Field of the invention

The present invention relates to a method to improve the production of a secondary metabolite catalyzed by a non-ribosomal peptide synthetase comprising contacting in a eukaryotic host a eukaryotic non-ribosomal peptide synthetase with an MbtH-like protein. The present invention further relates to a composition comprising a eukaryotic non-ribosomal peptide synthetase that is not a hybrid and a prokaryotic MbtH and to a eukaryotic host cell comprising a non-ribosomal peptide synthetase and a polynucleotide allowing the expression of an MbtH-like protein.

Background of the invention

Secondary metabolites are compounds produced in microorganisms through the modification of primary metabolite synthases. They do not play a role in growth, development, and reproduction like primary metabolites, but many have a role in ecological function, including defense mechanism(s), by serving as antibiotics and by producing pigments. Today, many secondary metabolites have high value for society and are routinely produced on an industrial scale in fermentation processes. Some examples of secondary metabolites with importance in industrial microbiology include atropine, bleomycin and antibiotics such as bacitracin, erythromycin, penicillin and vancomycin.

As is the case with any other production process, also industrial fermentations producing secondary metabolites are the ongoing subject of yield improvement programs. This increases unit productivity, reduces cost and in many cases improves product isolation and purification and thus ultimately, product quality. Next to multitudes of obvious strategies to improve yields of fermentation processes, such as tuning of nutrient compositions, optimizing conditions like pH and temperature, genetically modifying microbial pathways and perfecting downstream processing, there remains an ongoing need to further improve by implementing new technologies that can further stretch productivity.

MbtH-like proteins are small (8-10 kD) proteins with exceptionally conserved sequence motifs resembling MbtH from Mycobacterium tuberculosis. The function of MbtH-like proteins is, to a large extent, still unknown although recent studies indicate a role in the biosynthesis of peptides. The genes encoding MbtH-like proteins, mbtH-Wke genes, are often found in non-ribosomal peptide synthetase (NRPS) gene clusters. Non-ribosomal peptides (NRP) are an important class of secondary metabolites. Many mbtH-Wke genes are deposited in GenBank. In order to identify MbtH-like proteins a BLASTP study shows homologues encoded by members of Actinobacteria, Firmacutes and Proteobacteria, however not by Archaea (R.H. Baltz, J. Ind. Microbiol. Biotechnol. (201 1 ) 38, 1747-1760). There are no reports of mbtH-like genes in naturally occurring eukaryotic NRPS gene clusters, their function is exclusively related to prokaryotic NRPS's.

Nevertheless, in WO 2013/113646 the use of an MbtH-like protein in the preparation of semi-synthetic β-lactam antibiotics in Penicillium is described. However, in this case the NRPS is a non-natural hybrid (comprising both eukaryotic and prokaryotic modules) and the MbtH-like protein in question positively influences only the adenylation reaction catalyzed by the prokaryotic module of the NRPS. This confirms the prejudice that MbtH-like proteins only act on prokaryotic NRPS's.

However, eukaryotes (notably fungi like Aspergillus, Penicillium, and Trichoderma) are an important class of microorganisms used in industrial production processes. Several economically attractive secondary metabolites from eukaryotes are for example β-lactam antibiotics, chrysogenins, roquefortins, cyclosporine and echinocandins. They belong to the fungal non- ribosomal petides. The above general need for further improving productivity in industrial fermentations of secondary metabolites equally applies to those processes that are catalyzed by eukaryotic NRPS's.

Detailed description of the invention

In the context of the present invention, the term "heterologous" used in combination with modules refers to modules wherein domains, such as adenylation or condensation domains, are from different modules. These different modules may be from the same enzyme or may be from different enzymes.

The term "hybrid" refers to a NRPS that comprises modules from both eukaryotic and prokaryotic origin. Typically a hybrid NRPS is obtained by genetic construction and not naturally occurring. NRPS's that are not hybrid comprise exclusively modules from eukaryotic origin or comprise exclusively modules from prokaryotic origin.

The term "module" defines a catalytic unit that enables incorporation of one peptide building block, usually an amino acid, in the product, usually a peptide, and may include domains for modifications like epimerization and methylation.

