Bio-manufacturing of D-biotin

Technical Field of the Invention

The present invention relates to the bio-manufacturing of D-biotin. In particular, the invention relates to the genetic engineering of biotin-expressing cells and enzyme engineering of biotin synthase to enhance D-biotin production; and to optimised methods of culturing the genetically engineered cells to produce D-biotin, recovering the D-biotin from cultivation media, and purifying the D-biotin.

Background to the Invention

D-biotin (vitamin H or vitamin B7) is essential to all three domains of life. Consequently, D-biotin is used as a feed additive in the livestock industries and is increasingly finding application in the pharmaceutical and cosmetics industries.

Currently, D-biotin is produced in industry via an eleven-step chemical synthesis process, which has high energy input and generates large volumes of solvent waste. However, renewable carbon feedstocks and the application of biotechnology represent an important opportunity in transforming this inefficient, costly and environmentally harmful process.

It is known that microorganisms, including many species of bacteria, yeast and fungi, naturally produce D-biotin. However, microorganisms that produce D-biotin do so only at low levels. This low-level D-biotin production in microorganisms is a major hurdle in D-biotin bio-manufacture. Thus, interest has turned to the genetic engineering of microorganisms to enhance the biosynthesis of D-biotin in such microorganisms. Escherichia coli E. coli). is commonly used as an chassis in synthetic biology and industrial biotechnology as it is widely studied and has an ample catalogue of molecular tools.

The genes involved in the metabolism of D-biotin in E. coli are found within its chromosome. The first locus consists of the bio operon, which is split into bioA, bioB, bioF, bioC, and bioD. Each of these genes encodes a function or enzyme involved in the D-biotin pathway. For example, bioB encodes biotin synthase which is involved in the pathway of converting pimelyl-CoA to biotin. Expression of the bio operon is repressed by transcription repressor birA, which can also act as a D-biotin protein ligase. Furthermore, overexpression of the of the bio operon in E. coli is inhibitory for growth, which may be linked to bioB and the Fe-S biosynthesis regulator, iscR.

Genetic engineering methods aiming to enhance the biosynthesis of D-biotin must therefore focus on ensuring that the genes relevant to the D-biotin pathway are amplified effectively, and that any precursors or enzymes/enzyme co-factors are not limiting, for example as described in WO2021/254927.

In addition, there is a need to optimise the downstream methods of D-biotin manufacture with the aim of achieving higher yields, lower productions costs and reducing environmental impact.

It is an aim of embodiments of the invention to provide an improved and efficient method for producing D-biotin, using genetically engineered biotin-expressing cells to enhance the production of D-biotin in the cells, in addition to optimising downstream bio-manufacturing processes, such as cultivation of biotin-expressing cells, recovery of D-biotin from cultivation media and purification of D-biotin. Accordingly, it is also an aim of embodiments of the invention to provide an alternative method of producing D-biotin to current production processes that is more efficient, cost effective and more environmentally friendly.

It is also an aim of embodiments to the invention to overcome or mitigate at least one problem of the prior art, whether disclosed herein or not.

Summary of the Invention

“D-biotin” is used interchangeably with “biotin” and it is intended that both terms have the same meaning herein.

The terms “genetically modified”, “genetically engineered”, “genetic modifications” and “genetic engineering” are terms used interchangeably herein to describe lab-based technologies to alter the DNA of an organism.

The term “upstream processing” is used herein to describe the initial stages of fermentation, including the preparation of genetically and enzyme engineered cell factory that overproduces D-biotin.

The term “downstream processing” is used herein to describe the recovery and purification of D-biotin.

The “% identity” of the sequences throughout were calculated using the BLASTP program.

According to a first aspect of the invention there is a biotin-expressing cell comprising one or more genetic modifications selected from the group consisting of;

I. an inactivation or deletion of the native biotin synthase, bioB, and further comprising an overexpressed exogenous nucleic acid encoding a polypeptide having the activity of biotin synthase, II. an inactivation or deletion of the Fe-S biosynthesis regulator iscR,

III. a mutated biotin transcriptional repressor birA G57S from Escherichia coli,

IV. a mutated biotin transcriptional repressor birA Y58F from Escherichia coli,

V. any combination of I. to IV ; and wherein the genetically engineered cell produces D-biotin.

The genetic engineering of the biotin-expressing cells and the enzyme engineering of this invention relates to the upstream processing of D-biotin biomanufacture.

In some embodiments, the biotin-expressing cell comprising at least one modification selected from; (II.) an inactivation or deletion of the Fe-S biosynthesis regulator iscR, (III.) a mutated biotin transcriptional repressor birA G57S from E. coli, (IV.) a mutated biotin transcriptional repressor birA Y58F from E. coli; may further comprise an overexpressed exogenous nucleic acid encoding a polypeptide having the activity of biotin synthase. Thus, the genetically modified cell of this invention may also be subject to enzyme engineering. The inventors surprisingly found that the use of enzyme engineering according to this invention further enhances D-biotin production.

The native birA polypeptide sequence from E.coli is shown in SEQ ID NO:9, and thus (III) and (IV) comprise mutated versions comprising substitutions G57S and Y58F respectively, compared to SEQ ID NO:9.

In some embodiments, in the circumstances where the biotin-expressing cell has one or more of the modifications II. -IV., the genetically modified cell may not be subject to enzyme engineering in order to produce increased levels of D-biotin compared to the native cell. The genetic modifications I. to IV. may be a chromosome modification, which may be a modification of the bioB, IscR or birA genes. In some embodiments, genetic modifications of this invention relate solely to chromosome modifications of the biotinexpressing (host) cell.

In some embodiments, the biotin-expressing cell is a host cell.

The biotin-expressing cell may be a procaryote or eukaryote. Preferably, the biotin-expressing cell is a prokaryote.

