Development and Validation of a Fast and Optimized Screening Method for Enhanced Production of Secondary Metabolites Using the Marine Scopulariopsis brevicaulis Strain LF580 Producing Anti-Cancer Active Scopularide A and B

Abstract

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10–15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained.

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster

“…Corresponding clusters of supporting genes and their expression values are shown in Fig 8 . Our data indicate that this gene cluster, which is involved in the scopularide production and indeed, it is actively expressed under conditions supporting scropularides A and B production [ 49 , 50 ]. The nrps1 gene (g12932) is the best candidate gene to be responsible for production of the cyclic lipopeptide scopularide [ 51 ], which consists of five amino acids (glycine, L-valine, D-leucine, L-alanine and L-phenylalanine), and a reduced carbon chain [ 52 ].…”

Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

“…Production of scopularide A by S. brevicaulis has been partially optimized in stirred tank bioreactors, resulting in a 29-fold increase in volumetric production compared to non-agitated cultivation [ 16 ]. A random mutagenesis approach, based on UV-radiation, has also been applied, which identified one mutant (M26) that had improved scopularide A production characteristics [ 17 , 18 ]. In the present study, the recently sequenced genome of S. brevicaulis [ 19 ] was used to identify the putative scopularide biosynthetic gene cluster and the local transcription factor has been targeted for overexpression to enhance production of scopularide A.…”

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

“…The mutant strain LF580-M26 was obtained by UV radiation as described by Kramer et al . [ 10 ]. Ahead of the core experiment, the LF580-M26 strain was checked for its phenotypic traits by a simple cultivation test followed by an extraction and LC-MS analysis.…”

“…The wild type strain LF580 represents a non-model secondary metabolite producer, exhibiting high yields of the target metabolites (approximately 200 mg L -1 for scopularide A [ 9 ]). Using an optimised screening approach [ 10 ], a UV mutant strain ( M . brevicaulis strain LF580-M26) was detected, which showed an increased production level of the two scopularides under standard conditions, faster and different growth and changes in pellet formation.…”