Field of the invention

[0001] The present disclosure relates to methods for producing biotin in genetically modified microbial cell factories using a type 2 biotin synthase under controlled oxygen supply.

Background of the invention

[0002] Biotin (also known as vitamin B7 or vitamin H), is an essential dietary vitamin for humans, because in common with other metazoans, they cannot produce biotin. In the production of biotin biosynthetic methods are more cost effective and environmentally sustainable than chemical synthesis routes.

[0003] Biotin is an essential cofactor for enzymes catalyzing certain carboxylation reactions, such as acetyl-CoA carboxylase (ACC). ACC, which is present in all life forms, produces malonyl-CoA, a key building block for fatty acid biosynthesis. In nature, biotin is synthesized by a linear pathway involving the fatty acid biosynthetic pathway. The initial substrate of biotin synthesis in Escherichia coli (E. coli) is malonyl-ACP, which is also the starting metabolite for fatty acid synthesis. Prior to entering the fatty acid cycle, malonyl-ACP is masked by the SAM (S-adenosylmethionine)-dependent methyltransferase, BioC, thereby generating a malonyl-ACP methyl ester. Subsequently, two rounds of fatty acid chain elongation yield the molecule pimeloyl-ester-ACP. Hydrolysis of the O-methyl group of the pimeloyl- ester-ACP by a dedicated esterase, BioH, allows this molecule to exit the fatty acid elongation cycle. Subsequently, the intermediate, pimeloyl-ester-ACP, is converted to biotin via a biotin-specific pathway (figure 1A). In this pathway, BioF catalyzes the PLP-dependent decarboxylative aldol condensation of pimeloyl-ACP with alanine to yield KAPA (8-Amino-7-oxononanoate). BioA catalyzes the PLP-dependent transamination of KAPA to yield DAPA (7,8-diaminopelargonate), where the donor is SAM; with the by-product S-adenosyl-oxomethionine. BioK catalyzes the PLP-dependent transamination of KAPA to yield DAPA (7,8-diaminopelargonate), where the donor is lysine; with the by-product (S)-2-amino-6-oxohexanoate. BioD catalyzes the ATP-d riven carboxylation and ring closure of DAPA to form the thiophane ring in desthiobiotin (DTB). The final step in the biotin synthesis pathway is one of the most complex reactions known, since it involves the introduction of a sulfur bridge between two hydrocarbons by biotin synthase (BioB), to yield biotin.

[0004] The use of microorganism-based cell factories is a potential route for the biosynthetic production of biotin. The advantages of a recombinant microorganism such as E. coli as a cell factory for production of bio-products are widely recognized due to the fact that: (i) it has unparalleled fast growth kinetics; with a doubling time of about 20 minutes when cultivated in glucose-salts media and under optimal environmental conditions, (ii) it easily achieves a high cell density; where the theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/I or roughly 1 x 10 ¹³ viable bacteria/mL Additionally, there are many moleculartools and protocols at hand for genetic modification of E. coli; as well as it being an organism that is amenable to the expression of heterologous proteins; both of which may be essential for obtaining high-level production of desired bio-products.

[0005] In E. coli, the biotin operon structure is split into bioA and bioBFCD under the control of overlapping promoters on opposite strands [bioO locus), while bioH is located elsewhere on the E. coli chromosome. Expression of the biotin operon is down-regulated by a biotin-bound repressor (BirA); which binds to an operator in the biotin operon. BirA also functions as a biotin ligase, transferring biotin to cellular carboxylases. The switch in BirA function from biotin ligase to transcriptional repressor is regulated by the respective intracellular biotin and apo-carboxylase pools. Over expression of the biotin operon {bioA and bioBFCD) in E. coli was reported to be inhibitory for growth (Ifuku, O. et al., 1995). Since the cause of this inhibition was unknown, this creates a stumbling block to enhancing biotin synthesis. One contributor to this inhibition is contemplated to BioB and its consumption of FeS clusters as disclosed in e.g. in W02019012058

[0006] In general, there exists a need to explore and optimize these complex biosynthetic pathways such as to facilitate and increase the production of biotin in microorganism-based cell factories (e.g. E. coli), which are tailor-made to overcome the diversity of factors that may limit their ability to both grow and produce elevated levels of their respective pathway enzymes.

[0007] A key step in biosynthetic production of biotin is the conversion of desthiobiotin into biotin by biotin synthase (BioB). One type of BioB (herein named as Type I BioB or TIBioB) is an S-adenosyl- L-methionine (SAM or AdoMet) radical enzyme, which is found as a dimer; and comprises two iron- sulfur clusters: [2Fe-2S] ²⁺ and [4Fe-4S] ²⁺ in its active site. The sulfur atom, needed to create the thiophane ring in biotin, is believed to be recruited from the [2Fe-2S] ²⁺ cluster in TIBioB. As a consequence, the iron-sulfur cluster in the TIBioB dimer consumed in biotin synthesis is believed to require regeneration after each round of catalysis and this regeneration is believed to limit the cells capacity for producing biotin.

[0008] Another, hitherto undisclosed type of BioB (herein named as Type II BioB or T2BioB) is a BioB wherein a holo-protein of the T2BioB comprises per polypeptide chain a first [4Fe-4S] cluster (radical SAM (RS) cluster) coordinated to a CxxxCxxC motif in the polypeptide chain and a second [4Fe-4S] cluster. Genes encoding T2BioB may be introduced into a host cell for the cell to produce biotin and using T2BioB for producing biotin in genetically modified microbial cells is contemplated to possess improved versatility compared to or in addition to using the known Type 1 BioB (TIBioB). As this protein does not contain the 2Fe2S cluster of TIBioB it represents a new and different mechanism of producing biotin when compared to TIBioBs.

[0009] However, there remains a need for improving and optimizing process condition when cultivating genetically modified cells expressing T2BioB to produce biotin to exploit the properties of

T2BioB.

Summary of the invention

[0010] It has been found that T2BioB in genetically modified cells performs very poorly or not at all at producing biotin when the oxygenation of the culture medium is above certain levels of oxygenation. While the cause of this property is still unclear, the present inventors have found that keeping the level of oxygenation below a certain level will keep T2BioB active in producing biotin [0011] Accordingly, the invention provides a method for producing biotin comprising cultivating a genetically modified host cell in a growth medium allowing the cell to produce the biotin, wherein the genetically modified host cells comprise an operative metabolic pathway producing biotin comprising a transgene encoding a Type II biotin synthase (T2BioB) , wherein a holo-protein of the T2BioB comprises per polypeptide chain a first [4Fe-4S] cluster (radical SAM (RS) cluster) coordinated to a CxxxCxxC motif in the polypeptide chain and a second [4Fe-4S] cluster; wherein the cultivation comprises at least one step of microaerobic cultivation provided for by limiting oxygen supply to the growth medium; and optionally recovering and/or isolating the biotin.