In some embodiments, the biotin-expressing cell is a bacterium. The bacterium may be selected from the group consisting of; Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, Halomonas bluephagenesis and Bacillus subtillis. Preferably, the biotin-expressing cell is Escherichia coli (E. coll).

In other embodiments, the biotin-expressing cell is a yeast. The yeast may be selected from the group consisting of; Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica.

The biotin-expressing cell may be a recombinant host cell wherein DNA or one or more polynucleotide sequence is transferred into the biotin-expressing cell. The polynucleotide sequence(s) may be transferred into the host cell by an expression vector, which in some embodiments may be as described below. In some embodiments, the polynucleotide sequence(s) to be transferred is a polynucleotide encoding a polypeptide having the activity of biotin synthase, and preferably overexpressing the polynucleotide.

In some embodiments, the polypeptide having the activity of biotin synthase is a variant of an isolated polypeptide from E. coli. Preferably, the polynucleotide and/or polypeptide is from E. coli BW25113. Polypeptides having biotin synthase activity with E.coli origin are set out in SEQ ID NO: 1 to 5, with SEQ ID NO: 1 being a native sequence.

The polypeptide having the activity of biotin synthase may have at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:1. Preferably, the polypeptide having the activity of biotin synthase may have at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO:1.

The polypeptide having the activity of biotin synthase may have an amino acid residue substitution at the amino acid position corresponding to position 189 of SEQ ID NO:1. The amino acid residue substitution at the amino acid position corresponding to position 189 may be selected from the group consisting of: SI 89V, S189Q, and S189N.

The polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at the amino acid position corresponding to position 189 of SEQ ID NO:1, an amino acid substitution at one or more amino acid positions corresponding to positions selected from: 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 214, 215, 218, 220, 222, 224, 242, 243 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 136, 149 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to amino acid 189 of SEQ ID NO: 1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 186, 187, 188 and 289 of SEQ ID NO:l.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 218, 242, 243 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to amino acid position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 214, 215, 218, 220, 222, 224, 242, 243 and 289 of SEQ ID NO:1.

The amino acid residue substitution at the amino acid position corresponding to position 59 of SEQ ID NO: 1 may be Y59V or Y59I. The amino acid residue substitution at the amino acid position corresponding to position 85 of SEQ ID NO: 1 may be A85E. The amino acid residue substitution at the amino acid position corresponding to position 105 of SEQ ID NO:1 may be P105T. The amino acid residue substitution at the amino acid position corresponding to position 107 of SEQ ID NO: 1 may be E107R or E107C. The amino acid residue substitution at the amino acid position corresponding to position 136 of SEQ ID NO:1 may be E136A. The amino acid residue substitution at the amino acid position corresponding to position 149 of SEQ ID NO: 1 may be Y 1491. The amino acid residue substitution at the amino acid position corresponding to position 184 of

SEQ ID NO:1 may be G184N. The amino acid residue substitution at the amino acid position corresponding to position 186 of SEQ ID NO:1 may be K186T. The amino acid residue substitution at the amino acid position corresponding to position 187 of SEQ ID NO:1 may be V187E. The amino acid residue substitution at the amino acid position corresponding to position 188 of SEQ ID NO:1 may be C188N. The amino acid residue substitution at the amino acid position corresponding to position 214 of SEQ ID NO:1 may be T214P. The amino acid residue substitution at the amino acid position corresponding to position 215 of SEQ ID NO:1 may be P215A. The amino acid residue substitution at the amino acid position corresponding to position 218 of SEQ ID NO:1 may be S218I or S218V. The amino acid residue substitution at the amino acid position corresponding to position 220 of SEQ ID NO:1 may be P220H. The amino acid residue substitution at the amino acid position corresponding to position 222 of SEQ ID NO:1 may be N222L. The amino acid residue substitution at the amino acid position corresponding to position 224 of SEQ ID NO: 1 may be L224A. The amino acid residue substitution at the amino acid position corresponding to position 242 of SEQ ID NO:1 may be D242K. The amino acid residue substitution at the amino acid position corresponding to position 243 of SEQ ID NO:1 may be F242I. The amino acid residue substitution at the amino acid position corresponding to position 289 may be K249N.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:2. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:2, more preferably at least 80% sequence identity with SEQ ID N0:2, or most preferably at least 90% sequence identity with SEQ ID NO:2.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:3. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:3, more preferably at least 80% sequence identity with SEQ ID NO:3, or most preferably at least 90% sequence identity with SEQ ID NO:3.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:4. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:4, more preferably at least 80% sequence identity with SEQ ID NO:4, or most preferably at least 90% sequence identity with SEQ ID NO:4.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:5. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:5, more preferably at least 80% sequence identity with SEQ ID NO:5, or most preferably at least 90% sequence identity with

SEQ ID NO:5. In a further embodiment, the polypeptide having the activity of biotin synthase may be a variant of an isolated polypeptide from Streptomyces. Preferably, the polypeptide is from Streptomyces sp. 769. Polypeptides having biotin synthase activity with Streptomyces sp. 769 origin are set out in SEQ ID NO: 6 to 8, with SEQ ID NO: 6 being a native sequence.

The polypeptide having the activity of biotin synthase may have at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:6. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:6, more preferably at least 80% sequence identity with SEQ ID NO:6, or most preferably at least 90% sequence identity with SEQ ID NO:6.

The polypeptide having the activity of biotin synthase may have an amino acid substitution at one or more amino acid positions corresponding to positions selected from: 295, 306, 314 and 317 of SEQ ID NO:6. The amino acid residue substitutions at the amino acid positions corresponding to positions 295, 306, 314, 317 of SEQ ID NO:6, may be selected from the group consisting of: I295A, Q306L, M3141 and D317R.

The polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution(s) at amino acid positions corresponding to positions 295, 306, 314 and/or 317 of SEQ ID NO:6, an amino acid residue substitution at amino acid position corresponding to position 254 and/or 255 of SEQ ID NO:6. The further amino acid residue substitutions may be substitutions corresponding to R254K and/or C255I. In some embodiments, the polypeptide having the activity of biotin synthase may have an amino acid residue substitution at amino acid positions corresponding to positions 295, 306, 314 and 317 of SEQ ID NO:6. The polypeptide may have amino acid residue substitutions at amino acid positions corresponding to the following positions of SEQ ID NO:6: 254, 255, 295, 306, 314 and 317.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO: 7. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:7, more preferably at least 80% sequence identity with SEQ ID NO:7, or most preferably at least 90% sequence identity with SEQ ID NO:7.

The polypeptide having the activity of biotin synthase may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO: 8. Preferably, the polypeptide having the activity of biotin synthase may have at least 70% sequence identity with SEQ ID NO:8, more preferably at least 80% sequence identity with SEQ ID NO:8, or most preferably at least 90% sequence identity with SEQ ID NO:8.

In some embodiments, the biotin-expressing cell is transformed with an expression vector, wherein the expression vector comprises the polynucleotide encoding the polypeptide having the activity of biotin synthase as described above. The polynucleotide encoding the polypeptide having the activity of biotin synthase may be a variant of an isolated enzyme that has been subjected to enzyme engineering. Thus, in some embodiments, the biotin-expressing cells have been subjected to genetic and enzyme engineering.

In some embodiments, the genetically and enzyme engineered cells perform as cell factories for the overexpression of D-biotin.

In some embodiments, the expression vector may be a plasmid, a virus, or any other known means suitable for recombinant DNA methods.

In some embodiments, the expression vector is a plasmid. The plasmid may be a pBAD24 plasmid with ColEl, pMB l or pBR322 origin of replication, or a pCON- v23 plasmid with pl5A origin of replication, or any other suitable plasmid, as would be known to the skilled person or addressee.

The expression vectors may comprise selection genes to promote the transformation of the vector into the biotin-expressing cells. The selection genes may be an ampicillin resistance gene or a tetracycline resistance gene, or any other suitable antibiotic resistance gene or other suitable selection system.

The expression vectors may further comprise promotors of varying strength, and/or Ribosome Binding Sites (RBS) in order to control the growth rates of the cells relative to biotin synthase expression and the resulting product profile and yield. Suitable promotors and/or RBS are well-known in the art.

The transfer of the vector into the biotin-expressing cells may occur using any known methods in the art. The biotin-expressing cells may be prepared for DNA transfer prior to transforming the vector into the biotin-expressing cells, using any known methods in the art.

According to a second aspect of the invention, there is provided an isolated polypeptide having biotin synthase activity and having at least 70% identity with SEQ ID NO: 1 and comprising a substitution at amino acid position corresponding to position

189 of SEQ ID NO: 1.

The isolated polypeptide having biotin synthase activity may have at least at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:1.

In some embodiments, the isolated polypeptide is a variant of an isolated polypeptide from E. coli, more preferably from E. coli BW25113.

The isolated polypeptide may comprise one of the following amino acid residue substitutions at the amino acid position corresponding to position 189 of SEQ ID NO: 1 ; S189V, S189Q, or S189N.

The isolated polypeptide may comprise, in addition to the substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, a substitution at one or more amino acid positions corresponding to positions selected from; 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 214, 215, 218, 220, 222, 224, 242, 243 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 136, 149 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitutions at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 186, 187, 188 and 289 of SEQ ID NO:l.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 218, 242, 243 and 289 of SEQ ID NO:1.

In some embodiments, the polypeptide having the activity of biotin synthase may further comprise, in addition to the amino acid substitution at amino acid position corresponding to position 189 of SEQ ID NO:1, amino acid substitutions at amino acid positions corresponding to positions 59, 85, 105, 107, 136, 149, 184, 186, 187, 188, 214, 215, 218, 220, 222, 224, 242, 243 and 289 of SEQ ID NO:1.

The amino acid residue substitution at the amino acid position corresponding to position 59 of SEQ ID NO: 1 may be Y59V or Y59I. The amino acid residue substitution at the amino acid position corresponding to position 85 of SEQ ID NO: 1 may be A85E. The amino acid residue substitution at the amino acid position corresponding to position 105 of SEQ ID NO:1 may be P105T. The amino acid residue substitution at the amino acid position corresponding to position 107 of SEQ ID NO: 1 may be E107R or E107C. The amino acid residue substitution at the amino acid position corresponding to position 136 of SEQ ID NO:1 may be E136A. The amino acid residue substitution at the amino acid position corresponding to position 149 of SEQ ID NO: 1 may be Y 1491. The amino acid residue substitution at the amino acid position corresponding to position 184 of SEQ ID NO:1 may be G184N. The amino acid residue substitution at the amino acid position corresponding to position 186 of SEQ ID NO:1 may be K186T. The amino acid residue substitution at the amino acid position corresponding to position 187 of SEQ ID NO:1 may be V187E. The amino acid residue substitution at the amino acid position corresponding to position 188 of SEQ ID NO:1 may be C188N. The amino acid residue substitution at the amino acid position corresponding to position 214 of SEQ ID NO:1 may be T214P. The amino acid residue substitution at the amino acid position corresponding to position 215 of SEQ ID NO:1 may be P215A. The amino acid residue substitution at the amino acid position corresponding to position 218 of SEQ ID NO:1 may be S218I or S218V. The amino acid residue substitution at the amino acid position corresponding to position 220 of SEQ ID NO:1 may be P220H. The amino acid residue substitution at the amino acid position corresponding to position 222 of SEQ ID NO:1 may be N222L. The amino acid residue substitution at the amino acid position corresponding to position 224 of SEQ ID NO: 1 may be L224A. The amino acid residue substitution at the amino acid position corresponding to position 242 of SEQ ID NO:1 may be D242K. The amino acid residue substitution at the amino acid position corresponding to position 243 of SEQ ID NO:1 may be F242I. The amino acid residue substitution at the amino acid position corresponding to position 289 may be K249N.

