Cellular factories with improved transport of iron-sulfur clusters

Abstract

To provide a genetically modified bacterium for enhancing production biotin or lipoic acid or thiamine.SOLUTION: Provided is a genetically modified bacterial cell capable of improving iron-sulfur cluster transportation characterized by a modified gene encoding a variant iron-sulfur cluster regulator (IscR), and one or more transgenes encoding polypeptide which enhances biosynthesis of any one of biotin, lipoic acid and thiamine. The present invention further provides a method for producing any one of biotin, lipoic acid and thiamine by using a genetically modified bacterium of the present invention, and use of the genetically modified bacterial cell for producing biotin, lipoic acid or thiamine.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an engineered gene encoding a mutant iron-sulfur cluster regulator (IscR). and a polypeptide that enhances the biosynthesis of either biotin, lipoic acid, or thiamine. The present invention relates to a method for improving iron-sulfur cluster transport, the method comprising: The present invention further relates to a genetically modified bacterial cell of the present invention. The method of production of either biotin, lipoic acid, or thiamine used and the genetically modified It also relates to the use of the bacterial cells for the production of biotin, lipoic acid, or thiamine. [Background technology]

[0002] Biotin (also known as Vitamin B7 or Vitamin H) and Thiamin (Vitamin The thymus gland (also known as thymus gland B1) is a nutrient that allows humans, like other metazoans, to synthesize biotin or thiamine. Lipoic acid (LA) is an essential dietary vitamin for humans because the body cannot synthesize it. Vitamin-like antioxidants that are synthesized in small amounts in bacteria, plants, and animals. These vitamins and vitamin-like compounds are currently widely used as dietary supplements. The production of these substances relies on chemical synthesis, which is expensive. Biosynthetic approaches offer new, more cost-effective means to meet current and future needs. do.

[0003] Biotin acts as a carboxylase in certain enzymes, such as acetyl-CoA carboxylase (ACC). ACC, present in all living organisms, is an essential cofactor for the enzymes that catalyze fatty acid biosynthesis. In nature, biotin is essential for fatty acid synthesis, producing malonyl-CoA, a key building block in the synthesis of fatty acids. Biotin is synthesized by a linear pathway that includes the synthetic pathway. It is the first metabolite in fatty acid synthesis, which starts the fatty acid cycle. Previously, malonyl-ACP was used as a SAM (S-adenosylmethionine)-dependent methyltransferase. This is masked by the enzyme BioC, which produces malonyl ACP methyl ester. Then, two cycles of fatty acid chain elongation produce the molecule pimeloyl ester AC P is produced by the intrinsic esterase BioH, which is a pimeloyl ester ACP. Hydrolysis of the O-methyl group enables this molecule to complete the fatty acid elongation cycle.

[0004] The intermediate pimeloyl ester ACP is then converted to biotin via a biotin-specific pathway. In this pathway, BioF is converted to pimeloyl-ACP and alanine. It catalyzes the PLP-dependent decarboxylative aldol condensation of KAPA (8-amino-7-oxononaphthalene). BioA (and BioK) produces KAPA through the PLP-dependent transamination of KAPA. It catalyzes the transfer of 7,8-diaminopelargonic acid (DAPA), where the donor is The by-product is S-adenosyl-oxomethionine. BioD is ATP Catalyzes the carboxylation and ring closure of DAPA into desthiobiotin (DTB) The final step in the biotin synthesis pathway is the synthesis of BioB (biotin cinnamate). This involves the introduction of a sulfur bridge between two carbohydrates by glyceryl esterase (GlcNAc) to produce biotin. BioB is one of the most complex reactions known, because it converts S-adenosyl-L-methionine ( SAM or AdoMet) radical enzyme, which is found as a dimer and has a [2 Fe-2S] 2+and [4Fe-4S] 2+ It contains two iron-sulfur clusters. The sulfur atom required to form the thiophene ring is [2Fe-2S] in BioB. 2+ cluster As a result, BioB dimers consumed in DTB synthesis are thought to be replenished from The iron-sulfur clusters are thought to be regenerated after each catalytic cycle.

[0005] Lipoic acid (LA) is an important antioxidant because it is a potent scavenger of reactive oxygen species. In addition to being a cofactor for α-ketoacid dehydrogenase, LA is an intermediate in fatty acid metabolism. The three enzymes involved in LA synthesis in E. coli are LplA ( Lipoate-protein ligase), LipB (octanoyl protein ACP carrier) protein:protein transferase, and LipA (lipoic acid synthase). LplA, encoded by the lplA gene, is a non-binding site for the E2 subunit of the target enzyme. Catalyzes the binding of exogenous octanoate to lipoylated apolipoyl domains in an ATP-dependent manner LipB, encoded by the lipB gene, is a catalyzer that converts ACP into a target enzyme. It is possible to catalyze the transfer of an octanoyl residue to the apolipoyl domain of the E2 subunit. The AceF gene encodes the lipoyltransferase E2 subunit of pyruvate dehydrogenase. LipA, encoded by the lipA gene, encodes two CS domains. LipA ​​is involved in the formation of the iron-sulfide bond. The LipA-driven reaction requires iron sulfide to carry out its function. The yellow cluster (4Fe-4S) and SAM (produced by the metK gene) are required. Lipoic acid acts primarily as a protein-bound lipoamide in various multienzyme complexes within cells. exists as a part.

[0006] Thiamine biosynthesis has been studied in bacteria, some protozoa, plants and fungi. The thiazole and pyrimidine moieties of the amine are synthesized separately (Figure 3). 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP A-P) is a 5-aminoimidazole ribotide (A-P), an intermediate in the de novo purine biosynthetic pathway. In gram-negative bacteria, it is derived from the radical S-adenosyl-L-methionine (SA M)-dependent conversion of AIR to HMP-P occurs via one [ to HMP-P synthase, the product of the thiC gene, which binds the [4Fe-4S] cluster. This is catalyzed.

[0007] HMP-P is then converted to thiazole by ThiD kinase before it is attached to the thiazole unit. It is phosphorylated to form HMP-PP. The thiazole moiety, 5-(2-hydroxyethyl )-4-Methylthiazole phosphate (HET-P) is a phosphate derivative of L-tyrosine and 1-deoxy It is derived from cysteine ​​and D-xylulose phosphate (DXP), where sulfur The atom is thought to be derived from L-cysteine. Lysine lyase binds one [4Fe-4S] cluster per subunit and It catalyzes the dicarbamic acid-mediated cleavage of tyrosine to 2-iminoacetic acid and 4-cresol. At least five genes, thiF, thiS, thiG, and th Expression of iH and thiI is required.

[0008] The pyrimidine and thiazole moieties are then converted to the thiamine esterase encoded by thiE. -phosphate synthase (EC 2.5.1.3) to form TMP. Thus, TMP is the first product of all known thiamine biosynthetic pathways. In Enterobacteriaceae, in the presence of ATP, the thiamine phosphate kinase encoded by thiL is activated. TMP can be phosphorylated to the cofactor TPP by thiolase (EC 2.7.4.16). Cells containing a transgene expressing amine monophosphate phosphatase (E.C3.1.3.-) The strain is capable of enhancing thiamine production by converting TMP to thiamine. be.

[0009] The use of bacterial cell factories for the biosynthetic production of biotin, lipoic acid, and thiamine The advantages of recombinant E. coli as a cell factory for the production of bioproducts are: (i) When cultured under optimal environmental conditions in glucose-salt medium, the doubling time was approximately 20 minutes, and (ii) the theoretical density limit for E. coli liquid cultures is much higher than that of dry cultures. Dry cell weight about 200 g / l or approximately 1 x 10 13 High cell density estimated as viable bacteria / mL It is widely recognized that the gene Many molecular tools and protocols are available for the modification and synthesis of heterologous proteins. Both of these easy-to-express microorganisms allow for high levels of the desired bioproduct. It will be essential for

[0010] In E. coli, the biotin operon structure consists of overlapping promoters on opposing strands (the bioO locus). BioA and bioBFCD are split under the control of the chromosome DNA. BioH is a member of the E. coli chromosome. Expression of the biotin operon is initiated by the expression of biotin, which binds to the operator of the biotin operon. It is downregulated by the biotin-binding repressor (BirA), which is a biotin ligase. BirA also functions as a carboxylase, transferring biotin to cellular carboxylases. The switch from a ligase to a transcriptional repressor is mediated by the intracellular biotin and apoA receptors. The biotin operon (bioA and Overexpression of the β-lactamase (bioBFCD) has been reported to inhibit proliferation (Ifuku, The cause of this inhibition is unknown, and it is thought that this is due to the inhibition of biotin synthesis. This has become an obstacle to the development of the industry.

[0011] In general, biotin is used in tailor-made cell factories using bacteria (e.g., E. coli). To facilitate the production of lipoic acid and thiamine, the bottleneck of these complex biosynthetic pathways Identifying bottlenecks and enabling these pathways to increase levels of proliferation and production of the respective pathway enzymes There is a need to overcome various factors that may limit performance. Summary of the Invention [Problem to be solved by the invention]

[0012] [Means for solving the problem]

[0013] According to a first embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of atopic dermatitis, comprising administering to the patient ... A genetically modified bacterium for enhancing production of A genetically modified endogenous iscR gene encoding a mutant IscR polypeptide. Thus, the amino acid sequences of the mutant IscR polypeptides are as set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 2 and 14, and the amino acid sequence is ○ X is an amino acid residue other than the corresponding amino acid residue of SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14 Any of the amino acids L15X, C92X, C98X, C104X and H107X An endogenous iscR gene having at least one amino acid substitution selected from the group consisting of: With the legend, ○Having biotin synthase activity (EC2.8.1.6) to enhance biotin production and a polypeptide ○Has lipoic acid synthase activity (EC2.8.1.8) to enhance lipoic acid production A polypeptide, ○ HMP-P synthase activity (EC 4.1.99.17) for enhancing thiamine production and a polypeptide having the formula: ○ Tyrosine lyase activity (EC4.1.99.19) to enhance thiamine production and a polypeptide having ● at least one transgene encoding a polypeptide selected from; The present invention provides a genetically modified bacterium comprising:

[0014] Preferably, at least one amino acid substitution in the mutant IscR polypeptide is ○L15X, where X is one of F, Y, M, and W; ○C92X, where X is one of Y, A, V, I, G, L, M, F, and W; 98X, where X is one of A, V, I, G, L, F, and W; ○C104X, where X is any one of A, V, I, G, L, F and W; and ○H107X where X is any one of A, Y, M, F, W, V, I, G, and L is selected from the group consisting of:

[0015] 1 encoding a polypeptide having biotin synthase activity (EC 2.8.1.6) A genetically modified cell line according to the present invention for enhancing biotin production, comprising one transgene. The bacteria is ○SAM (S-adenosylmethionine)-dependent methyltransferase activity (Bio C;EC 2.1.1.197); 7-Keto-8-aminopelargonic acid (KAPA) synthase activity (BioF; EC2 .3.1.47) a polypeptide having the formula: 7,8-Diaminopelargonic acid (DAPA) synthase activity (BioA; EC: 2. 6.1.62) or L-lysine:8-amino-7-oxononanoate aminotransferase a polypeptide having ferrase activity (BioK:EC:2.6.1.105); ○Has dethiobiotin (DTB) synthetase activity (BioD; EC6.3.3.3) A polypeptide which Pimeloyl-[acyl carrier protein] methyl esterase activity (Bi oH;EC3.1.1.85) or 6-carboxyhexanoate - a polypeptide having CoA ligase activity (BioW; EC 6.2.1.14); and further comprising an additional transgene encoding one or more polypeptides selected from the group consisting of It may include.

[0016] Preferably, a polypeptide having biotin synthase activity (EC 2.8.1.6) is used. A gene according to the invention for enhancing biotin production, comprising one transgene encoding The engineered bacteria express SAM (S-adenosylmethionine)-dependent methyltransferase Polypeptide with enzyme activity (BioC; EC2.1.1.197); 7-keto-8-acetylglucosidase Possessing minopelargonic acid (KAPA) synthase activity (BioF; EC2.3.1.47). and 7,8-diaminopelargonic acid (DAPA) synthase activity (B A further transgene encoding a polypeptide having the formula (IoA; EC: 2.6.1.62) Further includes:

[0017] One encoding a polypeptide having lipoic acid synthase activity (EC 2.8.1.8) A genetically modified bacterium according to the present invention for enhancing the production of lipoic acid, comprising a transgene of teeth, Polypeptides with octanoyltransferase activity (EC2.3.1.181) Chid and Pyruvate dehydrogenase activity (EC 2.3.1.12) a polypeptide comprising an acetyltransferase portion; ○Polymers with lipoate protein ligase A (LplA; EC:6.3.1.20) Lipeptides and and further comprising an additional transgene encoding one or more polypeptides selected from the group consisting of It may include.

[0018] ThiC polypeptide having HMP-P synthase activity (EC 4.1.99.17) and / or one transgene encoding tyrosine lyase activity (EC 4.1.99. 19), comprising one transgene encoding a ThiH polypeptide having the The genetically modified bacteria according to the present invention for enhanced production include ○Th with ThiS adenylyltransferase activity (EC2.7.7.73) iF polypeptide, ThiE polypeptide with thiamine phosphate synthase activity (EC 2.5.1.3) And, ThiG polypeptide with thiazole synthase activity (EC 2.8.1.10) and, ○T with phosphohydroxymethylpyrimidine kinase activity (EC2.7.4.7) a hiD polypeptide; a ThiS polypeptide having sulfur carrier protein activity; Polypeptides with thiamine monophosphate phosphatase activity (EC 3.1.3.-) and, ThiO polypeptides having glycine oxidase activity (EC 1.4.3.19) and, Further transgene sequences encoding one or more polypeptides selected from the group consisting of to, optionally, ThiM polypeptide with hydroxyethylthiazole kinase activity (2.7.1.50) Lipeptides The vector may further comprise a transgene encoding

[0019] Preferably, the genetically modified bacterial cell contains the polypeptide ThiC (thiC gene) ThiD (encoded by the thiD gene); ThiE (encoded by the thiE gene); ThiF (encoded by the thiF gene) sulfur carrier protein (encoded by the thiS gene); ThiG (th iG gene); TMP phosphatase (TMP phosphatase gene) and ThiH (encoded by the thiH gene) or The vector contains a transgene encoding ThiO (encoded by the thiO gene). According to an embodiment, the cell is capable of expressing the enzyme ThiM (encoded by the thiM gene). It may further comprise a transgene encoding

[0020] Preferably, at least one transgene in the genetically modified bacterium of the invention and Each of the one or more additional transgenes is operably linked to a constitutive promoter. (The promoter may be operably linked to an operon containing the transgene).