Description of figures

Figure 1 shows the pathway for microbial production of biotin from malonoyl-CoA in bacterial cells (E. coli).

Figure 2 shows biotin complementation of BS3308 expressing B. obeum BioB on mMOPS agar plates cultivated anaerobically, but no complementation is achieved at aerobic cultivation.

Figure 3 shows d-biotin production and growth of BS3308 cultivated microaerobically and aerobically in a gradient of IPTG induction for B. obeum expression.

Figure 4 shows biotin production from DTB by recombinant E. coli cells expressing a B. obeum T2BioB as a function of oxygen uptake rate. Figure 5 shows biotin production from glucose by recombinant E. coli cells expressing a B. obeum T2BioB and pathway genes producing DTB from glucose.

Incorporation by reference

[0012] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.

Detailed Description of the invention

Definitions

[0013] Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1- 5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. http://enzvme.expasv.org/.

[0014] The term "FabH" as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] synthase 3 (EC2.3.1.-eg. EC2.3.1.180) capable of converting acetyl-CoA and malonyl-[ACP] into 3-oxobutanoyl- [ACP],

The term "FabG" as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] reductase ECl.1.1.-/ ECl.3.1.- e.g. ECl.1.1.100) capable of converting a (3R)-hydroxyacyl-[ACP] into 3-oxoacyl-[ACP], [0015] The term "FabZ" as used herein refers to a 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (EC4.2.1.59) capable of converting (3R)-hydroxyacyl-[ACP] into (2E)-enoyl-[ACP],

[0016] The term "Fab I" as used herein refers to an enoyl-[acyl-carrier-protein] reductase (EC1.3.1.- e.g. ECl.3.1.9) capable of converting 2,3-saturated acyl-[ACP] into (2E)-enoyl-[ACP],

[0017] The term "FabB" as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] synthase (EC2.3.1.- e.g. EC2.3.1.41) capable of converting fatty acyl-[ACP] and malonyI-[ACP] into 3-oxoacyl-[ACP] and holo-[ACP],

[0018] The term "FabF" as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] synthase (EC2.3.1.- e.g. EC2.3.1.179) capable of converting (llZ)-hexadecenoyl-[ACP] and malonyl-[ACP] into 3-oxo-(13Z)- octadecenoyl-[ACP] and holo-[ACP],

[0019] The term "BioFI" as used herein refers to a pimeloyl-acyl carrier protein methyl ester esterase (EC3.1.1.85) converting O-methylpimeloyl-acyl carrier protein to pimeloyl-acyl carrier protein [0020] The term "BioB" as used herein both in context of type I and type II BioB refers to a biotin synthase capable of converting desthiobiotin into biotin, optionally under co-conversion of S- adenosyl-l-methionine (SAM) to 5'-deoxyadenosine (5'-DOA), comprising a first polypeptide comprises at least two prosthetic [4Fe-4S] cluster moieties. Such BioB's are referred to herein as T2BioB's.

[0021] The term "BioC" as used herein refers to a malonyl-acyl carrier protein methyltransferase (EC2.1.1.197) capable of converting Malonyl-acyl carrier protein into malonyl-acyl carrier protein methyl ester.

[0022] The term "BioF" as used herein refers to an 8-amino-7-oxononanoate synthase (EC2.3.1.47) capable of converting a pimeloyl-acyl carrier protein into KAPA.

[0023] The term "BioA" as used herein refers to an adenosylmethionine-8-amino-7-oxononanoate transaminase (EC2.6.1.62) capable of converting KAPA into DAPA using SAM as amino donor.

[0024] The term "BioK" as used herein refers to an adenosylmethionine-8-amino-7-oxononanoate transaminase (EC2.6.1.62) capable of converting KAPA into DAPA using lysine as amino donor.

[0025] The term "BioD" as used herein refers to a desthiobiotin synthase (EC6.3.3.3) capable of converting DAPA into DTB.

[0026] The term "Biol" as used herein refers to a biotin biosynthesis cytochrome P450, (pimeloyl- [acp] synthase (ECl.14.14.46) capable of converting long-chain acyl-[acyl-carrier protein into pimeloyl- [acp]

[0027] The term "BioW" as used herein refers a 6-ca rboxy hexa noate-CoA ligase (EC6.2.1.14) converting pimelate into pimeloyl-CoA

[0028] The term "KAPA" as used herein refers to 7-keto-8-aminopelargonic acid.

[0029] The term "DAPA" as used herein refers to 7,8-Diaminopelargonic Acid.

[0030] The term "DTB" as used herein refers to desthiobiotin.

[0031] The term "SAM" as used herein refers to S-adenosyl-L -methionine.

[0032] The term "SAH" as used herein refers to S-Adenosyl-L-homocysteine

[0033] The term "CoA" as used herein refers to coenzyme A

[0034] The term "ACP" as used herein refers to Acyl Carrier Protein

[0035] The term "AMTOD" as used herein refers to S-adenosyl-2-oxo-4-thiomethylbutyrate

[0036] The term "5'DOA" as used herein refers to 5'-deoxyadenosine.

[0037] The term "[2Fe-2S] cluster"" as used herein refers to rhombic iron-sulfur cluster cofactors of proteins comprising two iron ions bridged by two sulfide ions and liganded by amino acid residues of a protein. [0038] The term "[4Fe-4S] cluster"" as used herein refers to cubic iron-sulfur cluster cofactors of proteins comprising four iron ions bridged by four sulfide ions and liganded by amino acid residues of a protein.

[0039] The terms "heterologous" or "recombinant" or "genetically modified" and its grammatical equivalents as used herein interchangeably refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0040] The term "substrate" or "precursor", as used herein refers to any compound that can be converted into a different compound. For example, IPP can be a substrate for "I PI converting into DMAPP. For clarity, substrates and/or precursors include both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic carbon molecules which the host cell can metabolize into a desired compound.

[0041] The term "metabolic pathway" as used herein is intended to mean two or more enzymes acting in a chain of reaction (sequentially or interrupted by intermediate steps) in a live cell to convert chemical substrate(s) into chemical product(s). Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds such as proteins for example enzymes (co enzymes).