The isolated polypeptide may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:2. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:2, more preferably at least 80% sequence identity with SEQ ID NO:2, or most preferably at least 90% sequence identity with

SEQ ID NO:2. The isolated polypeptide may have at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:3. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:3, more preferably at least 80% sequence identity with SEQ ID NO:3, or most preferably at least 90% sequence identity with SEQ ID NO:3.

The isolated polypeptide may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:4. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:4, more preferably at least 80% sequence identity with SEQ ID NO:4, or most preferably at least 90% sequence identity with SEQ ID NO:4.

The isolated polypeptide may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO:5. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:5, more preferably at least 80% sequence identity with SEQ ID NO:5, or most preferably at least 90% sequence identity with SEQ ID NO:5.

The isolated polypeptide according to the second aspect of the invention may be inserted into an expression vector using any known methods in the art.

In some embodiments, the expression vector containing the isolated polypeptide may be transformed into a cell. In further embodiments, the expression vector containing the isolated polypeptide may be transformed into the biotin-expressing cell of the first aspect of the invention. According to a third aspect of the invention, there is provided an isolated polypeptide having biotin synthase activity and having at least 70% identity with SEQ ID NO:6 and comprising a substitution at one or more amino acid positions selected from: 295, 306, 314 and 317 of SEQ ID NO:6.

In some embodiments, the isolated polypeptide may be a variant of an isolated polypeptide from Streptomyces. Preferably, the polypeptide is from Streptomyces sp. 769. Polypeptides having biotin synthase activity with Streptomyces sp. 769 origin are set out in SEQ ID NO: 6 to 8, with SEQ ID NO:6 being a native sequence.

The isolated polypeptide may have at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or 100% sequence identity with SEQ ID NO:6. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:6, more preferably at least 80% sequence identity with SEQ ID NO:6, or most preferably at least 90% sequence identity with SEQ ID NO:6.

The isolated polypeptide may have one or more amino acid residue substitutions at amino acid positions corresponding to positions selected from: 295, 306, 314 and 317 of SEQ ID NO:6. The amino acid residue substitutions at the amino acid positions corresponding to positions 295, 306, 314, 317 of SEQ ID NO:6, may be selected from the group consisting of: I295A, Q306L, M3141 and D317R.

The isolated polypeptide may further comprise, in addition to the amino acid substitution at amino acid positions corresponding to positions 295, 306, 314 and/or 317 of SEQ ID NO:6, an amino acid residue substitution(s) at amino acid position(s) corresponding to position(s) 254 and/or 255 of SEQ ID NO:6. The further amino acid residue substitutions may be substitutions corresponding to R254K and/or C255I. In some embodiments, the isolated polypeptide may have an amino acid residue substitution at amino acid positions corresponding to positions of SEQ ID NO: 6: 295, 306, 314 and 317. The isolated polypeptide may have amino acid residue substitutions at the amino acid positions corresponding to the following positions of SEQ ID NO:6: 254, 255, 295, 306, 314 and 317.

The isolated polypeptide may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO: 7. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:7, more preferably at least 80% sequence identity with SEQ ID NO:7, or most preferably at least 90% sequence identity with SEQ ID NO:7.

The isolated polypeptide may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% sequence identity with SEQ ID NO: 8. Preferably, the isolated polypeptide may have at least 75% sequence identity with SEQ ID NO:8, more preferably at least 80% sequence identity with SEQ ID NO:8, or most preferably at least 90% sequence identity with SEQ ID NO:8.

The isolated polypeptide according to the third aspect of the invention may be inserted into an expression vector using any known methods in the art.

In some embodiments, the expression vector containing the isolated polypeptide may be transformed into a cell. In further embodiments, the expression vector containing the isolated polypeptide may be transformed into the biotin-expressing cell of the first aspect of the invention. According to a further aspect of the invention there is provided a cell comprising a nucleotide sequence encoding a polypeptide of any one of the isolated polypeptide sequences defined in the second or third aspects of this invention (i.e. sequences corresponding to SEQ ID NO: 1 to 8). In further embodiments, the cell may comprise an expression vector wherein the expression vector comprises a nucleotide sequence encoding a polypeptide of any one of the isolated polypeptide sequences defined in the second or third aspects of this invention (i.e. sequences corresponding to SEQ ID NO: 1 to 8).

The biotin-expressing cell of the first aspect of this invention may comprise a nucleotide sequence encoding a polypeptide of any one of the isolated polypeptide sequences defined in the second or third aspects of this invention (i.e. sequences corresponding to SEQ ID NO: 1 to 8). In further embodiments, the biotin-expressing cell of the first aspect of this invention may comprise an expression vector wherein the expression vector comprises a nucleotide sequence encoding a polypeptide of any one of the isolated polypeptide sequences defined in the second or third aspects of this invention (i.e. sequences corresponding to SEQ ID NO: 1 to 8). The cell and/or vector comprising a nucleotide sequence encoding a polypeptide of any one of the isolated polypeptide sequences defined in the second or third aspect of this invention may be for use in, or used for, D-biotin production.

According to a fourth aspect of the invention, there is provided a method of biomanufacturing D-biotin using the biotin-expressing cell of this invention comprising the steps of:

I. Culturing the genetically engineered cells to produce D-biotin,

II. Recovering the D-biotin from aqueous media, III. Purifying the D-biotin.

Culturing the genetically engineered cells to produce D-biotin (step I.) comprises upstream processing. Recovering (step II.) and purifying (step III.) the D- biotin comprises downstream processing.

The genetically engineered cells may be cultured using any known methods in the art. In some embodiments, step I. comprises a method of fermentation. The biotinexpressing cells may be fermented under aerobic or micro-aerobic conditions. Aerobic or micro-aerobic conditions are particularly effective for ease of production.