[0021] The genetically modified bacteria of the present invention are preferably selected from the group consisting of Escherichia, Bacillus, Brevibacterium and the like. Bacterium, Burkholderia, Campylobacter, Corynebacterium, Lactia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter and Pseudomonas spp., more preferably, e.g. For example, Escherichia or Corynebacterium species such as E. coli or glutamic acid producing bacteria. be.

[0022] According to a second embodiment, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: ○Encodes a polypeptide having biotin synthase activity (EC 2.8.1.6) A genetically modified bacterium of the invention containing a transgene is introduced into a growth medium to produce a culture. and culturing the culture; Recovering the biotin produced by the culture and optionally purifying the recovered biotin. The process and The present invention provides a method for producing biotin, comprising the steps of:

[0023] According to a third embodiment, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: ○1 encoding a polypeptide having lipoic acid synthase activity (EC 2.8.1.8) A genetically modified bacterium of the invention containing one or more transgenes is introduced into a growth medium to produce a culture. A process of manufacturing the culturing the culture; recovering the lipoic acid produced by the culture and optionally purifying the recovered lipoic acid. and, The present invention provides a method for producing lipoic acid, comprising the steps of:

[0024] According to a fourth embodiment, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: ○ Coding a polypeptide having HMP-P synthase activity (EC4.1.99.17) and / or tyrosine lyase activity (EC4.1.99.19). The genetically modified bacteria of the invention containing a transgene encoding a polypeptide are added to the growth medium. introducing the cells into the culture to produce a culture; culturing the culture; Recovering the thiamine produced by the culture and optionally purifying the recovered thiamine. The process and The present invention provides a method for producing thiamine, comprising:

[0025] Preferably, the method is used in a method for producing any one of biotin, lipoic acid, and thiamine. The growth medium used is glucose, maltose, galactose, fructose, sucrose, and alpha- Labinose, xylose, raffinose, mannose, lactose, or any of them The carbon source may be selected from the following combination:

[0026] According to a fourth embodiment, the present invention provides a method for producing a transgene encoding a biotin synthase. The mutant iscR polypeptide is used to enhance biotin production in bacterial cells expressing the The use of a genetically modified gene for encoding a mutant iscR polypeptide comprising the amino acid sequence of the mutant iscR polypeptide. The sequence has at least 80% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14. and the amino acid sequence is: Any amino acid other than the amino acid residue L15X, Cys92X, Cys98X, Cy At least one amino acid substitution selected from the group consisting of s104X and His107X The present invention provides a use having the following structure:

[0027] According to a fourth embodiment, the present invention relates to a method for the production of biotin, lipoic acid or thiamine in bacteria. A genetically engineered polypeptide encoding a mutant iscR polypeptide for enhancing the production of either one of the The use of the gene, a polypeptide having biotin synthase activity (EC 2.8.1.6), a polypeptide having lipoic acid synthase activity (EC 2.8.1.8), A polypeptide having HMP-P synthase activity (EC 4.1.99.17) and a chimeric polypeptide having HMP-P synthase activity (EC 4.1.99.17). A polypeptide having rosin lyase activity (EC 4.1.99.19); and comprising at least one transgene encoding a polypeptide selected from Appear, The genetically modified gene is an endogenous iscR gene encoding a mutant IscR polypeptide. The amino acid sequence of the mutant IscR polypeptide is SEQ ID NO: 2, 4, 6, 8, 1 0, 12 and 14, The amino acid sequence is represented by the formula: X is the corresponding amino acid of SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14. Any amino acid other than the acid residue L15X, Cys92X, Cys98X, Cys10 At least one amino acid substitution selected from the group consisting of His107X and His4X. Provide for the use of.

[0028] According to a fifth embodiment, the present invention provides a method for enhancing the production of biotin, lipoic acid or thiamine. The present invention also provides the use of a genetically modified bacterium according to the present invention for the treatment of cancer.

[0029] According to a sixth embodiment, the present invention relates to a method for the preparation of a medicament for the treatment of atopic dermatitis, comprising administering to the patient a therapeutically effective amount of any one of biotin, lipoic acid, or thiamine. A genetically modified bacterium according to the invention for enhanced production, e.g., flavodoxacin. Polypeptides of the synthase / ferredoxin reductase and flavodoxin reduction systems or Polypeptides of the pyruvate flavodoxin / ferredoxin oxidoreductase system. The electron donor NADPH is converted to the SAM radical by the iron-sulfur cluster enzyme [4Fe-4S ] 2+ A polypeptide encoding a polypeptide capable of mediating enhanced electron transfer to the cluster. The bacterium further comprises one or more genes encoding [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows A) the bacterial biotin pathway intermediates and the enzymatic steps leading to the synthesis of biotin, SAM: S-adenosyl-L-methionine, SAH: S-adenosyl-L-homocysteine, CoA: coenzyme A, ACP: acyl carrier protein, KAPA: 7-keto-8-aminopelargonic acid, AMTOD: S-adenosyl-2-oxo-4-thiomethylbutyrate, DAPA: 7,8-diaminopelargonic acid, DTB: desthiobiotin, 5'DOA: 5'-deoxyadenosine, and B) the isc operon structure of IscR, its role in Fe-S cluster formation, and its regulatory mechanism. The isc operon contains the iscR gene, which encodes IscR, which controls the expression of the genes: iscS (cysteine ​​desulfurase), iscU (scaffold), iscA (A-type protein), HscB (Dnaj-like co-chaperone), HscA (DnaK-like chaperone), and fdx (ferredoxin). IscR also regulates over 40 genes, including the genes hyaA, ydiU, erpA, and sufA. [Diagram 2]Schematic diagram of bacterial lipoic acid pathway intermediates and the enzymatic steps leading to the lipoylated lipoyl domain (lipoic acid synthesis). Key enzymes in the pathway include LipA (lipoic acid synthase), LipB (octanoyl protein ACP carrier protein:protein transferase) and the substrate SAM: S-adenosyl-L-methionine. LplA is lipoate protein ligase A; EC: 6.3.1.20. [Diagram 3] FIG. 1 shows the bacterial thiamine pathway intermediates and the enzymatic steps leading to the synthesis of thiamine (THI); thiamine monophosphate (TMP) and thiamine diphosphate (TPP). Abbreviations for intermediates: 5-aminoimidazole ribonucleotide (AIR), 4-amino-2-methyl-5-(phosphooxymethyl)pyrimidine (HMP-P), 4-amino-2-methyl-5-(diphosphomethyl)pyrimidine (HMP-PP), 1-deoxy-D-xylulose 5-phosphate (DXP), dehydroglycine (DHG), 4-methyl-5-(2-phosphooxyethyl)thiazole (THZ-P), adenosine triphosphate (ATP), adenosine monophosphate (AMP), S-adenosyl-L-methionine (SAM), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), reduced ferredoxin (Fdx red), oxidized ferredoxin (Fdx ox). [Figure 4] Graph of cell density (measured by OD600) over time for the (E. coli BW25113) ΔbioB strain containing an IPTG-inducible bioB expression plasmid (right panel) and the reference (E. coli BW25113) strain containing an IPTG-inducible frameshifted bioB (premature stop codon) expression plasmid (left panel). OD620 was measured and converted to OD600 using Multiskan for replicates of four organism strains grown in 200 μL mMOPS containing 0.1 g / L DTB, 50 μg / mL kanamycin, 0 mM (circles), 0.01 mM (triangles) or 0.1 mM (squares). Respective log growth rate values ​​are shown in the adjacent boxes. [Diagram 5]Figure 1 is a scatter plot graph showing the final cell density (measured at OD600) of cultures of E. coli BS1011 containing plasmid pBS451 grown in 150 µL of mMOPS medium supplemented with 40 µg / ml Zeocin and zero or increasing concentrations of biotin up to 0.244 µg / mL after 20 hours of incubation at 37 °C with shaking at 275 rpm (as described in Biotin Quantification Methods). The dotted grey vertical line indicates the optimal biotin concentration range for the biotin bioassay, which is 0.024-0.24 µg / L. [Figure 6] 1 is a bar graph showing biotin production (see Table 1 for strains) of three different iscR mutant strains (BS1377, L15F), (BS1375, C92Y) and (BS1353, H107Y) expressing mutant IscR with amino acid mutations and the E. coli BW25113 ΔbioB strain (BS1011, ref.) in four biological replicates, each containing an IPTG-inducible bioB expression plasmid (pBS412). Strains were grown in 400 μL mMOPS containing 0.1 g / l DTB and 50 μg / mL kanamycin at 37° C. with 275 rpm shaking for 24 h. Bars show the average biotin production values ​​(height) and IPTG induction levels (grey shading), black dots show biotin production from individual replicate cultures and horizontal dotted line shows maximum biotin production from the reference wild-type strain. It should be noted that none of the strains produced detectable levels of biotin when grown in the absence of IPTG. [Figure 7]Graph of cell density and biotin production for iscR mutant strains (BS1353, H107Y) expressing mutant IscR and E. coli BW25113 ΔbioB strain (BS1011, reference). Each strain contains an IPTG-inducible bioB gene expression plasmid (pBS412). Data represent the average measured OD600 of three biological replicates of each of the iscR H107Y mutant strain (dark solid line) and the reference E. coli BW25113 ΔbioB strain (light grey dotted line) observed over a 25-hour period, as well as biotin production by each of the iscR H107Y mutant strain (dark circle) and the reference E. coli BW25113 ΔbioB strain (light grey circle). Strains were grown in 50 mL mMOPS containing 0.1 g / l DTB, 0.01 mM (A) or 0.5 mM (B) IPTG, and 50 μg / mL kanamycin in 250 mL baffled shake flasks at 37° C. with 275 rpm shaking. Growth rates are shown in black boxes. [Figure 8] FIG. 1 shows an annotated IscR coding sequence showing the locations of nucleotide and amino acid sequence mutations in the iscR gene of the identified mutant strains. [Figure 9] Diagram showing the crystal structure (PDB entry 4HF1) of an IscR dimer (gray) bound to the hya DNA binding site (black), with the L15F and H107Y iscR mutations shown as sticks (wild-type amino acids in gray, mutated amino acids in black) and a zoomed-in image highlighting the mutated residues. [Figure 10]Figure 1 is a bar graph showing biotin production of E. coli strains containing an IPTG-inducible bioB expression plasmid and a plasmid containing an isc operon (iscSUA-hscBA-fdx, corresponding to the native E. coli isc operon structure lacking the iscR gene) operably linked to a strong ribosome binding site (RBS) or an E. coli suf operon (sufABCDSE) and a T5 LacO repressible promoter from a medium copy number plasmid (p15A ori) or a control plasmid. The control plasmid contained an IPTG-inducible gene encoding GFP in place of the suf or isc operon. Biological triplicates of each strain were grown under low (0.01 mM IPTG) and high (0.1 mM IPTG) induction in mMOPS containing 100 μg / mL ampicillin and 50 μg / mL spectinomycin with 0.1 g / L (DTB) as substrate. Each strain was grown in deep-well plates at 37°C and 275 rpm for 24 hours and then assessed for biotin production using a growth-based bioassay. Bars indicate mean biotin production values ​​(height) and IPTG induction levels (grey shading), black dots indicate biotin production from individual replicates, and crosses indicate the endpoint (final) cell density of each strain measured as OD600. Note that none of the strains produced detectable levels of biotin when induced with 0.01 mM IPTG. [Figure 11] Figure 1 shows the correlation between BioB protein expression levels and biotin production in four different samples run in triplicate. Strains were BS1013 (E. coli BW25113, background strain) harboring pBS430 (bioB frameshift IPTG inducible plasmid), BS1011 (BS1013 containing ΔbioB) containing pBS412 (bioB IPTG inducible plasmid), and BS1353 (BS1011 containing an iscR H107Y mutation) containing pBS412. Strains were grown in mMOPS containing 0.1 g / l DTB and IPTG as indicated on the graph. [Figure 12]1 is a bar graph showing biotin production of E. coli ΔbioB strains containing an IPTG-inducible bioB expression plasmid and either the following genomic mutations in iscR: wild type (iscR WT), a knockout mutation encoding an E22*glutamic acid mutated to a stop codon at position 22 (iscR KO), or a mutation encoding a cysteine ​​to tyrosine substitution at position 92 (iscR C92Y). Bars indicate the average biotin production values ​​(height) at a given level of IPTG induction (grey shading), and dots indicate biotin production from individual replicates. Biological triplicates of each strain were grown in mMOPS containing 100 μg / mL ampicillin with no induction (0 mM IPTG), low induction (0.01 mM IPTG), and high induction (0.1 mM IPTG) and with 0.1 g / L DTB as substrate. Each strain was grown in deep-well plates at 37°C and 275 rpm for 24 h, after which biotin production was assessed using a growth-based bioassay. [Figure 13] FIG. 1 is a bar graph showing biotin production by E. coli ΔbioA ΔbioBFCD strains containing the biotin operon plasmid and either the wild type (iscR WT), the mutation iscR H107Y (encoding a histidine to tyrosine substitution at position 107), or the mutation encoding a cysteine ​​to tyrosine substitution at position 92 (iscR C92Y). Biological quadruplicates of each strain were cultured in mMOPS containing 10 μg / mL tetracycline with or without 0.1 g / L desthiobiotin (DTB) as substrate. Strains were grown in deep-well plates at 37° C. and 275 rpm for 24 h, after which biotin production was assessed using a growth-based bioassay. Bars indicate the average biotin production values ​​(height) and the presence or absence of DTB (gray shading), and black dots indicate biotin production from individual replicates. [Figure 14]Figure 1 is a bar graph showing lipoic acid production (grey bars) and final (final) OD600 (cross marks) after 24 hours of production for three biological replicates of two different iscR mutant strains expressing mutant IscR with amino acid mutations (BS1375, C92Y) and (BS1353, H107Y) and a control strain (BS1011, IscR WT) (see Table 1 for strains), each containing a plasmid expressing IPTG-inducible lipA (pBS993, see Table 4). Strains were grown in 400 μL mMOPS containing 100 μg / mL ampicillin, 0.1 mM biotin, 0.6 g / L octanoic acid, and 0.01 mM IPTG for 24 hours at 37°C with 275 rpm shaking. Bars indicate average lipoic acid production values ​​(height) and black dots indicate lipoic acid production from individual replicate cultures. It can be seen that the average production of lipoic acid increases by 1.79 fold even though the final OD600 remains the same. [Figure 15] Graph of cell density (measured by OD620) measured over time for a reference strain (E. coli BW25113) ΔlipA (WT, triangles) and a ΔlipA strain with mutant iscR(C92Y) (C92Y, squares), both containing an IPTG-inducible lipA expression plasmid (pBS1037). OD620 was measured using a Multiskan for replicates of six biological strains grown in 200 μL mMOPS containing 0.6 g / L octanoic acid, 100 g / mL ampicillin, and IPTG from 0 to 0.04 mM (increasing shades of grey). Respective growth rates (GR) are shown on the right. [Figure 16]Figure 1 is a bar graph showing thiamine production of four biological replicates of two different iscR mutant strains expressing mutant IscR with amino acid mutations (BS2019, C92Y) and (BS2020, H107Y) and an E. coli BW25113 ΔthiP, thiL* strain (BS750, ref.) (see Table 5 for strains), each containing a plasmid expressing the entire thiamine pathway genes thiCEFSGHMD (pBS140). Strains were grown in 400 μL mMOP containing 50 μg / mL kanamycin for 24 h at 37 °C with shaking at 275 rpm. Bars indicate the average thiamine production value (height) of the supernatant measured by a modified thiochrome assay (containing thiamine, TMP, TPP) at final OD600, and black dots indicate thiamine production from individual replicate cultures. It can be seen that the OD normalized titer is improved by 1.43-fold for the mutant strains (BS2019 and BS2020) compared to the reference strain (BS750). [Figure 17] 1 is a bar graph showing biotin production by E. coli ΔbioABFCD iscR H107Y (encoding a histidine to tyrosine substitution at position 107) strains containing the IPTG-inducible BioB overexpression plasmid pBS679 alone (BS1937) or additionally with pBS1112 with constitutive overexpression of FldA-Fpr (BS2185) or with pBS1054 with constitutive overexpression of GFP (BS2707). Each strain was grown in mMOPS containing 100 μg / ml ampicillin, 0.1 g / L desthiobiotin (DTB) as substrate, and 0, 0.01, 0.025, 0.05, 0.075, or 0.1 mM IPTG. The media for BS2185 and BS2707 were the same except that they contained 50 μg / ml kanamycin. Strains were grown in deep-well plates at 275 rpm and 37° C. for 24 hours, after which biotin production was assessed using a growth-based bioassay. Bars indicate biotin production values ​​(height) by each strain: BS1937 (black bars); BS2185 (grey bars); and BS2707 (checkered grey bars). [Figure 18]1 is a bar graph showing biotin production by E. coli ΔbioABFCD iscR H107Y (encoding a histidine to tyrosine substitution at position 107) strain containing IPTG-inducible BioB overexpression plasmid pBS679. BS2185 further contains pBS1112 with constitutive overexpression of FldA-Fpr. BS1937 was grown in mMOPS containing 100 μg / ml ampicillin, 0.1 g / L desthiobiotin (DTB) as substrate, and 0.025 mM IPTG induction. The medium for BS2185 was the same except that it also contained 50 μg / ml kanamycin. Strains were grown in deep-well plates at 275 rpm and 37° C. for 24 hours, after which biotin production was assessed using a growth-based bioassay. Dark grey bars indicate average biotin production values ​​(height) (n=6 for BS1937 and n=8 for BS2185) and light grey bars indicate final OD600. Black dots indicate biotin production and final OD600 from individual replicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Definition: Amino acid sequence identity: As used herein, the term "sequence identity" refers to a sequence that is substantially identical to It shows a quantitative measure of the degree of homology between two amino acid sequences of similar length. The sequence can be adjusted to fit the best fit by inserting gaps or making truncations at the ends of the protein sequence. To achieve this, alignment is required. The sequence identity is calculated as ((Nref-Nd It can be calculated as if)100) / (Nref), where Ndif is the a The total number of non-identical residues in two sequences when aligned. Nref is the number of non-identical residues in one sequence. The sequence identity is calculated using, for example, the BLASTP program (Pearson n WR and DJ Lipman (1988) (www.ncbi.nlm.n ih.gov / cgi-bin / BLAST) It is preferable to perform multiple sequence alignment using the ClustalW sequence alignment method. The default parameters are as described by Thompson J et al. 1994. and available at http: / / www2.ebi.ac.uk / clustalw / It is Noh.