The term "non-ribosomal peptide" or "NRP" refers to peptide secondary metabolites, usually produced by microorganisms like bacteria and fungi. NRP's are synthesized by NRPS's. NRP's often have cyclic and/or branched structures, can contain non-proteinogenic amino acids including D-amino acids, carry modifications like /V-methyl and /V-formyl groups, or are glycosylated, acylated, halogenated, or hydroxylated. Cyclization of amino acids against the peptide "backbone" is often performed, resulting in oxazolines and thiazolines; these can be further oxidized or reduced. On occasion, dehydration is performed on serines, resulting in dehydroalanine. NRP's are often dimers or trimers of identical sequences chained together or cyclized, or even branched. NRP's are often toxins, siderophores, or pigments. Non-ribosomal peptide antibiotics, cytostatics, and immunosuppressants are in commercial use. Examples of NRP's are antibiotics (such as actinomycin, bacitracin, cephalosporin C, daptomycin, gramicidin, penicillin G, penicillin V, teixobactin, tyrocidine, vancomycin, zwittermicin A), antifungals (such as echinocandins or aculeacins), antibiotic precursors (such as ACV tripeptide), cytostatics (such as bleomycin, epothilone), immunosuppressants (such as ciclosporin), nitrogen storage polymers (such as cyanophycin), phytotoxins (such as AM-toxin, HC-toxin), pigments (such as indigoidine), siderophores (such as enterobactin, myxochelin A) and toxins (such as microcystins, nodularins, cyanotoxins)

The term "non-ribosomal peptide synthetase" or "NRPS" refers to a class of modular multi- domain enzymes found in the cytoplasm of bacteria and fungi that synthesize a large variety of highly diverse peptides and which are, unlike ribosomes, independent of messenger RNA. NRPS's are organized in multi-subunit clusters and each subunit in turn is composed of modules, capable of carrying out one cycle of chain elongation. A typical module consists of an adenylation (A) domain, a peptidyl carrier protein (PCP) domain and a condensation (C) domain. A domains (-550 residues) and C domains (-450 residues) are responsible for loading PCP domains with the cognate amino acid and catalyzing the peptide bond formation between the upstream aminoacyl or peptidyl PCP and downstream peptidyl PCP, respectively. During the entire process, the growing peptide chain is covalently linked to a phosphopantetheine cofactor which itself is attached to a conserved serine by a dedicated Ppan transferase (Pptase).

The term "secondary metabolite" refers to compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group and often play an important role in defense systems. Humans use secondary metabolites as colorings, flavorings and medicines. Examples of secondary metabolites are alkaloids (such as atropine, cocaine, codeine, morphine, tetrodotoxin), natural phenols (such as polyphenols), monoterpenoids (such as geranyl diphosphate, limonene, pinene), diterpenoids (such as aphidicolin, geranylgeranyl diphosphate, pimaradiene, taxol), NRP's, pigments (such as chrysogenin), mycotoxins (such as roquefortin) and antibiotics (such as a β-lactam like 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid, adipyl-7-aminodesacetoxycephalosporanic acid, cephalosporin C, penicillin G or penicillin V, streptomycin, tetracyclin).

In a first aspect of the invention there is disclosed a method to improve the production of a secondary metabolite or a precursor occurring in the pathway leading to said secondary metabolite catalyzed by a NRPS comprising contacting in a eukaryotic host said NRPS with an MbtH-like protein, characterized in that said NRPS is from eukaryotic origin and is not a hybrid.

Surprisingly it is found that MbtH-like proteins introduced in eukaryotic hosts positively influence the production levels of NRPS-dependent intermediates and other NRPS-dependent secondary metabolites. The present invention demonstrates successful results with a selection of MbtH proteins covering a variety of different sources and grades of homology on the one hand, combined with a range of NRPS's on the other hand that are all fully eukaryotic, i.e. are not hybrids.