In some embodiments, step I. comprises batch fermentation, fed-batch fermentation, and/or continuous fermentation. Fed-batch methods may be preferred because of the controlled addition of substrate that allows for increased yields and productivity of the industrial process.

In some embodiments, step I. comprises repeated fermentations. Preferably, step I. comprises duplicate or triplicate fermentations.

Additionally, or alternatively, the cell culture may be cultivated using shake flask cultivation.

During step I., the biotin-expressing cells may be cultured in minimal media comprising carbon and nitrogen sources and inorganic salts. In some embodiments, the cells are cultured in M9 media. The principal carbon source fed to the cell culture may be at least one compound selected from the group consisting of: monosaccharides, disaccharides, cellulose, hemicellulose, levulinic acid, glycerol, triglycerides, fatty acids, derivatives thereof and any combination thereof. The cell culture may be cultivated at a temperature of less than 45 °C, less than 40 °C, less than 35 °C, less than 30 °C, or less than 25 °C. The cell culture may be cultivated at a temperature of between 25-45 °C, or more preferably 30-40 °C. Preferably, the cell culture may be cultivated at a temperature of around 37 °C.

The cell culture may be cultivated for less than 1 week, less than 120 hours, less than 108 hours, less than 96 hours, less than 84 hours, less than 72 hours, less than 60 hours, less than 48, less than 24 hours, less than 12 hours, less than 6 hours, less than 3 hours, or less than 1 hour. The cell culture may be cultivated for between, 6-120 hours, or 12-80 hours.

The cell culture was cultivated at a pH of between 5 and 9, between 6 and 9 or preferably between 6.5 and 8. In some embodiments the pH is between 6.5 and 7.5, or between 6.8 and 7.5, or between 6.9 and 7.4.

In some embodiments, step I. may comprise retaining the biotin-expressing cells using a ceramic membrane to maintain a high cell density during cultivation. This invention is not limited to the type of separation membrane used and other suitable known membranes in the art may be used.

The fermentation method described hereinabove may increase D-biotin production compared to other fermentation methods known in the art and reduces production costs.

During step II., D-biotin may be recovered from aqueous cultivation media using known methods in the art.

D-biotin may be recovered using an ion exchange resin. In some embodiments,

D-biotin is recovered by adsorption onto an anion exchange resin, followed by elution. The anion exchange resin may be a strongly basic anion exchange resin or a weakly basic anion exchange resin. Preferably, a weakly basic resin is selected. A weakly basic anionic resin has the advantage that less expensive reagents and eluents can be used, thus reducing manufacturing costs. The anion exchange resin may be in the bicarbonate form. Any suitable anionic exchange resin, such as a weak or strong anionic resin, may be usedD-biotin may be recovered using the anion exchange resin, Amberlite® IRA- 96 or Amberlite® IRA-400, for example.

The ion exchange methods may be carried out in a fluidised bed with a linear velocity.

D-biotin may be eluted from an anion exchange resin using a weakly acidic, neutral or weakly basic eluent. The eluent may have a pH in the range 5-11 or between 6-11 or between 7-10. Preferably the anion exchange resin is neutral or weakly basic. The eluent may be selected from: ammonium bicarbonate, sodium hydroxide and ammonium hydroxide.

An additional further step may be included following recovery of the D-biotin using an anion exchange mechanism. This additional step may comprise recovering the excess eluent from the eluate as ammonia via stream stripping (which may be in a reboiled absorber) or via direct steam injection. This additional method aims to improve the efficiency and environmental impact of the overall method of D-biotin production.

In some embodiments, the purification of D-biotin may comprise crystallisation of the D-biotin, such as from an aqueous solution by crystallisation. Alternative methods of purification known in the art may also be used, such as extraction methods, ultrafiltration and/or solvent evaporation. D-biotin may be crystallised by attaining a pH less than 7, less than 6, less than 5, less than 4, less than 3. Preferably, D-biotin may be crystallised by attaining a pH of around 2-5. More preferably, by attaining a pH of around 3. The pH may be attained using a mineral acid, an organic acid, or carbon dioxide.

The purification of D-biotin may comprise one or more rounds of crystallisation.

The crystallisation may be a cooling crystallisation. A cooling crystallisation further enhances the recovery of D-biotin.

The crystallisation may proceed at a temperature of less than 20 °C, less than 15 °C, less than 12 °C, less than 10 °C, less than 8 °C, or less than 6 °C. Preferably, the cooling crystallisation may proceed at a temperature between 5-9 °C.

The method of bio-manufacturing D-biotin may further comprise an additional step(s) to those of I. to III. Additional step(s) may comprise but are not limited to separation, centrifugation and/or drying processes. The additional steps may occur before, during and/or after any of steps I.-III. mentioned above.

Detailed Description of the Invention

In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 illustrates a process block diagram of the D-biotin bio-manufacturing process comprising upstream and downstream processing unit operations, according to this invention. Figure 2 illustrates the improvement in D-biotin production in shake flask culture after each round of enzyme engineering to the biotin synthase of E. coli BW25113 and Streptomyces sp. 769., according to the genetically engineered biotin-expressing cells of this invention.

Figure 3 illustrates the performance of the D-biotin overproducing cell factory as a consequence of genetic engineering entailing modification of the host strain and overexpression of an enzyme engineered biotin synthase, according to the genetically engineered biotin-expressing cells of this invention.

Figure 4 illustrates the accumulation of D-biotin and shows the specific yield on biomass in duplicate fed-batch fermentations, according to the method of bio-manufacturing D-biotin of this invention.

Figure 5 illustrates the dynamic adsorption capacity for an anion exchange resin, Amberlite IRA-96, for different D-biotin feed concentrations with a biotin: acetate mass ratio of 1:2, according to the method of biomanufacturing D-biotin of this invention. The Figure demonstrates the selectivity of the solid phase for D-biotin obtained from the breakthrough curve experiments.