[0032] Preferably, the substitution, insertion, addition or deletion of one or more amino acid residues in the polypeptide. The number is restricted relative to the comparison polypeptide, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 0 or less insertions, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less additions, and 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer deletions. Substitutions are conservative amino acid substitutions, Group 1: glycine, alanine, valine, leucine Group 2: serine, cysteine, selenocysteine, threonine, methionine, Group 3: Proline; Group 4: Phenylalanine, Tyrosine, Tryptophan Group 5: Aspartic acid, glutamic acid, asparagine, and glutamine Exchanges are limited to within the bar.

[0033] Amino acid abbreviations: leucine (L), cysteine ​​(C) and histidine (H).

[0034] endogenous gene: a gene within the genome of a bacterial cell that is homologous in origin to the host bacterium (i.e., Native genes). Endogenous genes can be transfected using tools known in the art. By modifying the endogenous gene, the genetically modified endogenous gene becomes identical to the parent endogenous gene from which it was derived. A mutant polypeptide that differs in amino acid sequence at one or more positions from the polypeptide encoded by It may code for a peptide.

[0035] Genome: The genetic material present in a cell or organism. The genome is the building block for that cell or organism. It contains all the information necessary to establish and maintain the chromosomes and It contains both the genetic material of the vector and the plasmid.

[0036] GFP: green fluorescent protein.

[0037] gi number: (geninfo identifier) ​​is DDBJ / EMBL / Ge Nucleotide sequences from nBank, SWISS-PROT, PIR and many others All sequences processed by Entrez, including protein sequences from A unique integer assigned to a sequence that identifies the particular sequence, independent of the database source. do.

[0038] Isc pathway: Iron-sulfur cluster pathway; encoded by the isc operon containing the iscR gene It will be downloaded.

[0039] Multiskan: Filter-based microplate photometer. 340-850nm (600-620 nm inclusive) wavelength range in 96 or 384 well plate format The plate is then incubated in a photometer at selected temperatures up to 50°C. The photometer was manufactured by Thermo Scientific.

[0040] Native gene: an endogenous gene in a bacterial cell genome that is homologous to that of the host cell.

[0041] Non-native promoter: In the context of the genetically modified bacteria of the present invention, a promoter operably linked to the gene or transgene of operably linked to this gene or a transgene in a bacterial cell found in nature. There is none.

[0042] OD: optical density

[0043] Transgene: A foreign gene that has been introduced into the genome of a bacterium by genetic engineering. In a nutshell, the genome contains both chromosomal and episomal genetic elements.

[0044] Detailed Description of the Invention A common feature of the biosynthetic pathways for the synthesis of biotin, lipoic acid, and thiamine is the complex ligation of the nucleosomes. one or more SAM or AdoMet radical enzymes to catalyze radical-mediated molecular rearrangements Biotin synthase, lipoic acid synthase, HMP-P synthase and tyrosine lyases catalyze these essential steps in these pathways. These are enzymes known to be involved in the synthesis of β-actin and β-actin. We isolated a mutant biotin operon that is less susceptible to feedback regulation by the irA repressor. The failure of previous attempts to enhance biotin biosynthesis in E. coli by using The observed growth inhibition was due to strong growth inhibition (Ifuku, O et al. 1995). The lack of an evidence-based explanation for the toxicity of biotin synthase overexpression is difficult to explain. To identify possible cellular factors, an alternative approach was needed.

[0045] The solution to this problem provided by the present invention is to transform bacterial cell factory cells (e.g., colon Biotin synthase, lipoic acid synthase, HMP-P synthase, and tyrosine synthase in Bacillus subtilis It has been shown that the results are equally applicable to enhancing the expression of sucrose lyase. The approach pursued to address this problem is to use a method that uses a defective error-correcting polymerase to A library of Escherichia coli cells with evolved genomic diversity due to the accumulation of background mutations The aim of this study was to generate a library of such cells by inducing IPTG-inducible bioB gene expression. The candidate mutants were transformed with a plasmid containing a gene expression cassette. BioB expression was increased in the presence of IPTG at a concentration sufficient to induce toxicity in the parent E. coli strain. These were library cells that could be propagated.

[0046] The genetic basis of the growth of the selected BioB-expressing mutants was identified by whole genome sequencing. Surprisingly, three strains expressed the pleiotropic transcription factor (IscR) [SEQ ID NO: No. 2], a mutation was introduced into the native iron-sulfur cluster regulator gene (iscR) that encodes It was found that Fe-S clusters are cofactors for many proteins and essential enzymes. It is necessary not only for the synthesis of S-containing compounds such as biotin, but also for the synthesis of redox or iron-related These proteins also confer various biochemical capabilities necessary for use as sensors of stress conditions.

[0047] IscR exists as an Fe-S cluster holoprotein or as an adipocyte lacking an Fe-S cluster. As a polyprotein, it exists in two states. Assembly of the Fe-S cluster in IscR is catalyzed by the Isc pathway, which is encoded by the isc operon. The gene first encodes a regulator (IscR), followed by a cysteine ​​desulfurase (I scS), scaffold (IscU), A-type protein (IscA), Dnaj-like cochaperone ( HscB), DnaK-like chaperone (HscA) and ferredoxin (Fdx) are encoded In addition to being essential for the assembly of the IscR holoenzyme, the Isc pathway also regulates the This is the major pathway for Fe-S cluster biosynthesis in fungi (Fig. 1B).

[0048] The ratio of the two forms of IscR is determined by the cellular levels of [2Fe-2S] clusters and is influenced by several factors, including iron and oxygen levels (Py, B. & Barr Under iron-rich conditions, IscR exists mainly as a holoenzyme, and its It then acts as a transcriptional repressor of the isc operon. However, under low iron conditions (low levels In the presence of the [2Fe-2S] cluster of iscR, IscR reverts to its apoprotein state and In its apoprotein form, IscR is sufABCD It functions as an activator of the SE operon and catalyzes Fe-S cluster biosynthesis under oxidative stress. To mediate.

[0049] IscR regulates the expression of two Fe-S cluster assembly systems in Escherichia coli. In addition to oxidative stress mechanisms (e.g., sodA), specific and global regulation data (e.g. yqjI and soxS, etc.), amino acid biosynthesis (e.g. argE) and unknown functions It regulates a range of functional genes, including over 40 genes involved in various mechanisms of action. The role of IscR is that the regulatory context of IscR changes between aerobic and anaerobic conditions. This is further complicated by the fact that l et al. 2006).

[0050] Given the role of IscR in homeostasis and global gene regulation, The consequences of altering the regulatory properties of are unpredictable and will have profound effects on cellular metabolism. Furthermore, the expression of both the sulfur formation (suf) and isc pathways was enhanced, leading to the formation of Fe-S clusters. The cellular state in which cluster biosynthesis is increased is characterized by the accumulation of Fe-S clusters in the Fenton reaction. This creates a risk of generating peroxide radicals.

[0051] In this light, we hypothesize that IscR mediates the function of the cellular biosynthesis pathway, as shown by three isolated individual mutants. It was totally unexpected that the Fe-S class is so important for the activity and toxicity of B. Overexpression of the isc operon or suf operon enhances the ability to synthesize and assemble β-terminal fragments. Since it was not found to enhance biotin production in cells overexpressing ioB (e.g. 1, see Figure 10), the effect of the mutant IscR protein on biotin biosynthesis was unexpected. Furthermore, the elimination of cellular iscR regulation by knocking out the iscR gene also reduced the did not enhance biotin production (see Example 1, Figure 12).

[0052] Three different mutations of the IscR protein eliminated the toxicity of BioB expression in mutant cells. The differences are amino acids L15 [SEQ ID NO: 16], C92 [SEQ ID NO: 18] and H107 [SEQ ID NO: No. 20] (Fig. 8). Two of the three mutations were at residues in IscR. and that each is essential for the formation of the IscR holoprotein. It is known that IscR contains an unusual Fe-S cluster ligase, as seen in E. coli. Therefore, the essential residue for Fe-S cluster ligation is C9. 2, C98, C104 and H107. This unusual ligation is similar to other Fe-S Reduces the stability of the holoenzyme state of IscR compared to the protein, and reduces its activity under low Fe-S conditions This causes a switch to the apoprotein state (Fleischhacker et al. 2012 ).

[0053] Without wishing to be bound by theory, this is In cells, enzymes (biotin synthase, lipoic acid synthase, HMP-P synthase, Promotes the assembly of iron-sulfur clusters containing tyrosine lyase while stimulating the assembly of excess iron-sulfur clusters. required for homeostatic control of Fe-S cluster biosynthesis and cell proliferation even during expression This suggests that global gene regulation remains unique.

[0054] In summary, we demonstrated that the expressed mutant IscR protein is in the form of an apoprotein. One or more amino acid residues required for ligation of the Fe-S cluster are present only in We identified a mutant iscR gene that encodes a mutant IscR protein characterized by the absence of a .alpha.-amino acid residue. The synthesis of iron-sulfur cluster containing enzymes was shown to be dependent on the synthesis of biotin, lipoic acid or thiamine in bacteria. This problem has been shown to be a major bottleneck in efforts to increase production. As presented by the present invention, a mutant IscR protein that exists in the apoprotein state These enzymes can be easily overexpressed in cell factories that contain genes encoding these enzymes. Various embodiments of the invention are described in more detail below.