[0042] The term "operative metabolic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0043] The term "in vivo", as used herein refers to within a living cell, including, for example, a microorganism or a plant cell.

[0044] The term "in vitro", as used herein refers to outside a living cell, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.

[0045] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, using these deviating terms can also include a range deviation plus or minus such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a specified value. [0046] The term "and/or" as used herein is intended to represent an inclusive "or". The wording X and/or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0047] The term "isolated" or "recovered" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0048] The term "non-naturally occurring" as used herein about a substance, refers to any substance that is not normally found in nature or natural biological systems. In this context the term "found in nature or in natural biological systems" does not include the finding of a substance in nature resulting from releasing the substance to nature by deliberate or accidental human intervention. Non-naturally occurring substances may include substances completely or partially synthetized by human intervention and/or substances prepared by human modification of a natural substance.

[0049] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences. "% identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues

- z 100

Length of alignment — total number of gaps in alignment

"% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm

(Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides

- - - z 100

Length of alignment — total number of gaps in alignment

The protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing

BLAST analyses is publicly available through the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:

- Cost to open gap: default= 5 for nucleotides/ 11 for proteins

- Cost to extend gap: default = 2 for nucleotides/ 1 for proteins

- Penalty for nucleotide mismatch: default = -3

- Reward for nucleotide match: default= 1

- Expect value: default = 10

- Wordsize: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins.

Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.

[0050] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNAthat is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0051] The term "codon optimized sequence" refers to a genetic sequence whose codon bias has been changed to better match the codon bias of the host organism. Since there are 64 possible codons (nucleotide triplets) but only 20 amino acid and three stop codons encoded by these codons multiple codons can encode one amino acid. Different organisms exhibit bias towards use of some codons over others for the same amino acid (codon bias) and the codons used can impact expression of a given polypeptide. A codon optimized sequence encodes the same protein as the original sequence, but the nucleotide sequence more closely matches the codon bias of the host organism to improve polypeptide expression.

[0052] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, orTGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0053] The term "control sequence" as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.

[0054] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.

[0055] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a polynucleotide construct or expression vector comprising a polynucleotide of the present disclosure. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0056] The term "polynucleotide construct" refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0057] The term "expression vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.

[0058] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.

[0059] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably. [0060] The term "comprise" and "include" as used throughout the specification and the accompanying claims as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0061] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0062] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the claimed disclosure or to imply that certain features are critical, essential, or even important to the structure or function of the claimed disclosure. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0063] The term "cell culture" as used herein refers to a growth medium comprising a plurality of genetically modified host cells. The growth medium containing the plurality of genetically modified host cells may also interchangeably be referred to as "culture medium" A cell culture may comprise a single strain of genetically modified host cells or may comprise two or more distinct strains of genetically modified host cells. The growth medium may be any medium suitable for the genetically modified host cells, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B. [0064] The term "[XYZ]" as used herein about amino acids in a motif is to be understood as an amino acid being either X, Y or Z. As an example if the motif is [HSRG]-[FY]-[SGD]-[ILM]-[VG]-[ATSV]-[AS]- [WGE]-[KRTYEL] the motif can be for example H-F-S-l-V-A-A-W-K or it can be G-Y-G-L-G-S-S-G-Y. [0065] The term "cultivation" as used herein refers to growing a culture of cells at conditions to increase biomass and/or to have the cell produce a desired metabolite such as biotin.

[0066] The term "mixed acid fermentation metabolite" as used herein means a metabolite resulting from fermentative metabolism instead of respiration, i.e. from a metabolic process in the absence of oxygen. Well known fermentation metabolites include various organic acids, such as lactic acid or alcohols such as ethanol.

[0067] The term "microaerobic conditions" as used herein refers to cultivation conditions where, due to oxygen limitation, the specific growth rate of the cultivated cells is less than the maximum specific growth rate in the presence of excess oxygen. Microaerobic conditions also refers to cultivation conditions where, due to oxygen limitation, the specific oxygen uptake rate (spOUR) of the host cells is less than the maximum spOUR of the cultivated cells in the presence of excess oxygen. spOUR is defined as the oxygen uptake rate divided by the cell dry weight (mmol 0 ₂ hour ¹ CDW ¹ ). Microaerobic conditions also refers to cultivation conditions where, due to oxygen limitation, the C0 ₂ generation of the cultivated cells is greater than the 0 ₂ uptake of the cells.

[0068] The term "anaerobic conditions" as used herein refers to cultivation in the absence of oxygen in a culture medium. It will be understood that in many fermentation processes, an initial amount of oxygen is present at the onset of the process, but such oxygen may be depleted over the course of the fermentation such that the majority of the process may take place in the absence of oxygen.

Further embodiments of the methods of producing biotin using controlled oxygen supply [0069] In consequence of the discovered oxygen sensitivity of the T2BioB to high oxygenation levels in the culture medium, an important feature of the methods provided herein is that oxygen supply to the culture medium is, at least periodically, kept at a level low enough for the cultivation to be microaerobic and for the T2BioB to be active in producing biotin. In some embodiments the oxygen supply is limited so that the dissolved oxygen concentration (DOC) in the culture medium is less than 20% of DOC for a culture medium fully saturated with oxygen (DOCMAX)· In further embodiments the DOC in the culture medium which is less than less than 15%, such as less than 10%, such as less than 5%, such as 0% of DOCMAX- While at a DOC of 0% the cultivation can be anaerobic (no oxygen supplied) it can also mean that the cells in the culture are metabolizing oxygen as fast as it is being supplied. In other embodiments the oxygen supply is limited so that the specific oxygen uptake rate (spOUR) of the host cells is between 1 and 5 mmol 0 ₂ per hour per grans cell dry weight (mmol 0 ₂ hour ¹ gCDW ^x ), particularly between 2 to 5, such as between 3 to 5 or 4 to 5 mmol 0 ₂ hour ¹ gCDW ¹ .