Figure 6 illustrates the D-biotin recovery and crystal purity (mass basis) for pH crystallisation as a function of pH, according to the method of biomanufacturing D-biotin of this invention.

Figure 7 illustrates the time course of further D-biotin recovery owed to cooling crystallisation at 7 °C from the mother liquor obtained after pH crystallisation at pH 2, according to the method of bio-manufacturing D- biotin of this invention.

Figure 1 shows a block diagram for the bio-manufacture of D-biotin from renewable feedstocks, according to the upstream and downstream processes of this invention. Upstream processing entails fermentation, underpinned by a genetically and enzyme engineered cell factory that overproduces D-biotin. Downstream processing entails primary recovery via anion exchange and ammonia stripping, purification by pH & cooling crystallisation, centrifugation and drying.

Example 1 - Genetically modified construct for D-biotin production

(a) A parent strain of E. coli BW25113 was constructed through knock-out of the native biotin synthase, bioB. The modified (bioB knockout) parent strain is named E. coli BW25113 bioB.

From the parent strain, E. coli BW25113 bioB, three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113, the isolated polypeptide form of which is shown in SEQ ID NO:1 (2) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal expression vector (e.g. pBAD24 and pCON) constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted bioB E. coli. (b) Using the E. coli BW25113 AbioB parent strain from (a), a second strain was constructed through knock-out of the Fe-S biosynthesis regulator, iscR. From this second strain, E. coli BW25113 AbioB AiscR. three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113 (SEQ ID NO:1), (2) a codon optimised biotin synthase from E. coli BW25 113 (SEQ ID NO: 1) mutated in S 189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal vector constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted by AbioB AiscR E. Coli.

(c) Using the E. coli BW25113 AbioB strain from (a), a third strain was constructed through knock-in of a mutated biotin transcriptional regressor, birA G57S. From this third strain, E. coli BW25113 AbioB birA G57S, three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113 (SEQ ID NO: 1), (2) a codon optimised biotin synthase from E. coli BW25113(SEQ ID NO:1) mutated in S189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal vector constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted by AbioB S57 birA E. coli. (d) Using the E. coli BW25113 AbioB parent strain from (a), a fourth strain was constructed through knock-in of a mutated biotin transcriptional regressor, birA Y58F. From this fourth strain, E. coli BW25113 AbioB birA Y58F, three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113 (SEQ ID NO: 1), (2) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal vector constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted by AbioB F58 birA E. coli.

(e) Using the E. coli BW25113 AbioB AiscR strain from (b), a fifth strain was constructed through knock-in of a mutated biotin transcriptional regressor, birA G57S. From this fifth strain, E. coli BW25113 AbioB AiscR G57S, three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113 (SEQ ID NO: 1), (2) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal vector constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted by AbioB S57 birA AiscR E. coli. (f) Using the E. coli BW25113 AbioB iscR strain from (b), a sixth strain was constructed through knock-in of a mutated biotin transcriptional regressor, birA Y58F. From this sixth strain, E. coli BW25113 AbioB AiscR Y58F, three cell factories were constructed respectively overexpressing, (1) a codon optimised, wild-type biotin synthase from E. coli BW25113 (SEQ ID NO: 1), (2) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189V and (3) a codon optimised biotin synthase from E. coli BW25113 (SEQ ID NO:1) mutated in S189Q, using an episomal vector constitutively expressing the single gene operon.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 2 summarises the overproduction of D-biotin for these three cell factories depicted by AbioB F58 birA AiscR E. coli.

All D-biotin concentrations were analysed by HPLC using a C18 column.

FIG 2 demonstrates the overproduction of D-biotin by all constructed strains (a) to (f).

Example 1 shows the benefit of genetically modifying cells according to this invention to the biosynthesis of D-biotin; the benefit being the overproduction of D- biotin compared to cells in their native environment. Thus, helping to overcome one of the major hurdles in D-biotin bio-manufacture.

Example 2 - Overexpression of D-biotin by recombinant host cells with bio synthase enzyme engineering

(a) The first recombinant strain E. coli BW25113 AbioB (a) from Example 1 was used as host background and transformed with an episomal vector (pBAD24 and pCON) overexpressing enzyme variants of the biotin synthase from E. coli BW25113. The isolated polypeptide form of these enzyme variants, obtained from five rounds of enzyme engineering of the isolated polypeptide of SEQ ID NO: 1, are shown in SEQ ID NO:2 - SEQ ID NO:5.

The enzyme variant, SEQ ID NO:2, has a sequence identity of 98% with SEQ ID NO:1. The enzyme variant, SEQ ID NO:3, has a sequence identity of 97% with SEQ ID NO: 1. The enzyme variant, SEQ ID NO:4, has a sequence identity of 96% with SEQ ID NO: 1. The enzyme variant, SEQ ID NO:5, has a sequence identity of 94% with SEQ ID NO:1.

Sequence % identity was calculated using the BLASTP programme.

Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 3a summarises the overproduction of D-biotin for these four cell factories, noting that Round 5 overexpresses the same sequence as SEQ ID NO: 5.

(b) The first recombinant strain E. coli BW25113 bioB (a) from Example 1 was used as host background and transformed with an episomal vector overexpressing enzyme variants of the biotin synthase from Streptomyces sp. 769. The isolated polypeptide form of these enzyme variants, obtained from two rounds of enzyme engineering of the isolated polypeptide of SEQ ID NO: 6, are shown in SEQ ID NO:7 - SEQ ID NO:8.

The enzyme variant, SEQ ID NO:, has a sequence identity of 99% with SEQ ID NO:6. The enzyme variant, SEQ ID NO:8, has a sequence identity of 98% with SEQ

ID NO:6.

Sequence % identity was calculated using the BLASTP programme. Shake flask cultures were undertaken in triplicate using M9 minimal media and incubated at 37 °C for 24 hours. FIG 3b summarises the overproduction of D-biotin for these two cell factories.