Table 1 is a summary of the many examples that have been investigated with three different NRPS's and a range of MbtH-like proteins in two different strains, clearly showing that any combination of the investigated NRPS's and MbtH-like proteins results is a positive effect at least at one point in the pathway of the secondary metabolite. Overview of the average number of metabolites effected in productivity by the presence of MbtH-like proteins from Tables 4-9 (+: number of metabolites with >10% increased productivity; +/-: number of metabolites with +/- 10% productivity; -: number of metabolites with >10% decreased productivity)

In a first embodiment, the eukaryotic host is a fungus or a yeast as in industry these are routinely employed eukaryotic microorganisms. Suitable examples are Aspergillus, Kluyveromyces, Penicillium, Pichia, Saccharomyces, Trichoderma, and Yarrowia and preferably Penicillium chrysogenum, Aspergillus nidulans, Aspergillus niger, Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae or Yarrowia lipolytica.

In a second embodiment of the invention the secondary metabolite is as defined above, is preferably a β-lactam, a pigment or a mycotoxin. More preferably, the β-lactam is

6- aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid, adipyl

7- aminodesacetoxycephalosporanic acid, cephalosporin C, penicillin G or penicillin V, the pigment is a chrysogenin and the mycotoxin is a roquefortin.

In a third embodiment the eukaryotic host strains are high level production strains. It has been found that e.g. high level penicillin production in some strains of Penicillium chrysogenum is due to presence of amplified tandem repeats of the penicillin gene cluster (reviewed in Martin F. (2000) J. Bacteriol 182:2355-2362.). High level production of secondary metabolites can be the result of amplified biosynthesis gene clusters and so the eukaryotic host is a multi copy strain with respect to the secondary metabolite cluster of interest.

In a fourth embodiment, preferred MbtH-like proteins are the ones described in R.H. Baltz (J. Ind. Microbiol. Biotechnol. (201 1 ) 38, 1747-1760). More preferred MbtH-like proteins are the ones comprising invariant amino acids N17, E19, Q21 , S23, W25, P26, P32, G34, W35, L48, W55, T56, D57, R59 and P60, also suitably referred to with the amino acid code NXEXQXSXWP-Xs-PXGW-X ₁₂ - L-X ₆ -WTDXRP (SEQ ID NO: 17). In the above annotation the letters D, E, G, L, N, P, Q, R, S, T, W and X refer to the commonly known single letter codes for amino acids (whereby X denotes one unspecified amino acid, X ₅ denotes 5 unspecified amino acids, Χβ denotes 6 unspecified amino acids and X ₁₂ denotes 12 unspecified amino acids). In preferred embodiments said MbtH-like protein comprises the amino acid code NXEXQXSXWP- X ₅ -PDGW-X ₁₂ -L-X ₆ -WTDXRP or NXEXQXSXWP-Xs-PAGW-X ₁₂ -L-X ₆ -WTDXRP or NXEXQXSXWP-X5-PGGW-X ₁₂ -L-X ₆ -WTDXRP or NXEXQXSXWP-X5-PQGW-X ₁₂ -L-X ₆ -WTDXRP wherein X5 is chosen from the list consisting of AFAEV, AFAAV, AFAEI, TFAEV, TFAAV, TFAEI, VFAEV, VFAAV and VFAEI (SEQ ID NO: 18 - SEQ ID NO: 53). In more preferred embodiments said MbtH-like protein comprises the amino acid code NXEXQXSLWP-Xs-PDGW-X ₁₂ -L-X ₆ - WTDXRP or NXEXQXSLWP-Xs-PAGW-X ₁₂ -L-X ₆ -WTDXRP or NXEXQXSLWP-X ₅ -PGGW-X ₁₂ -L- X ₆ -WTDXRP or NXEXQXSLWP-X ₅ -PQGW-X ₁₂ -L-X ₆ -WTDXRP wherein X ₅ is chosen from the list consisting of AFAEV, AFAAV, AFAEI, TFAEV, TFAAV, TFAEI, VFAEV, VFAAV and VFAEI (SEQ ID NO: 57 - SEQ ID NO: 92).