[0070] In further embodiments the oxygen supply is limited so that the respiratory quotient (RQ) is greaterthan 1, such as greater than 2, or greater than 3, orgreaterthan 4, or greater than 5, or greater than 10 or greater that 25 or even greater than 50. At RQ greater than 1 the cells do not produce energy solely via aerobic respiration. Instead mixed acid fermentation is used, and the production and excretion of acetate, formate, ethanol, hydrogen, lactate, and/or succinate. Accordingly, in further embodiments the host cells during the at least one step of microaerobic cultivation produces a detectable amount of a fermentation metabolite, optionally selected from one or more of acetic acid lactic acid, formic acid, succinic acid and ethanol. Specific C0 ₂ generation rate (Sp. CER, millimoles/g/hr) and specific oxygen uptake rate (Sp. OUR, millimoles/g/hr) can be calculated by measuring flow rate, inlet and exhaust gas composition of air (C0 ₂ , 0 ₂ , etc.), using, for example, mass spectrometry and/or cell density measurements. Specific carbon dioxide generation rate is the ratio of C0 ₂ produced (air flow rate multiplied by difference between outlet and inlet C0 ₂ concentration) to cell density per unit time. Specific oxygen uptake rate is the ratio of 0 ₂ consumed (air flow rate multiplied by difference between inlet and outlet 0 ₂ concentration) to cell density per unit time. Respiratory quotient (RQ) is ratio of CER and OUR. Only the inlet and outlet gas composition from mass spectrometry are required to calculate RQ for a given constant air flow rate. RQ can be used as a control variable that couples the oxygen uptake rate with the carbon flux through the bioreactor system. RQ is intrinsically independent of scale. RQ can be measured, for example, using exhaust gas analysis. In further embodiments the oxygen supply to the growth medium during the at least one step of microaerobic cultivation is less than 5 mmol 0 ₂ hour ¹ gCDW ¹ , such as between 1 and 5 mmol 0 ₂ hour ¹ gCDW ¹ , such as between 1 and 4 mmol 0 ₂ hour ¹ gCDW ¹ , such as between 1 and 3 mmol 0 ₂ hour ¹ gCDW ¹ , such as between 1 and 2 mmol 0 ₂ hour ¹ gCDW ¹ .

[0071] Performing at least one step of microaerobic cultivation in this context is to be understood as limiting the oxygen supply to the culture medium during one or more phases of the cultivation. Such phases can have a time span which is longer or shorter depending on the specific cultivation and the specific host cell.

[0072] In further embodiments and in addition to steps of microaerobic cultivation during one or more phases, the method provided for herein may comprise further phases of cultivation, wherein the oxygen supply is e.g. increased. For example, during the build stage or the biomass accumulation stage higher oxygen supply may be applied, and cultivation may be performed at fully aerobic conditions

[0073] Oxygen can be added to the culture medium using methods known in the art, through agitation and aeration of the medium by stirring, shaking or sparging. Although aeration of the medium has been described herein in relation to the use of air, other sources of oxygen can be used. Particularly useful is the use of an aerating gas that contains a volume fraction of oxygen greater than the volume fraction of oxygen in ambient air. In addition, such aerating gases can include other gases, which do not negatively affect the culture. In some embodiments, microaerobic conditions are achieved by bubbling the culture with nitrogen, e.g., high purity nitrogen (99.8%). In some embodiments, microaerobic conditions are achieved by cultivating the cells in air-tight vessels, for example, screw-capped vials and flasks, and the like. Because residual dissolved oxygen is consumed during cell growth, these conditions sharply lower, but do not completely deplete, oxygen availability during the course of the cell growth. In some embodiments, microaerobic conditions can be achieved by means of mixing air in an appropriate amount with a carrier gas. Alternatively, an appropriately low flow rate of air can be sparged. The oxygen level in the cultivation medium can be monitored using an oxygen electrode or any other suitable device, and the flow rate of the gas mix is adjusted to assure that the level of oxygen in the cultivation medium is maintained at a constant level. In addition to variations in the inlet gas flow rate or the composition of the inlet gas, microaerobic conditions can also be produced by decreasing the stir rate (thus decreasing the oxygenation of a large culture), or by adding more feedstock to increase the cell density (and hence higher oxygen demand), or combinations thereof. In some embodiments, dissolved oxygen can be controlled by feeding sugar to the host cells to keep the dissolved oxygen concentration at undetectable levels through most of the cultivation. In some embodiments, oxygen can be supplied via compressed gas sparging and mechanical agitation of the fermentation broth.

[0074] When cultivating cells expressing T2BioB, the T2BioB can assemble into a dimer consisting of two T2BioB monomer polypeptide moieties each comprising two prosthetic [4Fe-4S] cluster moieties and in a particular embodiment the T2BioB contains no [2Fe-2S] clusters.

[0075] Further it has been found that some T2BioB genes contain signature sequence that differentiates them from TIBioB's, while others may deviate from this signature. Forthe archaeal and extremophile prokaryotic clade, TIBioB's contain the motif [HSRG]-[FY]-C-[ILM]-[VG]-[ATSV]-[AS]- [WGE]-[KRTYEL], while T2BioB's contain the different motif [HSRG]-[FY]-[SGD]-[ILM]-[VG]-[ATSV]-[AS]- [WGE]-[KRTYEL] In particular for this clade the T2BioB's contain the motif [HSRG]-[FY]-S-[ILM]-[VG]- [ATSV]-[AS]-[WGE]-[KRTYEL] For the cyanobacterial clade TIBioB's contain the motif [HSRG]-[FYVIL]- C-[ILM]-[VG]-[WATSV]-[AS]-[WGE]-[KRTYEL], while the T2BioB's contain the different motif [HSRG]- [FYVIL]-C-[ILM]-[VG]-[WATSV]-Q-[WGE]-[QKRTYEL]

[0076] Accordingly, in some embodiments in the methods provided for herein the T2BioB monomer comprises a signature amino acid sequence motif, which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to a sequence selected from: a) [HSRG]-[FY]-[SGD]-[ILM]-[VG]-[ATSV]-[AS]-[WGE]-[KRTYEL]; and/or b) [HSRG]-[FYVIL]-C-[ILM]-[VG]-[WATSV]-Q-[WGE]-[QKRTYEL]

[0077] In other embodiments in the methods provided for herein the T2BioB monomer comprises a sequence motif which is at least 50% identical, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to [HSRG]-[FY]-S-[ILM]-[VG]-[ATSV]-[AS]- [WGE]-[KRTYEL]

[0078] In a preferred embodiment the signature amino acid sequence motif is selected from: c) [HSRG]-[FY]-[SGD]-[ILM]-[VG]-[ATSV]-[AS]-[WGE]-[KRTYEL]; and/or d) [HSRG]-[FYVIL]-C-[ILM]-[VG]-[WATSV]-Q-[WGE]-[QKRTYEL]; wherein the -[SGD]- moiety of a) and/or the -C- moiety of b) is conserved. In particular the signature sequence may be [FISRG]-[FY]-S-[ILM]-[VG]-[ATSV]-[AS]-[WGE]-[KRTYEL], wherein the -[S]- moiety is conserved.

