WO2017037629A1

WO2017037629A1 - Improved microorganisms for the biosynthesis of pamamycins

Info

Publication number: WO2017037629A1
Application number: PCT/IB2016/055194
Authority: WO
Inventors: Lutz Petzke; Andrea Herold; Christian Fleck; Laurent Marty; Peter OEDMAN; Joachim Dickhaut; Melanie WEINGARTEN; Andriy LUZHETSKY; Yuriy REBETS; Niko MANDERSCHEID
Original assignee: Basf Se; Basf (China) Company Limited
Priority date: 2015-09-04
Filing date: 2016-08-31
Publication date: 2017-03-09

Abstract

Provided are improved microorganisms for the biosynthesis of pamamycins. Also provided are methods for the production of pamamycins using these microorganisms.

Description

IMPROVED MICROORGANISMS FOR THE BIOSYNTHESIS OF PAMAMYCINS

FIELD OF THE INVENTION

The invention pertains to the field of production of natural products and, in particular, in the field of production of pamamycins. It provides improved microorganisms for the biosynthesis of pamamycin as well as methods for the production of pamamycins using these

microorganisms.

BACKGROUND OF THE INVENTION

Pamamycins are a group of macrolide compounds and have been described as early as 1979 (McCann and Pogell, JOURNAL OF ANTIBIOTICS (1979) Vol.: 32(7) pages: 673 to 678). They are produced by several Streptomyces species and have raised scientific interest, because they stimulate the formation of aerial mycelium in streptomycete species which produce pamamycins, as well as in other streptomycetes which are not known to produce pamamycins. They also enhance the production of secondary metabolites in several streptomycete species and have antibiotic activity against gram-positive bacteria, fungi and mycobacteria (Pogell, BM ; CELLULAR AND MOLECULAR BIOLOGY (1998); 44(3); pages 461 to 463; Natsume, M. et al. JOURNAL OF ANTIBIOTICS (1995)

Vol.:48(10); pages: 1 159 to 1 164; Hashimoto et al. in BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY. 2003, Vol 67(4), pages 803 to 808 and Hashimoto, M. et a.

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY (201 1 ) Vol. :75(9) pages: 1722 to 1726). Further investigations showed, that the different side chains located on the ring structure of pamamycins, as well as their dimethylamino group have different effects on the capacity of pamamycins to induce aerial mycelia, to inhibit growth of Streptomyces albonigeror to work as antibiotic on Bacillus subtilis, see Natsume, M. et al. JOURNAL OF ANTIBIOTICS (1995) Vol.:48(10); pages:1 159 to 1 164 and Natsume, M. et al.

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY (1995) Vol.:59(9) pages: 1766 to 1768.

The use of C-13- and N-15-labeled precursor units provided further information on the metabolic pathway leading to the production of pamamycins. N-15-labeling suggests that the nitrogen atom of the dimethylamino group present in many pamamycins was derived from the alpha-amino group of an amino acid which has been introduced into the pamaycin structure by transamination, followed by N-methylation. Feeding experiments with C-13- labeled acetate or propionate indicates that the carbon skeleton of pamamycins is derived from six acetate, four propionate and three succinate units, see Hashimoto, M. et al.

JOURNAL OF ANTIBIOTICS (2005) Vol.:58(1 1 ); pages:722 to 730 and Hashimoto, M. et al. BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY (2005) Vol.:69(2) pages:315 to 320.

The attractive features and potential uses of pamacins have spured attempts to provide sufficient amounts of pamamycins to enable technical applications. Thus, numerous methods to chemically synthesize pamamycins have been developed. However, the complex structure of pamamycins require complex and lenthy synthesis methods, which make these approaches unattractive. Alternative approaches relied on the use of natural producers of pamamycins. Organisms which produce pamamycins, as well as fermentation methods for the production of pamamycins and methods to purify pamamycins have been disclosed for example in US4283391 , JP62135476A, DE4134168, DE4316836, Natsume, M. et al. JOURNAL OF ANTIBIOTICS (1995) Vol.:48(10); pages:1 159 to 1 164 and HAERTL et al. THE JOURNAL OF ANTIBIOTICS (1998) VOL. 51 , NO. 1 1 , pp. 1040-1046.

These approaches include also the use of recombinant recombinant producer organisms as for example disclosed in WO2015/092575. However, the available methods to produce pamamycins via use of their naturaly occurring producer organisms or via recombinant producer organisms are limited by the production level of these organism. Thus, there is still a need to develop further methods for the enhanced production of pamamycins.

Accordingly, the technical problem underlying the present invention can be seen as the provision of additional means and methods for the production of pamamycins. The technical problem is solved by the embodiments characterized in the claims and herein below.

SUMMARY OF THE INVENTION

The invention comprises a pamamycin producer organism comprising at least one of:

a) a decreased methylmalonyl-CoA mutase activity, b) a decreased crotonyl-CoA carboxylase activity, c) a decreased acetyl/propionyl CoA carboxylase activity, d) a combination of at least two of a), b) and c), wherein the pamamycin producer organism comprises an intact katabolic pathway from isobutyryl-CoA to (2S)-methylmalonyl-CoA. Preferably these pamamycin producer organisms comprise a decreased methylmalonyl- CoA mutase activity and a decreased crotonyl-CoA carboxylase activity. But may also comprise a decreased methylmalonyl-CoA mutase activity, a decreased crotonyl-CoA carboxylase activity and a decreased acetyl/propionyl CoA carboxylase activity.

Another embodiment of the invention is a pamymcin producer organism as described above, wherein the decreased methylmalonyl-CoA mutase activity is due to a decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, or 6, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, 8, or 9, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 7, 8 or 9. A further embodiment comprises a pamamycin producer organism wherein the decreased crotonyl-CoA carboxylase activity is due to a decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, 1 1 , 12, or 13, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14 or 15, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 14 or 15, or, preferably, both. Further pamamycin producer organisms have features as described above, wherein the decreased acetyl/propionyl CoA carboxylase activity is due to decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, 17, 18, 19, 20, 21 , 25, 26, 27, 28, 29, or 30, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, 23, 24, 31 , 32, or 33, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 22, 23, 24, 31 , 32, or 33.

Preferred pamamycin producer organisms comprise: a) at least one polypeptide having pamC activity, and b) at least one polypeptide having pamG activity, and c) at least one polypeptide having pamF activity, and d) at least one polypeptide having pamA activity, and e) at least one polypeptide having pamB activity, and f) at least one polypeptide having pamD activity, and g) at least one polypeptide having pamE activity, and h) at least one polypeptide having pamO activity, and i) at least one polypeptide having pamK activity, and j) at least one polypeptide having pamJ activity, and k) least one polypeptide having pamM activity, and I) at least one polypeptide having pamN activity, and m) at least one polypeptide having pamL activity, and n) at least one polypeptide having pamX activity, and o) at least one polypeptide having pamY activity, and p) at least one polypeptide having pamS activity, preferably having at least one of those activities a) to p) up-regulated. These different activities can be encoded in a gene cluster. Accordingly, the invention does also comprise pamamycin producer organism as described above, comprising i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39, ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39 and comprising at least one expression cassette for a) at least one polypeptide having pamC activity, and b) at least one polypeptide having pamG activity, and c) at least one polypeptide having pamF activity, and d) at least one polypeptide having pamA activity, and e) at least one polypeptide having pamB activity, and f) at least one polypeptide having pamD activity, and g) at least one polypeptide having pamE activity, and h) at least one polypeptide having pamO activity, and i) at least one polypeptide having pamK activity, and j) at least one polypeptide having pamJ activity, and k) at least one polypeptide having pamM activity, and I) at least one polypeptide having pamN activity, and m) at least one polypeptide having pamL activity, and n) at least one polypeptide having pamX activity, and o) at least one polypeptide having pamY activity, and p) at least one polypeptide having pamS activity, iii) two or more fragments of the polynucleotides of i) or ii) wherein the fragments comprise functional expression cassettes for one or more of the polypeptides of a) to p) and wherein the fragments cover at least the whole sequence of a polynucleotide of i) or ii), if the fragments are combined.

Preferably all the pamamycin producer organisms described above are of the genus Streptomyces, preferably being of the species Streptomyces alboniger, Streptomyces aurantiacus or Streptomyces albus. Even more preferred they are of the species Streptomyces albus.

The pamamycin producer organisms described above can be used in methods to produce pamamycins, in particular pamamycin 579, 607 and 621. Accordingly, the invention comprises methods for the production of pamamycin comprising the steps of: i) cultivating a pamamycin producer organism as described above under conditions which allow for the production of pamamycin by said recombinant microorganism; ii) obtaining produced pamamycin, preferably obtaining pamamycin 607 and/or pamamycin 621.

Further methods of the invention are methods to enhance the production of pamamycin 579 in a pamamycin producer organism comprising i) decreasing the crotonyl-CoA carboxylase activity of said pamamycin producer organism and ii) cultivating the pamamycin producer organism of i) under conditions which allow for the production of pamamycin by said pamamycin producer organism.

Another embodiment of the invention is a method for the production of pamamycin 579 comprising the steps of, i) cultivating a pamamycin producer organism as described above, comprising a decreased crotonyl-CoA carboxylase activity under conditions which allow for the production of pamamycin by said pamamycin producer organism and ii) obtaining produced pamamycin 579.

A further method of the invention is a method to enhance the production of pamamycin, preferably the production of pamamycin 607 or pamamycin 621 or both, in a pamamycin producer organism comprising the steps of: i) cultivating a recombinant microorganism as described above, comprising a decreased crotonyl-CoA carboxylase activity under conditions which allow for the production of pamamycin by said pamamycin producer organism and ii) obtaining produced pamamycin, preferably obtaining produced pamamycin 607 or pamamycin 621 or both. Preferably the methods described above use a pamamycin producer organism comprising a decreased methylmalonyl-CoA mutase activity.

