CN118957003A - Construction and application of genetically engineered bacteria for producing NMN using basic carbon source fermentation - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to construction and application of genetically engineered bacteria for producing Nicotinamide Mononucleotide (NMN). The invention provides construction and application of genetically engineered bacteria for producing NMN by utilizing basic carbon source fermentation. The yeast engineering bacteria capable of producing NMN are obtained by knocking out catabolism related genes and introducing exogenous synthesis paths into the yeast engineering bacteria by a gene recombination technology, and the synthesis of NMN is promoted by increasing the gene dosage and precursor supply. The yeast engineering bacteria have the characteristics of low metabolic background, strong heterologous expression capability, capability of utilizing basic carbon sources to ferment and produce NMN and the like, and provide a new idea for industrial production of NMN.

Description

Construction and application of genetically engineered bacteria for producing NMN by utilizing basic carbon source fermentation

Technical Field

The invention belongs to the technical field of biology, and particularly relates to construction and application of genetically engineered bacteria for producing Nicotinamide Mononucleotide (NMN).

Background

Nicotinamide Mononucleotide (NMN) is a naturally occurring substance in the human body that can be converted into the important coenzyme NAD ⁺ associated with the metabolism of the human body in cells. Studies have demonstrated that intracellular NAD ⁺ levels decrease with age and exogenous supplementation of NMN is believed to have the effect of enhancing human cellular function.

The current NMN raw material production method mainly comprises the following three steps:

one is a chemical synthesis method. The method has simple process and low cost, is mainly completed through the reaction between the compounds, and needs a plurality of organic solvents as medium. The use of NMN raw materials produced by chemical synthesis is not recommended as new food raw materials are generally not approved in china for the chemical synthesis process to obtain purer compounds.

And secondly, a biological enzyme catalysis method. The process does not rely on microorganisms, but rather is produced by means of enzyme-catalyzed synthesis by means of specific engineered enzymes. The NMN raw material produced by the method has high purity, the production mode is relatively mild, and the whole process is slow and durable. The biological enzyme catalysis method is a current production mode, but the threshold is higher, and the price of several key catalytic enzymes is not enough, which accounts for about 80% of the cost of the whole production process.

Thirdly, fermentation method. Whole cell fermentation production is carried out by means of microorganisms. The method has the advantages of mild generation process, environment friendliness, no introduction of heavy metal ions and the like, but low production efficiency, complex fermentation process, high raw material cost and limitation of high-efficiency industrial scale production. In some expression systems, due to the limitation of expression efficiency, cell collection and concentration are required after the expression of the enzyme, and then a substrate is added for catalytic synthesis (a production method combining fermentation and enzyme catalysis), so that the production process is complex and is easy to pollute.

The microbial fermentation method used at present also needs to add substrates such as nicotinamide, nicotinic acid, nicotinamide ribose and the like, and the addition of the substrates increases the cost of NMN production. In addition, the existing microbial fermentation mainly uses escherichia coli as a produced chassis cell, and NMN produced by the chassis strain has safety problems such as endotoxin and the like in the application of the food industry, so that the difficulty and the cost of a purification process are increased. Pichia pastoris is used as a GARS strain and is widely applied to the production of proteins and small molecular compounds at present.

In summary, there is also a need in the art to enrich the production methods of NMN, optimize the production process of NMN, so as to improve the production efficiency and reduce the production cost.

Disclosure of Invention

The invention aims to provide construction and application of genetically engineered bacteria for producing NMN by taking a basic carbon source as a substrate through fermentation.

In a first aspect of the invention, a method for producing Nicotinamide Mononucleotide (NMN) is provided, wherein whole cells are used for de novo biosynthesis of NMN using a basic carbon source as a substrate. The method comprises the following steps: the PncA, pncC, nadR, ushA gene is knocked out from the yeast engineering bacteria. Further, a yeast engineering bacterium for synthesizing nicotinamide mononucleotide from the head is provided, and the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA, pncC, nadR, ushA gene in the strain; the method uses a basic carbon source and a nitrogen source, and does not need to additionally add a substrate to generate nicotinamide mononucleotide product.

