sp. strain HF627 using the uridine auxotrophic pyrG mutant strain as host and endogenous pyrG as marker. pUC19 harboring endogenous pyrG successfully restored the uridine auxotrophy of the host strain, and the integration of the vector DNA was confirmed by Southern hybridization. An autonomously replicating vector harboring an AMA1 sequence was constructed and applied to the constructed transformation system, which improved the transformation efficiency 16.7-fold. Southern hybridization revealed replication of the AMA1-harboring vector with an average copy number of 2.4. A ku80 knock-out strain was created to improve the efficiency of gene targeting. Deletion of the pyrG locus, which is homologous to the marker gene, from the ku80 knock-out strain achieved a targeting efficiency of 62.5 % against both trp1 and his3; the levels of these genes were 3.2- and 5-fold higher, respectively, than the ku80-intact strain. With the pyrG-deleted and ku80-inactivated strain constructed in this study, transformation and targeted homologous recombination were highly enhanced, by which genetic analysis in Lecanicillium spp. will be performed quickly and efficiently.

Aurovertins F-H from the entomopathogenic fungus Metarhizium anisopliae.

Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to isolation of aurovertin D ( 1) and three new aurovertin-type metabolites, aurovertin F ( 2), aurovertin G ( 3), and aurovertin H ( 4). Their structures were determined on the basis of spectroscopic analyses and chemical conversions.