It is noted that in R.H. Baltz (J. Ind. Microbiol. Biotechnol. (201 1 ) 38, 1747-1760) and in WO 2013/113646 erroneously the amino acid code NXEXQXSXWP-X ₅ -PXGW-X ₁₃ -L-X ₇ - WTDXRP is mentioned where in fact this refers to and should be NXEXQXSXWP-X ₅ -PXGW-X ₁₂ - L-X ₆ -WTDXRP. Preferably, the MbtH-like proteins of the present invention are Tcp13 (SEQ ID NO: 1 ) or Tcp17 (SEQ ID NO: 2) obtained from the teicoplanin biosynthesis cluster from Actinoplanes teichomyceticus (Sosio ei. al., Microbiology (2004) 150, 95-102), or the MbtH-like homologue identified in the Veg biosynthesis cluster obtainable from an uncultured soil bacterium (Banik J.J. and Brady S.F., Proc. Natl. Acad. Sci. USA (2008) 105, 17273-17277) encoded by nt 33826-34035 of GenBank: EU874252 (SEQ ID NO: 3) called VEG8 or the MbtH- like homologue identified in the Teg biosynthesis cluster obtainable from an uncultured soil bacterium (Banik J.J. and Brady S.F., Proc. Natl. Acad. Sci. USA (2008) 105, 17273-17277) encoded by nt 33949-33158 of GenBank: EU874253 (SEQ ID NO: 4) called TEG or the MbtH-like homologue (SEQ ID NO: 5) identified in the balhimycin biosynthesis cluster from Amycolatopsis balhimycina (Recktenwald ei a/., Microbiology (2002) 148, 1105-1 118, Stegman ei a/., FEMS Microbial Lett. (2006) 262, 85-92) called BPS or the MbtH-like homologue (SEQ ID NO: 6) identified in the complestatine biosynthesis cluster from Streptomyces lavendulae (Chiu et al., Proc. Natl. Acad. Sci. USA (2001 ) 98, 8548-8553) called COM or MbtH like homologue SCO0489 (SEQ ID NO: 7) identified in the calcium dependent antibiotic (CDA) biosynthesis cluster from Streptomyces coelicolor (Hojati et al. (Chem. & Biol. (2002) 9, 1175- 1187) called CDAI or MbtH-like proteins having an amino sequence with a percentage identity of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% to said sequences. Such polypeptide modules with a percentage identity of at least 70% are also called homologous sequences or homologues.

In a second aspect the invention provides a composition comprising a eukaryotic NRPS that is not a hybrid and a prokaryotic MbtH. Preferred combinations of eukaryotic NRPS's and MbtH-like proteins are the NRPS N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase PcbAB (UniProtKB - P26046 (ACVS2_PENCH, SEQ ID NO: 54) with COM, PcbAB with CDAI, PcbAB with TCP13, PcbAB with TEG, PcbAB with VEG8, the NRPS Pc21g12630 protein ChyA (UniProtKB - B6HLP9 (B6HLP9_PENRW, SEQ ID NO: 55) with COM, ChyA with CDAI, ChyA with TCP13, ChyA with TEG, ChyA with VEG8, the NRPS Roquefortine/meleagrin synthesis protein A RoqA (UniProtKB - B6HJU6.1 ROQA_PENRW, SEQ ID NO: 56) with COM, RoqA with CDAI, RoqA with TCP13, RoqA with TEG or RoqA with VEG8. Thus, preferred combinations are SEQ ID NO: 54 with SEQ ID NO: 1 , SEQ ID NO: 54 with SEQ ID NO: 3, SEQ ID NO: 54 with SEQ ID NO: 4, SEQ ID NO: 54 with SEQ ID NO: 6, SEQ ID NO: 54 with SEQ ID NO: 7, SEQ ID NO: 55 with SEQ ID NO: 1 , SEQ ID NO: 55 with SEQ ID NO: 3, SEQ ID NO: 55 with SEQ ID NO: 4, SEQ ID NO: 55 with SEQ ID NO: 6, SEQ ID NO: 55 with SEQ ID NO: 7, SEQ ID NO: 56 with SEQ ID NO: 1 , SEQ ID NO: 56 with SEQ ID NO: 3, SEQ ID NO: 56 with SEQ ID NO: 4, SEQ ID NO: 56 with SEQ ID NO: 6, SEQ ID NO: 56 with SEQ ID NO: 7, or sequences that are at least 90% homologous to any or both.