[0055] I. Genetically engineered bacterial cells for biotin production The present invention provides genetically modified bacterial cells capable of enhancing biotin production levels. Bacterial cells are genetically modified to express the mutant IscR instead of the wild-type IscR. The enzyme was modified to encode biotin synthase (biotin synthase having EC 2.8.1.6). Optionally, the genetically modified bacterial cell comprises a transgene that modifies the biotin pathway. The method further comprises one or more additional transgenes encoding polypeptides that catalyze the steps of: (Figure 1A) Increased levels of polypeptides that catalyze steps in the biotin pathway The bacterial cell then synthesizes both the intermediates of the biotin pathway and the final product of the pathway (biotin). This will promote growth.

[0056] The mutant IscR polypeptide expressed by the genetically engineered bacterial cell is The IscR polypeptide family is characterized by a tide backbone (apoprotein). The amino acid sequence of the wild-type member of the IscR polypeptide family is The sequence is selected from any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. and at least 70, 75, 80, 85, 90, 95, 96, 98, 100% amino acid sequence The amino acid sequence of the mutant IscR polypeptide according to the present invention has at least The amino acid sequence of the corresponding wild-type IscR polypeptide is The substitutions are L15X, C92X, C98X, C104X, and H107. X, where X is the corresponding amino acid of the wild-type IscR from which the mutant is derived. It is any amino acid other than the amino acid found in position.

[0057] In another embodiment, the amino acid substitution in the mutated IscR is More preferably, X is phenylalanine (F), tyrosine (Y), methionine (M ), and tryptophan (W); X is any amino acid other than C; More preferably, X is tyrosine (Y), alanine (A), methionine (M), phenyl C92X selected from arylalanine (F) and tryptophan (W); X is other than C X is any amino acid, more preferably alanine (A), valine (V), isoleucine (I), from isine (I), leucine (L), phenylalanine (F), and tryptophan (W) The selected C98X; X is any amino acid other than C, more preferably, X is alanine (A ), valine (V), isoleucine (I), leucine (L), phenylalanine (F), and and tryptophan (W); X is any amino acid other than H. More preferably, X is alanine (A), tyrosine (Y), valine (V), isoleucine (I), His107X selected from His107C, His2 ... For example, the amino acid substitutions in the mutant IscR are L15F, C92Y, C92A, 98A, Cys104A, H107Y, and H107A.

[0058] The mutant IscR expressed by the genetically modified bacterial cell of the invention (instead of the wild-type IscR) IscR is a gene located in the genome of a bacterial cell, either on the chromosome or on an autonomously replicating plasmid. The modified iscR gene in the chromosome is encoded by , may be located in the same genomic location as the wild-type iscR gene in the native genome The genome of the genetically modified bacterial cell of the present invention contains the native wild-type iscR gene. It has been deleted or directly replaced by a genetically modified iscR gene. Therefore, it lacks the native wild-type iscR gene. The promoter driving the expression of the gene is derived from or placed in a genetically modified iscR gene. It may be the native promoter of the wild-type iscR gene that has been replaced. The promoter may be a heterologous constitutive promoter or an inducible promoter. If is a heterologous constitutive promoter, suitable promoters include those from the apFab family [SEQ ID NO: No. 230-232], and suitable inducible promoters include pBad (arabinose-inducible The appropriate terminators include nucleotides 0, 1, 2, and 3. The terminator contains members of the apFAB terminator family, including [SEQ ID NOs: 235-237]. Includes:

[0059] The polypeptide having biotin synthase activity (EC 2.8.1.6) according to the present invention is , which catalyzes the conversion of desthiobiotin (DTB) to biotin. Members of this family of biotin synthases belong to a wide variety of genera. It is encoded by a gene found in bacteria belonging to the genus Biotin synthase. The amino acid sequences of the polypeptides are SEQ ID NO:22 (origin: E. coli); SEQ ID NO:27 (origin: Source: Candidate name Chlorachidobacterium thermopyrum B; SEQ ID NO: 29 (Origin: Strep Tomyces leidicus); SEQ ID NO:31 (origin: Paracoccus denitrificans ); SEQ ID NO: 33 (origin: Paracoccus denitrificans PD1222); SEQ ID NO: No. 35 (origin: Agrobacterium vitis); No. 37 (origin: Ruegeria pomeroy); SEQ ID NO: 39 (origin: Agrobacterium fabrum); SEQ ID NO: 41 ( Origin: Wolbachia endosymbiont of bedbugs); SEQ ID NO:43 (Origin: Sphingomonas paucimobilis; SEQ ID NO: 45 (origin: Acidithiobacillus ferrivorans (Ac idithiobacillus ferrivorans); SEQ ID NO: 47 (origin: Gallionella capsiferr iformans); SEQ ID NO: 49 (Origin: Ralstonia eutropha (Ralst onia eutropha); SEQ ID NO: 51 (origin: Bordetella parapertussis); SEQ ID NO: 53 (Origin: Psilimonas genus); SEQ ID NO:55 (Origin: Kenarcaeum shumbiosum genus) ; SEQ ID NO:57 (origin: Alicyclobacillus acidocaldarius); SEQ ID NO:59 (origin: Geobacillus thermoglucosidasius); SEQ ID NO:61 (origin: Bacillus subtilis); Sequence number 63 (origin: Lysinibacillus sphaericus); sequence number 65 (origin: Methylococcus Cass capsulatus); SEQ ID NO: 67 (Origin: Leclercsia adecarboxylata) ; SEQ ID NO: 69 (origin: Chromohalobacter salexigens); SEQ ID NO: 71, 73, One of 75, 77, 79, 81, 83, 85, 87 (origin: Pseudomonas) and at least 70, 75, 80, 85, 90, 95, 96, 98, 1 00% amino acid sequence identity.

[0060] The activity of the gene encoding the additional transgene in the genetically modified bacterial cell is determined by the biochemical properties of the gene encoding the additional transgene. Polypeptides that serve to enhance synthesis of both intermediates and products of the sucrose pathway include the following: As follows: a) SAM (S-adenosylmethionine)-dependent methyltransferase activity (Bi oC; EC 2.1.1.197); for example, SEQ ID NOs: 89 and 80, A polypeptide having an amino acid sequence with 85, 90, 95, or 100% sequence identity. D; b) 7-keto-8-aminopelargonic acid (KAPA) synthase activity (BioF;EC 2.3.1.47) polypeptides having the sequence of SEQ ID NO: 91, 80, 85, 90, a polypeptide having an amino acid sequence with 95 or 100% sequence identity; c) 7,8-diaminopelargonic acid (DAPA) synthase activity (BioA; EC: 2 6.1.62) polypeptides having the sequence of SEQ ID NO: 93 and SEQ ID NO: 80, 85, 90, 9 a polypeptide having an amino acid sequence with 5 or 100% sequence identity; or For example, amino acids having 80, 85, 90, 95, or 100% sequence identity to SEQ ID NO:97. Polypeptides having the amino acid sequence L-lysine: 8-amino-7-oxononanoate amine notransferase (BioK; EC:2.6.1.105); d) an amine having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 95; Desthiobiotin (DTB) synthetase activity (B ioD; E.C6.3.3.3); and, optionally, f) Amino acids having 80, 85, 90, 95, or 100% sequence identity to SEQ ID NO: 99 pimeloyl-[acyl-carrier protein]methyl, such as a polypeptide having a .alpha.-acyl sequence Polypeptides having a leucyl esterase (BioH; EC:3.1.1.85); Or, g) 6-carboxyhexanoate-CoA ligase activity (BioW; EC 6.2.1 14) a polypeptide having the sequence of SEQ ID NO: 101 and 80, 85, 90, 95, or is a polypeptide having an amino acid sequence with 100% sequence identity.

[0061] The transgene encoding BioB expresses a polypeptide that catalyzes a further step in the biotin pathway. The genome of the genetically modified bacterial cell is then transduced with one or more additional transgenes encoding a peptide. They are located within the genome and integrated into the bacterial cell chromosome or on autonomously replicating plasmids. Encodes BioB and one or more enzymes in the enzyme pathway (BioABFCD and H or W) The transgenes may be present in the genome in one or more operons.

[0062] A promoter driving the expression of a transgene encoding BioB and one or more further transgenes. The promoter is preferably a non-native promoter, and may be a heterologous constitutive promoter or an inducible promoter. If the promoter is a heterologous constitutive promoter, then a suitable promoter The motors include the apFab family [SEQ ID NOs: 230-232], which express the appropriate inducible promoter. The motor contains pBad (arabinose inducible [SEQ ID NO: 233] and LacI [SEQ ID NO: Suitable terminators include ap Members of the FAB terminator family are included. The data includes the coding sequence of BioB and the coding sequences of the BioC, BioD, BioA, and BioF polypeptides. and a coding sequence for one or more of the BioW or BioH polypeptides. The sequence may be operably linked to a polypeptide encoding the selected polypeptide. It may be operably linked to one or more operons.

[0063] II. Methods for Producing and Detecting Biotin Using the Genetically Modified Bacteria of the Present Invention Biotin can be used in the genetically modified bacterial cells of the invention (e.g., The modified E. coli cells were used to support growth and to promote biotin biosynthesis. The cells were then introduced into a medium containing a carbon source of their own choosing, and the biotin produced by the culture was finally recycled. It can be produced and extracted by collecting it.

[0064] The genetically modified plant of the present invention contains a transgene encoding biotin synthase (BioB). The bacterial cells were transformed to biotin when the carbon source provided contained desthiobiotin (DTB). Increase the production level of BioA, BioF, BioC, BioD and BioH or Bio The genetically modified bacterial cell of the invention further comprises a transgene encoding each of the oWs. The carbon sources supplied are glucose, maltose, galactose, fructose, and sucrose. saccharide, arabinose, xylose, raffinose, mannose, and lactose. When the β-actin is activated, biotin is produced (Example 1, Figure 13).

[0065] Methods for quantifying extracellular biotin produced by genetically modified bacterial cells of the invention The method is described in Example 1.5. The method comprises culturing extracellular growth media derived from the culture of the cells of the invention. BS1011 containing plasmid pBS451 in biotin-deficient growth medium supplemented with PBS. This bioassay is based on measuring the growth of overnight cultures of B. elegans under biotin-depleted conditions. The calibration curve for the biotin bioassay is shown in Figure 5. This is done by measuring the growth of overnight cultures lacking added biotin.

[0066] III. Genetically engineered bacterial cells for the production of lipoic acid The present invention provides genetically modified bacterial cells capable of enhancing lipoic acid production levels. The bacterial cell expresses a mutant IscR according to the invention instead of the wild-type IscR. It has been genetically modified to express lipoic acid synthase (EC 2.8. 1.8) is a transgene encoding LipA, which promotes the formation of two sulfur bonds. This catalyzes the conversion of the covalently linked octanoyl domain to the lipoyl domain. If necessary, the genetically modified bacterial cells can catalyze further steps in the lipoic acid synthesis pathway. A polypeptide that mediates the expression of the lipase, more specifically the encoded polypeptide LipB; e.g., the lipase EC: 2.3.1.181 of the B gene and the encoded polypeptide E2; e.g., ac Further comprising one or more additional transgenes encoding EC:2.3.1.12 of the eF gene, etc. The levels of these polypeptides that catalyze steps in the lipoic acid pathway may be increased by The increase promotes synthesis of both the pathway intermediates and the pathway end product in the bacterial cell. A further derivative encodes the end protein ligase A, LplA; EC:6.3.1.20 The transgene increased the synthesis of lipoic acid in cells fed with octanoic acid by increasing the octanoyl moiety. It helps facilitate this by catalyzing the transfer to the activated lipoyl domain.

[0067] Lipoic acid synthase is encoded by genes found in a wide range of bacteria and fungi belonging to a wide range of genera. The amino acid sequence of a polypeptide having lipoic acid synthase activity is No. 103 (origin: Escherichia coli); No. 105 (origin: Bacillus subtilis); No. 107 (origin: SEQ ID NO:109 (origin: Pseudomonas putida); SEQ ID NO:111 (origin: source: Bacteroides fragilis); and SEQ ID NO:113 (origin: Streptomyces coelicolor) and at least 70, 75, 80, 85 , 90, 95, 96, 98, and 100% amino acid sequence identity.

[0068] The activity of the lipoprotein is encoded by an additional transgene in the genetically modified bacterial cell. Polypeptides that play a role in enhancing synthesis of both intermediates and products of the acid pathway are as follows: This is: a) (Octanoyl residues are converted from ACP to the apolipoyl group of the E2 subunit of the target enzyme activity) Octanoyltransferase activity for transferring to domain; LipB; EC:2 .3.1.181, SEQ ID NO: 115 (origin: E. coli) or SEQ ID NO: 117 (origin: Flex Amino acids with 80, 85, 90, 95, or 100% sequence identity with Shigella flexneri a polypeptide having a sequence; b) For example, SEQ ID NO: 119 (origin: Escherichia coli) or SEQ ID NO: 121 (origin: Klebsiella La oxytoca) or SEQ ID NO: 239 (hybrid sequence) and 80, 85, 90, 95, or a polypeptide having an amino acid sequence with 100% sequence identity to pyruvate. Dehydrogenase (E2; EC:2.3.1.12) dihydrolipoyl lysine residue acetone A polypeptide comprising a thioltransferase component. c) For example, SEQ ID NO: 123 (origin: Escherichia coli) or SEQ ID NO: 125 (origin: Klebsiella Amino acids with 80, 85, 90, 95, or 100% sequence identity to La oxytoca Lipoate protein ligase A (LplA; EC: 6.3.1.20) A polypeptide having an activity.

[0069] A transgene encoding lipoic acid synthase catalyzes a further step in the lipoic acid pathway The genetically modified bacterial cell is then subjected to a step of carrying out the ... They are located in the genome of the bacterial cell, integrated in the bacterial cell chromosome or on autonomously replicating plasmids. A transgene encoding LipA and a transgene encoding an enzyme in the lipoic acid pathway (IpB, IplA, and AceF) are present in the genome in one or more operons. It may be possible.

[0070] A promoter driving the expression of a transgene encoding LipA and one or more further transgenes. The promoter is preferably a non-native promoter, and may be a heterologous constitutive promoter or an inducible promoter. If the promoter is a heterologous constitutive promoter, then a suitable promoter The motors include the apFab family [SEQ ID NOs: 230-232], which express the appropriate inducible promoter. The motor contains pBad (arabinose inducible [SEQ ID NO: 233] and LacI [SEQ ID NO: Suitable terminators include ap Members of the FAB terminator family are included. The data can be applied to each gene for individual gene regulation or operon regulation. They can be linked together.