FIG 3 a and FIG 3b demonstrate enhanced D-biotin production as a consequence of the enzyme engineering to the respective biotin synthase (bioB) from either E. coli BW25113 or Streptomyces sp. 769.

Example 2 highlights the advantages of using the enzyme engineering of biotin synthase according to this invention. The invention shows that the genetically modified biotin-expressing cells of this invention that are also subjected to enzyme engineering of biotin synthase further enhance D-biotin production. Surprisingly, amino acid substitutions at selected amino acid positions within an isolated polypeptide of known biotin synthases give rise to this enhanced D-biotin production.

Example 3 - Culturing the genetically modified cells by fermentation for D-biotin production

Using one of the high overexpressing cell factories from Example 1, E. coli BW25113 bioB iscR G57S overexpressing a codon optimised biotin synthase from E. coli BW25113 mutated in S189V (e); duplicate fed-batch fermentations were undertaken in M9 minimal media using glucose as carbon source.

Upon completion of the batch phase, a constant glucose feed was started and continued until completion of the fed-batch phase at 76 hours.

FIG 4 depicts the accumulation of D-biotin and the specific yield on biomass during the course of the duplicate fermentations, demonstrating consistent and appreciable D-biotin overproduction on a biomass specific basis. All D-biotin concentrations were analysed by HPLC using a C18 column.

Example 3 demonstrates the advantage of using a cell culturing method according to this invention for the overproduction of D-biotin.

Example 4 - Anionic exchange break-through for the recovery of D-biotin from aqueous media using an anionic exchange resin

Aqueous feed solutions of D-biotin and acetate were prepared at physiological pH (around pH 7.4) for anionic exchange break-through curve experiments, having a mass ratio of biotin: acetate of 1:2. Five feed solutions were prepared with D-biotin concentration ranging from 200 mg/L to 1400 mg/L.

Break-through curves were determined for each of the five feed solutions using a packed bed of anionic exchange resin, Amberlite IRA-96, in the HCO3’ form. The adsorption capacity of the Amberlite IRA-96 was calculated on a free settled resin basis by analysing the D-biotin in each of the collected column fractions.

FIG 5 summarises the favourable selectivity of the anion exchange resin for D- biotin as a function of the D-biotin concentration in the feed containing acetate as a typical by-product of fermentation.

All D-biotin concentrations were analysed by HPLC using a C18 column.

Example 4 demonstrates the benefits of using an anion exchange resin for the recovery of D-biotin in that more D-biotin is recovered from an aqueous solution when compared to known methods in the art. In addition, Example 4 highlights how the anion exchange recovery method of this invention uses relatively cheap eluents, therefore also providing the advantage of reducing costs. Example 5 - pH crystallisation experiments for the purification of D-biotin

An aqueous feed solution of D-biotin and acetate was prepared at physiological pH (around pH 7.4) for pH crystallisation experiments, having a biotin concentration of 5 g/L and an acetate concentration of 2 g/L. The feed solution was divided into five fractions and the pH was adjusted to pH 5, 4, 3, 2 and 1 using sulphuric acid respectively. The pH crystallisation experiments were undertaken at 16 °C for 24 hours. Crystal product was recovered via centrifugation and washed with a concentrated biotin solution and dried for 24 hours. The crystallisation experimental design was repeated in triplicate.

FIG 6 summarises the calculated D-biotin recovery and D-biotin crystal purity results as a function of pH, having solubilised the crystals using a 0.4 M NH4HCO3 solution. The results demonstrate high recoveries of D-biotin of greater than 80% (w/w) and high crystal purities of greater than 90% (w/w) below a pH of 4.

All D-biotin concentrations were analysed by HPLC using a C18 column.

Example 6 - Cooling crystallisation experiments for the purification of D-biotin

The mother liquor collected from the pH crystallisation experiments from Example 5 undertaken at pH 3 was further subjected to cooling crystallisation at 7 °C for approximately 25 minutes in triplicate.

FIG 7 summarises the time source of the batch cooling crystallisation experimental design, demonstrating greater than 10% (w/w) additional recovery of D- biotin from the residual D-biotin in the mother liquor. The crystallisation methods of this invention provide optimised methods for purifying D-biotin, which is typically relatively insoluble in water at pH 7. The inventors surprisingly found that crystallisation of D-biotin from an aqueous solution occurs by reducing the pH, and optionally cooling, producing D-biotin with a relatively high yield and purity.

The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

tin synthase BioB [Enterobacteriaceae]; NCBI Reference 0951213.1): VTELFEKPLLDLLFEAQQVHRQHFDPRQVQVSTLLSIKTGA SRYKTGLEAERLMEVEQVLESARKAKAAGSTRFCMGAAWK

NPHERDMPYLEQMVQGVKAMGLEACMTLGTLSESQAQRLANAGLDYYNHN

LDTSPEFYGNIITTRTYQERLDTLEKVRDAGIKVCSGGIVGLGETVKDRAGLLL

QLANLPTPPESVPINMLVKVKGTPLADNDDVDAFDFIRTIAVARIMMPTSYVR

LSAGREQMNEQTQAMCFMAGANSIFYGCKLLTTPNPEEDKDLQLFRKLGLNP

QQTAVLAGDNEQQQRLEQALMTPDTDEYYNAAAL

SEQ ID NO 2:

MAHRPRWTLSQVTELFEKPLLDLLFEAQQVHRQHFDPRQVQVSTLLSIKTGA

CPEDCKVCPQSSRYKTGLEAERLMEVEQVLESERKAKAAGSTRFCMGAAWK

NPHERDMPYLEQMVQGVKAMGLEACMTLGTLSASQAQRLANAGLDIYNHN

LDTSPEFYGNIITTRTYQERLDTLEKVRDAGIKVCQGGIVGLGETVKDRAGLL

LQLANLPTPPESVPINMLVKVKGTPLADNDDVDAFDFIRPIAVARIMMPTSYV

RLSAGREQMNEQTQAMCFMAGANSIFYGCNLLTTPNPEEDKDLQLFRKLGL

NPQQTAVLAGDNEQQQRLEQALMTPDTDEYYNAAAL

SEQ ID NO 3:

MAHRPRWTLSQVTELFEKPLLDLLFEAQQVHRQHFDPRQVQVSTLLSIKTGA

CPEDCKICPQSSRYKTGLEAERLMEVEQVLESERKAKAAGSTRFCMGAAWK

NTHCRDMPYLEQMVQGVKAMGLEACMTLGTLSASQAQRLANAGLDIYNHN LDTSPEFYGNIITTRTYQERLDTLEKVRDAGITENVGGIVGLGETVKDRAGLLL

QLANLPTPPESVPINMLVKVKGTPLADNDDVDAFDFIRPIAVARIMMPTSYVR

ESAGREQMNEQTQAMCFMAGANSIFYGCNEETTPNPEEDKDEQEFRKEGENP

QQTAVLAGDNEQQQRLEQALMTPDTDEYYNAAAL

SEQ ID NO 4:

MAHRPRWTLSQVTELFEKPLLDLLFEAQQVHRQHFDPRQVQVSTLLSIKTGA

CPEDCKICPQSSRYKTGLEAERLMEVEQVLESERKAKAAGSTRFCMGAAWK

NTHRRDMPYLEQMVQGVKAMGLEACMTLGTLSASQAQRLANAGLDIYNHN

LDTSPEFYGNIITTRTYQERLDTLEKVRDANITENVGGIVGLGETVKDRAGLLL

QLANLPTPPEVVPINMLVKVKGTPLADNDDVDAFKIIRPIAVARIMMPTSYVR

LSAGREQMNEQTQAMCFMAGANSIFYGCNLLTTPNPEEDKDLQLFRKLGLNP

QQTAVLAGDNEQQQRLEQALMTPDTDEYYNAAAL

SEQ ID NO 5:

MAHRPRWTLSQVTELFEKPLLDLLFEAQQVHRQHFDPRQVQVSTLLSIKTGA

CPEDCKICPQSSRYKTGLEAERLMEVEQVLESERKAKAAGSTRFCMGAAWK

NTHRRDMPYLEQMVQGVKAMGLEACMTLGTLSASQAQRLANAGLDIYNHN

LDTSPEFYGNIITTRTYQERLDTLEKVRDANITENVGGIVGLGETVKDRAGLLL

QLANLPPAPEIVHILMAVKVKGTPLADNDDVDAFKIIRPIAVARIMMPTSYVR

LSAGREQMNEQTQAMCFMAGANSIFYGCNLLTTPNPEEDKDLQLFRKLGLNP

QQTAVLAGDNEQQQRLEQALMTPDTDEYYNAAAL

SEQ ID N0:6: MDLLNTLVDKGLRREVPTRDEALAVLATSDDELLDVVAAAGKVRRTWFGR

RVKLNYLVNLKSGLCPEDCSYCSQRLGSKAEILKYTWLKPDDASKAAAAGV

AGGAKRVCLVASGRGPTDKDVDRVSETIATIKEQNEGVEVCACLGLLNDGQ

AERLRAAGADAYNHNLNTSEATYGDICTTHDFSDRVETVQQAQAAGMSACS

GLIAGMGETDADLVDVVFALRELDPDSVPVNFLIPFEGTPLAKEWNLTPQRCL

RILAMVRFVCPDVEVRLAGGREVHLRSMQPLALHLVNSIFLGDYLTSEGQAG

KDDLAMIADAGFEVEGTDTTTLPAHRRDTADATEPADAAAPTDPADAESAT

VPAPSEETRRDLVAVRRRGAGTDLPPNA

SEQ ID NO 7:

MDLLNTLVDKGLRREVPTRDEALAVLATSDDELLDVVAAAGKVRRTWFGR

RVKLNYLVNLKSGLCPEDCSYCSQRLGSKAEILKYTWLKPDDASKAAAAGV

AGGAKRVCLVASGRGPTDKDVDRVSETIATIKEQNEGVEVCACLGLLNDGQ

AERLRAAGADAYNHNLNTSEATYGDICTTHDFSDRVETVQQAQAAGMSACS

GLIAGMGETDADLVDVVFALRELDPDSVPVNFLIPFEGTPLAKEWNLTPQRCL

RILAMVRFVCPDVEVRLAGGREVHLRSMQPLALHLVNSAFLGDYLTSEGLAG

KDDLAIIARAGFEVEGTDTTTLPAHRRDTADATEPADAAAPTDPADAESATVP

APSEETRRDLVAVRRRGAGTDLPPNA

SEQ ID NO 8:

MDLLNTLVDKGLRREVPTRDEALAVLATSDDELLDVVAAAGKVRRTWFGR

RVKLNYLVNLKSGLCPEDCSYCSQRLGSKAEILKYTWLKPDDASKAAAAGV

AGGAKRVCLVASGRGPTDKDVDRVSETIATIKEQNEGVEVCACLGLLNDGQ

AERLRAAGADAYNHNLNTSEATYGDICTTHDFSDRVETVQQAQAAGMSACS GLIAGMGETDADLVDVVFALRELDPDSVPVNFLIPFEGTPLAKEWNLTPQKIL

RILAMVRFVCPDVEVRLAGGREVHLRSMQPLALHLVNSAFLGDYLTSEGLAG

KDDLAIIARAGFEVEGTDTTTLPAHRRDTADATEPADAAAPTDPADAESATVP

APSEETRRDLVAVRRRGAGTDLPPNA

SEQ ID NO 9 (birA from E.coliy.

MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFT

VPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD

ACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVM

AEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGIN

MAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAP

YLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEIS

LRSAEK