Also included by the invention is every use of a pamamycin producer organism as described herein for the production of pamamycins

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schows a schematic drawing of the metabolic pathway leading to pamamycins. The first step involves the activity of pamA, followed by the activity of pamB, pamD and the activity of pamE. The pathway splits in order to produce the compound S-chain via the activities of pamO, pamM, pamN, pamS and to procude the compound K-chain via the activities of pamF, pamG, pamO, pamM, pamN and pamS. Which is then further modified to produce the compound L-chain via the activities of pamX and pamY. The produced compounds S-chain and L-chain are combined with Coenzyme A (CoASH) via the activitiy of pamL. The final pamamycins are then produced by the combination of one compound S- chain-SCoA with one compound L-chain-SCoA, mediated by the activity of pamJ and pamK. The residues described as R1 , R2, R3, R4 and R5 of all formulas are the same residues as listed in the definition of Formula (I) given below.

Figure 2a depicts the relative production level of pamamycins in different Streptomyces albus strains. S. albus J 1074 R2 is a Streptomyces albus J 1074 strain comprising the cosmid R2, which contains the pamamycin biosynthesis gene cluster of SEQ ID NO: 39. S. albus Dell DelCCR2 DelMCM I R2 is a Streptomyces albus J 1074 strain comprising the cosmid R2, but also deletions of both crotonyl-CoA carboxylase genes (CCR1 and CCR2) and a deletion of the methylmalonyl-CoA mutase (MCM 1 ) gene, see Example 3. S. albus penta k.o. R2 is a Streptomyces albus J 1074 strain comprising the cosmid R2, but also deletions of both crotonyl-CoA carboxylase genes (CCR1 and CCR2), a deletion of the methylmalonyl-CoA mutase (MCM 1) gene and deletions of both acetyl/propionyl CoA carboxylase genes (PCC1 and PCC2), see Example 6. The pamamycin production level of the strain S. albus J 1074 R2 is set to 100%.

Figure 2b depicts the relative production level of pamamycins in different Streptomyces albus strains with or without high amounts of NH4+ in the growth medium.

The strains S. albus J 1074 R2 and S. albus penta k.o. R2 have been described in the description of Figure 2a. Both strains have been cultivated with normal amounts of NH4+ and with the high amount of 50 mM NH4+. Both strains produce less pamamycins when the level of NH4+ is enhanced to 50 mM. The pamamycin production level of the strain S. albus J 1074 R2 without additional NH4+ is set to 100%.

GENENERAL DEFINITIONS

The term "pamamycin" means a compound of Formula (I)

Table 1 :

Pamamycin R1 R2 R3 R4 R5

593 CH₃ H CHs CHs H

607 CHs CHs CHs CHs H

621A CHs CHs CHs CHs CHs

621 B CH2CH3 H CHs CHs CHs 621 C CH₃ CH₃ CH2CH3 CH₃ H

621 D CH2CH3 CHs CH₃ CHs H

635A CH₃ CH₃ CH2CH3 CH₃ CH₃

635B CHs CHs CHs CH2CH3 CHs

635C CH2CH3 CH₃ CH₃ CHs CH₃

635D CH2CH3 CHs CHs CH2CH3 H

635E CHs CH₃ CH2CH3 CH2CH3 H

635F CH2CH3 CHs CH2CH3 CH₃ H

649A CH2CH3 CH₃ CH2CH3 CH2CH3 H

649B CH2CH3 CHs CH2CH3 CHs CHs

Formula (I)

The term "pamamycin 579" means a compound of Formula (I), having the respective residues R1 , R2, R3, R4 and R5, of which two are CHs and three are H.

The term "pamamycin 593" means a compound of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 as defined in Table 1. The term "pamamycin 607" means a compound of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 as defined in Table 1.

The term "pamamycin 621 " means compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621 C, or 621 D as defined in Table 1. Usually the term pamamycin 621 refers to a mixture of at least two of 621 A, 621 B, 621 C and 621 D. The terms "pamamycin 621 A", "pamamycin 621 B", "pamamycin 621 C"and "pamamycin 621 D" mean compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621 C, or 621 D as defined in Table 1. The term "pamamycin 635" means compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 635A, 635B, 635C, 635D, 635E, or 635F as defined in Table 1. Usually the term pamamycin 635 refers to a mixture of at least two of 635A, 635B, 635C, 635D, 635E, and 635F. The terms "pamamycin 635A", "pamamycin 635B",

"pamamycin 635C", "pamamycin 635D", "pamamycin 635E"and "pamamycin 35F" mean compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621 C, 621 D, 621 E or 621 F as defined in Table 1.

The term "pamamycin 649" means a mixture of compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 649A or 649B as defined in Table 1. The terms "pamamycin 649A"and "pamamycin 649B" mean compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 649A, or 6249B as defined in Table 1. The term "S-chain" means compounds of Formula (II)

Formula (II)

R4 and R5 can be H, CH₃ or CH2CH3 in combinations as shown for R4 and R5 in Table 1. SCoA, representing bound Coenzyme A, can also be OH, preferably it is OH.

The term "L-chain" means a compound of Formula (III),

AND Enantiomer

Formula (III)

R1 , R2 and R3 can be H, CH3 or CH2CH3 in combinations as shown for R1 , R2 and R3 in Table 1. SCoA, representing bound Coenzyme A, can also be OH, preferably it is OH. The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values-set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower), preferably 15 percent, more preferably 10 percent and most preferably 5 percent.

The term "genome" or "genomic DNA" is referring to the heritable genetic information of a host organism. Said genomic DNA comprises the entire genetic material of a cell or an organism, including the DNA of the nucleus (chromosomal DNA), extrachromosomal DNA, and organellar DNA (e.g. of mitochondria). Preferably, the terms genome or genomic DNA is referring to the chromosomal DNA of the nucleus.

The term "chromosomal DNA" or "chromosomal DNA sequence" is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chromatids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated and analyzed by various methods known in the art like e.g., polymerase chain reaction (PCR) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), in situ PCR and next generation sequencing (NGS).

The term "promoter" refers to a polynucleotide which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5' region of a gene/operon, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter.

The term "enhancer" refers to a polynucleotide. An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription. Usually an enhancer is located close to a promoter, a 5'-untranslated sequence or in an intron.

A polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype or variety).

"Transgene", "transgenic" or "recombinant" refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man. For instance, a transgenic expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.

In case the term "recombinant" is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene",

"transgenic" or "recombinant" polynucleotide, which is usually specified later on. The term "recombinant" comprises also "man-made mutations" or "man-made mutants" Both are not isolated from nature, but are the result of artificial mutagenesis and/or selection pressure created by interference from man. Usually such mutations are produced by exposing the respective organisms to mutagenic chemicals or mutagenic irradiation . Man-made mutants are microorganisms comprising man-made mutations.

A polynucleotide "exogenous to" an individual organism is a polynucleotide which is introduced into the organism by any means other than by a sexual cross. The terms "operable linkage" or "operably linked" are generally understood as meaning an arrangement in which a genetic control sequence, e.g. a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide. Function, in this context, may mean for example control of the expression, i.e. transcription and/or translation, of the nucleic acid sequence. Control, in this context, encompasses for example initiating, increasing, governing or suppressing the expression, i.e. transcription and, if appropriate, translation. Controlling, in turn, may be, for example, growth stage- and / or cell type-specific. It may also be inducible, for example by certain chemicals, stress, pathogens and the like.

Preferably, operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed. An operably linkage does not necessarily require a direct linkage in the chemical sense. Genetic control sequences such as, for example, enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked. Preferred arrangements are those in which the nucleic acid sequence to be expressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another. The distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette, is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. The skilled worker is familiar with a variety of ways in order to obtain such an expression cassette. However, an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion. Such constructs are likewise understood as being expression cassettes for the purposes of the invention.

The term "expression cassette" means those construct in which the nucleic acid sequence encoding an amino acid sequence to be expressed is linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation). The expression may be, for example, stable or transient, constitutive or inducible.

The terms "express," "expressing," "expressed" and "expression" refer to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at a level that the resulting enzyme activity of this protein encoded for, or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene/pathway is expressed. The expression can be done by genetic alteration of the microorganism that is used as a starting organism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism or in a comparable microorganism which has not been altered. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).

In some embodiments, a microorganism can be physically or environmentally altered to express a gene product at an increased or lower level relative to level of expression of the gene product unaltered microorganism. For example, a microorganism can be treated with, or cultured in the presence of an agent known, or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.

The terms "deregulate," "deregulated" and "deregulation" refer to alteration or modification of at least one gene in a microorganism, wherein the alteration or modification results in increasing efficiency of production of a given compound in the microorganism relative to production in absence of the alteration or modification. In some embodiments, a gene that is altered or modified encodes an enzyme in a biosynthetic pathway, or a transport protein, such that the level or activity of the biosynthetic enzyme in the microorganism is altered or modified, or that the transport specificity or efficiency is altered or modified. In some embodiments, at least one gene that encodes an enzyme in a biosynthetic pathway, i.e. a polypeptide bringing about a specific activity in the biosynthetic pathway, is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene.

Deregulation also includes altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity. Also, deregulation further encompasses genetic alteration of genes encoding transcriptional factors (e.g., activators, repressors) which regulate expression of genes coding for enzymes or transport proteins. The terms "deregulate," "deregulated" and "deregulation" can further be specified in regard to the kind of deregulation present.

In case the particular activity is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene, the term "up-regulated" is used. In case particular activity is altered or modified such that the level or activity of the enzyme is lowered or decreased relative to the level in presence of the unaltered or wild type gene, the term "down -regulated" is used.