[0071] IV. Methods for Producing and Detecting Lipoic Acid Using the Genetically Engineered Bacteria of the Invention Lipoic acid can be used in the treatment of genetically modified bacterial cells of the invention (e.g., as shown in Example 2 and FIG. 14). For example, genetically modified E. coli cells are used, the cells are introduced into a suitable medium, and the The lipoic acid produced can be finally recovered.

[0072] The genetically modified organism of the present invention comprises a transgene encoding lipoic acid synthase (LipA). The bacterial cells produce lipoic acid when the carbon source supplied contains octanoic acid (OA). The cells are fed with a suitable carbon source, e.g., glucose, maltose, galactose, fructose, Sucrose, arabinose, xylose, raffinose, mannose, and lactose When provided with a source selected from among

[0073] The method for quantifying cellular lipoic acid produced by the genetically modified bacterial cells of the present invention comprises: The method is described in Example 2. The method is carried out by culturing cells in minimal medium supplemented with lipoic acid extracted from the cells of the invention. This is a bioassay based on measuring the growth of a lipoic acid-dependent auxotrophic E. coli strain in soil.

[0074] V. Genetically engineered bacterial cells for thiamine production The present invention provides genetically modified bacterial cells capable of enhancing thiamine production levels. The bacterial cell expresses a mutant IscR instead of the wild-type IscR according to the invention. It has been genetically engineered to encode the thiC gene (see Section I). , also known as HMP-P synthase (EC 4.1.99.17) 2-iminoacetic acid synthase or thiH (EC4 The plasmid contains an introduced gene encoding a tyrosine lyase, also known as tyrosine lyase (.1.99.19).

[0075] The genetically engineered bacterial cells express polypeptides that catalyze further steps in the thiamine synthesis pathway. The thiamine transporter may further comprise one or more additional transgenes encoding thiamine transporters (Figure 3). Increasing the levels of these polypeptides that catalyze steps in the pathway increases the rate of progression of the pathway in bacterial cells. The synthesis of both intermediates and end products of the pathway is promoted. For example, bacterial cells The vector may further comprise one or more transgenes that encode: ThiE thiamine phosphate synthase (EC 2.5.1.3); (e.g., encoded by the thiF gene) [ThiS] a Denylyltransferase (EC 2.7.7.73); ThiG thiazole synthase (EC2.8.1.10); ThiS sulfur carrier protein; ThiD phosphohydrin 3-Hydroxymethylpyrimidine kinase (EC 2.7.4.7) and thiamine monophosphate phosphatase ThiOglycine oxidase (EC 1.4.3. 19); and ThiM hydroxyethylthiazole kinase (2.7.1.50).

[0076] HMP-P synthase is found in a wide range of bacteria and fungi belonging to a wide variety of genera. The amino acid sequence of a polypeptide having -P synthase activity is SEQ ID NO: 201 (origin: Enterococcus; SEQ ID NO:203 (origin: Synechococcus elongatus) cus_elongatus)); SEQ ID NO: 205 (Origin: Corynebacterium glutathi micum); SEQ ID NO: 207 (origin Candidate name Baumannia cicadellinicola (Bauman A sequence selected from either one of the following: At least 70, 75, 80, 85, 90, 95, 96, 98, and 100% amino acid sequence identity It has oneness.

[0077] Tyrosine lyase is also called 2-iminoacetate synthase (EC 4.1.99.19). The amino acid sequence of a polypeptide having HMP-P synthase activity is set forth in SEQ ID NO:21. 7 sequences and at least 70, 75, 80, 85, 90, 95, 96, 98, and 100% They have amino acid sequence identity.

[0078] The activity of the genetically modified bacterial cell is encoded by one or more additional transgenes. Polypeptides that play a role in enhancing synthesis of both intermediates and products of the thiamine pathway are , as follows: a) an antibody having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 211; [ThiS] adenylyltransferase (E C2.7.7.73) Polypeptides having activity; b) an antibody having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 209; Thiamine phosphate synthase (EC 2.5.1.3), such as a polypeptide having the amino acid sequence ) polypeptides having activity; c) an antibody having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 215; Thiazole synthase (EC 2.8.1.1), such as a polypeptide having an amino acid sequence 0) a polypeptide having an activity; d) an antibody having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 225; phosphohydroxymethylpyrimidine kinase (E), such as a polypeptide having the amino acid sequence C2.7.4.7) A polypeptide having activity; e) a sequence selected from SEQ ID NOs: 219, 221 and 223, and 80, 85, 90, A polypeptide having an amino acid sequence with 95 or 100% sequence identity. Polypeptides having synthase (EC 1.4.3.19) activity; f) an antibody having 80, 85, 90, 95, or 100% sequence identity with SEQ ID NO: 213; A polypeptide having ThiS sulfur carrier activity, such as a polypeptide having an amino acid sequence. ; g) SEQ ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, 1 43, 145, 147, 149, 151, 153, 155, 157, 159, 161, 1 63, 165, 167, 169, 171, 173, 175, 177, 179, 181, 1 83, 185, 187, 189, 191, 193, 195, 197, 199 The sequence having 80, 85, 90, 95, or 100% sequence identity with the sequence selected from the above. Thiamine monophosphate phosphatase (EC3. 1.3.-) A polypeptide having an activity; and h) an amino acid sequence having 80, 85, 90, 95 or 100% sequence identity with SEQ ID NO: 227; ThiM hydroxyethylthiazole kinase activity, such as a polypeptide having the amino acid sequence A polypeptide having the formula (2.7.1.50).

[0079] Preferably, the genetically modified bacterial cell expresses the enzyme (encoded by the thiC gene). ThiC (encoded by the thiD gene) ThiD (encoded by the thiE gene) ThiE (encoded by the thiF gene); ThiF; sulfur carrier protein (encoded by the thiG gene); ThiG (encoded by the TMP phosphatase gene); and ThiH (encoded by the thiH gene) and ThiO (encoded by the thiO gene). The transgene includes either ThiO or According to this embodiment, the cell is provided with the enzyme Th (encoded by the thiM gene). It may further comprise a transgene encoding iM.

[0080] The level of thiamine synthesis in the genetically modified bacteria of the present invention was determined by the addition of thiamine-phosphate quinine. This can be further enhanced by mutation of the endogenous thiL gene, which encodes the thiL enzyme. The gene has the nucleotide sequence of SEQ ID NO: 228 and, when compared to the parent wild-type gene, Nucleotide 133 encoding a polypeptide having a 133D substitution [SEQ ID NO:229] It has a mutation at ~135 (GGT to GAC).

[0081] HMP-P synthase (EC 4.1.99.17) encoded by thiC or th A nucleotide sequence encoding a tyrosine lyase (EC 4.1.99.19) encoded by iH The transgene may include one or more polypeptides encoding additional steps in the thiamine pathway. The gene is located in the genome of the genetically modified bacterial cell together with the further transgenes described above, It is integrated into the cytoplasmic chromosome or on an autonomously replicating plasmid. One or more of the transgenes encoding the thiamine pathway enzymes are It may be present in the genome within the peron.

[0082] A promoter driving the expression of the thiC or thiH transgene and one or more further transgenes. The promoter is preferably a non-native promoter, and may be a heterologous constitutive promoter or an inducible promoter. If the promoter is a heterologous constitutive promoter, then a suitable promoter The motors include the apFab family [SEQ ID NOs: 230-232], which express the appropriate inducible promoter. The motor contains pBad (arabinose inducible [SEQ ID NO: 233] and LacI [SEQ ID NO: Suitable terminators include ap Members of the FAB terminator family are included. The data can be applied to each gene for individual gene regulation or operon regulation. They can be linked together.

[0083] VI. Methods for Producing and Detecting Thiamine Using the Genetically Engineered Bacteria of the Invention Thiamine, thiamine monophosphate (TMP), and thiamine diphosphate (TPP) are described in Examples 3 and 4. As shown in FIG. 16, the genetically modified bacterial cells of the invention (e.g., genetically modified bacterial cells of the Enterococcus coli cells) to introduce the cells into a suitable culture medium and extract the thiamin produced by the cells. It can be produced by finally recovering dimethyl ether, TPP, and TMP.

[0084] The genetically modified bacterial cell of the present invention contains a transgene encoding HMP-P synthase. The cells are supplied with a carbon source of glucose, maltose, galactose, fructose, and sucrose. Selected from sucrose, arabinose, xylose, raffinose, mannose and lactose When activated, it produces thiamin, TPP, and TMP.

[0085] A method for quantifying thiamine produced by the genetically modified bacterial cells of the present invention is described in Example 3. and involves the use of high pressure liquid chromatography to compare thiamine standards. It can be rare.

[0086] VII. Engineering Genetically Modified Bacterial Cells for the Production of Biotin, Lipoic Acid, or Thiamine method Enzyme activity related to the synthesis of biotin, lipoic acid or thiamine in the bacterial cell of the present invention One or more transgenes encoding one or more polypeptides having the Suitable integrative and self-replicating vectors for inserting the vector into a mammalian cell are commercially available and known to those skilled in the art (e.g., For example, Sambrook et al., Molecular Cloning: A Laboratory Manual. tory Manual,Second Edition,Cold Spring H (See Arbor Laboratory Press, 1989.) Bacterial cells are cells The bacterial cells of the present invention are genetically engineered by introducing heterologous DNA into the The present invention provides a method for the preparation of a polypeptide having an enzyme activity related to the synthesis of thiamine, lipoic acid or thiamine. Heterologous expression of the coding genes is shown in Examples 1, 2 and 3, respectively.

[0087] One or more enzymes having an enzymatic activity related to biotin, lipoic acid or thiamine synthesis according to the present invention The nucleic acid molecule encoding the polypeptide may be introduced into a host cell by a self-replicating vector. Alternatively, the vector may be integrated into the host cell genome using standard methods and techniques in the art. For example, the nucleic acid molecule can be transformed by chemical transformation, electroporation, transduction, or the like. The vector may be introduced by standard protocols such as transformation, including particle bombardment, etc. Expression of the nucleic acid molecule encoding the enzyme is achieved by integrating the nucleic acid molecule into the genome. Good too.

[0088] The genetic modification of the native endogenous iscR gene in the bacterial cell of the present invention can be carried out by suitable The parent bacterial cells were subjected to standard recombinant techniques to delete (knock out) the endogenous iscR gene. (b) a transgene encoding a mutant IscR polypeptide as defined in Section I; This can be achieved by inserting / substituting .;2000).

[0089] Genetically modified bacterial cells according to the invention for the production of biotin, lipoic acid or thiamine may be a bacterium, a non-exhaustive list of suitable bacteria is as follows: Escherichia coli A, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Pseudomonas, Serratia, Lactobacillus, Lactococcus, Acetobacter, Acinetobacter A species belonging to a genus selected from the group consisting of Tobacter, Pseudomonas, and the like.

[0090] Preferred bacterial species of the present invention are Escherichia coli, Pseudomonas putida, Serratia marcescens, ance and Corynebacterium glutamicum.

[0091] VIII. The biotin-producing ability of the genetically modified bacterial cells of the present invention is due to increased electron transport. It will be further enhanced. Oxidized [4Fe-4S] 2+ Cluster-containing SAM radical iron-sulfur cluster enzyme, For example, BioB, ThiC, and LipA are [4Fe-4S] + To give back to the cluster Requires electron transfer to reduce [4Fe-4S] + Only the clusters are required for catalysis. The necessary SAM radical can be generated from the electron donor NADPH. ] 2+ Electron transfer to the cluster is mediated by flavodoxin / ferredoxin reductase (Fpr ) and flavodoxin (FldA) reduction system, or pyruvate flavodoxin / Mediated by the ferredoxin oxidoreductase system.

[0092] In a further embodiment, the present invention provides a method for producing biotin, lipoic acid, or thiamine. The genetically engineered bacterial cells express flavodoxin / ferredoxin-NADP redox regulators. Genes encoding ectases (EC: 1.18.1.2 and EC 1.19.1.1); The gene encoding rubinyl flavodoxin / ferredoxin oxireductase (EC 1.2.7);Flavodoxin-encoding gene;Ferredoxin-encoding gene ; consisting of genes encoding flavodoxin and ferredoxin-NADP reductase The present invention further comprises one or more genes selected from the group consisting of: The linked promoter can enhance expression of one or more of these genes in bacteria. Each of these one or more genes may be a native gene or a transgene. Preferably, the operably linked promoter is a promoter that is capable of expressing a gene in the bacterium. The expression of one or more of these genes can be determined in the parent bacterium from which the genetically modified bacterium of the invention is derived. Preferably, the gene is modified according to the present invention to enhance the expression level of the gene. Bacterial cells express flavodoxin / ferredoxin-NADP reductase (EC: 1.18 EC 1.19.1.1) and the gene encoding flavodoxin or both the flavodoxin and ferredoxin-NADP reductase genes In addition, the genetically modified bacterial cell contains a single gene that contains a coding sequence. It may further comprise a gene encoding a doxin.

[0093] The expression of genes expressing components of the electron transport pathway in the genetically modified bacterial cells of the present invention Overexpression of these SAM labels (as shown in Example 4 for biotin-producing cells of the present invention) Dicarin A enhances the cellular activity of iron-sulfur cluster enzymes.

[0094] Preferably, the native or transgene in the genetically modified bacterial cell of the invention The polypeptide encoded by the polypeptide has flavodoxin / ferredoxin reductase activity. (EC:1.18.1.2 and EC1.19.1.1), SEQ ID NO: 241 ( Origin: fpr gene from E. coli; SEQ ID NO: 243 (Origin: yum from Bacillus subtilis 168) C gene); SEQ ID NO: 245 (origin: fpr -I gene); SEQ ID NO: 247 (origin: Streptomyces venezuelae ATCC SVEN_0113 gene from 10712; SEQ ID NO: 249 (origin: Corynebacterium Cgl2384 gene from U. glutamicum ATTCC 13032) and sequence no. No. 251 (Origin: SJN 15614.1 from Sphingobacterium sp. JB170) The sequence was selected from one of the three genes and was compared to 80, 85, 90, 95, or 100% of the original sequence. The amino acid sequence has sequence identity.

