CN118374429A - Coenzyme Q10-producing genetically engineered bacterium and application thereof - Google Patents

Abstract

The invention provides a coenzyme Q10-producing genetically engineered bacterium and application thereof.

Description

Coenzyme Q10-producing genetically engineered bacterium and application thereof

Technical Field

The invention belongs to the field of biological fermentation, and in particular relates to a coenzyme Q10-producing genetically engineered bacterium and application thereof.

Background

Ubiquinone (Ubiquinone, abbreviated UQ), also known as Coenzyme Q (Coenzyme Q), is a fat-soluble quinone compound that exists in nature. Ubiquinone molecules contain a side chain consisting of a plurality of isoprene units and linked to a p-benzoquinone core, the length of the side chain varies depending on the source of ubiquinone, and generally contain n=6 to 10 isoprene units. For humans and mammals, n=10, and therefore also known as coenzyme Q10 (Coenzyme Q, coQ 10), the structural formula is shown in formula (I):

Coenzyme Q10 is involved in energy metabolism of cells as an important hydrogen carrier in the respiratory chain of biological cells. Is an effective antioxidant component for preventing arteriosclerosis, and is involved in energy production and activation in human somatic cells. In recent years, the traditional Chinese medicine composition has been widely applied to the treatment of various heart diseases, diabetes, cancers, acute and chronic hepatitis, parkinsonism and other diseases. In addition, the composition has obvious effect in treating scurvy, duodenal ulcer, necrotic periodontitis, promoting pancreas function and secretion, and the like. Recently, researchers find that coenzyme Q10 has an anti-aging effect, so that the application of the coenzyme Q10 is expanded to the fields of cosmetics and health care products, and the demands of the coenzyme Q10 at home and abroad are further expanded.

At present, three production methods of coenzyme Q10 are mainly adopted, namely an animal and plant tissue extraction method, a chemical synthesis method and a microbial fermentation method. The animal and plant coenzyme Q10 content in the animal and plant tissue extraction method is low, various chemical components are complex and limited by raw materials and sources, so that the product cost is high, the price is high, and the large-scale production is limited. The chemical synthesis method is mature in technology, mainly uses the solanesol with rich sources as a raw material to synthesize, but the product is a mixture of cis-trans isomers, the biological activity is low, and the synthesized coenzyme Q10 with high biological activity does not reach the degree of industrial production yet. The coenzyme Q10 synthesized by the microbial fermentation method has the advantages of low cost, no optical isomer, good biological activity, high yield, good mass production and application effects and the like, and gradually becomes the main method for the industrial production of the coenzyme Q10.

The variety of coenzyme Q10-producing microorganisms is large, and rhodobacter sphaeroides (Rhodobacter sphaeroides) belonging to photosynthetic bacteria are important strains for the industrialized mass production of coenzyme Q10 at present because of the high content of intracellular synthesized coenzyme Q10 and relatively simple extraction steps. Currently, genetic modification and reformation of the biosynthesis pathway of rhodobacter sphaeroides coenzyme Q10 by metabolic engineering means has been reported in the prior art to increase the yield of rhodobacter sphaeroides coenzyme Q10. For example, chinese patent No. CN103509729B discloses that the key genes DXS and DDS that enhance the synthesis of polydodecenyl pyrophosphate in the rhodobacter sphaeroides intracellular MEP pathway can be utilized, thereby increasing the yield of engineered coenzyme Q10. However, the product yields of coenzyme Q10 obtained in the prior art are not ideal.

At present, there is still a need to provide genetically engineered bacteria with increased coenzyme Q10 yields, thereby facilitating more industrial mass production and downstream applications of coenzyme Q10.

Disclosure of Invention

The inventors have surprisingly found that in the parent strain of rhodobacter sphaeroides, the coenzyme Q10 yield of rhodobacter sphaeroides can be effectively increased by simultaneously expressing UbiF gene and regA gene, and the strain has good stability.

Accordingly, in a first aspect, the present invention provides an engineering bacterium of rhodobacter sphaeroides which is modified to express UbiF gene and RegA gene, wherein: the UbiF gene is integrated into the genome of the rhodobacter sphaeroides engineering bacterium, and the RegA gene is expressed through a plasmid vector.

QMP (quinone modification pathway) is the last few steps of modification of the coenzyme Q10 synthesis pathway to the cyclic quinone, involving UbiA, ubiD, ubiX, ubiI, ubiG, ubiH, ubiE, ubiF, ubiB isogenic. The enzymes encoded by the genes are as follows: ubiA, octenyl 4-hydroxybenzoate transferase; ubiD, 3-octenyl-4-hydroxybenzoic acid decarboxylase; ubiX xanthine pretransferase; ubiI, 2-octenyl phenol hydroxylase; ubiG, 2-octenyl-6-hydroxyphenol/2-octenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinone methyltransferase; ubiH, 2-octenyl-6-methoxyphenol hydroxylase; ubiE ubiquinone/cerebrospinal fluid quinone biosynthetic methyltransferase; ubiF, 2-octenyl-3-methyl-6-methoxy-1, 4-benzoquinone hydroxylase; ubiB, possibly protein kinases, are of unknown function.

In embodiments of the invention, the UbiF gene may be derived from a variety of species, provided that the UbiF protein encoded by the UbiF gene has 2-octenyl-3-methyl-6-methoxy-1, 4-benzoquinone hydroxylase activity. In some embodiments, the UbiF gene is derived from escherichia coli (e.coli).

In some preferred embodiments, the UbiF gene encodes a UbiF protein of E.coli, said UbiF protein having 2-octenyl-3-methyl-6-methoxy-1, 4-benzoquinone hydroxylase activity and comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 2. In some more preferred embodiments, the UbiF gene encodes the amino acid sequence of SEQ ID NO. 2.

In some preferred embodiments, ubiF gene is the UbiF gene of E.coli, preferably it comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 1. In some more preferred embodiments, the UbiF gene comprises the nucleotide sequence of SEQ ID NO. 1.

RegA and RegB are two-component transcriptional regulatory systems present in a variety of photosynthetic and non-photosynthetic bacteria that are capable of regulating expression of downstream genes in response to redox status in the bacteria. In an embodiment of the present invention, the RegA gene may be derived from various species as long as the RegA gene has transcriptional regulatory activity. In some embodiments, the RegA gene is derived from rhodobacter sphaeroides, such as rhodobacter sphaeroides KD131 (KCTC 12085), rhodobacter sphaeroides 2.4.1 (ATCC 17023), rhodobacter sphaeroides ATCC 17029, rhodobacter sphaeroides strain AB24, rhodobacter sphaeroides strain AB25, rhodobacter sphaeroides strain AB27, rhodobacter sphaeroides strain AB29, rhodobacter sphaeroides strain CH10, rhodobacter sphaeroides strain DSM158, rhodobacter sphaeroides strain MBTLJ-13, rhodobacter sphaeroides strain MBTLJ-20, rhodobacter sphaeroides strain MBTLJ-8, rhodobacter sphaeroides strain HJ, or rhodobacter sphaeroides strain derived therefrom.

In some preferred embodiments, the RegA gene encodes a RegA protein of rhodobacter sphaeroides, said RegA protein having a transcriptional activation function and comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 4. In some preferred embodiments, the RegA gene encodes the amino acid sequence of SEQ ID NO. 4.

In some preferred embodiments, the RegA gene is a RegA gene of rhodobacter sphaeroides, preferably comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 3. In some preferred embodiments, the RegA gene comprises the nucleotide sequence of SEQ ID NO. 3.

To achieve expression of UniF genes and RegA genes in rhodobacter sphaeroides engineering bacteria, promoters capable of initiating gene expression in rhodobacter sphaeroides, e.g., the promoters provided in table 1 of the present application, may be selected.

In some embodiments, ubiF gene is expressed from a strong promoter. In some embodiments, the UbiF gene is expressed from a strong promoter having a relative strength of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, or at least 120% relative to the Tac promoter in rhodobacter sphaeroides engineering bacteria. In some preferred embodiments, the UbiF gene is expressed from a promoter selected from T334-6(SEQ ID NO:10)、T334-36(SEQ ID NO:9)、NOLALANC_02773(SEQ ID NO:11)、T167-31(SEQ ID NO:12)、NOLALANC_02328(SEQ ID NO:13)、T334-7(SEQ ID NO:14)、NOLALANC_02693(SEQ ID NO:15)、T167-27(SEQ ID NO:16)、T167-29(SEQ ID NO:17) or T334-26 (SEQ ID NO: 18). In some more preferred embodiments, the UbiF gene is expressed as initiated by the T334-6 promoter (SEQ ID NO: 10).

In some embodiments, the RegA gene is expressed from a weak promoter. In some embodiments, the RegA gene is expressed from a weak promoter having a relative strength of at most 40%, at most 30%, at most 25%, at most 20%, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% relative to the Tac promoter in rhodobacter sphaeroides engineering bacteria. In some preferred embodiments, the RegA gene is expressed from a promoter selected from NOLALANC_04400(SEQ ID NO:60)、NOLALANC_03111(SEQ ID NO:55)、NOLALANC_00059(SEQ ID NO:56)、NOLALANC_01751(SEQ ID NO:57)、NOLALANC_03340(SEQ ID NO:58)、NOLALANC_03519(SEQ ID NO:59)、NOLALANC_03841(SEQ ID NO:61)、NOLALANC_03925(SEQ ID NO:62)、NOLALANC_03961(SEQ ID NO:63)、NOLALANC_01082(SEQ ID NO:64)、NOLALANC_01569(SEQ ID NO:65) or NOLALANC _02822 (SEQ ID NO: 66). In some more preferred embodiments, the RegA gene is expressed from the NOLALANC _04400 promoter (SEQ ID NO: 60).

In an embodiment of the invention, the UbiF gene is expressed by integration into the genome of the rhodobacter sphaeroides engineering bacterium.

Methods for integrating a gene fragment of interest into the genome of a host cell are well known to those skilled in the art. For example, the gene fragment of interest may be integrated into the host cell genome at a desired site by homologous recombination. The homologous recombination process relies on homology between DNA molecules. In the application of homologous recombination, it is often necessary to add upstream and downstream sequences, also called homology arms, of homology to the target site upstream and downstream of the foreign DNA sequence or gene fragment of interest.

In some embodiments of the invention, the target site for UbiF gene integration is the NOLALANC _00703 site in the rhodobacter sphaeroides genome, and the gene corresponding to that site is the flagellin matrix MS-loop/cycloprotein gene. In some embodiments, the UbiF gene is integrated into or upstream or downstream of the flagelliform matrix MS-loop/cyclopin gene in the genome of the rhodobacter sphaeroides engineering bacterium. In some preferred embodiments, the UbiF gene is integrated into the genome of the rhodobacter sphaeroides engineering bacterium within a range of 1000bp upstream or downstream of the flagelliform matrix MS-loop/cyclopin gene. In some more preferred embodiments, the UbiF gene replaces a fragment of 1000bp upstream to 1000bp downstream of the flagelliforme MS-loop/cycloprotein gene.

In some embodiments, the flagellin matrix MS-loop/cyclic protein gene comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 5. In some preferred embodiments, the flagellin matrix MS-loop/cycloprotein gene comprises the nucleotide sequence of SEQ ID NO. 5.

In some embodiments, the flagellin matrix MS-loop/cyclic protein gene encodes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 6. In some preferred embodiments, the flagellin matrix MS-loop/cycloprotein gene encodes the amino acid sequence of SEQ ID NO. 6.

In some embodiments, the 1000bp upstream of the flagelliforme MS-loop/cycloprotein gene comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 7. In some preferred embodiments, 1000bp upstream of the flagelliforme MS-loop/cycloprotein gene comprises the nucleotide sequence of SEQ ID NO. 7.

In some embodiments, 1000bp downstream of the flagelliforme MS-loop/cycloprotein gene comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 8. In some preferred embodiments, 1000bp downstream of the flagelliforme MS-loop/cycloprotein gene comprises the nucleotide sequence of SEQ ID NO. 8.

In some embodiments, the N-and C-termini of the gene fragment of interest for integration of UbiF gene into the site of interest each comprise a homology arm. In some embodiments, the homology arms comprise at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides of a flagellin matrix MS-loop/cycloprotein gene of rhodobacter sphaeroides. In some embodiments, the homology arm comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within the 1000bp sequence range upstream of the flagelliform matrix MS-loop/cycloprotein gene of rhodobacter sphaeroides. In some embodiments, the homology arm comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000bp sequence range downstream of the flagellum matrix MS-loop/cycloprotein gene of rhodobacter sphaeroides.