Preferably the recombinant microorganism having an "up-regulated" activity comprises additional expression cassettes for the expression of polypeptides having the respective up- regulated activity and/or may have an modified or exchanged promoter region for the respective endogenous expression cassette, in order to produce more of the polypeptide. However, the activities may be also up-regulated according to other technologies known to a person skilled in the art, for example but not excluding others, modifiying the mRNA sequence in order to enhance its stability or promote its translation, by using a codon optimized mRNA sequence, or by down-regulating an repressor of the respective activity or expression of the polypeptide providing the respective activity.

Similar means, but having the opposite effects, can be applied in order to "down-regulate" a particular activity.

The term "deregulated" includes expression of a gene product at a level lower or higher than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. In one embodiment, the microorganism can be genetically manipulated (e.g., genetically engineered) to express a level of gene product at a lesser or higher level than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein).

The term "deregulated gene activity" also means that a gene activity is introduced into a microorganism where the respective gene activity, has not been observed before, e.g. by introducing a recombinant gene, e.g. a heterologous gene, in one or more copies into the microorganism preferably by means of genetic engineering. The phrase "deregulated pathway or reaction" refers to a biosynthetic pathway or reaction in which at least one gene that encodes an enzyme in a biosynthetic pathway or reaction is altered or modified such that the level or activity of at least one biosynthetic enzyme is altered or modified. The phrase "deregulated pathway" includes a biosynthetic pathway in which more than one gene has been altered or modified, thereby altering level and/or activity of the corresponding gene products/enzymes. In some cases the ability to

"deregulate" a pathway (e.g., to simultaneously deregulate more than one gene in a given biosynthetic pathway) in a microorganism arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed a "cluster" or "gene cluster" In other cases, in order to deregulate a pathway, a number of genes must be deregulated in a series of sequential engineering steps.

To express the deregulated genes according to the invention, the DNA sequence encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into either microorganism. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.

The terms "overexpress", "overexpressing", "overexpressed" and "overexpression" refer to expression of a gene product, in particular to enhancing the expression of a gene product at a level greater than that present prior to a genetic alteration of the starting

microorganism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins,

suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). Another way to overexpress a gene product is to enhance the stability of the gene product to increase its life time.

The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).

Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31 , 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61 , AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1 ): 276-280 (2002) & The Pfam protein families database: R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman Nucleic Acids Research (2010) Database Issue 38:D21 1 -222). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31 :3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).

Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package

(Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1 ); 195-7).

Typically, this involves a first BLAST involving BLASTing a query sequence against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived. The results of the first and second BLASTS are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.

The term "sequence identity" between two nucleic acid sequences is understood as meaning the percent identity of the nucleic acid sequence over in each case the entire sequence length which is calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:

Gap Weight: 12 Length Weight: 4 Average Match: 2,912 Average Mismatch :-2,003

The term "sequence identity" between two amino acid sequences is understood as meaning the percent identity of the nucleic acid sequence over in each case the entire sequence length which is calculated by alignment with the aid of the program algorithm GAP

(Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:

Gap Weight: 8 Length Weight: 2

Average Match: 2,912 Average Mismatch:-2,003

The term "hybridisation" as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.

The term "stringency" refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt

concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below T_m, and high stringency conditions are when the temperature is 10°C below T_m. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.

The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The T_m is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16°C up to 32°C below T_m. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1 °C per % base mismatch. The T_m may be calculated using the following equations, depending on the types of hybrids: 1 ) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

T_m= 81 .5°C + 16.6xlogi₀[Na⁺]^a + 0.41x%[G/C^b] - 500x[L^c]-¹ - 0.61x% formamide

2) DNA-RNA or RNA-RNA hybrids:

T_m= 79.8°C+ 18.5 (logi₀[Na⁺]^a) + 0.58 (%G/C^b) + 1 1 .8 (%G/C^b)² - 820/L^c

3) oligo-DNA or oligo-RNA^d hybrids:

For <20 nucleotides: T_m= 2 (l_n)

For 20- 35 nucleotides: T_m= 22 + 1 .46 (l_n)

a or for other monovalent cation, but only accurate in the 0.01- 0.4 M range.

b only accurate for %GC in the 30% to 75% range.

c L = length of duplex in base pairs.

d oligo, oligonucleotide; l_n, = effective length of primer = 2^χ(ηο. of G/C)+(no. of A T).

Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.

Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from nonspecific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background.

Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions. For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass

hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 μ g/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3^rd Edition, Cold Spring Harbor

Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).

"Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. A deletion or insertion in context of a protein sequence refers to removal or addition of one or more amino acids from or to a given protein.

Typical sizes of intra-sequence deletions or insertions are in the order of about 1 to 10 amino acids, which can be distributes in several intra-sequence insertions or deletions of one or more amino acids or may even be a insertion or deletion of 10 consecutive amino acids.

Other typical additions to a protein are N- or C-terminal elongations by several amino acids in the order of about 1 to 10 consecutive amino acids, or N- or C-terminal fusion proteins or peptides. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag* 100 epitope, c-myc epitope, FLAG^®- epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a -helical structures or β -sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below). Table 2: Examples of conservative amino acid substitutions

Reference herein to an "endogenous" gene not only refers to the gene in question as found in an organism in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re) in traduced into a microorganism (a transgene). For example, a transgenic microorganism containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.

The term "vector", preferably, encompasses phage, plasmid, fosmid, cosmid, viral vectors as well as artificial chromosomes, such as bacterial artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector encompassing the polynucleotide of the present invention, preferably, further comprises selectable markers for propagation and/or selection in a recombinant microorganism. The vector may be incorporated into a recombinant microorganism by various techniques well known in the art. If introduced into a recombinant microorganism, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

The terms "transformation" and "transfection", conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a recombinant microorganism, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Methods for many species of

microorganisms are readily available in the literature, for example, in Turgeon (2010) Molecular and cell biology methods for fungi, p3-9, in Koushki, MM et al., (201 1 ), AFRICAN JOURNAL OF BIOTECHNOLOGY Vol.10 (41): p7939-7948, in Coyle et al. (2010) Appl Environ Microbiol 76:3898- 3903, in Current Protocols in Molecular Biology, Chapter 13. Eds Ausubel F.M. et al. Wiley & Sons, U.K., and in Genome Analysis: A Laboratory Manual, Cloning Systems. Volume 3. Edited by Birren B, Green ED, Klapholz S, Myers RM,

Riethman H, Roskams J. New York: Cold Spring Harbor Laboratory Press; 1999:297-565; Kieser et al. (2000), Practical Streptomyces Genetics, John Innes Foundation.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In a first aspect, the invention provides for improved pamamycin producer organisms.

Pamamycin producer organisms in general are organisms, preferably microorganisms, which have the capacity to produce pamamycins. Naturally occurring pamamycin producer organisms have been described in the literature. Some strains of pamamycin producer organisms have been deposited. Deposited strains are, for example but not excluding others, Streptomyces alboniger ATCC 12461 also deposited as DSM 40043 and IFO 12738 and isolated in 1952 from forest soil in Wisconsin (USA), Streptomyces aurantiacus ATCC 19822 also deposited as DSM 40412, Streptomyces aurantiacus I MET 43917,

Streptomyces aurantiacus JA 4570 and Streptomyces kitasatoensis JCM5001. These strains are deposited under the respective deposit numbers and disclosed in DE4134168, DE4316836, US4283391 , JP62135476A, in Hashimoto et al. in Biosci. Biotechnol. Biochm. 2003, Vol 67(4), pages 803 to 808 and/or HAERTL et al. THE JOURNAL OF ANTIBIOTICS (1998) VOL. 51 , NO. 1 1 , pp. 1040-1046.

However, pamamycin producer organism can also be created by producing recombinant microorganisms. Such recombinant microorganism and methods to produce them have been disclosed in WO2015/092575. WO2015/092575 discloses nucleic acid sequences which encode polypeptides being involved in pamamycin synthesis which could be used to create new pamamycin producer organism, to enhance the production of a naturally occurring pamamycin producer organism or to identiy new strains and species having the capacity to produce pamamycins. Suitable assays to identify and measure the amount of pamamycin produced by such organisms are known by the person skilled in the art and are described for example in Hashimoto et al (2004) Biosynthetic Origin of the Carbon Skeleton and Nitrogen Atom of Pamamycin-607, a Nitrogen-Containing Polyketide, Biosci.

Biotechnol. Biochem. 69; Hashimoto et al. (201 1 ) Effect of Pamamycin-607 on Secondary Metabolite Production by Streptomyces spp., Biosci. Biotechnol. Biochem. 75; Hashimoto et al. (2003) Relationship between Response to and Production of the Aerial Mycelium- inducing Substances Pamamycin-607 and A-factor, Biosci. Biotechnol. Biochem. 67;

Natsume et al. (1991 ) The Structures of Four New Pamamycin Homologues Isolated from Streptomyces alboniger, Tetrahedron Letters 32.

Pamamycin producer organisms preferably comprise a gene cluster for the production of pamamycins having a polynucleotide sequence, which is having at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39. Preferably this gene cluster comprises at least one expression cassette for each one of the (ORFs) listed in Table 3, or for variants of these ORF which encode an amino acid sequence being at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequences encoded by the ORFs of Table 3 and having the same activity.