In a third aspect the invention provides a eukaryotic host cell comprising a NRPS from eukaryotic origin that is not a hybrid and a polynucleotide allowing the expression of an MbtH-like protein. Preferably the MbtH-like proteins are those of the first aspect of the invention. Preferably the host cell is a fungus or a yeast. Suitable examples are Aspergillus, Kluyveromyces, Penicillium, Pichia, Saccharomyces, Trichoderma, and Yarrowia and preferably Penicillium chrysogenum, Aspergillus nidulans, Aspergillus niger, Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae or Yarrowia lipolytica. Preferably the host cell comprises an MbtH-like protein with SEQ ID NO: 17.

In a fourth aspect the invention provides a method for the preparation of the host cell of the third aspect of the invention. This may be achieved according to procedures known to the skilled person such as targeted or random integration of an expression cassette consisiting of a suitable promoter, the gene of interest and a terminator. Throughout this description the following three letter codes and one letter codes are used for amino acids:

HOMOLOGY & IDENTITY

The terms "homology" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent homology of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm can align both amino acid sequences and nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden.l. and Bleasby.A. Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Others can be specified. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

Global Homology Definition

The homology or identity is the percentage of identical matches between the two sequences over the total aligned region including any gaps. The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps. The identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as "IDENTITY".

Longest Identity Definition

The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as "longest-identity".

EXAMPLES

General materials and methods

Molecular and genetic techniques

Standard genetic and molecular biology techniques are known in the art (e.g. Maniatis et al. "Molecular cloning: a laboratory manual" (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller "Experiments in molecular genetics" (1972) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Sambrook and Russell "Molecular cloning: a laboratory manual" (3rd edition)" (2001 ) Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; Ausubel "Current protocols in molecular biology" (1987) Green Publishing and Wiley Interscience, New York).

Plasmids and strains

Escherichia coli: Cloning was performed using Escherichia coli DH5a.

Penicillium chrysogenum: DS 17690 (Harris D. et al., Metab. Eng. (2006) 8, 91 - 101 ) was used as a high level penicillin production strain of Penicillium chrysogenum, DS 47274 (Harris D. et al., Metab. Eng. (2006) 8, 91 - 101 ) was used as a low level penicillin production strain. They differ in the number of penicillin biosynthetic gene clusters. While the original strain DS17690 contains 8 copies, DS47274 is a one copy strain (Harris D. et al., Metab. Eng. (2006) 8, 91 - 101 ).

Media:

Escherichia coli: All cultures were grown using 2xPY (15 g/L Bacto-tryptone, 10 g/L Yeast extract, 10 g/L Sodium chloride, pH 7.0) at 37°C and 200 rpm. Antibiotics (25 g/mL Zeocin) were supplemented to maintain plasmid for plAT.

Penicillium chrysogenum:

Penicillium chrysogenum strains were grown on YGG medium (Bartoszewska M. et al., Appl. Environ. Microbiol. (2011 ) 77, 1413 - 14221 . Sporulation of mycelia was stimulated by growth on R agar (Bartoszewska M. et al., Appl. Environ. Microbiol. (2011 ) 77, 1413 - 14221 ) at 25°C for 10-11 days.

For genetic manipulation purposes,

Transformants were selected as cotransformants by their ability to grow on plates with acetamide as the only nitrogen source. Acetamide-agar comprises 0.04mM FeSO ₄ , 2.03mM MgSO ₄ , 51 .3mM NaCI, 55.5mM Glucose, 15g/l Agar Agar, 10g/I Acetamide, 5.1 mM,KH ₂ PO ₄ , 4.9mM K ₂ HPO ₄ , and 2ml/l Trace element solution (Na ₃ C ₆ H ₅ O ₇ , 149mM, FeSO ₄ , 89mM, MgSO ₄ , 1 .04mM, H3BO3, 0.2mM, Na ₂ MoO ₄ , 0.05mM, CuSO ₄ , 2.56mM, ZnSO ₄ , 8.76mM, CoSO ₄ , 2.28mM, MnSO ₄ , 17.99mM, CaCI ₂ , 10.88mM, EDTA, 107mM).

For production of secondary metabolites, penicillin production medium, PPM (Nijland J.G. et al., Appl. Environ. Microbiol. (2010) 76, 7109 - 7115) + 0.05% phenoxyacetic acid, was used.