In some preferred embodiments, the N-terminal homology arm of the gene fragment of interest for integration of UbiF gene into the site of interest comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 7. In some more preferred embodiments, the N-terminal homology arm comprises the nucleotide sequence of SEQ ID NO. 7. In some preferred embodiments, the C-terminal homology arm of the gene fragment of interest for integration of UbiF gene into the site of interest comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 8. In some more preferred embodiments, the C-terminal homology arm comprises the nucleotide sequence of SEQ ID NO. 8.

In some preferred embodiments, the N-terminal homology arm of the gene fragment of interest for integration of UbiF gene into the site of interest comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the reverse complement of SEQ ID NO. 8. In some more preferred embodiments, the N-terminal homology arm comprises a nucleotide sequence that is reverse complementary to SEQ ID NO. 8. In some preferred embodiments, the C-terminal homology arm of the gene fragment of interest for integration of UbiF gene into the site of interest comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the reverse complement of SEQ ID NO. 7. In some more preferred embodiments, the C-terminal homology arm comprises a nucleotide sequence that is reverse complementary to SEQ ID NO. 7.

In some embodiments of the invention, whether the UbiF gene is integrated into the site of interest can be confirmed by PCR identification of rhodobacter sphaeroides engineering bacteria using primer A (SEQ ID NO:78, TGAAGACGGGATCCATCACACACATATAC)/primer B (SEQ ID NO:79, GAGCGTGCTGGCGTTGA). And if the positive target fragment of 1576bp can be obtained through PCR identification, the UbiF gene is proved to be successfully integrated into the target site of the rhodobacter sphaeroides engineering bacterium genome.

In some embodiments of the invention, the RegA gene is expressed by a plasmid. The plasmid for expressing the RegA gene was constructed by inserting the RegA gene expression cassette into a plasmid vector suitable for rhodobacter sphaeroides. In some embodiments, the plasmid vector comprises an origin of replication capable of self-replication in rhodobacter sphaeroides. In some embodiments, the origin of replication is selected from the group consisting of: pUC, colE1, pBBR1, pMB1, pBR322, pSC101, R6K and p15A.

In some preferred embodiments, the plasmid vector comprises an origin of replication that is pBBR1. In some preferred embodiments, the vector used for plasmid expression of the RegA gene is the pBBR1MCS2 vector.

In an embodiment of the invention, the rhodobacter sphaeroides engineering bacteria are modified to express UbiF genes and RegA genes by modifying the parent rhodobacter sphaeroides. The parent rhodobacter sphaeroides may be selected from: rhodobacter sphaeroides KD131 (KCTC 12085), rhodobacter sphaeroides 2.4.1 (ATCC 17023), rhodobacter sphaeroides ATCC 17029, rhodobacter sphaeroides strain AB24, rhodobacter sphaeroides strain 25, rhodobacter sphaeroides strain AB27, rhodobacter sphaeroides strain AB29, rhodobacter sphaeroides strain CH10, rhodobacter sphaeroides strain DSM158, rhodobacter sphaeroides strain MBTLJ-13, rhodobacter sphaeroides strain MBTLJ-20, rhodobacter sphaeroides strain MBTLJ-8 and rhodobacter sphaeroides strain HJ.

In some preferred embodiments, the parent rhodobacter sphaeroides strain has a accession number of CGMCC No.7.275.

In a second aspect, the invention provides rhodobacter sphaeroides engineering bacteria, and the preservation number of the rhodobacter sphaeroides engineering bacteria strain is CGMCC No.30222.

In some embodiments, the rhodobacter sphaeroides engineering bacteria of the invention have increased coenzyme Q10 production compared to the parent rhodobacter sphaeroides. In some embodiments, the rhodobacter sphaeroides engineered strain of the invention has a coenzyme Q10 yield of at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, or at least 600% of the yield of the parent rhodobacter sphaeroides coenzyme Q10.

In a third aspect, the present invention provides a method of preparing coenzyme Q10, the method comprising:

(1) Culturing the rhodobacter sphaeroides engineering bacterium of the first or second aspect of the present invention under conditions suitable for production of coenzyme Q10;

(2) And recovering coenzyme Q10 from the culture of the rhodobacter sphaeroides engineering bacteria.

In a fourth aspect, the invention provides the use of an engineering bacterium of rhodobacter sphaeroides of the first or second aspect of the invention in the production of coenzyme Q10.

In a fifth aspect, the present invention provides a method of preparing an engineered rhodobacter sphaeroides of the first or second aspect of the invention, the method comprising:

(1) Integrating UbiF genes into the genome of a parent rhodobacter sphaeroides, thereby obtaining an intermediate rhodobacter sphaeroides strain; and

(2) And (3) transforming a plasmid vector containing the regA gene into the rhodobacter sphaeroides intermediate strain obtained in the step (1), thereby obtaining the rhodobacter sphaeroides engineering strain.

In some embodiments, the target site for UbiF gene integration is the NOLALANC _00703 site in the rhodobacter sphaeroides genome, and the gene corresponding to that site is the flagelliform matrix MS-loop/cycloprotein gene. In some embodiments, the UbiF gene is integrated into or upstream or downstream of the flagelliform matrix MS-loop/cyclopin gene in the genome of the rhodobacter sphaeroides engineering bacterium. In some preferred embodiments, the UbiF gene is integrated into the genome of the rhodobacter sphaeroides engineering bacterium within a range of 1000bp upstream or downstream of the flagelliform matrix MS-loop/cyclopin gene. In some more preferred embodiments, the UbiF gene replaces a fragment of 1000bp upstream to 1000bp downstream of the flagelliforme MS-loop/cycloprotein gene.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

For easier understanding of the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In this document, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to").

Herein, "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items. For example, composition comprising a and/or B may be interpreted as composition comprising a, composition comprising B, or composition comprising a and B.

All numerical designations, such as pH, temperature, time, concentration and molecular weight, including ranges, are approximations that may be varied by either 1.0 or 0.1 increments, or alternatively by +/-15%, 10%, 5%, 2% changes. It should be understood that all numerical designations are preceded by the term "about". It is also to be understood that the reagents described herein are merely exemplary and that equivalents thereof are known in the art. The term "about" as used herein when referring to a measurable amount, such as an amount or concentration, etc., is meant to include a change within 20%, 10%, 5%, 1%, 0.5% or 0.1% of the specified amount.

The terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length (ribonucleotides or deoxyribonucleotides). Thus, the term includes, but is not limited to, single-stranded, double-stranded or double-stranded DNA or RNA, genomic DNA, cDNA, DNA RNA hybrids, or polymers comprising, consisting of, or consisting essentially of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably and refer in their broadest sense to a polymeric form of two or more amino acid subunits, amino acid analogs, or peptidomimetics. "protein", "peptide", "polypeptide" and "amino acid sequence" contain at least two amino acids, and there is no limitation on the maximum number of amino acids. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

Equivalents having one or more amino acid modifications as compared to the protein or amino acid sequence described herein are also encompassed within the scope of the invention, provided that the one or more amino acid modifications do not affect or substantially affect the activity of the protein or amino acid sequence. In this context, an amino acid modification may be an amino acid substitution, an amino acid deletion or an amino acid insertion. Amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also known as conservative mutations, conservative substitutions or conservative variations) are amino acid substitutions in a protein that change a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, or size). In this context, "conservative substitution" refers to the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or one charged or polar residue for another, such as arginine for lysine, glutamic for aspartic acid, glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the following variations: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine and serine to threonine; threonine to serine; tryptophan changes to tyrosine; tyrosine to tryptophan or phenylalanine; etc.

The terms "equivalent" or "functional variant" when referring to a particular molecule, biological material, or cellular material, are used interchangeably and refer to those that have minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a reference polypeptide (e.g., ubiF or RegA proteins described herein); or a polypeptide encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a reference polynucleotide (e.g., ubiF gene or RegA gene described herein).

In this context, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, amino acid sequence, or protein. If the nucleic acid sequence is derived from genomic DNA, expression may include splicing mRNA in eukaryotic cells.

The term "encoding" when applied to a nucleic acid sequence refers to a nucleic acid sequence that is said to "encode" a polypeptide if it can be transcribed to produce mRNA and/or translated to produce the polypeptide in its natural state or when manipulated by methods well known to those of skill in the art. The antisense strand is the complement of such a nucleic acid and from which the coding sequence can be deduced.

"Homology" or "identity" refers to sequence similarity between two polypeptides or between two nucleic acid sequences. The percent identity may be determined by comparing the positions in each sequence, which may be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences depends on the number of matched positions that are shared. "unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences of the present invention. Means for comparing the similarity between sequences are well known to those skilled in the art, for example, by introducing nucleic acid or amino acid sequences into ClustalW (available from https:// genome. Jp/tools bin/ClustalW/and using the ClustalW, the alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined.

The term "promoter" as used herein refers to an expression control sequence that controls the initiation and rate of transcription of a gene or transgene. Promoters may be, for example, constitutive, inducible, repressible or tissue specific. Promoters may contain genetic elements that regulate the binding of proteins and molecules such as RNA polymerase and transcription factors.

When applied to a promoter, the term "relative strength" refers to the percentage of the strength of the test promoter to the strength of a control promoter (e.g., the Tac promoter used in the present application, the strength of which is defined as 100%) in the strain of interest or the cell of interest. The strength of the promoter may be determined by methods well known to those skilled in the art. For example, the promoter to be tested may be operably linked to a nucleotide sequence encoding a fluorescent protein such that expression of the fluorescent protein is initiated by the promoter to be tested. In this case, the expression level of the fluorescent protein reflects the intensity of the promoter to be tested.

"Operably linked" or "operably linked" as used herein refers to a linkage of nucleic acid sequences such that one sequence provides the desired function for the linked sequences. In the present invention, "operably linked" may be to connect a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. When a sequence of interest encodes a protein and expression of the protein is desired, "operably linked" means that the promoter is linked to the sequence in a manner that allows for efficient transcription and translation of the sequence.

The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host or capable of insertion of a gene segment of interest into the host genome, which transfers the carried gene segment of interest into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

The terms "genome insertion" and "genome integration" are used interchangeably herein to refer to the insertion of an exogenous DNA sequence or gene fragment of interest into the genome of a strain of interest.

The terms "insertion site" and "integration site" are used interchangeably herein to refer to a site of interest at which an exogenous DNA sequence or a fragment of a gene of interest is inserted into the genome.

In some embodiments, the target site upon genome insertion may be an endogenous gene, and upon insertion of exogenous DNA, the original endogenous gene coding sequence is disrupted or replaced. In some embodiments, the sequence upstream and downstream of the endogenous gene remains unchanged. In some embodiments, the sequence upstream and downstream of the endogenous gene can be altered, for example, by homologous recombination.

In some embodiments, the target site at the time of genome insertion may be upstream or downstream of the endogenous gene, preferably within 1000bp upstream or downstream. After insertion of the foreign DNA, the original endogenous gene coding sequence remains unchanged.

As used herein, "genome insertion overexpression" refers to the expression of a gene of interest in a strain of interest using a means of integrating exogenous DNA. In this case, the gene fragment of interest is inserted into the target site of the host cell genome and expressed.

Methods for inserting an exogenous DNA sequence or gene fragment of interest into a target site in the genome are well known to those skilled in the art. For example, an exogenous DNA sequence or a gene fragment of interest may be inserted into a target site of the genome by homologous recombination. The homologous recombination process relies on homology between DNA molecules. In the application of homologous recombination, it is often necessary to add upstream and downstream sequences, which may also be referred to as homology arms, of homology to the integration site of interest, upstream and downstream of the foreign DNA sequence or gene fragment of interest.

As used herein, "plasmid overexpression" refers to the use of a plasmid to express a gene of interest in a strain of interest. In this case, the gene fragment of interest is located on a plasmid that is capable of being present and/or self-replicating in the host cell, without integrating into the genome of the host cell.

As used herein, relative production (%) of coenzyme Q10 refers to the relative production of coenzyme Q10 relative to the parent strain as a control (defined as 100%).

As used herein, coenzyme Q10 increase (%) refers to the percentage of increase in coenzyme Q10 production, defined as 100% of the production of coenzyme Q10 by the parent strain.