Table 3: Listing of genes and encoded polypeptides of the polnucleotidesequence described by SEQ ID NO: 39:

Gene Gene Starting Endpoint Seq Function Correspond in

Number Name point in in SEQ ID I D g protein Seq

(ORF) SEQ ID No No 1 NO: ID NO:

1

0726 pamR2 18 755 129 TetR type 130 regulator

0727 pamW 843 2519 136 transporter 137

0729 pamC 2616 2846 3 ACP 4

0730 pamG 2900 3934 10 KS 1 1

0731 pamF 3946 5265 17 KS 18

0732 pamA 5477 6814 24 KS 25

0733 pamB 681 1 8673 31 Acyl-CoA transf. 32

0734 pamD 871 1 9670 38 KS 39

0736 pamE 9723 10757 45 KS 46

0737 pamO 1081 1 1 1605 52 KR 53

0738 pamK 1 1621 12700 59 KS 60

0740 pamJ 12717 14012 66 KS 67

0741 pamM 14038 15582 73 KR 74

0742 pamN 15579 16406 80 DH/KR 81

0743 pamL 16403 18106 87 Acyl-CoA ligase 88

0744 pamD 18090 19073 122 hydrolase 123

0745 pamX 19170 20516 94 amino 95

transferase

0746 pamY 20565 21386 101 methyl 102 transferase

0747 pamS 21604 22410 108 enoyl-CoA 109 hydratase

0749 pamR1 22435 23082 1 15 LuxR Transcrf. 1 16

Table 3 provides a listing of the polypeptide (protein) encoding sequences of SEQ ID NO: 39, the respective ORF names, the number of the nucleotides in SEQ ID NO: 39, which are starting and endpoints of the polypeptide encoding sequences, the likely function of the encoded polypeptides and the respective SEQ ID NOs: of the polynucleotide and amino acid sequences in the sequence listings. The starting points and end points given in Table 3 do not necessarily represent the 5-prime and 3-prime ends of the polypeptide encoding regions.

The pamamycin producer organisms described above, naturally occurring or recombinant, can be improved by decreasing the methylmalonyl-CoA mutase activity, the crotonyl-CoA carboxylase activity and/or the acetyl/propionyl CoA carboxylase activity being present in the unmodified version of the pamamycin producer organisms.

Accordingly, the invention comprises pamamycin producer organisms, comprising a decreased activity selected from at least one of a) to c)

a) a decreased methylmalonyl-CoA mutase activity,

b) a decreased crotonyl-CoA carboxylase activity,

c) a decreased acetyl/propionyl CoA carboxylase activity,

Preferably, the pamamycin producer organism comprises at least a decreased

methylmalonyl-CoA mutase activity, more preferred, the pamamycin producer organism comprises at least a a decreased methylmalonyl-CoA mutase activity and a decreased crotonyl-CoA carboxylase activity. In some embodiments, that pamamycin producer organism comprises a decreased methylmalonyl-CoA mutase activity, a decreased crotonyl-CoA carboxylase activity and a decreased acetyl/propionyl CoA carboxylase activity.

These decreased activities are preferably by modifying the genome of the pamamycin producer organism in order to reduce the amount or activity of the enzymes providing these activities in the cell. Such genome modifications are for example the exchange of the promoter driving the transcription of the genes of these enzymes, for an less active promoter, or by inducing the expression of a transcriptional repressor of these promoters, or by exchanging the protein coding region of these genes with polynucleotides which encode less active version of these enzymes.

The reduction of these methylmalonyl-CoA mutase, crotonyl-CoA carboxylase or

acetyl/propionyl CoA carboxylase activity in the improved versions of the pamamycin producer organisms is usually compared to the particular strain of the pamamycin producer organism which has been modified. Such a decrease in activity is, preferably, statistically significant. Whether a decrease is significant can be determined by statistical tests well known in the art including, e.g., Student^'s t-test. More preferably, the increase is an increase of the amount of pamamycin of at least 5%, at least 10%, at least 15%, at least 20% or at least 30% compared to said control.

A preferred way to reduce at least one of methylmalonyl-CoA mutase, crotonyl-CoA carboxylase or acetyl/propionyl CoA carboxylase activity is the deletion of all or parts of the expression cassette encoding the enzyme for the respective activity, so that no or no active enzyme can be produced. These deletions preferably comprises the deletion of all or important parts of the amino acide sequence encoding part of the expression cassette, but may also be limited to all or parts of the promoter of those expression cassettes. A skilled person is aware of various ways to prevent the expression of protein encoding sequences by deletions of genomic regions.

The expression cassettes encoding enzymes responsible for the methylmalonyl-CoA mutase, crotonyl-CoA carboxylase or acetyl/propionyl CoA carboxylase activity present in the genome of a given pamamycin producer organisms intended to be improved can be identified by amino acid or polynucleotide homology searches.

Polypeptides having methylmalonyl-CoA mutase activity:

Polypeptides having methylmalonyl-CoA mutase activity can mediate the production of (2R)-methylmalonyl-CoA from succinyl-CoA and vice versa in a given pamamycin producer organism. This activity is usually provided by an enzyme comprising an large and a small subjunit.Preferred polypeptides of the invention are polypeptides which can complement the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 1 or the or the small polypeptide having the amio acid of SEQ ID NO: 4 from the Streptomyces albus genome, or the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 2 or the or the small polypeptide having the amio acid of SEQ ID NO: 5 from the Streptomyces aurantiacus genome, or

the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 3 or the or the small polypeptide having the amio acid of SEQ ID NO: 6 from the Streptomyces fulvissimus genome.

It is clear to the person skilled in the art, that other microorganisms comprise homologs of these sequences in their genome and that those sequences will be need to be deleted in the genome of the respective microorganism.

Accordingly, the invention comprises pamamycin producer organisms which have a decreased methylmalonyl-CoA mutase activity usually provided by a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, or 6, preferably they have a decreased activity of both of the subunits of the enzyme.

In particular preferred are pamamycin producer organism having a genomic deletion of all or parts of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, 8, or 9, or which hybridises under medium stringency

hybridisation conditions, preferably high stringency hybridisation conditions, to a

complement of SEQ ID NO: 7, 8 or 9.

Polypeptides having crotonyl-CoA carboxylase activity:

Polypeptides having crotonyl-CoA carboxylase activity can mediate the carboxylation of crotonyl-CoA to (2S)-ethylmalonyl-CoA in a given pamamycin producer organism.

Preferred polypeptides of the invention are polypeptides which can complement the deletion of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 or 13 or, preferably, both from the Streptomyces albus genome, or

the deletion of the polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 1 from the Streptomyces aurantiacus genome, or

the deletion of the polynucleotide sequence encoding a polypeltide having the amino acid sequence of SEQ ID NO: 12 from the Streptomyces fulvissimus genome.

Accordingly, the invention comprises pamamycin producer organisms which have a decreased crotonyl-CoA carboxylase activity usually provided by a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, 1 1 , 12, or 13.

In particular preferred are pamamycin producer organism having a genomic deletion of all or parts of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14 or 15, or, preferably, both or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 14 or 15, or, preferably, both.

Polypeptides having acetyl/propionyl CoA carboxylase activity:

Polypeptides having acetyl/propionyl CoA carboxylase activity can mediate the production of (2S)-methylmalonyl-CoA from propionyl-CoA in a given pamamycin producer organism. This activity is usually provided by an enzyme comprising an large and a small subjunit. Preferred polypeptides of the invention are polypeptides which can complement the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 16 or 26, or the or the small polypeptide having the amio acid of SEQ ID NO: 19 or 28 from the Streptomyces albus genome,

or the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 17 or 26, or the or the small polypeptide having the amio acid of SEQ ID NO: 20 or 29 from the Streptomyces aurantiacus genome, or the deletion of the polynucleotide sequence encoding either the large polypeptide having the amino acid sequence of SEQ ID NO: 18 or 27, or the or the small polypeptide having the amio acid of SEQ ID NO: 21 or 30 from the Streptomyces fulvissimus genome.

Accordingly, the invention comprises pamamycin producer organisms which have a decreased methylmalonyl-CoA mutase activity usually provided by a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, 17, 18, 19, 20, 21 , 25, 26, 27, 28, 29, or 30, preferably they have a decreased activity of both of the subunits of the enzyme. In particular preferred are pamamycin producer organism having a genomic deletion of all or parts of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, 23, 24, 31 , 32, or 33, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ I D NO: 22, 23, 24, 31 , 32, or 33.

Katabolic pathway from Isobutyryl-CoA to to (2S)-methylmalonyl-CoA:

The pamamycin producer organisms having at least one of the above described decreased methylmalonyl-CoA mutase, crotonyl-CoA carboxylase or acetyl/propionyl CoA carboxylase activities comprise a functional kataboliy pathway from isobutyryl-CoA to (2S)- methylmalonyl-CoA.

The enzymes mediating this pathway are well known in the art and perform the pathway steps from isobutyryl-CoA to beta-hydroxyisobutyryl-CoA,

from beta-hydroxyisobutyryl-CoA to (2S)-beta-hydroxyisobutyryl-CoA,

from (2S)-beta-hydroxyisobutyryl-CoA to (2S)-methylmalonyl-CoA semialdehyde and from (2S)-methylmalonyl-CoA semialdehyde to (2S)-methylmalonyl-CoA.

One preferred way to produce isobutyryl-CoA in the pamamycin producer organism is to degrade L-Valine via keto-isobutyrate to isobutyryl-CoA, wherein the first step is mediated by the enzyme valine-dehydrogenase.

The activity of valine-dehydrogenase is in many species of pamamycin producer organisms repressed by the presence of high amounts of ammonium-ions in the growth medium, while its activity is usually induced by the presence of additional amounts of valine in the growth medium. A person skilled in the art will be able to identify the right proportion of ammonium- ions and valine in the growth medium, in order to achieve a high activity of valine- dehycrogenase in the pamamycin producer organism.

In addition or alternatively, it is possible to exchange the promoter region of the naturally occurring expression cassette of valine-dehydrogenase with a new promoter region, or with with recombinant expression cassettes for valine-dehydrogenase, in order to achieve higher expression of valine-dehydrogenase in the presence of high ammonium-ion concentration in the medium. Polypeptide and polynucleotide sequences which can be used to construct expression cassettes for valine-dehydrogenase are will known in the art.