[0079] The amino acid sequence of the two T2BioB monomers in the dimer may be the same a or different, while in most cases the same. The amino acid sequence of a T2BioB polypeptide monomer suitably comprises an amino acid sequence selected from the group consisting of a) an amino acid sequence which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the T2BioB comprised in anyone of sequences SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 120; b) an amino acid sequence encoded by a polynucleotide which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in anyone of SEQ ID NO: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 121 encoding the T2BioB comprised in of anyone of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 120 or genomic DNA thereof; c) a functional variant of the mature polypeptide of the amino acid sequences (a) or (b) having BioB activity.

[0080] In particular the amino acid sequence of the T2BioB monomer is at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the T2BioB comprised in anyone of SEQ ID NO: 18, 20, 22, 24, 26,

28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 120, more particularly SEQ ID NO: 18. [0081] In the methods provided for herein the genetically modified cell comprises a transgene encoding the T2BioB and in one embodiment the transgene has a nucleotide sequence which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in anyone of sequences SEQ ID NO: 19, 21, 23, 25, 27,

29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 or 121 encoding the T2BioB comprised in anyone of SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 120 or genomic DNA thereof.

[0082] In particular, T2BioB encoding transgene has a nucleotide sequence which is at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to T2BioB encoding gene comprised in SEQ ID NO: 19. [0083] The transgene encoding the T2BioB may not be naturally occurring and can for example can be codon optimized for bacterial or fungal expression and/or be cDNA.

[0084] For expressing the T2BioB in the cells, the gene encoding the T2BioB is linked in a polynucleotide construct to one or more control sequences, which direct the expression of theT2BioB. Conditions for the expression should be compatible with the control sequences. Genes and constructs may be manipulated in a variety of ways known to the skilled person to allow expression of a polypeptide. Manipulation of polynucleotide constructs prior to its insertion into an expression vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art. The T2BioB gene may be heterologous to the one or more control sequences and/orthe one or more control sequences may also be heterologous to the cell. The one or more control sequences can in particular be selected from constitutive promotors, regulated/inducible promotors, ribosomal binding sites, and terminators. Suitable constitutive promotors are known in the art. Suitable regulated promotors are also known in the art including but not limited to pLac, pT7, pBAD, pRha, pTrp, pT5, pT5lacO. Suitable ribosomal binding sites are known in the art. Suitable terminators are also known in the art including but not limited to tRrnB, tapFAB382, tT7, tTonB, tHis, and tLambdatO. It may also be desirable to add regulatory sequences that regulate expression of the T2BioB relative to the growth of a host cell in which the T2BioB is expressed. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The polynucleotide construct harboring the T2BioB and the control sequences may further be inserted into a recombinant expression vector further comprising various nucleotide sequences having one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide construct at such sites. The recombinant expression vector can be any vector (for example a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the T2BioB encoding polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini chromosome, or an artificial chromosome. The vector may contain any means for assuring self replication. Alternatively, the vector may, when introduced into the host cell, integrate into the genome and replicate together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used. The vector may contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene from which the product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like. Useful selectable markers include beta lactam resistance cassette, chloramphenicol resistance cassette, tetracycline resistance cassette, kanamycin/neomycin resistance cassette, spectinomycin / streptomycin resistance cassette, and UR A3 uracil prototrophy marker. The vector preferably contains element(s) that permits integration of the vector into the host cell's genome or permits autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 35 to 10,000 base pairs, such as 100 to 10,000 base pairs, such as 400 to 10,000 base pairs, and such as 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0085] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo. Useful origins of replication include pSClOl, pl5A, pBR322, pMBl, ColEl, R6K, pUC. More than one copy of a polynucleotide encoding the T2BioB may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number can be obtained by integrating one or more additional copies of the polynucleotide coding sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, so that cells containing amplified copies of the selectable marker gene - and thereby additional copies of the polynucleotide - can be selected by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0086] The operative metabolic pathway provided for herein can advantageously further comprise one or more native of heterologous pathway elements selected from a) one or more fatty acid synthesis enzymes selected from FabH, FabG, FabZ, Fabl, FabB and FabF; b) a malonyl-acyl carrier protein methyltransferase (BioC) converting Malonyl-acyl carrier protein to malonyl-acyl carrier protein methyl ester; c) a pimeloyl-acyl carrier protin methyl ester esterase ( BioH) converting O-methylpimeloyl-acyl carrier protein to pimeloyl-acyl carrier protein; d) a 8-amino-7-oxononanoate synthase (BioF) converting Pimeloyl-acyl carrier protein to KAPA; e) an adenosylmethionine-8-amino-7-oxononanoate transaminase (BioA) converting KAPA to DAPA using SAM as amino donor; f) an adenosylmethionine-8-amino-7-oxononanoate transaminase (BioK) capable of converting KAPA into DAPA using lysine as amino donor; g) a desthiobiotin synthase (BioD) converting DAPA to DTB; h) a biotin biosynthesis cytochrome P450, (pimeloyl-[acp] synthase (Biol) converting long-chain acyl-[acyl-carrier protein to pimeloyl-[acp]; i) a 6-carboxyhexanoate-CoA ligase (BioW) converting pimelate and CoA to pimeloyl-CoA; j) an Fe-S cluster Transcription factor polypeptide (IscR) capable of regulating [the isc operon] production of a Fe-S cluster co-factor; and/or k) a Type 1 biotin synthase (TIBioB) converting DTB to Biotin.

[0087] In the pathway, BioC may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to BioC comprised in of SEQ ID NO: 1. BioH may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to BioH comprised in SEQ ID NO: 3. BioF may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the BioF comprised in SEQ ID NO: 5. BioA may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the BioA comprised in SEQ ID NO: 7. BioK may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the BioK comprised in SEQ ID NO: 118. BioD may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the BioD comprised in SEQ ID NO: 9. Biol may have at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Biol comprised in SEQ ID NO: 12. BioW may have has at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the BioW comprised in SEQ ID NO: 14; IscR has at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IscR comprised in SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, or 117; and/or TIBioB has at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TIBioC comprised in SEQ ID NO: 16. In a special embodiment one or more polypeptides comprised in the operative metabolic pathway is heterologous to the genetically modified host cell.