In this context, the "parent strain" refers to a strain used for engineering or the like, and may also be referred to as "starting strain". On the basis of the parent strain, the strain obtained by rational design modification or modification by mutagenesis is called as "genetically engineered strain" or "engineering strain".

The invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the invention more apparent, and the advantages and features of the invention will be more apparent from the description. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, without specific conditions noted in the examples below, is by molecular cloning according to conditions conventional in the art, for example, sambrook and Russeii et al: laboratory manual (third edition) (2001), conditions described in CSHL press, or conditions recommended by the manufacturer. Unless otherwise indicated, the experimental materials and reagents used in the following examples are all commercially available.

Examples

Example 1 obtaining promoters of different intensities

The choice of promoters reported in the rhodobacter sphaeroides present literature is limited, and in order to realize the regulation of gene expression intensity and thus the transformation of rhodobacter sphaeroides, promoters with different intensities are studied in the embodiment so as to facilitate the construction and use of strains.

(1) Promoter selection

In this example, potential promoters in rhodobacter sphaeroides were selected for intensity testing, and the relative intensity of each tested promoter was calculated using the intensity of the commonly used promoter Ptac as 100%.

(2) Plasmid construction

Plasmid pK18mobsacB was used for constructing the vector required for integrative expression (purchased from ATCC, # 87097) or plasmid pBBR1MCS2 was used for constructing the vector required for episomal expression (purchased from Addgene, plasmid # 85168).

In plasmid construction, the above vector was used as a PCR template, and linearized vector DNA fragments were amplified with the following primers:

upstream and downstream primers of pK18 mobsacB:

F:TGCCGCAAGCACTC(SEQ ID NO:67)

R:ATTGCGTTGCGCTC(SEQ ID NO:68)

upstream and downstream primers for pBBR1MCS 2:

F1:TCATCCCAGGTGGCACTTTTCGGGG(SEQ ID NO:69)

R1:ATGTCAGCTACTGGGCTATCTGGAC(SEQ ID NO:70)

PCR use High-Fidelity 2X Master Mix. The method comprises the following steps: starting: 98 ℃ for 30 seconds; cycling 30 times: 98℃for 10 seconds, 57℃for 10 seconds, 72℃for 30 seconds/kb; final extension (final extension): 30 seconds at 72 ℃. After template digestion treatment of amplified PCR products with DpnI, clean recovery of DNA was performed using AxyPrep PCR clean kit, and finally the concentration of the cleaned DNA fragments was determined by Nanodrop.

Plasmid inserts (e.g., promoter, terminator, gene) amplify linearized DNA using the same PCR method. PCR templates were all genetically synthesized from Huada genes.

Linearized inserts and vector DNA fragments relevant gene fragments were inserted into the vector by the method of Gibson assembly (https:// www.nature.com/arotics/nmeth.1318) usingHiFi DNA Assembly (NEB E5520S) kit combination is connected with different fragments to complete construction of a target plasmid, and the construction method is a kit description method. The plasmid for testing the strength of each promoter was obtained by constructing a [ promoter-DASHER GFP-rrnB terminator ] expression cassette on the pBBR1MCS2 vector with pBBR1MCS2 as the vector and DASHER GFP as the over-expression gene. Wherein, the nucleotide sequence of DASHER GFP is shown as SEQ ID NO. 71, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 72; the rrnB terminator is derived from an E.coli rrnB gene, and the nucleic acid sequence of the rrnB terminator is shown as SEQ ID NO. 73. The constructed plasmid is verified by sequencing and identifying Huada genes.

(3) Conversion of conjugation

The hemA gene was knocked out from E.coli S17 strain (ATCC 47055) to obtain hemA knocked out E.coli S17 strain (hereinafter referred to as S17 hemA) (Thoma,S.,et al.An improved Escherichia coli donor strain for diparental mating.FEMS Microbiol Lett.2009May;294(2):127-32.https://doi.org/10.1111/j.1574-6968.2009.01556.x).

The target plasmid is transformed into S17 hemA, the transformation process is as follows: mu.l of the target plasmid was added to 50 mu l S hemA-competence, ice-bath for 30min, and rapidly placed on ice for 2 min after heat shock at 42 ℃. 5-aminolevulinic acid (5-aminolevulinic acid, ALA for short) was added to 400. Mu.l of SOB medium to form a liquid medium having a 5-aminolevulinic acid concentration of 50. Mu.g/ml; resuscitates at 37℃for 1 hour. Transfer 200. Mu.l of the resuscitated broth to 400. Mu.l of liquid medium containing kanamycin (26. Mu.g/ml) and ALA (50. Mu.g/ml) and incubate overnight at 37 ℃. Rhodobacter sphaeroides strains were picked on the same day and were monoclonal to 400. Mu.l of fermentation medium (see strain (4) for detailed formulation) and incubated overnight at 30℃at 800 rpm. SOB medium configuration methods are described in https:// cshprotocols.cshlp.org/content/2018/3/pdb.rec102723. Fullrss=1.

The next day OD600 was measured, and after transformation, cultured overnight with LB was washed S17 hemA 2 times and OD600 was adjusted to 0.4-0.6 with LB. Rhodobacter sphaeroides strain NHU5915 (the strain is classified and named as rhodobacter sphaeroides (Rhodobacter sphaeroides), and is preserved in China general microbiological culture collection center (CGMCC, no. 3 of the North Star West-Lu 1 of the Korean region of Beijing city) at 18/03/2024) with the preservation number of CGMCC No.7.275, and OD660 is regulated to 1.5 by using LB. S17 hemA is compared with rhodobacter sphaeroides strain NHU and 5915 according to volume ratio 1:1 mixing and then plating onto LB solid plates containing kanamycin (26. Mu.g/ml), culturing at 30℃for 3-5 days until rhodobacter sphaeroides strain monoclonal appears, and verifying.

(4) Strain culture medium

Seed medium (1L): 5g (NH 4) ₂SO₄, 1g Ajinomoto monosodium glutamate, 1g corn flour, 1g KH ₂PO4,1g K₂ HPO4, 10g dextrose monohydrate, 4g MgSO ₄·7H₂O,8g CaCO₃, 4g NaCl,2g yeast extract, 2mL trace elements stock1(0.1g/L CoCl₂·6H₂O,3g/L MnSO₄·H₂O),2.4g FeSO₄·7H₂O,, and 6.85-6.9 pH adjusted with 6M NaOH. If necessary, the corresponding antibiotic, such as 25mg kanamycin, is added after sterilization.

Fermentation medium (1L): 6g (NH ₄)₂SO₄, 6g Ajinomoto monosodium glutamate, 4g corn flour, 1.5g KH ₂PO₄, 50g dextrose monohydrate, 12.6g MgSO ₄·7H₂O,5g CaCO₃, 2.8g NaCl,0.5g yeast extract, 1mL trace element stock2 (7 g/L CaCl ₂,5g/L MnSO₄·H₂O),0.2g FeSO₄·7H₂ O, pH adjusted to 6.85-6.9 with 6M NaOH) if necessary, and the corresponding antibiotic, such as 25mg kanamycin, is added after sterilization.

(5) Fluorometry assay

Rhodobacter sphaeroides strains that were confirmed to be correct were selected and monoclonal into 2.2ml 96-well plates (hereinafter referred to as PC plates) containing 400 μl of seed medium, and the corresponding antibiotics were selected according to strain requirements.

Placing the PC board in a culture table, and culturing at 30deg.C, 1000rpm and 90% humidity for three days;

Transferring 60 μl of the bacterial liquid to a 2.2ml 96-well plate containing 600 μl of fermentation medium, and culturing at 30deg.C, 1000rpm and 90% humidity in a culture shaker for four days;

Transferring 10 mu l of bacterial liquid into 190 mu l of ELISA plate containing 190 mu l of sterile water, and fully and uniformly mixing;

the ELISA plate was placed in a BioTek spectrometer and OD660 and fluorescence intensity were measured. For DASHERGFP, the parameters were set to excitation 485nm, emission 528nm.

(6) Experimental results

The relative intensities of the promoters were calculated by measuring the expression intensities of the fluorescent proteins initiated by the respective promoters and using the commonly used strong promoter Tac promoter (SEQ ID NO: 74) as a control, and the results are shown in Table 1.

TABLE 1 relative intensities of the promoters relative to the Tac promoter were tested

EXAMPLE 2 enhancement of rhodobacter sphaeroides coenzyme Q10 production by genomic insertion overexpression UbiF

The NOLALANC _00703 locus is selected as an integration locus of the UbiF expression cassette, and a corresponding gene of the locus codes for a flagelliforme MS-ring/cycloprotein gene (FLAGELLAR BASAL-body MS-ring/collar protein), the coding sequence of the gene is shown as SEQ ID NO. 5, and the coded amino acid sequence of the gene is shown as SEQ ID NO. 6. The upstream 1000bp sequence of the gene is shown as SEQ ID NO. 7, and the downstream 1000bp sequence is shown as SEQ ID NO. 8.

(1) Plasmid construction

See example 1 for a description of the vectors.

In this example, using pK18mobsacB as a vector, T334-6 promoter was selected, and the [ homology arm 1-promoter-UbiF-rrnB terminator-homology arm 2] expression cassette was constructed on the vector by Gibson assembly method for transformation into rhodobacter sphaeroides strain NHU5915 and measurement of coenzyme Q10 production. Wherein UbiF gene is derived from Escherichia coli (E.coli), its nucleic acid sequence is shown in SEQ ID NO. 1, and its amino acid sequence of encoded protein is shown in SEQ ID NO. 2. The sequence of the T334-6 promoter is shown in SEQ ID NO. 10. The rrnB terminator is derived from an E.coli rrnB gene, and the nucleic acid sequence of the rrnB terminator is shown as SEQ ID NO. 73. The homology arm 1 is 1000bp upstream of the flagellum matrix MS-loop/cycloprotein gene, the nucleotide sequence of the homology arm is shown as SEQ ID NO. 7, the homology arm 2 is 1000bp downstream of the flagellum matrix MS-loop/cycloprotein gene, and the nucleotide sequence of the homology arm is shown as SEQ ID NO. 8.

(2) Conversion of conjugation

As in example 1.

(3) Strain culture

As in example 1.

(4) Coenzyme Q10 assay

① Instrument:

Agilent 1290infinity II UHPLC System with DAD.

Centrifuge (Eppendorf, 5810R;Thermo,Multifuge X pro)

Oscillator (OHRUS and DM)

② Chromatographic column:

Thermo Scientific Hypersil ODS(C18)Column,2.1x150 mm,3μm Cat#30103-152130

③ Chemical reagent:

CoQ10

ethanol (EtOH), sigma-Aldrich,

Methanol (MeOH), sigma-Aldrich

Isopropyl alcohol (IPA), sinopharm,

④ Consumable material

2ML vial, agilent

50ML ampoule volumetric flask, thermo

96-Well deep well plate, A-gen

Microplates, 0.5mL, U, agilent

⑤ Method of

Standard preparation

1) 25.00 Mg of coenzyme Q10 and 25.00 mg of 4-hydroxybenzoic acid were weighed into a 50 ml ampoule.

2) The volume was fixed with isopropyl alcohol (IPA).

Calibration standard preparation

The standard was diluted to 10, 50, 100, 200, 300, 500ppm with IPA. Transfer to sample vials.

Sample preparation

100 Microliters of rhodobacter sphaeroides culture was transferred to each well of a 96-well deep well plate. 500 microliters of IPA was added to each well. After shaking on a shaker at 2000rpm for 20 minutes. The plate was shaken on a shaker at 40℃and 800rpm for 90 minutes. For the fermentation samples, centrifugation was performed at 6000rpm/10 min. Transfer 200 microliters to 0.5 milliliters of Agilent microwell plate.

Analysis method

Mobile phase B1:1:1EtOH/MeOH

Mobile phase A1: h ₂ O

Sample injection amount 2. Mu.l

Pump cleaning: 10% IPA

Needle cleaning: 75% IPA

Column temperature: 55 DEG C

Flow rate: 0.55ml/min

Wavelength: 275nm and 256nm

TABLE 2 gradient dilution

Time (min)	A1(％)	B1(％)
			0	90	10
0.2	90	10
			0.5	0	100
2	0	100
			2.01	90	10
3	90	10

(5) Experimental results

After transformation of the constructed genomic integrative plasmid into rhodobacter sphaeroides strain NHU5915 (parent strain), strain [ NOLALANC _00703: [ pT334-6> UbiF_TrrnB ] Bota20101 was obtained. The coenzyme Q10 yields of Bota20101 and NHU5915 were tested and compared, and the results indicated that the coenzyme Q10 yield of Bota20101 reached 172% of NHU5915 yield, indicating that insertion of UbiF into the genome of the parent strain producing coenzyme Q10 (e.g., insertion of NOLALANC _00703 locus) was effective in improving the yield of coenzyme Q10 (see table 3 for results).