For example, polypeptides having the amino acid sequence of SEQ ID NO: 34, provide for the valine-dehydogenase in Streptomyces albus,

polypeptides having the amino acid sequence of SEQ ID NO: 35, provide for the valine- dehydogenase in Streptomyces aurantiacus genome, or

polypeptides having the amino acid sequence of SEQ ID NO: 36, provide for the valine- dehydogenase in Streptomyces fulvissimus genome. It is clear to the person skilled in the art, that other microorganisms comprise homologs of these sequences in their genome and that those sequences could be used to construct recombinant expression cassettes for valine-dehydrogenase in these microorganism and that homologs of valine-dehydrogenase from other organisms can be used to construct such expression cassettes for a given pamamycin producer orgaism as well.

Accordingly, the invention comprises pamamycin producer organisms which comprise recombinant expression cassettes for a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34, 35 or 36.

In particular preferred are pamamycin producer organism comprising a recombinant expression cassette comprising a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 37.

The invention comprises further pamamycin producer organisms which comprise a high activity of valine-dehydrogenase in the presence of high amounts of ammonium-ions in the growth medium, as well as a high metablic flux through the katabolic pathway leading from isobutyryl-CoA to (2S)-methylmalonyl-CoA, e.g. by overexpression of at least one of the enzymes mediating a step in this pathway.

Natural and recombinant pamamycin producer organisms: The polynucleotides and polypeptides provided above are suitable to improve pamamycin producer organisms.

Pamamycin producer organism have been described in WO2015/092575, included herein by reference in its entirety, and comprise at least one functional expression cassette for each one of the necessary polypeptides to establish the metabolic pathway for pamamycin production in such pamamycin producer organism.

Accordingly, the invention includes pamamycin producer organisms having at least one of the above described decreased methylmalonyl-CoA mutase, crotonyl-CoA carboxylase or acetyl/propionyl CoA carboxylase activities and

which comprise

a) at least one expression cassette encoding a polypeptide having pamC activity, and b) at least one expression cassette encoding a polypeptide having pamG activity, and c) at least one expression cassette encoding a polypeptide having pamF activity, and d) at least one expression cassette encoding a polypeptide having pamA activity, and e) at least one expression cassette encoding a polypeptide having pamB activity, and f) at least one expression cassette encoding a polypeptide having pamD activity, and g) at least one expression cassette encoding a polypeptide having pamE activity, and h) at least one expression cassette encoding a polypeptide having pamO activity, and i) at least one expression cassette encoding a polypepti de having pamK activity, and j) at least one expression cassette encoding a polypepti de having pamJ activity, and k) at least one expression cassette encoding a polypepti de having pamM activity, and I) at least one expression cassette encoding a polypepti de having pamN activity, and m) at least one expression cassette encoding a polypepti de having pamL activity, and n) at least one expression cassette encoding a polypepti de having pamX activity, and o) at least one expression cassette encoding a polypepti de having pamY activity, and p) at least one expression cassette encoding a polypepti de having pamS activity.

Preferably at least one of a) to p) is up-regulated.

Up-regulation can be achieved by providing more than one expression cassette for the activities of a) to p) or by providing an expression cassette of a) to p) with a strong promoter or with a terminator which provides for enhanced stability of the transcript.

The person of skill in the art knows further methods which lead to an enhanced activity of at least one of a) to p) in a given pamamycin producer organism.

The pamamycin producer organisms comprise preferably microorganisms which have been intentionally modified via molecular biology techniques, like transformation or gene knockout or gene replacement in order to establish all necessary steps in the metabolic pathway for the production of pamamycins, or in order to enhance the production of pamamycins by providing additional expression cassettes for one or more of the polypeptides having pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity, or by achieving higher activity of these polypeptides in the pamamycin producer organism, e.g. by providing more active promoter regions or more active polypeptides for these activities. However, the term pamamycin producer organism does also apply to variants of pamamycin producer organisms, which are non-naturally occurring microorganisms. Non-naturally occurring pamamycin producer organism are microorganisms which have been created by single or repeated mutagenesis and selection performed on pamamycin producer organism isolated from nature. Non- naturally occurring pamamycin producer organism comprise at least one up-regulated activity of a polypeptide having pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity, pamR1 activity, pamW activity, and/or at least one down -regulated pamR2 activity or pamH activity, in comparison to the un-modified version of such microorganism.

Preferably, the pamamycin producer organism belongs to the group of bacteria, preferably to the group of Streptomyces or Amycolatopsis.

Preferred bacteria of the group Streptomyces or Amycolatopsis are selected from the group consisting of: Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces achromogenes, Streptomyces acidiscabies, Streptomyces albus, Streptomyces ambofaciens, Streptomyces antibioticus, Streptomyces aureofaciens, Streptomyces aurantiacus, Streptomyces avermitilis, Streptomyces capreolus, Streptomyces carcinostaticus, Streptomyces cervinus, Streptomyces clavuligerus,

Streptomyces coelicolor, Streptomyces coeruleorubidus, Streptomyces cyaneofuscatus, Streptomyces davawensis, Streptomyces diastaticus, Streptomyces fradiae, Streptomyces fulvissimus, Streptomyces ghanaensis, Streptomyces griseus, Streptomyces griseoflavus, Streptomyces griseoviridus, Streptomyces hirsutus, Streptomyces hygroscopicus,

Streptomyces kanamyceticus, Streptomyces lavendulae, Streptomyces lividans,

Streptomyces loidensis, Streptomyces natalensis, Streptomyces noursei, Streptomyces nodosum, Streptomyces olivaceus, Streptomyces platensis, Streptomyces peuceticus, Streptomyces pristinaespiralis, Streptomyces rimosus, Streptomyces roseosporus,

Streptomyces sviceus, Streptomyces spectabilis, Streptomyces tauricus, Streptomyces tendae, Streptomyces thioluteus, Streptomyces toxytricini, Streptomyces venezuelae, Streptomyces violaceoniger, Streptomyces violaceoruber, Streptomyces violaceus,

Streptomyces viridochromogenes, Streptomyces violaceoruber and Streptomyces zaomyceticus.

In particular the Streptomyces species Streptomyces albus, Streptomyces auratus,

Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces clavuligerus,

Streptomyces fulvissimus, Streptomyces ghanaensis, Streptomyces griseoflavus,

Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces pristinaespiralis, Streptomyces roseosporus, Streptomyces roseosporus, Streptomyces sviceus

Streptomyces viridochromogenes, Streptomyces violaceoruber.

Preferred Streptomyces strains are: Streptomyces alboniger ATCC 12461, Streptomyces alboniger DSM 40043, Streptomyces alboniger DSM 40412, Streptomyces albus B24108, Streptomyces albus J 1074, Streptomyces albus DSM40313, Streptomyces avermitilis SUKA17 or Streptomyces avermitilis SUKA22, Streptomyces coelicolor DSM40233, Streptomyces kitasatoensis JCM5000, Streptomyces kitasatoensis JCM5001 Streptomyces lividans ATCC69444.

In particular preferred are microorganisms which already have the capacity to produce pamamycin such as, but not excluding others: Streptomyces alboniger ATCC 12461 Streptomyces alboniger DSM 40043, Streptomyces aurantiacus ATCC 19822 and

Streptomyces kitasatoensis JCM5001.

A person skilled in the art will understand that the deposit number provided for the description of the strains are only examples and that the same strains may also be accessible under different deposit numbers at other strain collections.

Polypeptides having pamC activity:

A polypeptide having pamC activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 40, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 41 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capable to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 40. Preferably it is the same promoter. The invention provides for polypeptides having pamC activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 41.

Guidance on how to produce further variants can be deduced by the information provided by Figure 3 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamG activity:

A polypeptide having pamG activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 42, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 43, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 42. Preferably it is the same promoter.

The invention provides for polypeptides having pamG activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 43.

Guidance on how to produce further variants can be deduced by the information provided by Figure 4 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamF activity:

A polypeptide having pamF activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 44, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 45, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 44. Preferably it is the same promoter.

The invention provides for polypeptides having pamC activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 45. Guidance on how to produce further variants can be deduced by the information provided by Figure 5 and its description disclosed in WO2015/092575, both of which are included herein by reference. Polypeptides having pamA activity:

A polypeptide having pamA activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 46, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 47, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 46. Preferably it is the same promoter.

The invention provides for polypeptides having pamA activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ I D NO: 47.

Guidance on how to produce further variants can be deduced by the information provided by Figure 6 and its description disclosed in WO2015/092575, both of which are included herein by reference. Polypeptides having pamB activity:

A polypeptide having pamB activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 48, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 49, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 48. Preferably it is the same promoter.

The invention provides for polypeptides having pamB activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 49.

Guidance on how to produce further variants can be deduced by the information provided by Figure 7 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamD activity:

A polypeptide having pamD activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 50, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 51 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 50. Preferably it is the same promoter.

The invention provides for polypeptides having pamD activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51 , or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 51.

Guidance on how to produce further variants can be deduced by the information provided by Figure 8 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamE activity:

A polypeptide having pamE activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 52, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 53, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 52. Preferably it is the same promoter.

The invention provides for polypeptides having pamE activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 53.

Guidance on how to produce further variants can be deduced by the information provided by Figure 9 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamO activity:

A polypeptide having pamO activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 54, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 55, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 54. Preferably it is the same promoter.

The invention provides for polypeptides having pamO activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ I D NO: 54, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 555, or

are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ I D NO: 55.

Guidance on how to produce further variants can be deduced by the information provided by Figure 10 and its description disclosed in WO2015/092575, both of which are included herein by reference. Polypeptides having pamK activity:

A polypeptide having pamK activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 56, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 57, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 56. Preferably it is the same promoter.