Example 1

Synthetic design and cloning of MbtH-like protein expression constructs

To express the MbtH-like proteins in Penicillium chrysogenum, expression cassettes comprising the Penicillium chrysogenum IPNS promoter and the Penicillium chrysogenum AT terminator were designed. The vector plAT comprising the promoter cassette from the isopenicillin N synthetase (IPNS, pcbC-gene) of Penicillium chrysogenum (Promoter pcbC) flanked by NotI / Ndel sites (SEQ ID No 8), a DNA fragment harbouring a cat-ccdB cassette (Chloramphenicol resistance and a toxicity gene for Escherichia coli), and a transcription terminator cassette from the acyl-CoA:isopenicillin N acyltransferase (AT, pcbDE-gene) of Penicillium chrysogenum flanked by Nsil/Notl-sites (SEQ ID No 9) was used for cloning of the MbtH encoding genes as Ndel/Nsil fragments between promoter and terminator.

Five different MbtH-like proteins were chosen, one from the teicoplanin biosynthetic cluster annotated as tcp13 (SEQ ID NO: 1 , GenBank: AJ605139 Genomic DNA; Translation: CAE53354.1 ) and called TCP13, one from the Veg biosynthetic clusters identified by a search for homologous MbtH-like sequences in the Veg Cluster (SEQ ID NO: 3, GenBank: EU874252, nt 33826-34035, between veg9 and veg10), called VEG8, one from the complestatine biosynthetic cluster annotated as hypothetical protein (SEQ ID NO: 6, GenBank: AF386507 Genomic DNA; Translation: AAK81828.1 ) and called COM, one from the Teg biosynthetic clusters identified by a search for homologous MbtH-like sequences in the Teg Cluster (SEQ ID NO: 4, GenBank: EU874253, nt 32949-33158, between teg8 and teg9), called TEG, and one from the calcium dependent antibiotic biosynthesis cluster, SCO0489 (SEQ ID NO: 7) from Streptomyces coelicolor (Hojati ef al. (Chem. & Biol. (2002) 9, 1175-1 187) called CDAI. Target genes encoding the selected proteins were constructed synthetically resulting in nucleotide SEQ ID NO: 10-14 and ordered at IDT as gBlocks (Integrated DNA technology, Coralville, Iowa, USA) flanked by restriction sites Ndel and Nsil for final cloning between IPNS promoter and AT terminator. The gene encoding VEG8 was used as wild type sequence, while the genes encoding TCP13, COM, CDAI, BPS and TEG were codon optimized for expression in Penicillium chrysogenum.

The final plasmids harbouring the expression constructs for overexpression of the MbtH-like proteins in Penicillium chrysogenum constructed by cloning the Ndel/Nsil fragments taken from the gBlocks into the Ndel/Nsil sites of expression vector plAT were named plAT-Tcp13, plAT- Veg8, plAT-COM, plAT-TEG, and plAT-CDAI. The final sequences of the MbtH expression constructs were confirmed using sequencing provided by Macrogen (Macrogen Europe, Amsterdam, The Netherlands)

Example 2

Transformation of MbtH-like protein expression constructs plAT-MbtH plasmids were cut with Notl, and run on an agarose gel. The Notl-fragments comprising the MbtH expression cassettes were cut from the agarose gels, and gel-cut fragments were purified and concentrated by desalting. Protoplast formation and transformations of two Penicillium chrysogenum strains, DS 17690 and DS 47274, was performed as described by Kovalchuk A et al. Methods Mol Biol. (2012); 835: 1 -16.

Transformation was performed as co-transformation with the amdS selection marker comprising the Aspergillus nidulans acetamidase encoding gene amdS under control of the Aspergillus nidulans gpdA promoter (US patent 5876988, Selten GCM, Swinkels BW, van Gorcom RFM. 1999. Selection marker gene free recombinant strains: method for obtaining them and the use of these strains) in a molar ratio of 10:1 (MbtH expression construct : amdS selection Marker). This transformation approach results in random integration of the MbtH expression construct and the amdS selection marker in the genome of the host organisms and the number of expression cassettes can vary per transformant obtained.

Because of this variation in most cases we have investigated multiple independent transformants per host strain / MbtH expression construct variation instead of limiting ourselves to one.