[0088] Further, in the methods provided for herein the one or more native or heterologous pathway elements of the pathway producing biotin are selected from the group of: e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the BioC comprised in SEQ ID NO: 1; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the BioH comprised in SEQ ID NO: 3; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the BioF comprised in SEQ ID NO: 5; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the BioA comprised in SEQ ID NO: 7; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 119 or genomic DNA thereof encoding the BioK comprised in SEQ ID NO: 118; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 10 or 11 or genomic DNA thereof encoding the BioD comprised in SEQ ID NO: 9; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 13 or genomic DNA thereof encoding the Biol comprised in SEQ ID NO: 12;

L) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 15 or genomic DNA thereof encoding the BioW comprised in SEQ ID NO: 14; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 98, 100, 102, 104, 106, 108, 110, 112, 114, or 116 or genomic DNA thereof encoding the IscR comprised in SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, or 117; and/or n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the polynucleotide comprised in SEQ ID NO: 17 or genomic DNA thereof encoding the TIBioB comprised in SEQ ID NO: 16.

[0089] The cell may be further genetically modified to provide an increased amount of a substrate for at least one polypeptide of the operative metabolic pathway as well as it may be further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, product molecules from the operative metabolic pathway or one or more of the proteins of the biosynthetic metabolic pathway. In another embodiment the cell may be further genetically modified to include a transporter polypeptide facilitating transport of any precursors of substrates and/or secretion or excretion of the formed biotin.

[0090] The genetically modified cell may be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells include cells of the genuses Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter and Pseudomonas, in particular the genuses Escherichia, Corynebacterium, Bacillus, Serratia, Pseudomonas. Preferred species of prokaryotic cells are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Serratia marcescens pseudomonas putida or Pseudomonas mutabilis.

[0091] Eukaryotic cells include mammalian, insect, plant, fungal or archaeal cells. Fungal cells include cells of the genuses Saccharomyces, Pichia or Ashbya. Particular species include Saccharomyces cerevisiae, Pichia pastoris, Ashbya gossypii.

[0092] Further, the cell may comprise at least 2 copies of the polynucleotide encoding the T2BioB of this disclosure and/ or it may be modified to attenuate, disrupt and/or delete one or more native genes.

[0093] In one embodiment of the methods provided for herein, further comprises feeding one or more exogenous biotin precursors to the cell culture, optionally selected from the group of, KAPA, DAPA, DTB and pimelatic acid.

[0094] The methods provided for herein entail cultivating cells in a growth medium suitable for propagating cell count and production of biotin. For example, the cultivation may be carried out as shake flask cultivation, or small-scale or large-scale cultivation and/or fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the metabolic pathway to operate to produce biotin. [0095] The growth medium suitably comprises carbon and nitrogen sources and inorganic salts known in the art and such media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and/or based on the regulatory requirements for the host cell. The medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable growth medium comprises a carbon source (e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.). [0096] Further, cultivation of the host cell is suitably performed over a period of from about 0.5 to about 30 days. The cultivation process may be a batch process, continuous or fed-batch process, and depending on the hist cell suitably be performed at a temperature in the range of 0-100°C or 0-80°C, for example, from about 0°C to about 50°C and/or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions are a temperature in the range of from about 25°C to about 55°C and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell. Accordingly, the methods provided for herein may further comprising one or more elements selected from: a) cultivating the cell culture under mixing, such as agitation; b) cultivating the cell culture at a temperature of between 25 to 50 °C; c) cultivating the cell culture at a pH of between 3-9; and/or d) cultivating the cell culture for between 10 hours to 120 days.

[0097] The method may further comprise feeding one or more exogenous biotin precursors to the cell culture.

[0098] Not all conversion steps of the pathway producing biotin provided for herein need to occur in vivo in the host cell, so in a particular embodiment one or more of these steps are carried out in vitro. Accordingly, in an embodiment the method provided for comprises at least one pathway step which is performed in vitro.

2. The produced biotin may be recovered and/or isolated using methods known in the art. For example, the biotin may be recovered from the culture medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The biotin may be further isolated and purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989). Accordingly, in a preferred embodiment the recovery and/or isolation comprises separating a liquid phase of the cell or growth medium from a solid phase of the cell or growth medium to obtain a supernatant comprising the biotin and subjecting the supernatant to one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced biotin, then optionally recovering the biotin from the resin in a concentrated solution prior to isolation of the biotin by crystallisation or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography resins in order to obtain at least a portion of the biotin, then optionally recovering the biotin from the resin in a concentrated solution prior to isolation of the biotin by crystallisation or solvent evaporation; and/or c) extracting the biotin from the supernatant, such as by liquid-liquid extraction into an immiscible solvent, then optionally isolating the biotin by crystallisation or solvent evaporation; thereby recovering and/or isolating the biotin.

[0099] Further, the method for producing biotin may comprise mixing recovered and/or isolated biotin with one or more pharmaceutical grade excipient, additives and/or adjuvants. Sequence listings

[0100] The present application contains a Sequence Listing prepared in Patentln version 3.5.1, which is also submitted electronically in ST25 format which is hereby incorporated by reference in its entirety.

Examples

Materials

Chemicals [0101] Chemicals used herein e.g. for buffers, media and substrates are commercial products of at least reagent grade. mMOPS medium

[0102] The minimal medium (mMOPS) used herein had the following composition in demineralized H ₂ 0 (dH ₂ 0): pH of the mMOPS medium was adjusted to 7.4±0.1 with NaOH or H ₂ S0 ₄ .

5 medium [0103] The minimal screening medium (S medium) used herein had the following composition in dH20: pH of the S medium was adjusted to 7.410.1 with NaOH or H ₂ S0 ₄ .

LB agar medium

[0104] The LB agar medium used herein had the following composition in dH ₂ 0: mMOPS agar medium

[0105] The mMOPS agar medium used herein had the following composition in mMOPS medium: B medium

[0106] The fermentation batch medium (B medium) used herein had the following composition in dH20:

F medium

[0107] The fermentation feed medium (F medium) used herein had the following composition in dH20:

Antibiotic solution

[0108] The antibiotics solution used herein had the following composition in dH20:

Strains and plasmids [0109] Strains of Escherichia coli used herein were the following:

¹ Commercially available strain e.g. from The Coli Genetic Stock Center; http://cgsc2.biology.yale.edu/KeioList.php

² Commercially available strain e.g. from The Coli Genetic Stock Center; http://cgsc2.biology.vale.edu/KeioList.php

³ W02019012058A1

⁴ Datsenko, K. A. and Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 97(12):6640-5, D0l:10.1073/pnas.l20163297

[0110] Plasmids used herein were as follows Analytical Procedures

Procedure I: Optical densities measurements

[0111] To measure optical densities (OD) of a cell culture as cuvette OD at 600 nm (cOD ₆ oo), the culture was diluted 10-fold with dH ₂ 0 to a final volume of 1 ml and transferred to a 1.5 ml transparent cuvette with 10 mm pathlength. The diluted culture was measured at 600 nm and 10 mm pathlength on a mySPEC (VWR). If the diluted culture was measured to cOD ₆ oo>0.4, the culture was further diluted 10-fold and remeasured.