The invention provides for polypeptides having pamK activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 57.

Guidance on how to produce further variants can be deduced by the information provided by Figure 1 1 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamJ activity:

A polypeptide having pamJ activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 58, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 59, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 58. Preferably it is the same promoter.

The invention provides for polypeptides having pamJ activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, or are expressed from a polynucleotide which hybridises under medium stringency

hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 59.

Guidance on how to produce further variants can be deduced by the information provided by Figure 12 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamM activity:

A polypeptide having pamM activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 60, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 61 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 60. Preferably it is the same promoter.

The invention provides for polypeptides having pamM activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61 , or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 61.

Guidance on how to produce further variants can be deduced by the information provided by Figure 13 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamN activity:

A polypeptide having pamN activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 62, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 63, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 62. Preferably it is the same promoter.

The invention provides for polypeptides having pamN activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 62, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 63, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 63.

Guidance on how to produce further variants can be deduced by the information provided by Figure 14 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamL activity:

A polypeptide having pamL activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 64, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 65, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 64. Preferably it is the same promoter.

The invention provides for polypeptides having pamL activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 65.

Guidance on how to produce further variants can be deduced by the information provided by Figure15 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamX activity:

A polypeptide having pamX activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 66, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 67, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 66. Preferably it is the same promoter. The invention provides for polypeptides having pamX activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 67.

Guidance on how to produce further variants can be deduced by the information provided by Figure 16 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamY activity: A polypeptide having pamY activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 68, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 69, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 68. Preferably it is the same promoter.

The invention provides for polypeptides having pamY activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ I D NO: 69.

Guidance on how to produce further variants can be deduced by the information provided by Figure 17 and its description disclosed in WO2015/092575, both of which are included herein by reference. Polypeptides having pamS activity:

A polypeptide having pamS activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 70, or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 71 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 70. Preferably it is the same promoter.

The invention provides for polypeptides having pamS activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71 , or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 71.

Guidance on how to produce further variants can be deduced by the information provided by Figure 18 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamR1 activity:

A polypeptide having pamR1 activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 72, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 73, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 72. Preferably it is the same promoter.

The invention provides for polypeptides having pamR1 activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 72, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, or are expressed from a polynucleotide which hybridises under medium stringency

hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 73.

Guidance on how to produce further variants can be deduced by the information provided by Figure 19 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamH activity:

A polypeptide having pamH activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 74, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 75, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a lower level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 74. Preferably it is the same promoter.

The invention provides for polypeptides having pamH activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 75.

Guidance on how to produce further variants can be deduced by the information provided by Figure 20 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamR2 activity:

A polypeptide having pamR2 activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 76, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 77, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to produce pamamycin 607. A successful

complementation is achieved, if the complemented strain produces pamamycin 607 on a lower level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 76. Preferably it is the same promoter.

The invention provides for polypeptides having pamR2 activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 77.

Guidance on how to produce further variants can be deduced by the information provided by Figure 21 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Polypeptides having pamW activity:

A polypeptide having pamW activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 78, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 79, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the

complemented strain for the capability to export pamamycin 607. A successful

complementation is achieved, if the complemented strain exportes pamamycin 607 on a higher level from its cells to the growth medium than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr.

40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 78.

The invention provides for polypeptides having pamW activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78, and/or

are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 79, or are expressed from a polynucleotide which hybridises under medium stringency

complement of SEQ ID NO: 79.

Guidance on how to produce further variants can be deduced by the information provided by Figure 22 and its description disclosed in WO2015/092575, both of which are included herein by reference.

Variants of the polypeptides having pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY, pamS, pamR1 , pamR2, pamW or pamH activity can be obtained by techniques known to a person skilled in the art from pamamycin producing organisms. Preferred pamamycin producing microorganism are: Streptomyces alboniger ATCC 12461 Streptomyces alboniger DSM 40043, Streptomyces alboniger I F012738, Streptomyces aurantiacus ATCC 19822 and Streptomyces

kitasatoensis J CM 5001. Polypeptides having pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY, pamS, pamR1 , pamR2, pamW or pamH activity have been found to be organized in gene clusters (Rebets, Y., et al., Insights into the pamamycin biosynthesis. (2015) Angew. Chem. Int. Ed. Engl. 54 : 2280-2284).

One of these gene clusters (SEQ ID NO: 38) can be found in Streptomyces alboniger DSM 40043. A longer version of this gene cluster comprising additional expression cassettes is provided herein as SEQ ID NO: 39.

Preferably the recombinant microorganisms comprise at least one expression cassette for each one of the polypeptides providing pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY and pamS activity, which has at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the respective polypeptide encoded by the respective polypeptide encoding region of SEQ ID NO: 38 and/or 39. One way to produce recombinant microorganisms of the invention is to transform

microorganisms with polynucleotides comprising expression cassettes for one or more of the polypeptides providing pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY and pamS activity. Accordingly, the invention comprises recombinant microorganisms comprising

i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39, ϋ) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39 and comprising at least one expression cassette for

at least one polypept de having pamC activity, and

at least one polypept de having pamG activity, and

at least one polypept de having pamF activity, and

at least one polypept de having pamA activity, and

at least one polypept de having pamB activity, and

at least one polypept de having pamD activity, and

at least one polypept de having pamE activity, and

at least one polypept de having pamO activity, and

at least one polypept de having pamK activity, and

at least one polypept de having pamJ activity, and

at least one polypept de having pamM activity, and

at least one polypept de having pamN activity, and

at least one polypept de having pamL activity, and

at least one polypept de having pamX activity, and

at least one polypept de having pamY activity, and

at least one polypept de having pamS activity

iii) two or more fragments of the polynucleotides of i) or ii) wherein the fragments

comprise functional expression cassettes for one or more of the polypeptides of a) to p) and wherein the fragments cover at least the whole sequence of a polynucleotide of i) or ii), if the fragments are combined.

Preferably the polynucleotide of ii) differs mainly in the intergenic regions, promoters and/or terminators from the sequence of SEQ ID NO: 38 and/or 39. Hence, the polynucleotide of ii) may have at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, sequence identity to SEQ ID NO: 1 and/or 2, and comprises polypeptide encoding regions for the polypeptides providing pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY and pamS activity, which have at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the

polypeptides described by the respective amino acid sequence of SEQ ID NOs: 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. Preferably the are encoded by polynucleotides which have at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotides described by the respective polynucleotide sequence of SEQ ID NOs: 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77 and 79.

Further variants of the disclosed polynucleotides can be constructed, e.g. by adapting the codon usage of polypeptide encoding nucleic acid sequences to the codon usage of a preferred species of microorganism, or by exchanging promoter regions and/or terminator regions or both of an expression cassette in order to adapt the expression of an encoded polynucleotide to a preferred species of microorganism or culture conditions.

Promoters, terminators and information about codon usage suitable to be used for a particular microorganism are known by a person skilled in the art.

Preferred promoters for microorganisms of the genus Streptomyces are the promoters known be a person skilled in the art. Examples of such promoters, but not excluding others, are:

the ermeE*p promoter (Schmitt-John and Engels, 1992, Promoter constructions for efficient expression in Streptomyces lividans, Appl Micobiol Biotechnol, 36, 493-498), having constitutive expression, the tipSp promoter (Holmes et al. 1993, Autogenous transcriptional activation of a thiostrepton-induced gene in Streptomyces lividans, EMBO J, 12, 3183- 3191 ), being inducible with thiostreptone,

the chi63p promoter (Ingram and Westpheling, 1995, ccrA1 : mutation in Streptomyces coelicolor that affects the control of catabolite repression, J Bacteriol, 177, 3579-3586) the gylCABp (Hindle et al, 1994, Substrate induction and catabolite repression of the Streptomyces coelicolor glycerol operon are mediated through the GylR protein, Mol Micobiol, 12, 737-745), being inducible with glycerol,

the mcrABp promoter (August et al., 1996, Inducible synthesis of the mitomycin C resistance gene product (MCRA) from Streptomyces lavendulae, Gene, 175, 261 -267), being inducible with mitomycin C

Other suitable promoters can for example be found in Siegl, T et al. METABOLIC

ENGINEERING (2013) Volume:19, Pages:98-106, disclosing also sequence variants of these promoters.

Methods for production of pamamycins and cultivating pamamycin producer organism:

The term "cultivating" as used herein refers maintaining and growing the pamamycin producer organism under culture conditions which allow the cells to produce pamamycin. This implies that the polynucleotides of the present invention are expressed in the pamamycin producer organism so that the encoded polypeptides are present in in a biologically active form. Suitable culture conditions for cultivating the pamamycin producer organism are well known in the art, some variants of these methods are also disclosed in WO2015/092575.

In particular, pamamycin producer organism as described herein can be cultured using, for example, glucose, sucrose, maltose, honey, dextrin, starch, glycerol, molasses, animal or vegetable oils and the like as the carbon source for the culture medium. Furthermore, soybean flour, wheat germ, corn steep solids, corn steep liquor, cotton seed waste, meat extract, polypeptone, malt extract, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used for the nitrogen source. The addition of inorganic salts which can produce sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid (di- potassium hydrogen phosphate and the like), sulfuric acid (magnesium sulfate and the like) and other ions as required is also effective. Furthermore, various vitamins such as thiamine (thiamine hydrochloride and the like), amino acids such as glutamine (sodium glutamate and the like), asparagine (DL-asparagine and the like), trace nutrients such as nucleotides and the like, and selection drugs such as antibiotics and the like can also be added as required. Moreover, organic substances and inorganic substances can be added

appropriately to assist the growth of the microorganism and promote the production of pamamycin and/or to promote the preduction of pamamycin precursors. The pH of the culture medium is, for example, of the order of pH 4.5 to pH 8. The culturing can be carried out with a method such as the solid culturing method under aerobic conditions, the concussion culturing method, the air-passing agitation culturing method or the deep aerobic culturing method, but the deep aerobic culturing method is the most suitable. The

appropriate temperature for culturing is from 15°C to 40°C, but in many cases growth occurs in the range from 20°C to 30°C. Usually a temperature of about 28°C is used. The production of pamamycin or precursors of pamamycin differs according to the culture medium and culturing conditions, or the host which is being used, but with any culturing method the accumulation of pamamycin reaches a maximum generally in from 5 to 20 days. The culturing is stopped when the amount of pamamycin or precursors of pamamycin in the culture reaches its highest level and the target material is isolated from the culture and refined for isolating pamamycin or precursor of pamamycin from the culture material.