TABLE 3 enhancement of coenzyme Q10 production by genomic insertion overexpression UniF

The strain Bota20101 is classified and named as rhodobacter sphaeroides (Rhodobacter sphaeroides), and is preserved in China general microbiological culture Collection center (CGMCC, no.3 of West-road 1 of the Korean area North Star of Beijing) in the China general microbiological culture Collection center (China) of 04 th and 01 th of 2024, and the preservation number is CGMCC No.30221.

EXAMPLE 3 enhancement of rhodobacter sphaeroides coenzyme Q10 production by plasmid overexpression of RegA

This example uses either the RegA endogenous promoter or NOLALANC _04400 promoter to overexpress RegA by plasmid and test the effect on coenzyme Q10 production in genetically engineered strain Bota20101 producing coenzyme Q10.

(1) Construction of the target plasmid

The plasmid used in this example was constructed as pBBR1MCS2 (available from Addgene, plasmid # 85168). The linearized vector DNA fragment was amplified using pBBR1MCS2 as PCR template and the following primers:

upstream primer and downstream primer of pBBR1MCS2

F2:GGATCGGTTGTCGAGTAAG(SEQ ID NO:75)

R2:ATTGCGTTGCGCTC(SEQ ID NO:76)

The rest of the procedure is as in example 1. The numbers and information of the constructed plasmids are shown in Table 4.

TABLE 4 plasmid information

Plasmid numbering	Insert carrier	Promoters	Overexpression of genes	Terminator
					pRSA1011	pBBR1MCS2	Endogenous sources	RegA[-775S:275E]	Endogenous sources
pRSA1012	pBBR1MCS2	NOLALANC_04400	RegA	rrnB

Note that: the coordinates within the RegA [ -7755S: 275E ] brackets "[ ]" refer to the upstream and downstream sequences based on the RegA gene. Wherein "S" represents the start position of the coding sequence of the RegA gene and "E" represents the end position of the coding sequence of the RegA gene. Wherein a negative "-" represents upstream and no negative represents downstream. In RegA [ -7755 S:275E ] -775S refers to 775bp (-775) upstream of the start position (S) of the coding sequence of the RegA gene. 275E refers to 275bp (275) downstream of the end position (E) of the coding sequence of the RegA gene. Thus, the whole meaning of the regA [ -775S:275E ] is "a DNA sequence, which starts from 775bp (-775) upstream of the start position (S) of the coding sequence of the regA gene and ends from 275bp (275) downstream of the end position (E) of the coding sequence of the regA gene", and which comprises the endogenous promoter and terminator of the regA gene, the sequence of which is shown as SEQ ID NO: 77. The regA over-expressed by plasmid pRSA is wild-type regA gene, and the sequence is shown as SEQ ID NO. 3.

(2) Conversion of conjugation

As in example 1.

(3) Strain culture

As in example 1.

(4) Coenzyme Q10 assay

As in example 2.

(5) Experimental results

The results of measuring the production of coenzyme Q10 are shown in Table 5. As can be seen from Table 5, the effect of endogenous promoter expressed RegA on increasing the production of coenzyme Q10 was limited and only 8%. Using NOLALANC _04400 promoter, a more than 6-fold increase in coenzyme Q10 production can be obtained. The NOLALANC _04400 promoter is much more effective than the endogenous promoter.

TABLE 5 Effect of overexpression of RegA on coenzyme Q10 production Using plasmids

Therefore, ubiF is inserted into a genome to carry out over-expression and is combined with plasmid to carry out over-expression of RegA, the yield of rhodobacter sphaeroides coenzyme Q10 can be obviously improved, and the obtained strain has good application prospect in industrialized mass production of the coenzyme Q10.

The strain Bota20102 is classified and named as rhodobacter sphaeroides (Rhodobacter sphaeroides), and is preserved in China general microbiological culture Collection center (CGMCC, no.3 of West-road 1 of the Korean area North Star of Beijing) in the China general microbiological culture Collection center (China) of the China, with a preservation number of CGMCC No.30222 in the 04 month 01 of 2024.

While the invention has been described with reference to certain embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims.

Sequence listing

SEQ ID NO. 1Ubif Gene coding sequence (E.coli)

atgacaaatcaaccaacggaaattgccattgtcggcggaggaatggtcggcggcgcactggcgctggggctggcacagcacggattt

gcggtaacggtgatcgagcacgcagaaccagcgccgtttgtcgctgatagccaaccggacgtgcggatctcggcgatcagcgcggctt

cggtatcattgcttaaagggttaggggtctgggatgcagtacaggctatgcgttgccatccttaccgcagactggaaacgtgggagtggg

aaacggcgcatgtggtgtttgacgccgctgaacttaagctaccgctgcttggctatatggtggaaaacactgtcctgcaacaggcgttgtg

gcaggcgctggaagcgcatccgaaagtaacgttacgtgtgccaggctcgctgattgcgctgcatcgccatgatgatcttcaggagctgg

agctgaaaggcggtgaagtgattcgcgcgaagctggtgattggtgccgacggcgcaaattcgcaggtgcggcagatggcgggaattg

gcgttcatgcatggcagtatgcgcagtcgtgcatgttgattagcgtccagtgcgagaacgatcccggcgacagcacctggcagcaattta

ctccggacggaccgcgtgcgtttctgccgttgtttgataactgggcatcgctggtgtggtatgactctccggcgcgtattcgccagttgcag

aatatgaatatggcacagctccaggcggaaatcgcgaagcatttcccgtcgcgtctgggttacgttacaccgcttgccgctggtgcgtttc

cgctgacgcgtcgccatgcgttgcagtacgtgcagccagggcttgcgctggtgggcgatgccgcgcataccatccatccgctggcggg

gcagggagtgaatcttggttatcgtgatgtcgatgccctgattgatgttctggtcaacgcccgcagctacggcgaagcgtgggccagttat

cctgtcctcaagcgttaccagatgcggcgcatggcggataacttcattatgcaaagcggtatggatctgttttatgccggattcagcaataat

ctgccaccactgcgttttatgcgtaatctcgggttaatggcggcggagcgtgctggcgtgttgaaacgtcaggcgctgaaatatgcgttag

ggttgtag

SEQ ID NO. 2Ubif Gene coding amino acid sequence (E.coli)

MTNQPTEIAIVGGGMVGGALALGLAQHGFAVTVIEHAEPAPFVADSQPDVRISAISAA

SVSLLKGLGVWDAVQAMRCHPYRRLETWEWETAHVVFDAAELKLPLLGYMVENTV 。

LQQALWQALEAHPKVTLRVPGSLIALHRHDDLQELELKGGEVIRAKLVIGADGANSQ

VRQMAGIGVHAWQYAQSCMLISVQCENDPGDSTWQQFTPDGPRAFLPLFDNWASLV

WYDSPARIRQLQNMNMAQLQAEIAKHFPSRLGYVTPLAAGAFPLTRRHALQYVQPGL

ALVGDAAHTIHPLAGQGVNLGYRDVDALIDVLVNARSYGEAWASYPVLKRYQMRR

MADNFIMQSGMDLFYAGFSNNLPPLRFMRNLGLMAAERAGVLKRQALKYALGL

SEQ ID NO. 3RegA gene coding sequence

ATGACTGAGGATCTGGTATTCGAACTCGGGGCCGACAGGTCCCTGCTTCTCGTGGA

CGATGACGAGCCCTTCCTGAAGCGGCTTGCCAAGGCGATGGAGAAGCGGGGTTTC

GTTCTCGAAACGGCACAGAGCGTCGCGGAAGGCAAGGCGATCGCCCAGGCGCGCC

CGCCGGCCTATGCAGTGGTGGACCTGCGGCTCGAGGACGGCAACGGGCTCGACGT

GGTCGAGGTGCTGCGCGAGCGCCGGCCCGATTGCCGCATCGTCGTACTGACGGGC

TACGGCGCCATTGCGACGGCGGTCGCTGCGGTGAAGATAGGCGCCACGGACTACC

TGTCCAAGCCCGCCGACGCCAACGAGGTGACGCATGCGCTGCTGGCCAAGGGCGA

GAGCCTGCCGCCGCCGCCGGAAAATCCGATGTCGGCGGACCGCGTGCGATGGGAG

CACATCCAGCGCATCTACGAAATGTGCGACCGCAACGTCTCGGAGACCGCCCGGA

GGCTCAACATGCACCGGCGGACGCTGCAACGCATCCTGGCCAAGCGCAGCCCGCG

CTGA

SEQ ID NO. 4RegA Gene encoding amino acid sequence

MTEDLVFELGADRSLLLVDDDEPFLKRLAKAMEKRGFVLETAQSVAEGKAIAQARPP

AYAVVDLRLEDGNGLDVVEVLRERRPDCRIVVLTGYGAIATAVAAVKIGATDYLSKP

ADANEVTHALLAKGESLPPPPENPMSADRVRWEHIQRIYEMCDRNVSETARRLNMHR

RTLQRILAKRSPR

SEQ ID NO. 5 flagellum matrix MS-ring/cycloprotein gene coding sequence (NOLALANC _ 00703)

ATGGATGGTCGGGCAAGGCAGAAGCCCGTGTCGGAGACCGAAGTGCAGAACCTCA

TCTCAATCTGGAACGATCTCGACGGACGCCGCCGGGCCGTCGTCGTGGGCGCGAC

GGTTGCCATGTTCCTGGCCGTGCTCGGTCTGTCGCAGCTCGCCGGTCAGCCGCCGA

TGGCGCTGCTCTATGCCGGTCTGCAGGGACCGGCGGCGGGCGAGGTCGTGACCGC

GCTCGAGGCGCAGGGCGCGGCCTACGAGGTGAGAGGCGATTCGGTCTATGTCGAG

GCGGCCCGCCGGGACGAGATCCGCATGAAACTCGCGGGCGAGGGGCTGCCCGCCA

CGGGCGCCGCGGGTTACGAGCTCCTCGACGGGCTCTCGGGCTTCGGCACCACGTC

GCAGATGTTCGACGCGGCCTATCTGCGCGCGAAGGAGGGCGAGCTTGCCCGGACA

ATCCTCGCCAGCCCGCAGGTCAAGGCGGCGCGGGTGCATATCGCGCAGCCGGCGG

CCCAGCCGTTCCGGCGGGAGCAGCGCCCGACGGCGAGCGTCACCGTGACCACATC

AGGAGGCCCGGTGACCGCGGATCAGGCGCGGGCCTTCAAACATCTGGTGGCCTCG

GCCGTGGCCGGGCTGCAGCCCGAAGACGTGTCGGTGATCGACAGCGTGATGGGCC

TGATCCGGTCCGGGGACGAGAAGGACAGCGCCACCGCCGCGGGCGAGAGCCGCG

CCGCCGAACTCCGGAGCAACGTCGAGCGGCTGCTCGAGGCGCGGGTCGGGCCGGG

CCGGGCGGTGGTCGAAGTCAGCGTCGATGTCGAGACCGCGCGCGAGTCGATCGTG

GAAAAGAGCTTCGACCCCAAGGGCCGCGTGCCGATCTCGTCGGAAACTCAGGAGA

AATCCAGCAACTCGACCGAACCCTCGCCCGACGTGACGGTGGCGTCGAACCTGCC

GGAAGGCGACGCGAAGGGAGGCACGGGCGGCAAGTCGAGCACCAGCGAAACTCG

CGAGACGGTCAATTTCGAGGTCTCCCAGACCCAACGCGAACTCATGCGCCAGCCG

GGTGCCATCCGGCGGCTCACGGTCGCCGTTCTGGTCGATGGGATCAAAACGGTTGC

GGACGATGGCACCGTGGGCTGGGAACCGCGCCCCGAGGAGGAGCTTGCCGTCCTG

CGCGAACTGGTCGGCTCAGCGGTGGGGCTCGATGAATCGCGTGGCGACAGGCTGA

CCTTGCGCTCGCTGGCCTTCGAGACGCCGCCCGAAGCGGGAACCCTGGCCGAGAC

CGGCTTCCTCAGCCGCCTCGGCGCCTTCGACCTGATGAGCCTGATCCAGATCGGCG

TGCTCGCTCTGGTGGCTCTGATCCTCGGCCTCTTTGTGGTGCGCCCGGTTCTGACGA

CGTCGCCAACGCCGCGGCTGCCGGCGGCCCCGCAGCCCCTGGCGCTCCCGGGTTA

CGAGGGCGGCGAGCAGGCGCTGACCGGCGAGATCGACGACAGCGGCGACCTGCC

GGACTTCCCGATGGTGTCCTCCGCGCCCCTCGACCTCGACGAGGAGGCGGATCCG

GTCGCGCGGCTCCGCCGTCTCATCGAGGAGCGCCAGGCCGAGTCGGTCGAGATCC

TGCGCGGCTGGATGGAACCGCAGCGGGAGGCGCCGTGA

SEQ ID NO. 6 flagellin matrix MS-ring/cyclic protein gene coded amino acid sequence