After 12 days, several selected colonies for each MbtH/amdS co-transformation were purified using three acetamide-agar to R-agar plate transfer cycles. During purification, colonies exhibiting insufficient growth on selective acetamide agar or too fast sporulation on R-agar- plates, were discarded, respectively. To confirm MbtH integration, pieces of mycelium from selected colonies were homogenized in milliQ water and used as template DNA in a PCR reaction setup. Primers were targeting the 5 ^' flanking IPNS promoter (SEQ ID No: 15) and the 3 ^' AT terminator (SEQ ID No: 16) of each MbtH expression cassette.

Finally, three to nine amdS positive and MbtH expression construct containing transformants per MbtH expression construct and strain background were obtained for further characterization, with the exception of expression construct VEG8; here one transformant only for strain background DS17690 and two transformants for strain background DS47274 were obtained. Table 2 gives an overview on the transformants obtained and the codes chosen for the different transformants.

Table 2 Overview on amdS positive and MbtH gene containing transformants for high and low level penicillin producing Penicillium chrysogenum strains DS17690 and DS47274.

Example 3

Small scale fermentations of of MbtH-like protein expressing Penicillium chrysogenum strains for secondary metabolite production

Positively tested Penicillium strains as summarized in Table 2 were subjected to small scale fermentation experiments in 100 ml shake flasks over a total course of 5 days. As reference strains, the non transformed strains DS17690 and DS47274 were taken along. Cultures were inoculated from spore crops in a volume of 25mL YGG medium. After 24 h of growth, the sporulated pre - culture was 10-fold diluted in a total volume of 2 x 30mL in PPM plus 0.25% phenoxyacteic acid. Two biological and two technical replicates were used per strain and experiment. All cultures were grown at 250°C and 200 rpm using Innova 44 shaker (Eppendorf, Hamburg, Germany). Sampling was conducted at day 2 and 5 after pre-culture transfer. Thereby, 1 mL of culture was taken, centrifuged at 40°C, 14000xg for 10 minutes. The supernatant is subsequently filtered using a PTFE syringe filter (0.2μΜ, No.514-0068, VWR, Radnor, Pennsylvania, USA), reduced with 10mM DTT (1 ,4-Dithiotreithol, No. 6908.2, Carl Roth GmbH, Karlsruhe, Germany ) and stored at -80°C up until analysis. The remaining volume of the culture after 5 days is additionally used in order to determine the dry weight.

Example 4

Analysis of secondary metabolite production

The genome of Penicillium chrysogenum encodes ten NRPS, twenty polyketide synthases (PKS), and two hybrid NRPS-PKS genes (van den Berg ef al. (Nature Biotech (2008) 26, 1161 -1168). Several of these genes have been associated with specific secondary metabolites . Three groups of secondary metabolitesfor which biosynthesis routes have been assigned to specific NRPSs were chosen for analysis of secondary metabolite production: Penicillin related secondary metabolites (NRPS: PcbAB - Pc21g21390), Roquefortine related metabolites (RoqA - Pc21g15480), and Chrysogine related metabolites (NRPS: ChyA - Pc21 g12630), respectively. Available standards were used to identify peaks according to retention time and accurate mass by LC/MS analyses. A total of 26 metabolites are associated with these three clusters (Salo O.V., BMC Genomics (2015), 16:937). Table 3 gives an overview on the detectable reference compounds by the LC/MS method applied. The biosynthesis cluster, compound names, monoisotopic ionized masses (M/Z [H]+), molecular composition and retention times for the applied LC program are indicated.