Examples for such conditions which allow for the production of pamamycin are disclosed in US4283391 , DE4134168 and DE4316836 which are included herein by reference in their entirety.

The term "obtaining" as used herein encompasses the provision of the cell culture including the recombinant microorganisms and the culture medium as well as the provision of purified or partially purified preparations thereof comprising pamamycin or a precursor thereof, preferably, in free form. More details on purification techniques can be found elsewhere herein below. The usual methods of extraction and refinement which are generally used in these circumstances, such as methods of isolation such as solvent extraction, methods involving ion exchange resins, adsorption or partition chromatography, gel filtration, dialysis, precipitation, crystallization and the like can be used either individually or in appropriate combinations. In particular, pamamycin can be isolated from a pamamycin containing medium or cell lysate, or from both, using any applicable method, for example but not excluding others, methods which have been described in the prior art for the isolation of pamamycin.

A particular group of pamamycin producer organism having a decreased crotonyl-CoA carboxylase activity produce less pamamycin 635, but produce more pamamycin 579.

Accordingly, the invention does further comprise methods for the production of pamamycin 579, which comprise the following steps:

i) cultivating a pamamycin producer organism, comprising at least a decreased crotonyl- CoA carboxylase activity under conditions which allow for the production of

pamamycin by said pamamycin producer organism and ii) obtaining produced pamamycin 579.

Preferably the pamamycin producer organism does further comprise a decreased methylmalonyl-CoA mutase activity and/or a decreased acetyl/propionyl CoA carboxylase activity.

The invention does further comprise a method to enhance the production of pamamycin 579 in a pamamycin producer organism, comprising the steps of

i) decreasing the crotonyl-CoA carboxylase activity of said pamamycin producer

organism and

ii) cultivating the pamamycin producer organism of i) under conditions which allow for the production of pamamycin by said pamamycin producer organism.

Another embodiment of the invention is a method to enhance the production of pamamycin, preferably the production of pamamycin 607 or pamamycin 621 or both, in a pamamycin producer organism comprising

i) cultivating a recombinant microorganism as claimed in any one of claims 1 to 10, comprising a decreased crotonyl-CoA carboxylase activity under conditions which allow for the production of pamamycin by said pamamycin producer organism and ii) obtaining produced pamamycin, preferably obtaining produced pamamycin 607 or pamamycin 621 or both.

Preferably the pamamycin producer organism used the methods disclosed above does further comprise a decreased methylmalonyl-CoA mutase activity and/or a decreased acetyl/propionyl CoA carboxylase activity.

The invention comprises also the use of the pamamycin producer organism discloced herein in a method for the production of pamamycin, as well as the use of any one of the polynucleotides, expression cassettes, vectors disclosed herein in a method to produce pamamycin, in a method to identify pamaycin producing microorganisms or microorganisms potentially capable to produce pamamycin, or in a method to produce a recombinant pamamycin producer organism, or in a method to enhance the production of pamamycin in a pamamycin producer organism.

The amino acid and polynucleotide sequences cited herein have been derived from W015092575 or public available databases the respective accession numbers are listed in Table 4: Table 4:

The information presented in the database entries of the listed accession numbers, in particular the information about conserved or essential amino acids, are included herein by reference. A skilled person will be able to transfer this information to sequence variants and homologs of the respective polypeptides in order to select enzymatically active sequence variants and homologs.

The amino acid and polynucleotide sequences of the pamamycin gene cluster have been disclosed in W015092575.

The original source of Streptomyces albus J 1074 is unknown (Zaburannyi et al. Insights into naturally minimised Streptomyces albus J 1074 genome; BMC Genomics 2014, 15:97), but it has been first mentioned in 1980 (Klappenbach JA, Dunbar JM, Schmidt TM: RRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 2000,

66: 1328- 1333. in which J 1074 was referred to as a Sail system-deficient strain derived from S. albus G. Although, the origin of S. albus G is also unknown, it was used as one of S. albus strains in 1970 (Leyh-Bouille M, Bonaly R, Ghuysen JM, Tinelli R, Tipper D: LL- diaminopimelic acid containing peptidoglycans in walls of Streptomyces sp. and of

Clostridium perfringens (type A). Biochemistry 1970, 9:2944- 2952).

Streptomyces alboniger ATCC 12461 has been deposited by Porter et al. in 1951 (US2,763,642) after isolation from soil.

Streptomyces aurantiacus J A 4570 has been described to be Streptomyces aurantiacus IMET 43917 in Graefe, U.; Schlegel, R.; Ritzau, M.; et al.;Aurantimycins, new depsipeptide antibiotics from Streptomyces aurantiacus IMET 43917 production, isolation, structure elucidation, and biological activity; Journal of Antibiotics (Tokyo) Volume: 48 Issue: 2 Pages: 1 19-125 Published: 1995. Graefe et al. discloses this strain to obtained from the IMET strain collection (a subdivision of Deutsche Sammlung Mikroorganismen, Gottingen (DSM), Germany), where it was classified as Streptomyces aurantiacus according to taxonomic studies and deposited in the DSM culture collection, Gottingen, Germany, with the accession No. DSM 8818. According to Shiring, E. B. &D. Gottlieb: Cooperative description of type cultures of Streptomyces IV. Species description from the second, third and fourth studies. Int. J. Syst.Bact. 19 (4): 391-512, 1969 it had originally been isolated by Krassilnikov in China.

Streptomyces fulvissimus DSM 40593 had originally been described by Waksman, SA, Henrici, AT; Genus I. Streptomyces; In Bergey's manual of determinative bacteriology, 6th ed, Williams & Wilkins, Baltimore , 929-977, 1948

All references cited in this specification are herewith incorporated by reference with respect to their entire content and the content specifically mentioned in this specification. EXAMPLES

Example 1 : Pamamycins were analyzed as described in Natsume, M. et al. JOURNAL OF ANTIBIOTICS (1995) Vol.:48(10); pages: 1 159 to 1 164 and HAERTL et al. THE JOURNAL OF ANTIBIOTICS (1998) VOL. 51 , NO. 1 1 , pp. 1040-1046.

Example 2: Knockout of crotonyl-CoA carboxylase (CCR2)

The Streptomyces albus strain used for this study comprises several deletions of genomic islands, which includes the deletion of the CCR1 gene. The strain has been described as S. albus Dell in Myronovsky M., Tokovenko B., Broetz E., Rueckert C, Kalinowski J.,

Luzhetskyy A., Genome rearrangements of Streptomyces albus J 1074 lead to the carotenoid gene cluster activation, Applied Microbiology and Biotechnology (2014), 98: 795- 806.

The knockout was performed using the IMES system Myronovsky M., Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied microbiology and

biotechnology (2014b), 98.10: 4557-4570. The primers "CCR2 redET for" and "CCR2 redET rev" (SEQ ID NO: 80 and 81 ) were used to perform the CCR2 replacement. The knockout was verified by isolation of chromosomal DNA, amplification of the targeted region by PCR with the primers "CCR2 seq for" and "CCR2 seq rev" (SEQ ID NO: 82 and 83) and sequencing of the PCR product. The resulting strain was S. albus Dell DelCCR2. The cosmid R2, containing the pamamycin biosynthesis gene cluster (SEQ ID NO: 39), was introduced into the mutant strain. The resulting strain S. albus Dell DelCCR2 R2 had a pamamycin metabolite spectrum differing from the reference strain (S. albus Dell R2, i.e. strain S. albus Dell + cosmid R2). The total pamamycin titer was increased in the supernatant and in the biomass of the culture. Additionally, pamamycin 579 could be detected in both the supernatant and the biomass of the culture. Pamamycin 593 could only be detected in the supernatant of the culture. PMM635 could only be detected in minor amounts. Example 3: Knockout of methylmalonyl-CoA mutase (MCM 1 )

The strain used for this study is S. albus Dell DelCCR2, as described in example 2.

The subsequent knockout was performed using the IMES (Myronovsky M., Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied microbiology and biotechnology (2014b), 98.10: 4557-4570). The primers "MCM redET for" and "MCM redET rev" (SEQ ID NO: 84 and 85) were used to perform the MCM replacement. The knockout was verified by isolation of chromosomal DNA, amplification of the targeted region by PCR with the primers "MCM seq for" and "MCM seq rev" (SEQ ID NO: 86 and 87) and

sequencing of the PCR product. The cosmid R2, containing the pamamycin biosynthesis gene cluster, was introduced into the mutant strain. The resulting strain had a pamamycin metabolite spectrum differing from the reference strain (S. albus Dell DelCCR2 R2, as produced in Example 2). The total pamamycin titer was increased in the supernatant and in the biomass of the culture. Example 4: Knockout of acetyl/propionyl-CoA carboxylase (PCC1 )

The subsequent knockout was performed using the IMES system (Myronovsky M.,

Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied

microbiology and biotechnology (2014b), 98.10: 4557-4570). The primers "PCC1 redET for" and "PCC1 redET rev" (SEQ ID NO: 88 and 89) were used to perform the PCC1

replacement. The knockout was verified by isolation of chromosomal DNA, amplification of the targeted region by PCR with the primers "PCC1 seq for" and "PCC1 seq rev" (SEQ ID NO: 90 and 91 )and sequencing of the PCR product. The cosmid R2, containing the pamamycin biosynthesis gene cluster, was introduced into the mutant strain. The resulting strain had a pamamycin metabolite spectrum differing from the reference strain (S. albus Dell DelCCR2 R2, as produced in Example 2). The total pamamycin titer was slightly increased in the supernatant and in the biomass of the culture.