[0095] Preferably, the native or transgene in the genetically modified bacterial cell of the invention The polypeptide encoded by the pyruvate flavodoxin / ferredoxin oxy If it has reductase activity (EC1.2.7), then SEQ ID NO: 253 (origin: E. coli K 12 ydbK gene from MG1655; SEQ ID NO: 255 (origin: Geobacter sp. por gene from Streptomyces rufredus AM-1); SEQ ID NO: 257 (origin: Streptomyces Sfla_2592 gene from Myces platensis ATCC 33331; sequence No. 259 (Origin: Propionibacterium früdenreichii DSM 202 71); SEQ ID NO: 261 (origin: Synechocystis sp. PCC 6803) and 80, 8 The amino acid sequence has 5, 90, 95, or 100% sequence identity.

[0096] Preferably, the native or transgene in the genetically modified bacterial cell of the invention If the polypeptide encoded by the above is flavodoxin, it is SEQ ID NO: 263 (origin : fldA gene from E. coli K12 MG1655; SEQ ID NO: 265 (origin: E. coli fldB gene from K12 MG1655; SEQ ID NO: 267 (origin: Bacillus subtilis 168) ykun gene of the past); SEQ ID NO: 269 (origin: Synechocystis sp. PCC 6803 isiB gene from Streptomyces venezuelae A TCC 10712); SEQ ID NO: 273 (origin: Methanococcus Methanococcus aeolicus Nank ai-3)) and 80), The amino acid sequence has 85, 90, 95 or 100% sequence identity.

[0097] Preferably, the native or transgene in the genetically modified bacterial cell of the invention When the polypeptide encoded by the polypeptide is a ferredoxin, it is represented by SEQ ID NO: 275 (the Source: fdx gene from E. coli; SEQ ID NO: 277 (origin: fer gene from Bacillus subtilis 168) gene); SEQ ID NO: 279 (origin: Corynebacterium glutamicum ATTCC 13 fdxB gene from 032); SEQ ID NO: 281 (origin: Synechocystis sp. PCC fdx gene from 6803); SEQ ID NO: 283 (origin: Streptomyces venezuelae SVEN_7039 gene from Rhee ATCC 10712); SEQ ID NO: 285 (origin :Methanococcus aeolus Nankai-3 a sequence selected from any one of the fdx genes derived from and has an amino acid sequence that has 80, 85, 90, 95 or 100% sequence identity with the above.

[0098] Native or introduced genes encoding polypeptides of the electron transport pathway of the bacterium Promoters capable of enhancing gene expression when operably linked to a gene are preferred. This promoter is a constitutive apFAB309 promoter. Preferably, the nucleic acid sequence of the present invention is a member of the data family [SEQ ID NO: 230-232]. The non-native promoter is operably linked to the native gene or transgene. If so, expression of the encoded polypeptide in the genetically modified bacterium is This native or introduced gene is enhanced to a higher level than the parent bacteria from which it was derived. Suitable terminators that can be operably linked to the nucleic acid include the apFAB378 terminator. The inonotus family [sequence numbers 235-237] is included. EXAMPLES

[0099] Example 1: Identification and characterization of genetically modified E. coli strains capable of enhancing biotin production Sexual evaluation

[0100] 1 method 1.1: The E. coli strains used in the examples are shown below.

[0101] Table 1: Strains [Table 1]

[0102] 1.2: The plasmids used in the examples are shown below.

[0103] Table 2: Plasmids [Table 2]

[0104] 1.3 Media and supplements: The growth medium (mMOPS) used in each example had the following composition: 1.32 mM K 2 HP0 4 2g / l D-glucose; 0.0476mg / l calcium pantothenate 0.0138mg / l p-aminobenzoic acid; 0.0138mg / l p-hydroxy Benzoic acid; 0.0154 mg / l 2,3-dihydroxybenzoic acid and 1x modified MOP S buffer.

[0105] 10x modified MOPS is 0.4 M MOPS (3-(N-morpholino)propanesulfonyl) 0.04M Tricine; 0.1mM FeSO 4 7H 2 95 mM NH 4 Cl; 2.76 mM K 2 SO 4 ; 5 μM CaCl 2 2H 2 O; 5.25 mM Mg Cl 2 0.5M NaCl; and a 5000-fold diluted stock solution of micronutrients .

[0106] Micronutrient stock solutions:

number

[0107] The following antibiotic stocks were used: ampicillin (amp, 100 mg / mL); Namycin (kan, 50 mg / mL), Zeocin (zeo, 40 mg / mL). Therefore, it was added to the growth medium to obtain a 1000-fold dilution.

[0108] 1.4 Preparation of E. coli strain library: The E. coli library with evolved genomic diversity was generated using a large strain of plasmid pBS412. From cells of the E. coli strain BS1011, mMOPS medium supplemented with kan (MOPS-kan) These cells were cultured overnight in a static culture medium at 4°C, and a 100-fold dilution of the resulting mMOPS-kan culture was The culture was prepared by repeating the successive steps of overnight incubation and dilution five times. Accumulation of background mutations generated by defective error-correcting polymerases After each round of incubation and dilution, the culture is diluted with IPTG to generate genetic diversity. Plate the cell culture samples on mMOPS plates (see below) and measure the expression of BioB. Next, we transformed each library cell line with a BioB overexpression plasmid. Transformed with pBS412.

[0109] 1.5 Selection of mutant strains IPT at concentrations of 0, 0.0001, 0.001, 0.01, 0.1, or 1 mM A series of 1.5% agar plates containing mMOPS (Φ=9 cm) containing G were 10 obtained from o / n culture of mMOPS-kan of BS1011 containing 4 , 10 5 , 1 0 6 , 10 7 Selection assays were performed by seeding each of the following cells: Plates were incubated at 37° C. for up to 36 hours and cell growth was assessed at intervals. Under these conditions, induction of BioB expression from pBS412 with 0.1 mM IPTG was achieved. Guidance is 10 5 It was found that 1 mM IPTG prevented the growth of up to 100 cells. The induction prevented the proliferation of at least 10 Λ7 cells when plated in a single Petri dish. 10 5 Selection pressure involving induction with 1 mM IPTG on a cell population of B This proved to be optimal for identifying strains with higher robustness to ioB expression. Therefore, the following was done:

[0110] 1) Approximately 10 copies of each library are cloned as described in section 1.4. 5 Each cell 1 mM OPS-kan was plated on IPTG agar plates and incubated at 37°C for up to 24 hours. Incubated. 2) A single colony was grown in mMOPS-kan liquid medium to create a preculture. Then, either 0.00, 0.01, or 0.1 mM IPTG was added to mMOPS- Biotin bioassays performed in kan (see section 1.6 below) confirmed this. The cells of each preculture were incubated in 20% glycerol for 1 h at 4°C for 1 h. It was stored as stock. 3) Colonies producing 1.5 mg / l or more of biotin (detected as extracellular biotin) were selected. Re-streak onto mMOPS-kan agar plates and incubate at 37°C for up to 24 hours. and bioassayed again for biotin production in biological replicates (see section below). (More details in Section 1.6.)

[0111] 4) The selected biotin-overproducing cells were evaluated as follows: a) Genes in the genome of cells of the selected strains compared to the genome of the parent strain BS1011 Whole genome sequencing to identify mutations was performed on D isolated from cells of the selected strains. The following was done with NA: Selected cells were grown in 5-10 mL of mMOPS-kan. The cells were then harvested. Order the kit (https: / / www.thermofisher.com / order / The genome was extracted from the harvested cells using the cataloq / product / K182001. Mutational DNA was isolated and whole genome sequencing of the extracted DNA was performed. b) Curing these pBS412 plasmids from cells of the selected strains. Then, the cells of the pBS412 plasmid-cured strain were transformed again. Cell cultures of the transformed strains were again bioassayed for biotin production. The process for curing these pBS412 plasmids from cells was carried out with 1 mM IPTG The cells were incubated at 37°C in Luria Broth (LB) rich medium containing no antibiotics. The cells from the resulting culture were grown overnight at 37 °C and streaked onto LB agar plates. The incubation was performed overnight. A single colony from the agar plate was removed and 50 μl Dilute in LB medium and spot onto LB and LB-amp agar plates using 5 μl The bacteria did not grow on the LB-amp plate, but the L These single colonies that grew on the B plate were re-streaked onto LB plates. Single colonies were obtained and these were used as cured strains for retransformation.

[0112] Biotin production by cells of the transformed strains was measured in biological replicates (see section 1 below). Briefly, 0.00, 0.01 or 0.1 mM IPTG was added. Biotin production was re-evaluated for biological replicates in mMOPS-kan. The cell growth rate of each transformant was then evaluated in a microplate sealed with a transparent, breathable seal. In a titer plate, 200 μL of mMOPS-kan medium was placed at 37°C for 24 hours. Measurements were performed using a Multiskan FC with "high speed shaking" for aerobic growth. 30 min Cell growth was monitored by measuring OD620 every 2 h.

[0113] 1.6 Bioassays to quantitate biotin production Precultures were prepared from selected single cell colonies in 96 deep well plates. 00 μL of mMOPS-kan and incubated at 37 °C with shaking at 275 rpm. The mixture was incubated for 16–18 hours. 40 wells were placed in a 96 deep-well plate containing IPTG to a final concentration of 1 mM. 0 μL of mMOPS-kan, initial OD 600 Sufficient preculture to make the Production cultures were made by inoculating 4 μL of the cultures. The cultures were then rotated at 275 rpm. The cultures were grown for 24 hours at 37°C with shaking. 600 After measuring, 4000 Pellet the cells in the 96 deep-well plate by centrifugation for 8 min at G. The supernatant from each culture was diluted with ultrapure water (Milli-Q) at biotin concentrations ranging from 0.05 to 100 mM. In parallel, 0.1 nM (biotin 0.024 Over 5 biotin standards ranging in concentration from 0.5μg / L to 1nM (biotin 0.24μg / L) A mixture of 15 μL of each diluted supernatant and each biotin standard solution was then prepared. Each well contained the plasmid pBS451. The overnight culture contained 135 μL of biotin-starved BS1011. Initial OD in mMOPS with Zeocin 620 The concentration was diluted to 0.01. The container was sealed with a breathable seal and incubated at 37°C for 20 hours with shaking at 275 rpm. After incubation, the OD 600 In this bioassay, various biotin standards were The biotin bioassay calibration curve obtained using the solution is shown in FIG.

[0114] 1.7 Identification of genomic mutations For all next generation sequencing (NGS) data, genomes of cells from selected strains The mutations in the genome of the parent strain were compared with those in the genome of the parent strain using CLC Genomic Workbench version 9.5. 3 (provided by Qiagen) (single or small number of substitutions, deletions, or insertions are identified using Varia nt Detection, Larger insertions / deletions are detected by InDels and Struc The genomic variants introduced by the sequencing procedure were identified. A cutoff of 85% was used to distinguish genomic variants caused by incorrect nucleotides. Using this method, more than 85% of the DNA molecules (genomes) isolated from cells of a particular bacterial strain were mutated. We defined "significant mutation" as meaning the presence of

[0115] Considering the Keio ΔbioB scar mutation, the sequence of which was confirmed by sequencing and using genome accession number CP009273 from NCBI as a reference sequence. did.

[0116] 1.8 Characterization of the proteomic landscape of iscR mutations BS1013+pBS430, BS1011 at 0.025mM IPTG induction level +pBS412 and BS1353+pBS412 and BS1 at 1 mM IPTG induction The protein content of 353+pBS412 was determined using LC-MS and efficient protein extraction. This was achieved by a recently developed combined approach (Schmidt et al., In addition to a peptide threshold of 2.0% FDR, three peptides were identified for analysis. The most significant changes in protein expression were observed in the Scaffold 1000 and 10000 peptides. Benjamini-Hochb multiple test with dViewer4.7.5 Based on analysis of variance (ANOVA) with erg correction, a 0.5% confidence interval was observed. do.

[0117] The strains were then incubated at OD 600 The β-amyloides was induced by IPTG in mMOPS until the β-amyloides reached 0.5 (log phase). The cells were grown for approximately 10 generations. The 10 8 The cells were harvested and washed once with ice-cold PBS buffer. The PBS buffer was removed. They were then repelleted by centrifugation at maximum speed at 4°C and flash frozen in liquid nitrogen.

[0118] 2.Results 2.1 Overexpression of BioB is toxic It retains the native biotin synthase gene (bioB) but contains a low copy number of IPTG-inducible frameshifted colon derived from smid (replication origin Sc101) The fungal bioB gene (encoding a nonfunctional biotin synthase with a premature stop codon) E. coli expressing the β-actin was grown aerobically in mMOPS-kan medium with or without IPTG. This was confirmed by the results of the analysis of E. coli BW25113, a BioB frameshift mutant, The logarithmic growth curve of E. coli knockout strains is shown in Figure 4 (left panel). Overexpression of a functional biotin synthase gene (bioB) in strain ΔbioB resulted in increased inhibition of growth. This is because the low copy number Escherichia coli biosynthesis from an IPTG-inducible T5 promoter on the mide (origin of replication Sc101) Figure 4 (right panel) shows the growth of the E. coli knockout strain ΔbioB expressing the oB gene. As shown in Figure 4, biotin-induced phenotypes in response to increasing IPTG levels (gray darkening). Induction of increased B expression has a large effect on the lag phase but only a small effect on the growth rate (Black border).

[0119] 2.2 Isolation of an iscR mutant with enhanced biotin production titer E. coli library with evolved genomic diversity (see sections 1.4 and 1.5) The strains with improved resistance to bioB gene expression and increased biotin production were screened. Whole genome sequencing of the selected strains revealed that L15F, C92Y , H107Y or H207Y. Three unique mutants containing the cluster regulator (iscR) gene were identified. The amino acid sequences of the encoded regulators are SEQ ID NOs: 16, 18 and 20, respectively. As described in section 1.6 (and Figure 5), a bioassay was used to detect the The levels of riboflavin produced by the iscR mutant and E. coli BW25113 Δbio The biotin production titers of each of the B reference strains were measured at two different IPTG concentration levels (the higher mMOP with 0.1 g / l DTB in the absence or presence of 0.1 g / l IPTG (grey, more IPTG). Shown are four biological replicates (black dots) grown in S. Biotin production in the reference strain was inhibited at IPTG levels above 0.01 mM, which was consistent with the growth of the reference strain. This corresponds to a toxic IPTG level (see Figure 4). In contrast, the iscR mutant The strain was grown at IPTG levels of 0.01 to 0.1 mM and produced biotin. All mutant strains produced approximately 1.5-fold more virions than the reference strain (dotted line) at an IPTG concentration of 0.01 mM. The production of the iscR mutant strain was the highest at 0.1 mM IPTG compared to the reference strain. The production titer is up to twice as high (approximately 3.2 mg / l biotin) as the production titer (approximately 1.5 mg / l biotin). there were.