Procedure II: Bioassav for d-biotin quantification

[0112] For quantification of d-biotin in supernatant samples from small-scale screening and fermentations, a bioassay involving d-biotin auxotrophic strain BS1093, mMOPS (excluding d-biotin) supplemented with zeocin and >5 of d-biotin standards in the dynamic growth range of BS1093 was used following the assay described in section 1.6 of the methods in the examples in W02019012058. Specifically, the supernatant from each culture was diluted alongside >5 biotin standards in the concentration range of 0 mM (mg/L) to 40 mM (12.9 mg biotin/L) prepared in Milli-Q water. 15 pL of each diluted supernatant and each of the biotin standards was then added to a well of a 96-well microtiter plate. Each well contained prior to addition 135 mί of mMOPS medium without d-biotin but with supplementation of zeocin and inoculated to an initial ODeoo of 0.01 with a biotin-starved overnight culture of BS1093. The plate was sealed with a breathable seal and incubated at 37°C with 275 rpm shaking for 20 hours before ODeoo was measured. A biotin bioassay calibration curve was obtained using the dynamic range of growth response of BS1093 to the biotin standards. For sample wells, the calibration curve was used to calculate concentrations of d-biotin produced based on the growth response of BS1093 in the individual wells.

Example 1 - Cloning genetic construct for T2BioB (over)expression

[0113] DNA encoding T2BioB was amplified using the primers indicated in table 1 using Phusion U polymerase (Thermo Fischer Scientific) following manufacturer's protocol. Similarly, plasmid backbones of pBS682 (pSClOl) and pBS1779 (pBR322) were amplified using primers oBS1587 and oBS1591 (GFP gene was not amplified). The correct size of fragments was confirmed by gel electrophoresis and fragments were gel purified using Monarch ^® DNA Gel Extraction Kit (New England Biolabs) using manufacturer's protocol. T2-BioB fragments were mixed with either the pSClOl or the pBR322 origin plasmid backbone fragments from pBS682 and pBS1779, respectively and digested and ligated using USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) following the manufacturers' protocols. These mixtures were transformed by electroporation into BS1575 and transformed cells were grown on selective LB agar supplemented with ampicillin overnight at 37°C resulting in the strains BS3306, BS3307, BS3308, BS3328, BS3329, BS3331, BS3448, BS3449 and BS3477 carrying the individual plasmids shown in table 1. Table 1: Cloning of plasmids

Example 2 - Screening E. coli AbioB complementation by T2BioBs at aerobic and microaerobic/anaerobic conditions

[0114] A single colony of each strain found in table 1 was taken from an LB agar plate supplemented with ampicillin and streaked onto two identical mMOPS agar plates supplemented with ampicillin. Both plates of each strain were incubated at 37°C for 40 hours; one at aerobic conditions in atmospheric air and the other in anaerobic conditions using the Anaerocult ^® A (Merck) system. After 40 hours of incubation, both mMOPS plates cultured either aero- or anaerobically for each strain were inspected for formation of separated, single colonies. Separated, single colonies indicated complementation of the AbioB in the background strain BS1575 by the T2BioB doing catalysis and producing biotin from the DTB supplemented in the mMOPS agar plate. As an example, figure 2 shows complementation on mMOPS agar plates of B. obeum BS3308 (BS1575+pBS1676) at aerobic and anaerobic conditions. Single colonies, showing biotin complementation and thus catalysis by B. obeum T2BioB, is clearly seen under anaerobic cultivation, but not under aerobic cultivation. Table 2 below shows complementation observed for BS3306-8, BS3328-9, BS3331, BS3448 and BS3449.

Table 2: Complementation of T2BioBs at aerobic and anaerobic conditions.

Example 3 - Screening in vivo catalysis of T2BioBs in E. coli [0115] Precultures for production of each strain of example 1 were prepared by inoculating a colony into 400 mI. mMOPS medium with ampicillin in a 96 deep-well plate, incubated at 37°C with shaking at 275 rpm for 16-18 hours. For microaerobic condition, production cultures were established by inoculating with 8 pL from the precultures into 800 pL S medium with ampicillin and further supplemented with 0.0107 g/L desthiobiotin (DTB), and optionally comprising isopropyl b-D-l- thiogalactopyranoside (IPTG) at a final concentration of up to 0.1 mM, in a 96 deep-well plate (DWP) (EnzyScreen, CR1496a). For aerobic condition, the same medium composition as for the microaerobic condition was used, but the medium volume was 400 pi and the inoculum volume was 4 pi. The inoculated wells of the DWP were sealed with an aluminum seal. Cultures were then grown at 37°C with 275 rpm shake for 48 hours. After incubation, cultures in the 96 deep-well plate were pelleted by centrifuging at 4000 G for 5 minutes, after measuring cODeoo of the cultures, d-biotin concentrations of supernatants were determined using the bioassay described in the analytical section above. Figure 2 shows the in vivo biotin production in response to IPTG induction for strain BS3477 expressing B. obeum T2BioB in response to IPTG induction. The figure show that this strain expressed T2BioB and producing d-biotin in quantity in vivo under microaerobic cultivation, but not under aerobic cultivation. Similar screening of E. coli strains BS3306, BS3307, BS3328, BS3329, BS3331, BS3448 and BS3449 expressing otherT2BioBs showed significant biotin production under microaerobic cultivation.