Example 5: Knockout of acetyl/propionyl-CoA carboxylase (PCC2)

The subsequent knockout was performed using the IMES system (Myronovsky M.,

Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied

microbiology and biotechnology (2014b), 98.10: 4557-4570). The primers "PCC2 redET for" and "PCC2 redET rev" (SEQ ID NO: 92 and 93) were used to perform the PCC2

replacement. The knockout was verified by isolation of chromosomal DNA, amplification of the targeted region by PCR with the primers "PCC2 seq for" and "PCC2 seq rev" (SEQ ID NO: 94 and 95) and sequencing of the PCR product. The cosmid R2, containing the pamamycin biosynthesis gene cluster, was introduced into the mutant strain. The resulting strain had a pamamycin metabolite spectrum differing from the reference strain (S. albus Dell DelCCR2 R2, as produced in Example 2). The total pamamycin titer was slightly increased in the supernatant and in the biomass of the culture. Example 6: Penta knockout of CCR1 , CCR2, PCC1 , PCC2, and MCM

The subsequent knockouts were performed using the IMES system (Myronovsky M., Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied

microbiology and biotechnology (2014b), 98.10: 4557-4570). The primers for the CCR2, PCC1 , PCC2 and MCM deletions, as described in Examples 2 to 5, were used. Each knockout was performed separately, the marker was excised as described, followed by the subsequent knockout. The cosmid R2, containing the pamamycin biosynthesis gene cluster, was introduced into the mutant strain. The resulting strain had a pamamycin metabolite spectrum differing from the reference strain (S. albus Dell DelCCR2 R2, as produced in Example 2). The total pamamycin titer was increased in the extract of the whole culture, additionally pamamycin 593 could be detected, PMM635 could only be detected in minor amounts. Example 7: Blocking of valine dehydrogenase (VDH) by feeding ammonium ions

50 mM of NH₄+ was added to a culture of Streptomyces albus J 1074 containing the R2 cosmid (cultivation conditions as described in our pamamycin patent I). This resulted in a massive decrease of pamamycin production. VDH activity is inhibited by ammonium ions as described by Navarette R. M., Vara J. A., and Hutchinson C. R., Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2), Journal of General Microbiology (1990), 136: 273-281 and Tang L, Zhang Y.-Z., and Hutchinson C. R., Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae, Journal of

Bacteriology (1994), 176.19: 6107-61 19.

50 mM NH4+ was added to a culture of the Streptomyces albus penta knockout strain, as described in Example 5, containing the R2 cosmid. Pamamycins 593, 607 and 621 could be detected in slightly decreased amounts compared to S. albus Dell DelCCR2 + cosmid R2, as produced in example 2.

Example 8: Knockout of valine dehydrogenase (VDH)

The knockout was performed using the IMES system (Myronovsky M., Rosenkraenzer B., and Luzhetskyy A., Iterative marker excision system. Applied microbiology and

biotechnology (2014b), 98.10: 4557-4570). in S. albus J 1074 and S. albus penta knockout as described in Example 5. The primers "VDH redET for" and "VDH redET rev" (SEQ ID NO: 96 and 97) were used to perform the VDH replacement. The knockout was verified by isolation of chromosomal DNA, amplification of the targeted region by PCR with the primers "VDH seq for" and "VDH seq rev" (SEQ ID NO: 98 and 99) and sequencing of the PCR product. The cosmid R2, containing the pamamycin biosynthesis gene cluster, was introduced into the mutant strains. The S. albus J 1074 VDH knockout strain was not able to produce pamamycins. The VDH knockout in the penta knockout strain (being described in Example 5) with cosmid R2 was not able to produce pamamycins.

Claims

A pamamycin producer organism comprising at least one of:

a) a decreased methylmalonyl-CoA mutase activity,

b) a decreased crotonyl-CoA carboxylase activity,

c) a decreased acetyl/propionyl CoA carboxylase activity,

d) a combination of at least two of a), b) and c),

wherein the pamamycin producer organism comprises an intact katabolic pathway from isobutyryl-CoA to (2S)-methylmalonyl-CoA.

A pamamycin producer organism as claimed in claim 1 ,

comprising a decreased methylmalonyl-CoA mutase activity and a decreased crotonyl-CoA carboxylase activity.

A pamamycin producer organism as claimed in claim 1 or 2,

comprising a decreased methylmalonyl-CoA mutase activity, a decreased crotonyl-

CoA carboxylase activity and a decreased acetyl/propionyl CoA carboxylase activity.

A pamamycin producer organism as claimed in any one of claims 1 to 3

wherein the decreased methylmalonyl-CoA mutase activity is due to a decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 , 2, 3, 4, 5, or 6, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, 8, or 9, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 7, 8 or 9.

A pamamycin producer organism as claimed in any one of claims 1 to 4,

wherein the decreased crotonyl-CoA carboxylase activity is due to a decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, 1 1 , 12, or 13, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14 or 15, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 14 or 15, or, preferably, both.

A pamamycin producer organism as claimed in any one of claims 1 to 5,

wherein the decreased acetyl/propionyl CoA carboxylase activity is due to decreased activity of a polypeptide having an amino acid sequence which is at least 70%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, 17, 18, 19, 20, 21 , 25, 26, 27, 28, 29, or 30, preferably being due to a complete or partial deletion of a polynucleotide having a polynucleotide sequence which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, 23, 24, 31 , 32, or 33, or which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 22, 23, 24, 31 , 32, or 33.

A pamamycin producer organism as claimed in any one of claims 1 to 6, comprising a) at least one polypeptide having pamC activity, and

b) at least one polypeptide having pamG activity, and

c) at least one polypeptide having pamF activity, and

d) at least one polypeptide having pamA activity, and

e) at least one polypeptide having pamB activity, and

f) at least one polypeptide having pamD activity, and

g) at least one polypeptide having pamE activity, and

h) at least one polypeptide having pamO activity, and

i) at least one polypeptide having pamK activity, and

j) at least one polypeptide having pamJ activity, and

k) at least one polypeptide having pamM activity, and

I) at least one polypeptide having pamN activity, and

m) at least one polypeptide having pamL activity, and

n) at least one polypeptide having pamX activity, and

0) at least one polypeptide having pamY activity, and

p) at least one polypeptide having pamS activity,

preferably having at least one of those activities a) to p) up-regulated.

A pamamycin producer organism as claimed in any one of claims 1 to 7, comprising

1) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39,

ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%,

82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and/or 39 and comprising at least one expression cassette for

a) at least one polypeptide having pamC activity, and

b) at least one polypeptide having pamG activity, and

c) at least one polypeptide having pamF activity, and

d) at least one polypeptide having pamA activity, and

e) at least one polypeptide having pamB activity, and f) at least one polypeptide having pamD activity, and

g) at least one polypeptide having pamE activity, and

h) at least one polypeptide having pamO activity, and

i) at least one polypeptide having pamK activity, and

j) at least one polypeptide having pamJ activity, and

k) at least one polypeptide having pamM activity, and

1) at least one polypeptide having pamN activity, and

m) at least one polypeptide having pamL activity, and

n) at least one polypeptide having pamX activity, and

o) at least one polypeptide having pamY activity, and

P) at least one polypeptide having pamS activity

iii) two or more fragments of the polynucleotides of i) or ii) wherein the fragments comprise functional expression cassettes for one or more of the polypeptides of a) to p) and wherein the fragments cover at least the whole sequence of a polynucleotide of i) or ii), if the fragments are combined.

9. A pamamycin producer organism as claimed in any one of claims 1 to 8, being of the genus Streptomyces, preferably being of the species Streptomyces alboniger, Streptomyces aurantiacus or Streptomyces albus.

10. A pamamycin producer organism as claimed in claims 7 or 8, preferably claim 8, being of the species Streptomyces albus.

1 1. A method for the production of pamamycin comprising the steps of:

i) cultivating a pamamycin producer organism as claimed in any one of claims 1 to

10 under conditions which allow for the production of pamamycin by said recombinant microorganism;

ii) obtaining produced pamamycin, preferably obtaining pamamycin 607 and/or pamamycin 621.

12. A method to enhance the production of pamamycin 579 in a pamamycin producer organism comprising the steps of:

i) decreasing the crotonyl-CoA carboxylase activity of said pamamycin producer organism and

13. A method for the production of pamamycin 579 comprising the steps of:

i) cultivating a pamamycin producer organism as claimed in any one of claims 1 to 10, comprising a decreased crotonyl-CoA carboxylase activity under conditions which allow for the production of pamamycin by said pamamycin producer organism and ii) obtaining produced pamamycin 579.

A method to enhance the production of pamamycin, preferably the production of pamamycin 607 or pamamycin 621 or both, in a pamamycin producer organism comprising the steps of:

i) cultivating a recombinant microorganism as claimed in any one of claims 1 to 10, comprising a decreased crotonyl-CoA carboxylase activity under conditions which allow for the production of pamamycin by said pamamycin producer organism and

ii) obtaining produced pamamycin, preferably obtaining produced pamamycin 607 or pamamycin 621 or both.

A method as claimed in any one of claims 1 1 to 14, wherein the pamamycin producer organism comprises a decreased methylmalonyl-CoA mutase activity.