In the culture samples, 23 out of the 26 metabolites were sufficiently abundant, to allow for comparison with the wild type strain. The 23 relevant metabolites can be assigned to the three clusters in the following manner; 3 metabolites for Penicillin biosynthesis, 10 metabolites for Chrysogine biosynthesis, and 10 metabolites for Roquefortine biosynthesis. Metabolite levels were further evaluated by measuring the peak area, normalizing for dry weight and finally calculating ratios, relative to wildtype metabolite abundance. A complete list of all metabolites and intermediates which were identified in the MbtH expressing transformants and their relative abundance compared to the untransformed wildtype strains is summarized in Tables 4 - 9, whereby Table 4, Table 5 and Table 6 show relative productivity of secondary metabolites in the penicillin, roquefortin and chrysogine cluster, respectively, in high level penicillin production strain DS17690 in the presence of the MbtH-like proteins investigated after 2 and 5 days of cultivation, while Tables 7-9 show this for the low level penicillin production strain DS47274. For each metabolite of the respective cluster measured at the same cultivation time point and for the same strain background, the average of the relative productivity for all MbtH expressing transformants was calculated to visualize the observed trends. Metabolites were classified as a) increased, when the average value was above 1 .1 (>10% increased productivity), b) decreased, when the average value was below 0.9 (>10% decreased productivity), and as unchanged, when the average value x was 1 .1 > x > 0.9 (+/- 10% productivity). Finally, the total number of metabolites classified as increased, decreased or unchanged per biosynthesis cluster, strain and cultivation day was determined and is given as overall summary in Table 1 .

5μL of every culture supernatant sample obtained from Penicillium fermentations were subjected to LC/MS analysis. Two technical replicates were run per sample. Analysis was performed using a LC/MS Orbitrap machine (Thermo Scientific) in combination with a RP-C18 column (Shimadzu Shim pack XR-ODS 2.2; 3.0x75mm) in positive mode. A gradient program with MiliQ water (A), Acetonitrile (B) and 2% Formic acid (D) was run; 0 min; A 90%, B 5%, C 5%; 4 min, A 90%, B 5%, C 5%; 13 min, A 0%, B 95%, C 5%; 16 min A 0%, B 95%, C 5%; 16 min, A 90%, B 5%, C 5%; 21 min A 90%, B 5%, C 5% at a flow rate of 0.3ml min ^-1 .

Legend to Tables 3-9

Table 3: Overview on secondary metabolites and their corresponding biosynthetic pathways measured with the applied LC program.

Table 4: Relative productivity of secondary metabolites in the penicillin cluster in strain

DS17690 in the presence of MbtH-like proteins indicated after 2 and 5 days of cultivation. Productivity is compared to the non modified strain DS17690 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 5a: Relative productivity of secondary metabolites in the chrysogenin cluster in strain

DS17690 in the presence of MbtH-like proteins after 2 days of cultivation. Productivity is compared to the non modified strain DS17690 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 5b: Relative productivity of secondary metabolites in the chrysogenin cluster in strain

DS17690 in the presence of MbtH-like proteins after 5 days of cultivation. Productivity is compared to the non modified strain DS17690 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 6a: Relative productivity of secondary metabolites in the roquefortine cluster in strain

DS17690 in the presence of MbtH-like proteins after 2 days of cultivation. Productivity is compared to the non modified strain DS17690 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 6b: Relative productivity of secondary metabolites in the roquefortine cluster in strain

DS17690 in the presence of MbtH-like proteins after 5 days of cultivation. Productivity is compared to the non modified strain DS17690 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 7: Relative productivity of secondary metabolites in the penicillin cluster in strain

DS47274 in the presence of MbtH-like proteins after 2 and 5 days of cultivation.

Productivity is compared to the non modified strain DS47274 (without presence of

MbtH-like proteins), for which all values are set to 1.0.

Table 8a: Relative productivity of secondary metabolites in the chrysogenin cluster in strain

DS47274 in the presence of MbtH-like proteins after 2 days of cultivation. Productivity is compared to the non modified strain DS47274 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 8b: Relative productivity of secondary metabolites in the chrysogenin cluster in strain

DS47274 in the presence of MbtH-like proteins after 5 days of cultivation. Productivity is compared to the non modified strain DS47274 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 9a: Relative productivity of secondary metabolites in the roquefortine cluster in strain

DS47274 in the presence of MbtH-like proteins after 2 days of cultivation. Productivity is compared to the non modified strain DS47274 (without presence of MbtH-like proteins), for which all values are set to 1.0.

Table 9b: Relative productivity of secondary metabolites in the roquefortine cluster in strain

DS47274 in the presence of MbtH-like proteins after 5 days of cultivation. Productivity is compared to the non modified strain DS47274 (without presence of MbtH-like proteins), for which all values are set to 1.0. Table 3