MDGRARQKPVSETEVQNLISIWNDLDGRRRAVVVGATVAMFLAVLGLSQLAGQPPM

ALLYAGLQGPAAGEVVTALEAQGAAYEVRGDSVYVEAARRDEIRMKLAGEGLPATG

AAGYELLDGLSGFGTTSQMFDAAYLRAKEGELARTILASPQVKAARVHIAQPAAQPFR

REQRPTASVTVTTSGGPVTADQARAFKHLVASAVAGLQPEDVSVIDSVMGLIRSGDEK

DSATAAGESRAAELRSNVERLLEARVGPGRAVVEVSVDVETARESIVEKSFDPKGRVP

ISSETQEKSSNSTEPSPDVTVASNLPEGDAKGGTGGKSSTSETRETVNFEVSQTQRELM

RQPGAIRRLTVAVLVDGIKTVADDGTVGWEPRPEEELAVLRELVGSAVGLDESRGDR

LTLRSLAFETPPEAGTLAETGFLSRLGAFDLMSLIQIGVLALVALILGLFVVRPVLTTSPT

PRLPAAPQPLALPGYEGGEQALTGEIDDSGDLPDFPMVSSAPLDLDEEADPVARLRRLI

EERQAESVEILRGWMEPQREAP

SEQ ID NO. 7 flagellum matrix MS-loop/cyclic protein gene upstream 1000bp nucleotide sequence

AGGAGCGCCAGAAGCGCAAGAGCGCCGCGGGTGCGCGATTTGCGGCTCATGCGTC

GGTCTCCGAGCTTTCGCGCAGGCTGCGGCGGCGGACGAACCGCCTGCGGCGCTCG

GCCTCGATCTCGGGATCCGGCTGCGGATCCGGCGCTCTGGCCGGGATGTCGTGCAT

CGAGGCGAGCATCAGTTCAAGCCGTGCCGCGACCGACTCGGCCCGTTCAGTCAAC

CCATCGAGCGAGCTTGCCGAGTGGGTCGCCGCGGCCCGCGCCTTTTCCAGCGCGCG

CGTCATCTCGTCGACCTGCGCGGAGAGGACCGCTATGGCGCCGCCCATCCCGCCCT

CGAGCGTGGAGAAGCGCTTCAGCCTTTGACCGAGAACGTAGCAGTAGAGCGCCGC

TCCGAAGGTGCCGCCCGCCATCAGGATATTCGCAATGAAATCCACTGCCACCTCCT

AGTTCAGGACGAATTCCGTCACGAGCAGGTCGCGGACGCGGCCCTCACCGGTGAC

GATCTGGATGCGGCGCAGCATCTGCGCCCGCAGCTCGACGAGCGCGGTCGGTTCC

TCCAGCCGCGAGACGGCCACTGCCCGCAGGTAGCCGTTGATGACATCGACGATCC

GCGGCATCAGCATCGTGACCTCGGCCGCATTCGCCTTCGGCACCTCGAGCTGCGCC

GCGAATCTGAGGTGAGAGAGGTCCGACCCGCGGCCAAGGCTGATCACGATCGGGT

CGATCGGCACGAAGGCGATGTCGGGAAGGGCCGCCGGCGCCTCATGTGCGGGCTC

GGCCTCGTGCCCGCCGGACGCGCCCAGAATCAGGCCCGACCAGGTGGCGTAGAAC

CCGCCGCCCCCCAGAACCAGGGCCAGCACGACGCCGAGGATCAGCGGCAGCAGC

GACCGCTTCTTTGTCGTTTCAATCTGATCCGCGCCCGCATCCGCCATGACCGACTCC

TTTCCGTCCGTCGATCTATAGGCCGGATCCCCCTAACCGATTGTTAAGCCACATCC

GTC

SEQ ID NO 8 flagella matrix MS-ring/cycloprotein gene downstream 1000bp nucleotide sequence

TGGCGCTCCGGCTCGAGGTCTTCGAAGTCGCGGCACCGGAACCCGACATTGTCGTGACCGACGCGCTGGCCGTGGAGGAGGGCCGGCAGGCCTCGTACGAGGAAGGCTACCGCGCCGGATGGGAAGATGCGGTGAAGGCCGGTTCCGAGGAGGAGGCCCGGTTGCGGGCCGATCTTGCCAGGAACCTGCAGGCTCTGGGCTTCACCTTCCGCGAGGCGCGCATGCATGTCCTGCGGGGCATCGAGCCGCTGATGGAGGCGCTCGTCGGCCGCATCCTGCCCGACCTGGCGCGGCAGATGCTGGCGCCTGTTGTGCTCGAGACGCTGATGCCTCTGGTGGACGGGATGACCGAGGCGCCTGTCACGCTTCTCGTCCACCCGTCAGCGCGCGCCTCCCTTGAGAGCCTGCTCGACGGCGCCACGGCACTTCCCCTCGTCATCCACGACGAGCCGAGCCTCGACGAGGGGCAGGTGTCGATGCGTCTTGGCGCCACCGAGACGCAGGTCGACCTCAACCGGGCAGTGGCCGACATCGCGGCCGCCGTGCGCAGCTATTTCGATCTCACGCAACAGGAGGTCCGGCATGGATGAGGCCGACCAGAATCCCCTTACCCAGGTCCCGATCGAGATCACTATCTCGGTGGGCCGCGCCCGCCCTCCCGTTCGCGAACTGCTCCGCCTCCAGCGGGACGCCGTGCTGCCCCTCGACCGTCGGGTGGACGATCCGGTGGAGCTCTATGTCGGCGATCGGTTGATCGGACGCGGCGAACTGACGGAGCTCGAGGGCGAGCAGGCGGGCCAACTGGCTGTCCGGCTCACCGAGGTCGCGAACCTGCGCGGCGGATTGTGATGCGCCTCCTGCTGGTCCTGCTTGTCGTGCTCGGCGGCGTCGGCCCGGCCATGGCGCAGAGCCTCTCGCTCGACCTCGGCGACGGCGGCTCGCTCACCGCGCGCAGTGTGCAACTCATCGCCGTCATCACGCTCCTGAGCCTCGTGCCGGC

SEQ ID NO. 9T 334-36 promoter

GTCTCTCTCCTGCCGTCCGCCGGATCGCCTGCCCGGCTGTGTCCTGTCCGCCCGTACGGGTCCCCGTGCGCAGCGCCCCGGCCCATCCTGCGCCGAAGGCCGGGGCGACCGGAAGCCCCGCGCAGAGTGGGCAGTCCGAAGGCCGGCTTTGCCTGCGCGGGCTATGCCCGCAGCTATTCCTGCGCCCCTCCGCCGGGATGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGCGATGTCGCCCATGGGCCGCAGCGTCGCCCCTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO. 10T 334-6 promoter

GTCTCTCTCCTGCCGTCCGCCGGATCGCCTGCCCGGCTGTGTCCTGTCCGCCCGCACGGGTCCCCGTGCGCAGCGCCCCGGCCCATCCTGCGCCGAAGGCCGGGGCGACCGGAAGCCCCGCGCAGAGTGGGCAGTCCGAAGGCCGGCTTTGCCTGCGCGGGCTATGCCCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAGAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 11 NOLALANC_02773 promoter

CGGCGTGGGCCAGCGGCGCGGAGGCGAGGAAGAAGGCACAGATGATCGAGGTGAGGCGGGTCATGCTCAGGTCCTTGCGACGGCAGGCAGAAGGTGGCACGCCACGCCTCCCGCGCCAAGGCGGAGCTTGCATTTTGATGCATCCCCCCCGTTGACAGGTGCCGGACGCTCTCATATCTCTCGCGTCGTTGGCACTCGCCTTGGGTGAGTGCTAATCACATTCAACCTGGAACCTAGGGAGTGTTCTCAG

SEQ ID NO. 12T 167-31 promoter

GCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCATCGCCCTTCGGCTTGACGCACCCCTGCCGGGCGACAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 13 NOLALANC_02328 promoter

GCCTCTCATACGCGTCGCAATGCCGCGCTCGGCGTCGGTTTTCGACATGGTGCGCGCGCGGAGCGACGCCGCCCCTCCGGGCGGGGCCGACCGTCCCCCGTGCGCCACAATCGCAGGCGCTTCAACGGGGTCGGGGGGCGCCCGGGACCCTGCAGCGATGCGGCCCGCGGCGCGCGCCCTGCGACGATCCGACCTTGCGGCGGCAAGCTCGCGCGCATACTCTTGGAGCAACCATGAATGGAGGGCGACG

SEQ ID NO. 14T 334-7 promoter

GTCTCTCTCCTGCCGTCCGCCGGATCGCCTGCCCGGCTGTGTCCTGTCCGCCCGCACGGGTCCCCGTGCGCAGCGCCCCGGCCCATCCTGCGCCGAAGGCCGGGGCGACCGGAAGCCCCGCGCAGAGTGGGCAGTCCGAAGGCCGGCTTTGCCTGCGCGGGCCATGCCCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTCCGGCTTGACGCACCCCCTGCCGGGCGGCGACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 15 NOLALANC_02693 promoter

ACGCCCCGACCGCGCAGGCGGCGGAGGAGGACGCCCAGGACGAGGCCGGGCAGCAGGAGGCGGAGAAGGAGGCGGTAGAGCGTCACGCGGGTCCATCCCTTAGGGCGCGAGACTTGTCGCCGGGAACCGCGCTTCGTGCAAGCCGGGCTGCGACCAAACTGGCAGATTAGAATTGATCTAAGTCAAAGGTATCGGGTTGGAACCGTTCTAGCCTCCGGTCAATGCGTATCGATTGCACAGGAGACCGACCSEQ ID NO:16 T167-27 Promoter CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGACGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

17T 167-29 promoter of SEQ ID NO

CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGACGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCCGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO. 18T 334-26 promoter

GTCTCTCTCCTGCCGTCCGCCGGATCGCCTGCCCGGCTGTGTCCTGTCCGCCCGCACGGGTCCCCGTGCGCAGCGCCCCGGCCCATCCTGCGCCGAAGGCCGGGGCGACCGGAAGCCCCGCGCAGAGTGGGCAGTCCGAAGGCCGGCTTTGCCTGCGCGGGCTATGCCCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGCCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO. 19T 167-15 promoter

CCGCAGCTATTCCTGCGGCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGACGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 20 NOLALANC_02906 promoter

AGAGTGCGCGATCAAGGGCAATGTCTCGCGCAACGGCCGGATCTATCACATGCCCGGACAGGCCGACTATGCGCGCACCGTGATCTCGGGCCCCGAGGAGCAGTGGTTCTGCACCGCGGCCGAGGCCGAGTCTGCGGGCTTCCGGCCGGCGAAGCGCTGACATTGAGAAGCGCTGACATTGATACGCATCAAGGCGGCGAAAGAATCTGTGACGTAGCTTTTCCCTAACGAAACCAAGAGGGAGCACGGC

SEQ ID NO 21 NOLALANC_02132 promoter

CCTTTCCGTTCCGTTGCCGCCGGGCCGTCCGCAATTCCGCTTGCACTGAACCACGGCGGCACGTTGTCGCGCGAATCGTCTCCGCCCGGCGCTTTTCCCCGATCTTATCGCCGCTTGCCGACCGATGCGCCCCCCTTTGGCATGAGAAACTTGCCCGCGCGCTTGATCTTGCCGGTCGGGCTGGTATGCAGCCCAGAAGCCGGGCGCGCAAGAAGTCGCACCCCGGGGGGATCCAAGTCGGAGACATGCC