[0120] 2.3 Biotin production and growth of the IscR H107Y mutant The growth profile and biotin production titer of the iscR(H107Y) mutant were characterized in 5 0 mL of mMOPS plus 0.1 g / L DTB, at two different IPTG induction levels ( 7A) and 0.5 mM (Figure 7B) in 250 mL shake flask experiments. At low IPTG levels (Figure 7A), the IscR mutant (dark grey) and the reference The growth and biotin production titers were similar to those of the strain (light grey), with the final titer being approximately 1 However, at high IPTG induction levels (0.5 mM in Figure 7B), The growth of the control strain (light grey) was significantly suppressed, whereas the IscR mutant had low IPTG induction. The growth profile of the IscR mutant was similar to that of the control strain. increased approximately two-fold, reaching a maximum of approximately 2.2 mg / l after 25 h of growth.

[0121] 2.4 Mechanism of action of IscR mutations As shown in Figure 6, all three identified IscR mutants showed increased biotin resistance. The ability of the C92 mutation (C92Y) to enhance biotin resistance was clearly demonstrated. The force is due to the role of C92 in the [Fe-S] cluster binding properties of IscR It has been suggested that the loss of [Fe-S] cluster binding properties due to the C92Y mutation is due to I It has been proposed that scR inactivates the Isc operon repression behavior of C92 The Y mutant IscR maintains intact promoter function and upregulates multiple cellular processes. H107 has also been proposed to possess the ability to activate other pathways essential for I It also plays a key role in conferring the [Fe-S] cluster binding properties of scR. The Y mutation can also increase the biotin resistance of E. coli. However, the depletion of iron-sulfur clusters was partially overcome by breaking the iron-sulfur cluster bonds. It has been proposed that L15 and H10 of IscR bind to DNA. 7 (the hya binding site, PDB entry 4HF1), and L15 is the position of each Is It can be seen that it is located inside the cR subunit. Phenylalanine is more abundant than leucine. It is a significantly larger amino acid than α-amino acids and may interfere with the tertiary folding of proteins. do.

[0122] 2.5 Overexpression of the isc or suf operon in E. coli strains only results in biotin biosynthesis. not enough to boost production The isc operon (iscSUA-hscBA-fdx, is The suf operon (corresponding to the native E. coli operon structure lacking the cR gene) or the native Direct effects of overexpression of sufABCDSE, which corresponds to the active E. coli operon structure To determine the effect of each operon on the expression of the operon, we engineered the operon to be expressed under the strong RBS and IPTG-inducible T5 promoters. Cloned into a medium copy number plasmid (p15A ori) under the control of The superfolder green fluorescent protein was used instead of the isc operon or suf operon. A plasmid containing a gene encoding a protein (sfGFP) was used as a control. Each plasmid was transformed into cells of an E. coli strain containing a G-inducible bioB expression plasmid. Transformed: 1) IPTG-inducible iscB expression plasmid in addition to the IPTG-inducible bioB expression plasmid operon, 2) IPTG-inducible suf operon, or 3) IPTG-inducible GFP (control ) were incubated with 100 μg / mL ampicillin and spectinomycin 50 μg / mL in 400 μL of mMOPS at low (0.01 After cultivation at high (0.1 mM IPTG) induction and high (0.1 mM IPTG) induction (section The cells were assayed for biotin production (as described in 1.5).

[0123] The graph (Figure 10) shows that biotin production is IPTG inducible in all strains, but not detectable. The IPTG concentrations required to reach competent biotin production levels are shown in Figure 6 for the reference and mutant strains. Compared to the iscR strain, the concentration of IPTG increased from 0.01 mM to 0.1 mM. Furthermore, when compared to both sfGFP strains in Figure 10, the biotin productivity was significantly higher. The titer was significantly decreased by overexpression of the isc operon or the suf operon. Overexpression of the sc operon suppressed biotin production more than overexpression of the suf operon. Considering the increase in biotin production observed in the mutant strain with a single point mutation in iscR (Figure 6), Taken together, derepression of the isc operon in these strains may be responsible for the biogenesis of these strains. It is unlikely to be the sole / main reason for the improvement in otin production.

[0124] 2.6 BioB protein content correlates with biotin production Overexpression of BioB in wild-type and mutant background strains To investigate the molecular effects, a wild-type background strain was used: BS10 carrying pBS430. 13; wild-type iscR strain carrying the bioB production plasmid: BS1 carrying pBS412 and a mutant iscR strain carrying the bioB production plasmid: pBS412. We performed proteomic analysis of BS1353, which is a strain of The latter strain was grown in mMOPS containing 1 mM I Cells were harvested at log phase for proteomic analysis and the remaining The cell cultures were incubated for a total of 24 hours and then subjected to bioassays as described elsewhere. was used to measure biotin production.

[0125] From the graph (Figure 11), the measured BioB protein levels strongly correlated with biotin production. The linear correlation indicates that the promotion of BioB expression is related to the IscR mutation. This indicates that the key to improving biotin production in heterologous cell factories is the proteomics data. ANOVA analysis of the data revealed significant increases in expression of an additional 29 proteins (95 % confidence interval, p value 0.00166) were revealed. Among these, the isc operon (IscA and IscS) and members of the suf operon (SufB and SufS). Included.

[0126] 2.7 Biotin production is not enhanced in the iscR knockout mutant Using MAGE, we identified the glutamic acid-encoding codon at position 22 of iscR (E,G By converting the codon (AA) into a stop codon (*, TGA), translation of the iscR gene is terminated. The qout was introduced into the BW25113 ΔbioB strain. The success of the codon conversion was confirmed by The wild-type iscR was verified by PCR amplification followed by Sanger sequencing. , iscR knockout (E22*) and mutant iscR (C92Y) The resulting strain was transformed with IPTG-inducible bioB plasmid pBS412 and transformed into DTB0 The three different strains were cultured in mMOPS supplemented with 0.1 g / l and kanamycin 50 μg / l. Biological replicates grown at IPTG induction levels (0 mM, 0.01 mM and 0.1 mM) (n=3) were tested for biotin production.

[0127] When bioB expression was induced by IPTG induction, iscR knockout (iscR No significant difference in biotin production was observed between the iscR KO strain and the wild-type iscR WT strain. This indicates that knocking out iscR does not improve biotin production. For the mutant iscR encoding the IscR C92Y substitution, A significant improvement in biotin production compared to both the WT and iscR KO strains was again observed. Ta.

[0128] 2.8 De novo biotin production is enhanced in the iscR mutant of the present invention Both the bioA gene and the entire biotin operon (ΔbioB-bioD) were deleted. , iscR WT gene, iscR H107Y mutant gene, or iscR C92Y mutant gene The BW25113 E. coli strain containing either the native E. coli bioA or bioB gene was The strain was transformed with a tetracycline resistance plasmid that constitutively overexpresses the chin operon. There was a single point mutation at the bioO operator site (type 9 mutation, Ifuku et al. 1993 For these three different strains, we used 10 μg / ml tetracycline as described above. The results are shown in Table 1. The mMOPS (glucose 2 Biotin production in biological replicates (n=4) at 1000μg / l was evaluated (Figure 13).

[0129] When the substrate DTB was added to the growth medium, a significant increase in biotin titer was observed in all three strains. The bioB enzyme reaction that converts DTB to biotin is itself a biotin biosynthetic reaction in these strains. This indicates that the iscR WT strain is no longer a bottleneck for production (Fig. 13). Compared to the control, both iscR mutants showed a significant increase in de novo production of biotin from glucose. Considering these results, all of the mutant iscR strains of the present invention are directly related to the precursor We speculate that this may help enhance biotin production from both the host cell, DTB, and from glucose. It is possible.

[0130] Example 2: Engineering and characterization of genetically modified E. coli strains capable of enhancing lipoic acid production evaluation The strains of E. coli used in this example are shown below.

[0131] Table 3: Strains [Table 3]

[0132] The plasmids used in this example are shown below.

[0133] Table 4: Plasmids [Table 4]

[0134] LipA ​​[SEQ ID NO: 103] cloned on plasmid pBS1037 was encoded The IPTG-inducible transgene was then introduced into an E. coli host strain containing the native iscR gene or As described in Example 1, the native iscR gene contains a C92Y or H107Y substitution. The E. coli host was replaced by a mutant iscR gene encoding the IscR protein having strains; these two strains additionally contained knockouts of bioB or lipA. These strains were incubated with 0.1 mM biotin (for the ΔbioB strain), 0.1 mM α-glucose (for the ΔbioB strain) for induction of lipA expression, In mMOPS medium (section 1) supplemented with IPTG and 0.6 g / l octanoic acid as a substrate. After incubation at 37°C for 24 hours in the culture medium (described in Section 1.3), the free lipoic acid in the supernatant was measured. For the ΔlipA strains (BS1912 and BS2114, Table 3), 24 h at 37°C Growth was followed during the growth of the cells.

[0135] Lipoic acid produced by cultured cells of the described strains was cultured in BS191 containing pBS451. The supernatant was analyzed by a bioassay similar to that described in section 1.6 using 2 A growth-based bioassay for the quantification of lipoic acid was performed in mice unable to synthesize lipoic acid. A single ΔlipA mutant of Escherichia coli (Herbert and Guest, 1999) 75) (BS1912 containing pBS451) was used. The lipoic acid was added as the sole source of lipoic acid, and the carbon source was added at 500 mg / kg. By measuring the growth of lipoic acid auxotrophs in minimal medium containing 0 nM succinic acid A calibration curve for the lipoic acid bioassay was run in parallel, with lipoic acid standards of known concentration range. Auxotrophic strains were grown in minimal medium supplemented with broth.

[0136] The test was performed using mutant forms of the IscR protein (IscR with C92Y or H107Y substitutions). Overexpression of the lipA gene in an E. coli strain containing a gene encoding the I The lipA gene in the parental E. coli strain containing the gene encoding the native form of the scR protein Compared with overexpression of the α-glucose derivative, this resulted in both a more stable production and an 80% increase in lipoic acid titer. Therefore, the iscR WT strain (BS containing pBS993) The standard deviation of the titer of iscR C92Y (pBS99 3) was 1.42, and iscR H107Y (pBS993) was 1.51. 1353) was 0.11 (see Table 1 for strains). Based on the results, lipoic acid production in the mutant strain was improved by 1.79-fold compared to the WT strain (Figure 1). See 4.

[0137] Overexpression of LipA was performed in the WT iscR strain (triangles, BS191 harboring pBS1037). 2) and the iscR mutant (square, BS2114 containing pBS1037), IP showed a clear tendency to decrease the growth rate in response to increased induction of LipA by TG ( (dark grey, Figure 15). However, the growth rate of the WT iscR strain was 0.01 mM to 0.03 mM. At all IPTG induction levels tested in mM, the (See Figure 15.)

[0138] Example 3: Engineering and characterization of genetically modified E. coli strains capable of enhancing thiamine production Sexual evaluation The strains of E. coli used in this example are shown below.

[0139] Table 5: Strains [Table 5]

[0140] The plasmids used in this example are shown below.

[0141] Table 6: Plasmids [Table 6]

[0142] Thiamine pathway gene thiCEFSG cloned on plasmid pBS140 HMD was administered to an E. coli host strain (BS750 ) and the native iscR gene was C92Y or by a mutant iscR gene encoding an IscR protein with a H107Y substitution. The substituted nucleotides were introduced into the derivatives of this reference strain (BS2019 and BS2020). These strains were cultured in mMOPS medium at 37°C for 24 hours in each well of a deep culture plate. The cells were cultured in medium (as described in section 1.3).

[0143] Extracellular and intracellular thiamine, TMP and TP produced by cultured cells of the above strains. P was harvested and extracted as follows: 0.4 mL of each culture was placed in a culture plate and 40 The pellet was then collected by centrifugation at 100 x g for 5 min at 4 °C. All remaining steps were carried out on ice. For the analysis of extracellular TPP, TMP and thiamine, 40 μL of the supernatant was gently removed. After decanting the remaining supernatant, the culture plate was turned upside down to remove residual medium, Then, vortex the mixture. 100 μL of ice-cold HPLC grade methanol was added to the culture plate. The cells were then vortexed again and incubated on ice for a minimum of 20 minutes. After incubation, cell debris was pelleted by centrifugation at 4000 x g for 5 min. The supernatant was used as the intracellular extract for further analysis.

[0144] To detect TPP, TMP, and thiamine using a fluorescence detector, the amount of TPP, TMP, and thiamine produced in each culture was measured. The thiamine compound was derivatized into a highly fluorescent thiochrome. The procedure was carried out at room temperature. A volume of 40 μl of extracellular and intracellular extracts was added to 80 μl of 4 M potassium acetate. Freshly prepared potassium ferricyanide was added to 3. 40 μl of 7M NaOH containing 8 mM NaOH was added and mixed. Contains 0.06% saturated KH 2 PO 4 Quench the reaction by adding 40 μl of The extract was neutralized by adding 47 μL of 6 M HCl and then analyzed by HPLC or All derivatized compounds were analyzed by direct fluorescence measurement using a Multiskan. The material was then subjected to freshly prepared TP derivatization to thiochrome in parallel with the analyzed extracts. Quantification was performed using fluorescent standard curves of P, TMP and thiamine standards.

[0145] This study involved the use of mutant forms of the IscR protein (IscR with C92Y or H107Y substitutions). The host E. coli strains (BS2019 and BS2019) containing the gene encoding the scR protein 20) and the thiC and thiH genes and the TMP phosphatase gene (At Overexpression of thiamine pathway genes, including in combination with 5g32470, was associated with a reduction in the native Overexpression in a host E. coli strain (BS750) containing the gene encoding the IscR protein of the Enhancement of the biosynthesis of thiamine, TMP and TPP, especially thiamine, compared with overexpression of This shows that.