Example 4 - Fermentation of E-coli expressing T2BioB at different levels of oxygenation [0116] A single colony of BS3308 streaked on LB agar plate with ampicillin was inoculated into 50 ml mMOPS medium with ampicillin in a 250 ml shake flask and incubated at 37°C and 250 rpm shake for 16 hours resulting in an optical density of cOD ₆ oo=3.72. 200 ml B medium supplemented with ampicillin was added to an Applikon 500 ml MiniBio Reactor with temperature set to 37°C, pH controlled to pH=7 by addition of 5 M NH ₄ OH and dissolved oxygen (DO) set-point to DO=15% by controlling sparging of atmospheric air and agitation speed. 10 ml of the BS3308 culture was used to inoculate the B medium resulting in an initial cOD ₆ oo=0.186. 2.12 hours after inoculation, the culture was induced by addition of IPTG to a concentration of 0.119 g/L and from 3 hours after inoculation F medium was added to the culture at a feed rate of 0.14 ml/min. The DO control was terminated at 10.42 hours incubation, agitation fixed to between 200 and 1200 rpm (resulting in spOUR between 1 and 5), sparging fixed at 1 VVM and 1 ml of 1% antifoam solution added. Additional 1 ml antifoam solution was added 26.40 hours and fermentation terminated at 30.23 hours. Culture samples were taken from the fermenter at 0.00, 6.00, 11.58, 21.17, 25.00 and 30.00 hour after inoculation. From these samples optical density (cODeoo) was measured according to analytical procedure I and supernatants were obtained by spinning biomass down in a microcentrifuge at 17,000 G for 1 minute. Supernatants were screened for d-biotin using bioassay described in analytical procedure II. Growth and d-biotin production of BS3308 over time in cultivation can be seen in figure 4, which shows biotin production from DTB by recombinant E. coli cells expressing a B obeum T2BioB as a function of oxygen uptake rate and reveals both growth and d-biotin production over time at microaerobic cultivation conditions. The same procedure described here at optimal spOUR was also performed using strain BS3477 instead of BS3308. The difference in the strains was that BS3477 carried plasmid pBS1886 instead of pBS1676. pBS1886 encodes the same T2BioB as pBS1676, but at higher expression level. D-biotin was produced at titers of 50 mg/L.

Example 5 - Production of biotin from glucose by E. coli cell factory expressing B. obeum T2BioB in batch culture

[0117] E. coli strain BS3604 comprising plasmid pBS1886 encoding B. obeum T2BioB and plasmid pBS15656 encoding pathway genes required to produce desthiobiotin (DTB), from malonyl-CoA was prepared as described above. The pathway genes included genes encoding a malonyl-acyl carrier protein methyltransferase (EC2.1.1.197) converting Malonyl-acyl carrier protein to malonyl-acyl carrier protein methyl ester; a pimeloyl-acyl carrier protein methyl ester esterase (EC3.1.1.85) converting O-methylpimeloyl-acyl carrier protein to pimeloyl-acyl carrier protein; a 8-amino-7- oxononanoate synthase (EC2.3.1.47) converting Pimeloyl-acyl carrier protein to KAPA; a Adenosylmethionine-8-amino-7-oxononanoate transaminase (EC2.6.1.62) converting KAPA to DAP A; and a desthiobiotin synthase (EC6.3.3.3) converting DAPA to DTB. Precultures of production strain BS3604 were prepared by inoculating a colony into 400 pL mMOPS medium with ampicillin and kanamycin in a 96 deep-well plate, incubated at 37°C with shake at 275 rpm for 16-18 hours. Production cultures were established by inoculating with 4 pL from the precultures into 400 pL S medium with ampicillin and kanamycin and with 4 g rather than 10 g of glucose and comprising isopropyl b-D-l-thiogalactopyranoside (IPTG) at a final concentration of 10 pM, in a 96 deep-well plate (DWP) (EnzyScreen, CR1496a). The inoculated wells of the DWP were sealed with an aluminum seal. This step is critical for lowering oxygen conditions for T2BioB to function. Cultures were then grown at 37°C with 275 rpm shake for 24 hours. After incubation, cultures in the 96 deep-well plate were pelleted by centrifuging at 4000 G for 5 minutes, after measuring cOD ₆ oo of the cultures, d-biotin concentrations of supernatants were determined using the bioassay described in the analytical section above. Using the production conditions described here biotin was produced from glucose using an E. coli cell factory in batch culture as shown in figure 5.

Example 6 - Production of biotin from glucose by E. coli cell factory expressing B. obeum T2BioB in fed-batch culture

[0118] E. coli strain BS3604 comprising plasmid pBS1886 encoding B. obeum T2BioB and plasmid pBS1565 encoding pathway genes required to produce desthiobiotin (DTB), from malonyl-CoA is prepared as described above. The pathway genes include genes encoding a malonyl-acyl carrier protein methyltransferase (EC2.1.1.197) converting Malonyl-acyl carrier protein to malonyl-acyl carrier protein methyl ester; a pimelyl-acyl carrier protin methyl ester esterase (EC3.1.1.85) converting O-methylpimeloyl-acyl carrier protein to pimeloyl-acyl carrier protein; a 8-amino-7-oxononanoate synthase (EC2.3.1.47) converting Pimeloyl-acyl carrier protein to KAPA; a Adenosylmethionine-8- amino-7-oxononanoate transaminase (EC2.6.1.62) converting KAPA to DAP A; and a desthiobiotin synthase (EC6.3.3.3) converting DAPA to DTB. A single colony of BS3604 from streaking on LB agar plate with ampicillin and kanamycin is inoculated into 50 ml mMOPS medium with ampicillin and kanamycin in a 250 ml shake flask and incubated at 37°C and 250 rpm shake for 16 hours. 200 ml B medium but without any addition of desthiobiotin and supplemented with ampicillin and kanamycin is added to an Applikon 500 ml MiniBio Reactor with temperature set to 37°C, pH controlled to pH=7 by addition of 5 M NH ₄ OH and dissolved oxygen (DO) set-point to DO= 10-15% by controlling sparging of atmospheric air and agitation speed. 10 ml of the BS3604 culture is used to inoculate the B medium without DTB. 2 hours after inoculation, the culture is induced by addition of IPTG to a concentration of 0.119 g/L and starting at 3 hours after inoculation F medium is added to the culture at a feed rate of 0.14 ml/min. The DO control is terminated after 10 hours of cultivation and agitation is fixed to 1000 rpm (targeting spOUR of 4), sparging fixed at 1 VVM. Cultivation is continued for a total of 48 hours. Samples are taken at 6-hour intervals. From these samples optical density (cOD ₆ oo) is measured according to analytical procedure I and supernatants are obtained by spinning biomass down in a microcentrifuge at 17,000 G for 1 minute. Supernatants are screened for d-biotin using bioassay described in analytical procedure II. Using this cultivation setup, d-biotin is produced from glucose - in some instances where high T2BioB expression plasmids are applied more than 40 mg/L of d-biotin.