SEQ ID NO. 22T 167-18 promoter

CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTTGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO. 23T 167-22 promoter

CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCGCCCCCTGCCGGGCGGCAGCAGTGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 24 NOLALANC_01387 promoter

CTGCCCCGATTGGCGGGCCTCTGGCCTGCCGCGGCCGAGCCTTCTCGCCGCGCCGTCGGTCTGGCGCGGCGAGGCGCTGGTGGCCGCACCTCTCGCGGGACTGTCGAACGGCTGGACGGCCGTCGCGGATCCCCTGCAAAATGGGGATTTTCTTTCTCTACTATCGCATTGAACGGCCCGCGCTTATGTCTATCTTATGCCGCAATGACCTGCGGGTGGGCCCGCGGAGAGACTCTGCAAGGAGACATTT

SEQ ID NO 25 NOLALANC_00239 promoter

GCAGCGCCGAGCGTCCATCGCATCTGCATGAAGTCTCCCGAATGTCCTGATCCCGTTCTAGCCCTGCCTGCCTCGGCCGTTCCAATCAAGGTTTTGCGAGGCTCGTCCGCGAAAAAGCCGCAGAAGGGCTCTGGATTTTCGGCATACATCGGTATACAAGACCGCCATCTCCCTTGCGCGACCAGGCCCGGTGGGGCAAAACGCGCGCGGAGCACAAGCCCAACCCTCGCCACGGCGCCAGGAGGACCGC

26T 334-13 promoter of SEQ ID NO

GTCTCTCTCCTGCCGTCCGCCGGATCGCCTGCCTGGCTGTGTCCTGTCCGCCCGCACGGGTCCCCGTGCGCAGCGCCCCGGCCCATCCTGCGCCGAAGGCCGGGGCGACCGGAAGCCCCGCGCAGAGTGGGCAGTCCGAAGGCCGGCTTTGCCTGCGCGGGCTATGCCCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTCCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGCGAGGGCTGGTCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 27 NOLALANC_01665 promoter

AGGGGGCGTTCCTCCTCCTCGGTCTCGAAATCCACGGTTCCATCCCGCATGCGCTGCCTCCGACTCGCAAGCACTCCTCACGCGCAACGCCCCGACGGGCGTCCTGTTCCGGCGTGGGGGCAGGTTAATCACCCTGCGCCGGACTCATCTATTGCGATTTTCGGGCAGGGGCGACGGAAGCGGCGGGGCGTGGCGTTTGAGATGCATGAACCAGGCAGCAACGGCTGCCGCGACCCCAGGAGAGACCGCC

SEQ ID NO 28 NOLALANC_04177 promoter

ACCTCGTAGGCGCCGAGCGCCCGGTGCTCTTCGAGGAGGATCTCGAGCGTGCGCCGGCGCACCGGCGTCAGGCGGGCCCCGGCCGCGGCCGTGGCCTCCTCGGCATGGGCGAGGATCGAGGCCGCACAACGGCTGTGATCGTGCGGCGCGAATGCCGCGGTGCCTCCGGTATCGGCGGTCATCCTCTCCCCCATTGACGCGTTATGATATAACGTAATACATGTGCGCCCATCTCAAGGAAAGGCAGACC

SEQ ID NO 29 NOLALANC_02710 promoter

ATAGGCCTCCGTCTCGACGATCCGTCCGCCCACGCCGCGCACCTGCAGATGCGCCCCCAGAAGATCGACCGCTACCGCGGGCGCCTCGCGCGCGAAACAGGCTGCCTGCCCCTCCATCTGCACCCCTTCCTGCGCGGTCTGACGCCGGTGTGACATCCGCCTCCCGGAACATTCGTCCGCCGCTGCCGTTCCTTCCGCAACCCCGCACAGCCTGCGGGACCGGTGATGTTCACGAATGGAGAGACTGTCG

SEQ ID NO. 30T 167-40 promoter

CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGCGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO. 31T 167-11 promoter

CCGCAGCTATTCCTGCGGCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGCGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 32 NOLALANC_02369 promoter

AGTGGCTGACGCTCGAGGACAAGGCCAGCAAAGGTTAGCCGATGTCCTGTCCGCCTTGCGCTGGAATTGGCGCATGGCACTCTCCGCATATCACCTCCTCGTGAGCGGATCTGGCGAACCCCCCTTCCGAGAAATAGGACATGTGCACGGAAGGCGTCGTCATCCCCGGCGACGCGAGACCGTCAACGAGAGATCCGGCATCCCCGCAAAAGCAGCAGTGCGGACGTCGGAAGCTTAATTGGAGACAGAG

33T 167-19 promoter of SEQ ID NO

CCGCAGCTATTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCGCGAAAGCGTGAGAGGGGGCGGCGTGATGTCGCCCACGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCCTGCCGGGCGGCAACAGCGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 34 NOLALANC_01610 promoter

TGGGGCATCTGCCGATCCAGAGCTACCTCTGAGGCTGGTGCCCGAGGGGGTCGCTGGCAGACCCTCCCCCAAAATATTCGAGGATGAGCATAAGTCCGCTTTCGCCTGTAATGATTGGCAAATCTGGAGGTGCTCGAGGGTCGTGCGGCAAGCTGCCGCAGTTGAAACCTTGCCTGATCGCGACGGATTTCGGGGGTGTCACCGTGTGACGTTCGCCCGCCCGTTGTGGGCCTGTAGCAAGGAAAGACCA

SEQ ID NO 35 NOLALANC_00630 promoter

GCTCCACACGGCGGCATGTCCCGTGTGCCAGCTTTCCAGAAGCCCGCCGCGCCAGAGCTCCACCATCCTCGCCGGATCCGTCATGACATCCCCTCACAGCTGCGCGATTTTGTGCCCAAGAGGCCTTTTGTCCGGCGCAATGCTTGCGCTAAGAAGCGTTATCGGGTTGAAACGGCCTGCGCTACTCAAAAAGGTCGCCACAAGGCGGCGTGGGGCGGGCAGAGGCACTGACAGCTGGAGGCTGTGAACG

SEQ ID NO 36 NOLALANC_00965 promoter

AGGCGGTCCGCACCTCGTCCCAGTTTTCGAATCCCATGTCTGCCCCCGTCCGAATCGCGTGGAAGTGATGTGCGCCTGCGCACAGCATACTGGCCGAGCCGGGAACTTACACAGCTAACGCTCAGAGCCTACATCCTCCTCACGAGCCGCGGAGATCGCCCCAAGCCGGAGACGGAGGGAGCGCAAGCCACGGCCCGCAAAGGATAATCGGCCGATCCGCAGGGGCGGACGGCCCAGCAGGAGACATGAG

SEQ ID NO 37 NOLALANC_01799 promoter

TTCGTGCTGGACGAGGCCATGCGCCGCCGTCTGGCCGAACTGAACCCGGAGGCGAGCGTCCGCATGGCCGAGCGTCTTCTGGAAGCGAGTGCCCGTAACTATTGGCAGCCCGACGCGGAGACTTTGGCCGCGTTGCAGGGGGCTGCGGACGAGTTGGAAGACCGTCTCGAGGGGATTGCCGCCGAGTGAAGCGGACGGGACGGCAAGCCCTGCGGGACGCAGGGATGGCTTTAGCAATGGGGGGCCTCCG

SEQ ID NO 38 NOLALANC_02415 promoter

CTCCTCAACGGGCAATGGCCCGGTGGTTCCCACGCGATCCCGAAGCAGGCTGCTGCCTTGGACCATAGGCAGCATATCGGCGGCTTTTTGCCTATGAAATAGGCCAATATCGGCGGATTTCCGCATGATGCGCCACCGGCCTAATTCCTCGCTTTGAGAGGGGAAACCAACCCCGCCGCGACCCGTTGGACATGCAGCAGGCCAACACGGCTCTGTCCAATTAGTCTCCCAAGGCGAAAGGATCGCTACA