[0146] More specifically, when pBS140 was used, the strain with WT iscR (BS7 50) and strains encoding iscR mutations (BS2020, H107Y or BS2019, C OD normalized extracellular production of thiamin (thiamin, TMP, and TPP) between 92Y and Tests showed that the effect of the 1.43-fold increase in ...

[0147] Example 4: Flag to enhance the productivity of genetically modified E. coli strains capable of producing biotin Fpr and Fladoxin / ferredoxin reductase (FldA) Overexpression of the origin 1 method 1.1: The E. coli strains used in the examples are shown below.

[0148] Table 7: Strains [Table 7]

[0149] The plasmids used in this example are shown below.

[0150] Table 8: Plasmids [Table 8]

[0151] An IPTG-inducible transgene encoding BioB was cloned onto plasmid pBS679. A constitutively regulated transgene encoding GFP was introduced into the plasmid pBS The synthetic operon encoding FldA-Fpr was cloned onto 1054. A constitutively regulated transgene containing pBS679 was transformed with the IscR protein carrying the H107Y substitution as described in Example 1. The native iscR gene is replaced by a mutant iscR gene encoding Then, the gene was introduced into an E. coli host strain (BS1615) containing a knockout of the bioAFCD gene. The BS1937 strain was then transformed with the plasmid pBS1054 or pBS 1112, and the resulting strains were BS2707 (control strain) and BS21 We obtained 85 shares.

[0152] The strain was cultured with the appropriate antibiotic and 0.1 g / L of DTB as a substrate for BioB-mediated catalysis. , IPTG0, 0.01, 0.025, 0.0 to induce the expression of BioB gene The cells were cultured in mMOPS medium (described in Example 1.3) supplemented with 0.5, 0.075 or 0.1 mM Cells were incubated in individual wells of deep-well culture plates at 37°C for 24 h. Estimate the final OD, collect the supernatant by centrifugation, and eluate with biotin biotin as described in Example 1.6. Biotin was quantified from the supernatant by assay.

[0153] For strain BS1937, as shown in Figures 12 and 17, the genetically modified endogenous i BioB gene expression in E. coli cells containing the scR gene was increased by increasing the concentration of IPTG. Upon induction, cells show a corresponding gradual increase in biotin production. Biotin production in the engineered cells was confirmed by using the parent strain BS1937 and the FldA-Fpr subunit instead of FldA-Fpr. The FldA-Fp gene was significantly increased in the control strain expressing a transgene encoding GFP. This is further enhanced by coexpression of a transgene encoding r (strain BS2185).

[0154] Encodes a mutant form of the IscR protein (IscR protein with the H107Y substitution) The gene encoding the ribosomal RNA, the BioB gene (pBS679) and the FldA-Fpr gene (pBS1 Biotin production in the BS2185 strain containing the plasmid for overexpression of 112 was compared with that in the control strain. 2.12-fold increase compared to strain BS1937 (without overexpression of FldA-Fpr genes) This was done (Figure 18).

[0155] References: Datsenko KA, Wanner BL. (2000) One-step inactivation of chromosomal genes in E scherichia coli K-12 using PCR products. Proc Natl Acad Sci US A. ;97(12): 6640 -5. Fleischhacker, A. S. et a / . (2012) Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR. Biochemistry 51 : 4453-4462. Giel, J. L., Rodionov, D., Liu, M., Blattner, F. R. & Kiley, P. J. (2006) IscR -dependent gene expression links iron-sulphur cluster assembly to the control of 02-regulated genes in Escherichia coli. Mol. Microbiol. 60, 1058- 1075. Herbert AA, Guest JR. (1975) Lipoic acid content of Escherichia coli and other microorganisms. Arch Microbiol.; 106(3) :259-66. Epub 1975 / 12 / 31. pmid: 814874 Ifuku, O. et al. Sequencing analysis of mutation points in the biotin operon o f biotin-overproducing Escherichia coli mutants. Biosci Biotechnol Biochem 57, 7 60-765 (1993). Ifuku, O. et a / ., (1995) "Molecular analysis of growth inhibition caused by ov erexpression of the biotin operon in Escherichia coli." Bioscience, biotechnolog y, and biochemistry 59(2): 184-189. Martin, J. E. & Imlay, J. A. (2012) Replication during periods of iron starvat ion. 80, 319-334. Py, B. & Barras, F. (2010) Building Fe-S proteins: bacterial strategies. Nat. Rev. Microbiol. 8, 436-446. Schmitt, A, Kochanowski K., Vedelaar S., Ahrne E., Volkmer B., Callipo L., Kno ops K., Bauer M., Aebersold R., & Heinemann M., (2015) The quantitative and cond ition-dependent Escherichia coli proteome. Nature Biotechnology 2015; doi: 10.10 38

Claims

1. A genetically modified bacterium, the bacterium having enhanced production of biotin, lipoic acid, or thiamine; a. a genetically modified endogenous iscR gene encoding a mutant IscR polypeptide, wherein the amino acid sequence of the mutant IscR polypeptide has at least 90% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, wherein the amino acid sequence is i. L15X, C92X, C98X, C104X and H107X, where X is an amino acid other than the corresponding amino acid residues of SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14. A genetically modified endogenous iscR gene having at least one amino acid substitution selected from the group consisting of: b. at least one transgene, ii. a polypeptide having biotin synthase activity (EC 2.8.1.6); iii. A polypeptide having lipoic acid synthase activity (EC 2.8.1.8); iv. a polypeptide having HMP-P synthase activity (EC 4.1.99.17); v. a polypeptide having tyrosine lyase activity (EC 4.1.99.19); At least one transgene encoding a polypeptide selected from the group consisting of: A genetically modified bacterium comprising:

2. At least one amino acid substitution in the mutant IscR polypeptide is a. L15X, where X is any of F, Y, M, and W; b. C92X, where X is Y, A, M, F, or W; c. C98X, where X is A, V, I, L, F, or W; d. C104X, where X is A, V, I, L, F, or W; e. H107X, where X is any of A, Y, V, I, and L; The genetically modified bacterium for enhancing the production of biotin, lipoic acid or thiamine according to claim 1, selected from the group consisting of:

3. The at least one transgene encodes a polypeptide having biotin synthase activity (EC 2.8.1.6), and a. A polypeptide having SAM (S-adenosylmethionine)-dependent methyltransferase activity (EC 2.1.1.197); b. A polypeptide having 7-keto-8-aminopelargonic acid (KAPA) synthase activity (EC 2.3.1.47); c. A polypeptide having 7,8-diaminopelargonic acid (DAPA) synthase activity (EC:2.6.1.62 or EC:2.6.1.105); d. A polypeptide having dethiobiotin (DTB) synthetase activity (EC 6.3.3.3); e. A polypeptide having a pimeloyl-[acyl carrier protein] methyl ester esterase (EC 3.1.1.85), and f. A polypeptide having 6-carboxyhexanoate-CoA ligase activity (EC 6.2.1.14); and further comprising an additional transgene encoding one or more polypeptides selected from the group consisting of The bacterium is for enhancing production of biotin. A genetically modified bacterium according to claim 1 or 2.

4. the at least one transgene encodes a polypeptide having lipoic acid synthase activity (EC 2.8.1.8), a. a polypeptide having octanoyltransferase activity (EC 2.3.1.181); b. a polypeptide comprising the dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase (EC 2.3.1.12); c. a polypeptide having lipoate protein ligase A activity (EC:6.3.1.20); and further comprising an additional transgene encoding one or more polypeptides selected from the group consisting of The bacterium is for enhancing production of lipoic acid. A genetically modified bacterium according to claim 1 or 2.

5. the at least one transgene encodes a polypeptide having HMP-P synthase activity (EC 4.1.99.17) and / or a polypeptide having tyrosine lyase activity (EC 4.1.99.19), a. a ThiF polypeptide having ThiS adenylyltransferase activity (EC 2.7.7.73); b. a ThiE polypeptide having thiamine phosphate synthase activity (EC 2.5.1.3); c. a ThiG polypeptide having thiazole synthase activity (E.C.2.8.1.10); d. a ThiD polypeptide having phosphohydroxymethylpyrimidine kinase activity (EC 2.7.4.7); e. a ThiO polypeptide having glycine oxidase activity (EC 1.4.3.19); f. a ThiS polypeptide having sulfur carrier protein activity; g. a ThiM polypeptide having hydroxyethylthiazole kinase activity (2.7.1.50); h. a polypeptide having thiamine monophosphate phosphatase activity (E.C.3.1.3.-); and further comprising an additional transgene encoding one or more polypeptides selected from the group consisting of The bacterium is for enhancing the production of thiamine. A genetically modified bacterium according to claim 1 or 2.

6. The bacterium a. a ThiC polypeptide having HMP-P synthase activity (EC 4.1.99.17); b. a ThiH polypeptide having tyrosine lyase activity (EC 4.1.99.19) or a ThiO polypeptide having glycine oxidase activity (EC 1.4.3.19); c. a ThiF polypeptide having ThiS adenylyltransferase activity (EC 2.7.7.73); d. a ThiE polypeptide having thiamine phosphate synthase activity (EC 2.5.1.3); e. a ThiG polypeptide having thiazole synthase activity (E.C.2.8.1.10); f. A ThiD polypeptide having phosphohydroxymethylpyrimidine kinase activity (EC 2.7.4.7); g. a ThiS polypeptide having sulfur carrier protein activity; h. a polypeptide having thiamine monophosphate phosphatase (E.C.3.1.3.-) activity; The genetically modified bacterium of claim 5, further comprising an additional transgene encoding:

7. The genetically modified bacterium of any one of claims 1 to 6, wherein the at least one transgene and the one or more further transgenes are operably linked to a constitutive promoter.

8. The genetically modified bacterium according to any one of claims 1 to 7, wherein the bacterium is a species selected from the group consisting of Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Pseudomonas, Serratia, Lactobacillus, Lactococcus, and Acinetobacter, Pseudomonas, and Acetobacter.

9. a. introducing the genetically modified bacterium of any one of claims 1 to 3, 7, and 8 into a growth medium to produce a culture, wherein the at least one introduced gene encodes a polypeptide having biotin synthase activity (EC 2.8.1.6); b. culturing the culture; c. recovering the biotin produced by the culture and optionally purifying the recovered biotin; A method for producing biotin, comprising:

10. a. introducing the genetically modified bacterium of any one of claims 1, 2, 4, 7, and 8 into a growth medium to produce a culture, wherein the at least one introduced gene encodes a polypeptide having lipoic acid synthase activity (EC 2.8.1.6); b. culturing the culture; c. recovering the lipoic acid produced by the culture and optionally purifying the recovered lipoic acid; A method for producing lipoic acid comprising the steps of:

11. a. introducing the genetically modified bacterium of any one of claims 1, 2, 5-8 into a growth medium to produce a culture, wherein the at least one introduced gene encodes a polypeptide having HMP-P synthase activity (EC 4.1.99.17) and / or a polypeptide having tyrosine lyase activity (EC 4.1.99.19); b. culturing the culture; c. recovering the thiamin produced by the culture and optionally purifying the recovered thiamin; A method for producing thiamine, comprising:

12. 12. The method for producing any one of biotin, lipoic acid and thiamine according to any one of claims 9 to 11, wherein the growth medium comprises a carbon source selected from glucose, maltose, galactose, fructose, sucrose, arabinose, xylose, raffinose, mannose, and lactose or any combination thereof.

13. 1. Use of a genetically modified gene encoding a mutant IscR polypeptide to enhance production of any one of biotin, lipoic acid, or thiamine in a genetically modified bacterium, the bacterium comprising: i. a polypeptide having biotin synthase activity (EC 2.8.1.6); ii. a polypeptide having lipoic acid synthase activity (EC 2.8.1.8); iii. a polypeptide having HMP-P synthase activity (EC 4.1.99.17); iv. a polypeptide having tyrosine lyase activity (EC 4.1.99.19); and expressing at least one transgene encoding a polypeptide selected from the group consisting of: The genetically modified bacterium has an endogenous iscR gene encoding a mutant IscR polypeptide, wherein the amino acid sequence of the mutant IscR polypeptide has at least 90% amino acid sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, and wherein the amino acid sequence has at least one amino acid substitution selected from the group consisting of L15X, C92X, C98X, C104X, and H107X, where X is an amino acid other than the corresponding amino acid residue in SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

14. 14. Use of a genetically modified gene encoding a mutant IscR polypeptide for enhancing production of any one of biotin, lipoic acid, and thiamine in the genetically modified bacterium according to claim 13, wherein at least one amino acid substitution in the mutant IscR polypeptide is: a. L15X, where X is any one of F, Y, M, and W; b. C92X, where X is one of Y, A, M, F, and W; c. C98X, where X is any one of A, V, I, L, F, and W; d. C104X, where X is any one of A, V, I, L, F, and W; e. H107X, where X is any one of A, Y, V, I, and L; The use of any one of the following:

15. 9. Use of the genetically modified bacterium according to any one of claims 1 to 8 for enhancing production of any one of biotin, lipoic acid, or thiamine.

16. The bacterium a. a gene encoding flavodoxin / ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1); b. A gene encoding pyruvate flavodoxin / ferredoxin oxireductase (EC 1.2.7); c. a gene encoding flavodoxin; d. A gene encoding ferredoxin; e. Genes encoding flavodoxin and ferredoxin-NADP reductase; and further comprising one or more genes selected from the group consisting of: The one or more genes are operably linked to a non-native promoter capable of enhancing expression of the one or more genes in the bacterium, and the one or more genes may be native genes or transgenes. A genetically modified bacterium according to any one of claims 1 to 8.

17. 17. Use of the genetically modified bacterium of claim 16 for enhancing production of any one of biotin, lipoic acid, or thiamine.

18. The genetically modified bacterium is a. a gene encoding flavodoxin / ferredoxin-NADP reductase (EC: 1.18.1.2 and EC 1.19.1.1); b. A gene encoding pyruvate flavodoxin / ferredoxin oxireductase (EC 1.2.7); c. a gene encoding flavodoxin; d. A gene encoding ferredoxin; e. Genes encoding flavodoxin and ferredoxin-NADP reductase; and further comprising one or more genes selected from the group consisting of: The one or more genes are operably linked to a non-native promoter capable of enhancing expression of the one or more genes in the bacterium, and the one or more genes may be native genes or transgenes. A method for producing any one of biotin, lipoic acid, and thiamine according to any one of claims 9 to 12.

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