SEQ ID NO 39 NOLALANC_01312 promoter

CCGCGAACTGCGCCGCCCCGGCGATCACGATGATCGAGAAGCCCATCACCTCGACGATGTCGAGCCCCGCCTCGGTCCCCACGACACCGAAGAGAAGCCCGAAAGGCGCG

ATCACCAGCGAGAAGGGCGTGGCGGCCCAGAGACCGCGCAGGAAGGCCGAGCGT

CGGGTGGACATGGCAGAACCTTGTGGTTGTCGGACGCAACTCTTGCGCTTAGGGCT

TCTCGTCGGACGATGCAATCGGAGGCGGCC

SEQ ID NO 40 NOLALANC_00842 promoter

AGTGCCGAACTCGTCCGCGTGATAGACACGCTTCCCTGACGAGGCCGCAGCCTGC

GGAGGGACCGCGGACCCGCGGTCTTTGACATGATTTTCACGCCCGCGTGACGCTTG

CAGCCCCAAATCCCCCCCCCTATATACGCCCGGTAGCCGGAAGGGGCGCCACCCC

GGATGGCAGGAATTCGGGATCGGGGATCGGACGCCGACAGTCGGGCCTGACCCCC

ACAACATCGCCGCAAGAGAGGTGCACAAC

SEQ ID NO 41 NOLALANC_01129 promoter

TCTCGGGCCCGCTGCGGGAGAGGAGCGCGACGCGCGCGCCCGCCGCCGCGAAGAC

GCGCACCGCGGCCGCTCCGATCCCGCGGCTGGCCCCGGTGATTGCCACGACCTTGC

CCTGCATGTTGCCCATTGCGCTGCTCCTTCCCTGGCCTTCTGACAAACGTGTTAGCC

GATCCTTGCATTCGACGGAAAGGCTTGACCCTCCAGAACCCACGGAAGAATGTGG

GCCGATTATCCGCGTTGGGGAAGGCAT

SEQ ID NO 42 NOLALANC_01863 promoter

ACGGGGGGGATCGGAAGTTGCCTCCCTCTCCCTGACAGGGGCCTGAAGCGGCAGT

CACGAAAGGGCGAGGCCGAGCGACGGCGGGCCGGGGCGGCCATTCGGTGCGCCG

CGCGTCCGGCAGTCGGAGGCGGTGAGTGCGAGCCTCCGCCGGCACGCATGCGGTG

ATGAGGGGAGCCCCGTGCCGCTCCCGCATGTCCGACGGAAGAGGGGCGGCCCGAC

GTTCAAGCGCCATGTAACGAGGAGTTTGGTC

SEQ ID NO 43 NOLALANC_03935 promoter

CGCGTCCGGCCCAGGCGTCCCGCAGGCGGCAGGGGCGGTGCTGGCTCAGGTGCTG

CGCCAGACCGGGGGCAATGTCTCCGAAGCCGCGCGACGGCTGGGCGTCAATCGCT

CGACAATTCATCGCCAGATCCGCCGAATGGGCGTGACACGCTGAAAAGGCGGGCG

TTCGCGGTCATCCTGCACGCAGGTGCTTGACTTTGGAAACTGGTTGGTTTCCTAAG

CAGGCCACCGCTGCCGATGGAGTCTGCCC

SEQ ID NO 44 NOLALANC_00723 promoter

GCCATCAGGTCACGGAGCCTTATGATTTCACCCGCGCCATTGCGGTCGCCGCGCGG

GAATTCGCGCCCGACGCCTTCGTCGTGCTCGGCCCCGGCACCACGCTGGGCGGGG

CCGTGGCGCAGAGCTTGATCCTCGCCGGCTGGCGCGGGATGAAGGACCGCAAGGA

TTTCCAGACCCGTCAGGCCGAGAGCCCCCTGCTGATCGCATTGGGGCGCGAGGAC

CAGCGCGGGCGCGTCACAGGAGGACCCAG

SEQ ID NO 45 NOLALANC_03984 promoter

CGGTGGCGCAGCCCGGCAGTTCCGGCGGCAACCGCGGGGGCGGGCTGCTCGGGCT

CATCATGCCCCGGCCGCCGGGGCTTGCGACCGAGACCGACGCGGCGTCTGGCCCC

ACGGTCTGGGTCCTGCGCGACGGAAAGCCCGACCGGGTGGCGGTCGAGACCGGCG

AGACCGACGGGCGGCGGACCGAGATCCGCGGCGACGCGCTCGAGGAGGGGGATC

CGGTCATCACCGATCAGCGCGAGGCCGGAGC

SEQ ID NO 46 NOLALANC_03002 promoter

GCGGCCATGGCGCGCTGGCTGAGCGGCACGGCCGCCTCGAAGGCTTCGCTTTCATC

GAACTGGCTCATGCCCGCCAATCTAGCCTGCCTCTGCCCGGAGTTAAAGAAGTGTT

CCGCAATCGTTCACAAAACCTGTTCCGGCAGGCGCCGGATCGGTCAGATTTTCTTC

CCGGACGTCGCCCTTGAGCTATTGCGCCGCACCGCGGCACCACTAGAGTTCGCGCA

CCAGCCTGATGCCCGGAGGTGAACGC

SEQ ID NO. 47T 167-42 promoter

CAGCTACTCCTGCGCCCCTCCGCCGGGAAGCGCCCGCCACGAAAGCGTGAGAGAG

GGCGGCGTGATGTCGCCCATGGGCCGCAGCGTCGCCCTTCGGCTTGACGCACCCCC

TGCCGGGCGGCAACGGCGAGGGCTGGCCCAGCTTGCATCTGAAGGAGGCACC

SEQ ID NO 48 NOLALANC_00782 promoter

ACGAGGAGGGCCGCCCGGTCTGGCGCCATGCGGGCGAGGTGCGGCTCACGATGCG

CGACTTTTCCGACGGCTGGATCGACGGCTACATCGCCCGCAACTGGGAGGCCGTG

CGCCATTCGGTCGGAGGCTACCACCTCGAAGGCGAGGGAGTGCGGCTGTTCTCGG

CCGTCGAGGGGGATTATTTCACCGTCCTCGGCCTGCCGCTCCTGCCGCTCTTGAAT

TATCTGGGTCAAAGAGGCTTCATTCCGAC

SEQ ID NO 49 NOLALANC_01836 promoter

AAGGCGCGGTCGAGACGGTGCCGCAGGCGCTGCTCGATCAGCTGAAGGAAGGCG

GCCGGATCGGCTGCATCTTCATGGAAGGTCCGCTCGGCGTGGCCCGCATCGGATAC

AAGGTGGACGGGGCGATCACCTGGCGCCCGGTTTTCAACGCCAGTGCCCCGGTCC

TGCCGGGGTTCGAGGTGAAGCGCGATTTTGCGTTGTAAGGCAAGTCTTTCAGAAAC

AAGAGATTGACGGGACAGTGGAGAGGGCA

SEQ ID NO 50 NOLALANC_00054 promoter

AAGGGAATGGGCTTCGGGAAACTGGCCGCCCCCTTGCGCACAGCCCTTGCCGGGC

GCACGGTGACGCCCAGCGTCTACGACATGATGCTTGTCATCGGTCGGGACGAGAC

GATTGCGCGGCTGGAGGATGCCGCGGCGGCCTGAGCCGCCGCCCTCTCCTTCTGTC

CGAGCCCCGGTCCGTCGCGGAACCGGAAGCTATCGGCCCCGCGCCATCCCGTGCG

GAGCCGGCCCGAACGAGAGAGGGAGTAGC

SEQ ID NO 51 NOLALANC_02229 promoter

CGTTGGCAATTGAAAATCCATTTCATCGCGGCCTTTTGCCGCCGGTGGCACCGGCT

GTGCGGTAGGAGGTGCACAACAAGTCCGAACGGCCCCCAGGGCCCGAAGGCGCA

AGAACGAAGGGAGGAGGCCCGGCGGCACGGTGCGCCGCGGGGCAGTGACCTGAA

CCTTTGCCGTGATCCGCAGACCCAGGGCGGCCCCCGGCCGCATTCCGTGGGAGCA

GGCTCCCACGCCATAGATGGAGGCTCATGGC

SEQ ID NO 52 NOLALANC_04429 promoter

AGAAGCCCTCATGCTTCCCCGTCCGAAATCCCGGCGCCCGAGGGCGCGGGGAGCG

CGTCCTGCATGGGTCCGGCCCCGCACCGGGCCGGAATGCCCGGGCCGCGCTGATC

GGCTGAGGCGCGCGGCCTGAAGGGACCTGCCCAACGGGCGGCCTTTCGGGCGCAG

GACGGCTGTCCGGGTCTCCCCGGCCGCCCGGCCGTGGCCGGATCCGCTCGATCCCC

TTTTTCAGTCCTTTTACGGAGGTTTCCTC

SEQ ID NO 53 NOLALANC_03679 promoter

GAGGCGCAGCCCGAGCGTGCGGGTATAGAAGTCGAGGTTGCGGCGGGCCGGGCC

GGAGATGGCGGTGACATGGTGCAGGCCTTGGGTCATTTCGATCCTCCTCGGGGCCG

GGGCGGCCCCTTCGCGTTGACAGGAAGATGGGGAAGGCCGGCCGCCGCGTCACCC

CACTTCCCCTTGCATCCATGCAAGACTAACCTTGCACCGATGGCGCGCTTCTTGCG

CCGGAGCACAGACCGTGAGGGCATTCTCC

SEQ ID NO 54 NOLALANC_02260 promoter

AGCAGGGCGTGTCGGTCTGGCTCAAGGCCGACCTCGATGTGCTCTGGCACCGGGT

GCGGCACAAGGCCACCCGGCCGCTTTTGCGCACGCCCAACCCGCGCGAGACGCTG

CGCGCGCTTCTCGAGGCGCGCGATCCGGTCTATGCGCAGGCGGACCTCGCGGTGG

AGAGCGGCGAGGGAACGGTGGAACAGATGGCGGTGCGGGTGCGCGAGGCGCTGG

CCACCCGCCCCGATGTGCTGGAGACGGACGA

SEQ ID NO 55 NOLALANC_03111 promoter

GGCCTTTCCGCCAATGCCGATCGCCGACAGGATTATTCCGCGAACCGGAACGAAG

GGCCGCGCCTACCGTCGCGGGATCTGTACCCCCAAACCGCCCGGATCTGTGTCTGT

CGTCCCCGATCCTCTTCGCGCTAGGAGCAGGGTCAGACACCTGATCGACCCTGCTT

CCCGGTCGATCGATCCCCCCTTTTGCACCTAACGCGCGGACGACCGCACCCGGCGG

CCTCCCGCCCGGCGGATGGAGACGTGA

SEQ ID NO 56 NOLALANC_00059 promoter

CTGCTGCTGCAGATCGCGGCTCCCTCGACGCAGGTTCTCGATCAGGTGCTCGACGA

GGTGGGCGCCATGACCGGGGTCCGCAGCTCGGAAAGCCTCATCCATCTCTCGACC

CGGATCGACCGGGCCGTGTGACGGGCAGTTACCGCGCCTGCCGGAGATGCGACCG

CGACCGCGGCGGCCCGAGCGCGATGAGGCTTGGCCTCGGGCCGCGGCTTGGATAG

ACCGGGCGCAACGAACTGCGAGGCCCGAC

SEQ ID NO 57 NOLALANC_01751 promoter

CCCAAGGCCTACCGCGATTTCGCCTTACTCTTCGCCATTTTTGCGGAGACTGGTAA

GTGGACGCGTAAGGTGTGGTATGTATTCCTGGCGATGCTTCGCCTTTGGTATGCAC

GGGTGCAACAGTGGCGGATCGATAATTTATATCTCTTTGAGAAAGTGTGGCCTGCG

TGACGGCGAGATGGGATAGAAGGTCGCTTCGGCCGGCGCCTGCGGGGGCGGCTTG

GACAGCATCGGCACGGGCGGACTGAGG

SEQ ID NO 58 NOLALANC_03340 promoter

GGAGGAGGGTTTCGAGGGTGGCGCTGTGTTGGGGCATAGGAAGCATGGTAATTGG

TTAGCTTGCTATCGATATGGGGCGAATTTTGCCGGGACAAGCCCGGACAAGGCAG

ACATGCACGGGGTGCAAGGGTGCTTGGAGGGTTACGCCGACGCGCGTCCTGCGTC

ACCGCGATCCACATCCGGTTGACGCGGCGCATCGCGGGGGCGGCGCCCCTCGGTC

AAGCTGGGGCTCCCGAACGGAGGAACTGCC

SEQ ID NO 59 NOLALANC_03519 promoter

CAAAGCCGCGCACCTTCAGGCCATCGGCCAAGGGCGGGCTGCCGCTCACAGTTGC

GGGTGCAGTCGCGGAGAACGGTCTCCTTCACCGTTTTCGCTTTTCATCCTTTGGACC

TTGCGAGCCTCGAGAACCGGCGCGTGCGGGGGTGACACACCGTGGGCTCCCCCTC

ACCGCGCCGGCTGCCACAGGTGAGTTGATGTAGGATATGTTATAATATATCCTTCT

GGCACTTCGACGCTACAGGAGCCCGCC

SEQ ID NO 60 NOLALANC_04400 promoter

CCGCACGCTCCTGTCCTACAATCCGCTCACCCATGTGATCGCGAAGATGCGTCAGG

GCTTCTTCGCGACCTACGACGCCCGGCTGGTCGACCCGCTCTATGCCTATTCCGTA

AGCCTCGTCTGTCTCTTCTTCGGCATGCTGTTGCTGAACCGTCATCACCGGATGCTT

CTGAATGAAGGTGCCTGACCGCGTCCCTTTGCGTCTACCTCAGGTAGTTGTTAGGT

ATCGGCCATTCATTTGGAGACGGAC

SEQ ID NO 61 NOLALANC_03841 promoter

GCGCCTTCCCGGCGAACCGGTGGCCTCTTGCCCGGAGCATCCCCGTCACAGCGGCG

GGCCCGCGCCGGATTTCCACCGGCTTCCCGATTCTCCGCCCGAAGGCGGCACCTGA

CCCCTGCCACAAAGCAGAACCGGCGCGCCCGGGCAACCGTCCGCGGTCCAGCCGC

CCCTCAGAGCCTTGAAGGCCGCGGCGGCGCGCCCCATAGTCGCGCGAGGCGTCCG

GAAGGGGCGCGCTCCGCAGGCAGGTCTC

SEQ ID NO 62 NOLALANC_03925 promoter

GCAGGGGCACTTGCATCATGTCGCATGGGTTCTACAGCACCAGTTCGTCTTCCGGC

AGTATCAACTAAAGGTTGTAACCCGTCTATACTTTAGCGATAGAGTTTCATTAAGA

TACAATCAAGCGGGATTGTTCCTTCGAGACCGGAACACCGTCAAAAGTGTGGGAT

ATGGTCATTTTGACACAGTCCCTGCTTGCGTTGTCGCTTGCCCAGCCTAGTTTTTCG

CCAGTTTCGAAACGAGTGTTCCGATC

SEQ ID NO 63 NOLALANC_03961 promoter

TGCAGGTTTTTTCGGCGCCGGGAGAAGCCGCCAGCCGCGGTGCGCAAGCGACCCG

GTTATGGCCGGAGCCCCCGGTTGCAGAGCCTCGCCCGCAGTGGCGAGGAGCCCGC

GGGCCTTGCGTCCCGGGCAGGAGCTGGCAGGTCCCGGGCTGCGACAGCCCCGCAC

GGGAGTGCCGCGGCGGCGCCATCGGCGGTTGCCGCGCAGGCACCGGCCCGCCATT

GTATCCCTGCCTCCCTCTTACGGACCTTCC

SEQ ID NO 64 NOLALANC_01082 promoter

CGGCATGGCGATCCGCGACAATGTCTTGACCGACTGGGTGAAGGCCAACGGCATG

GGCAATATCGACCCCGAGCGCATGGCCCGCGCCATCGAACAGACGAAATCCGTCT

ACGAGTTCCAGAACGAACCCGACGCCGCCCTCTACTTCGATTCGCAGTGGCTGCCC

GCCGACGGCAGCCTGACCTTCGAGTGACGTCCGAGTGACCGGAGCGCGGGCCATG

GGCCCCGCCCCCTCCCCGACGGGAAGACC

SEQ ID NO 65 NOLALANC_01569 promoter

AGGCGGTGCTGGCCGCCCGTCGCCGGGGTCTCGTCTGAGACCTCCTGCCGGCTCGGCTGCGTTGCGAAAGAACGGGGCGGTTGCGAATGGCCGGGCTTCGCCGCTGGCCTGTCCGGGTTAAGGTTTCGGGAAAGGATCCGGGGCAGGATGCTGGGGTGACTCGCGCCTGCGGTGCGGGTCGTCCCCGGGAAGGAGACCTGCAGGTGACGAGTGCCGTTCCGACCCGACCCGGCCCGCGACGGGTGCGGCG

SEQ ID NO 66 NOLALANC_02822 promoter

GCGCCTCGCCTGCCTGCACGAGGAGATCGCGCAGGGGCTGGGGCTGGCCAACGACAGCCCGAACGCGCGGCCCTCGATCTTCAACGACGACGAGGAATTCGCGCTTCTGACCACGCAGGACGAGCTGATGCTGAAGATCCTCTACGACCGGCGCCTGAAGCCCGGCATGACGGTCGAGGAAGCGCGGCCCATCGTCCAGACGATCGCCTACGAGCTGCTCGGCGGCGAAAGCTGAAGGCACGGCCGGGCG

SEQ ID NO. 67 pK18mobsacB upstream primer F

TGCCGCAAGCACTC

SEQ ID NO. 68 pK18mobsacB downstream primer R

ATTGCGTTGCGCTC

SEQ ID NO. 69 pBBR1MCS2 upstream primer F1

TCATCCCAGGTGGCACTTTTCGGGG

70 PBBR1MCS2 downstream primer R1

ATGTCAGCTACTGGGCTATCTGGAC

The nucleotide sequence of SEQ ID NO 71 encoding DASHER GFP

atgaccgcactaacagaaggagctaaactattcgaaaaggagattccttacattacagaattagagggtgatgtcgaaggaatgaaattcattatcaagggcgagggtactggtgacgctactaccggtacgattaaagcaaagtacatctgtacaacaggtgaccttcctgttccgtgggctactctggtgagcactttgtcttatggagttcaatgttttgctaaatacccttcgcacattaaagactttttcaaaagtgcaatgcctgagggctatactcaggagagaacaatatctttcgaaggagatggtgtgtataagactagggctatggtcacgtatgaaagaggatccatctacaatagagtaactttaactggtgaaaacttcaaaaaggacggtcacatccttagaaagaatgttgcctttcaatgcccaccatccatcttgtacattttgccagacacagttaacaatggtatcagagttgagtttaaccaagcttatgacatagagggtgtcaccgaaaagttggttacaaaatgttcacagatgaatcgtcccctggcaggatcagctgccgtccatatcccacgttaccatcatatcacttatcataccaagctgtccaaagatcgtgatgagagaagggatcacatgtgtttggttgaagtggtaaaggccgtggatttggatacttaccaaggttga

The amino acid sequence of SEQ ID NO. 72 Dasher GFP

MTALTEGAKLFEKEIPYITELEGDVEGMKFIIKGEGTGDATTGTIKAKYICTTGDLPVPWATLVSTLSYGVQCFAKYPSHIKDFFKSAMPEGYTQERTISFEGDGVYKTRAMVTYERGSIYNRVTLTGENFKKDGHILRKNVAFQCPPSILYILPDTVNNGIRVEFNQAYDIEGVTEKLVTKCSQMNRPLAGSAAVHIPRYHHITYHTKLSKDRDERRDHMCLVEVVKAVDLDTYQG

SEQ ID NO. 73 rrnB terminator (E.coli)

ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc

SEQ ID NO. 74 Tac promoter

cacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaatTTGACAATTAATCATCGGCTCGTATAATGtgtggaattgtgagcggataacaa

SEQ ID NO. 75 pBBR1MCS2 upstream primer F2

GGATCGGTTGTCGAGTAAG

SEQ ID NO. 76 pBBR1MCS2 downstream primer R2

ATTGCGTTGCGCTC

SEQ ID NO. 77 RegA [ -7755 S:275E ] nucleotide sequence

GCTCATGCCACTTTGCCTGTATCGGAACCTTGTTAGGTTGCCGTGCCCGCGGGATCAATCGGCAACCGCAAGCAGGAGGATCATCTGATGACGAAGCTCTATGCCGGGGTCGCGGCGGCGGCCATCGCAGCACTTCTTGCGGGCTCGGCGGCGTGGGTCTTCCTCGGCAGATCCGAAGAGCGGTTCGCGGGGTGCGGCGCCAATCAGGTGGCGGGCGGCGCCATCGGCGGGCCCTTCACGCTGGTCGATCAGGAGGGCCGCACCGTGACCGACCGCG

AGGTTCTGGCAAAGCCCTCGCTCGTCTATTTCGGCTATACCTTCTGCCCCGACGTCT

GCCCCTTCGACATGGCGCGCAATGCGCAGGCCGTGGACATCCTGACGGAATGGGG

CATCGAGGTCACGCCGGTCTTCATCTCGATCGATCCCAAGCGCGACACGCCCGAAC

AGCTGAAGTTCTTCGCCGAGGCGATCCATCCCGACACGATCGCGCTGACCGGCAC

CGAGGCTCAGGTAAAGGCCGCCTCACAGGCCTACAAGACCTTCTACCGGGTGCAG

GAGTCCGACGACGACTATTACCTCATCGACCATTCGACCTTCACCTACTTCATGCT

GCCCGGCACGGGCTTCGTCGACTTCTTCAAGCGCGAAGACACGCCCGAACAGATC

GCCGAGCGAATTTCGTGCTTCGCGAATGACAGCCATGTGTCAACTTCCTTTGACGct

cgagCGCAGAAGTCCTATCAAGCATCCAGGGGAAAGCAGATGGGCGACAATCATGA

CTGAGGATCTGGTATTCGAACTCGGGGCCGACAGGTCCCTGCTTCTCGTGGACGAT

GACGAGCCCTTCCTGAAGCGGCTTGCCAAGGCGATGGAGAAGCGGGGTTTCGTTC

TCGAAACGGCACAGAGCGTCGCGGAAGGCAAGGCGATCGCCCAGGCGCGCCCGC

CGGCCTATGCAGTGGTGGACCTGCGGCTCGAGGACGGCAACGGGCTCGACGTGGT

CGAGGTGCTGCGCGAGCGCCGGCCCGATTGCCGCATCGTCGTACTGACGGGCTAC

GGCGCCATTGCGACGGCGGTCGCTGCGGTGAAGATAGGCGCCACGGACTACCTGT

CCAAGCCCGCCGACGCCAACGAGGTGACGCATGCGCTGCTGGCCAAGGGCGAGAG

CCTGCCGCCGCCGCCGGAAAATCCGATGTCGGCGGACCGCGTGCGATGGGAGCAC

ATCCAGCGCATCTACGAAATGTGCGACCGCAACGTCTCGGAGACCGCCCGGAGGC

TCAACATGCACCGGCGGACGCTGCAACGCATCCTGGCCAAGCGCAGCCCGCGCTG

ACCTATTCGCCTCGGCAGAAGAAAGGGCGGCGGCCGAGGGCCTGCCGCCTTTTTC

ATTGGCAGGCGGAGGGGAACTTGGATCGGCAAAGGGTATTCGACTACCCGAAAGT

GCAATAGATCCCTAAAATTCCTCGATGGACAGAGGGCTAAATGTCGAATTTCTCAA

GCCACCCGATTGACCTTTTTACGAAACCGTAAAATGAATTGGGGAATGGTTCGTTT

TAACGGGAAGAATGAATGGATATCGATCTCGACTCCATGAGCCTCAAGGAACTG

SEQ ID NO. 78 primer A

TGAAGACGGGATCCATCACATAC

SEQ ID NO. 79 primer B

GAGCGTGCTGGCGTGTTGA

Claims (15)

1. A rhodobacter sphaeroides engineering bacterium modified to express UbiF gene and RegA gene, wherein:

the UbiF gene is integrated into the genome of the rhodobacter sphaeroides engineering bacterium, and the RegA gene is expressed through a plasmid vector.

2. The rhodobacter sphaeroides engineering bacterium of claim 1, wherein the UbiF gene encodes a UbiF protein having 2-octenyl-3-methyl-6-methoxy-1, 4-benzoquinone hydroxylase activity.

3. The rhodobacter sphaeroides engineering bacterium of claim 1 or 2, wherein the UbiF gene is UbiF gene of escherichia coli.

4. The rhodobacter sphaeroides engineered strain of any of claims 1-3, wherein the UbiF gene is expressed starting from a strong promoter having a relative strength in the rhodobacter sphaeroides engineered strain of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115% or at least 120% relative to the Tac promoter.

5. The rhodobacter sphaeroides engineered strain of any of claims 1 to 4, wherein the UbiF gene is expressed from a promoter selected from T334-6(SEQ ID NO:10)、T334-36(SEQ ID NO:9)、NOLALANC_02773(SEQ ID NO:11)、T167-31(SEQ ID NO:12)、NOLALANC_02328(SEQ ID NO:13)、T334-7(SEQ ID NO:14)、NOLALANC_02693(SEQ ID NO:15)、T167-27(SEQ ID NO:16)、T167-29(SEQ ID NO:17) or T334-26 (SEQ ID NO: 18); preferably, the UbiF gene is expressed starting from the T334-6 promoter (SEQ ID NO: 10).

6. The rhodobacter sphaeroides engineered strain of any of claims 1 to 5, wherein the RegA gene encodes a RegA protein having transcriptional regulatory activity.

7. The rhodobacter sphaeroides engineering bacterium of any of claims 1 to 6, wherein the RegA gene is a RegA gene of rhodobacter sphaeroides.

8. The rhodobacter sphaeroides engineered strain of any of claims 1-7, wherein the RegA gene is expressed starting from a weak promoter having a relative strength in the rhodobacter sphaeroides engineered strain of at most 40%, at most 30%, at most 25%, at most 20%, at most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2% or at most 1% relative to the Tac promoter.

9. The rhodobacter sphaeroides engineering bacterium of any of claims 1 to 8, wherein the RegA gene is expressed from a promoter selected from NOLALANC_04400(SEQ ID NO:60)、NOLALANC_03111(SEQ ID NO:55)、NOLALANC_00059(SEQ ID NO:56)、NOLALANC_01751(SEQ ID NO:57)、NOLALANC_03340(SEQ ID NO:58)、NOLALANC_03519(SEQ ID NO:59)、NOLALANC_03841(SEQ ID NO:61)、NOLALANC_03925(SEQ ID NO:62)、NOLALANC_03961(SEQ ID NO:63)、NOLALANC_01082(SEQ ID NO:64)、NOLALANC_01569(SEQ ID NO:65) or NOLALANC _02822 (SEQ ID NO: 66); preferably, the RegA gene is expressed initially from the NOLALANC _04400 promoter (SEQ ID NO: 60).

10. The rhodobacter sphaeroides engineered strain of any of claims 1 to 9, wherein the UbiF gene is integrated into the flagelliform matrix MS-loop/cyclic protein gene locus in the rhodobacter sphaeroides engineered strain genome; preferably, the UbiF gene is integrated into or within 1000bp upstream or downstream of the flagelliforme MS-loop/cycloprotein gene; more preferably, the UbiF gene replaces a fragment of 1000bp upstream to 1000bp downstream of the flagelliforme MS-loop/cycloprotein gene.

11. The rhodobacter sphaeroides engineering bacterium of any of claims 1 to 10, wherein the plasmid vector for expressing the RegA gene is a pBBR1MCS2 vector.

12. Rhodobacter sphaeroides engineering bacteria with a preservation number of CGMCC No.30222.

13. A method of making coenzyme Q10, the method comprising:

(1) Culturing the rhodobacter sphaeroides engineered strain of any of claims 1 to 12 under conditions suitable for production of coenzyme Q10;

(2) And recovering coenzyme Q10 from the culture of the rhodobacter sphaeroides engineering bacteria.

14. Use of an rhodobacter sphaeroides engineering bacterium according to any of claims 1 to 12 for the production of coenzyme Q10.

15. A method of preparing an rhodobacter sphaeroides engineered strain of any of claims 1 to 12, the method comprising:

(1) Integrating UbiF genes into the genome of a parent rhodobacter sphaeroides, thereby obtaining an intermediate rhodobacter sphaeroides strain; and

(2) And (3) transforming a plasmid vector containing the regA gene into the rhodobacter sphaeroides intermediate strain obtained in the step (1), thereby obtaining the rhodobacter sphaeroides engineering strain.