Preferably, after obtaining the yeast engineering bacteria, the strain is directly cultivated and the production of the product is performed without cell collection and/or concentration.

Preferably, the method further comprises one or more of the following steps:

(1) Obtaining a yeast engineering bacterium I, wherein the yeast engineering bacterium I is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3;

(2) Obtaining yeast engineering bacteria II, wherein the yeast engineering bacteria II are transformed with expression cassettes of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA gene in the strain;

(3) Obtaining a yeast engineering bacterium III, wherein the yeast engineering bacterium III is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock out the PncA, pncC genes in the strain;

(4) Obtaining yeast engineering bacteria IV, wherein the yeast engineering bacteria IV are transformed with expression cassettes of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and the PncA, pncC, nadR gene was knocked out in the strain.

The PhzE gene has an amino acid sequence shown in SEQ ID NO. 1, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which has more than 70% of identity with the sequence of SEQ ID NO. 1 and codes for a functional protein;

the PhzD gene has an amino acid sequence shown in SEQ ID NO.2, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an isofunctional protein and has more than 70% of identity with the sequence of SEQ ID NO. 1;

The PnuC gene has an amino acid sequence shown in SEQ ID NO. 3, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 3;

The NadC gene has an amino acid sequence shown in SEQ ID NO. 4, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 4;

The NbaC gene has an amino acid sequence shown in SEQ ID NO. 5, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 5;

The DhbX gene has an amino acid sequence shown in SEQ ID NO. 6, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 6;

The NadE gene has an amino acid sequence shown in SEQ ID NO. 7, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 7;

The PRS gene has an amino acid sequence shown in SEQ ID NO. 8, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for an homologous functional protein and has more than 70% of identity with the sequence of SEQ ID NO. 8;

The ARO3 gene has an amino acid sequence shown in SEQ ID NO. 9, a coding nucleotide sequence thereof, or a degenerate sequence thereof, or a nucleotide sequence which codes for a homofunctional protein and has more than 70% of identity with the sequence of SEQ ID NO. 9.

PncA, pncC, nadR, ushA is the name of the knocked-out gene, and the core conserved sequence .PncA(Nicotinamidase,3.5.1.19)、PncC(nicotinamide-nucleotide amidase,3.5.1.42)、NadR(DNA-binding transcriptional repressor,2.7.1.22)、UshA(UDP-sugar hydrolase,3.1.3.5) thereof needs to be knocked out, and can refer to the base sequences of XM_002492537.1, XM_002491470.1, XM_002491781.1 and XM_002491567.1 in Genebank databases with IDs respectively.

Preferably, the yeast engineering is Pichia pastoris (Pichia pastoris), more preferably the yeast engineering is Pichia pastoris GS115 or Δku70. For example, in a preferred embodiment of the invention, the yeast engineering is pichia pastoris GS115 or Δku70.

Specifically, the method for producing NMN comprises the following steps:

(1) Providing a yeast engineering bacterium, wherein the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3;

(2) Providing a yeast engineering bacterium, wherein the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA (Nicotinamidase, 3.5.1.19) gene in the strain;

(3) Providing a yeast engineering bacterium, wherein the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock out PncA, pncC (nicominamide-nucleotide amidase, 3.5.1.42) genes in the strain;

(4) Providing a yeast engineering bacterium, wherein the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA, pncC, nadR (DNA-binding transcriptional repressor, 2.7.1.22) gene in the strain;

(5) Providing a yeast engineering bacterium, wherein the yeast engineering bacterium is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and the PncA, pncC, nadR, ushA (UDP-sugar hydrolase, 3.1.3.5) gene was knocked out in the strain.

Culturing the yeast engineering bacteria of (1), (2), (3), (4) and (5), except a basic carbon source and a nitrogen source, generating nicotinamide mononucleotide product without adding extra substrate, and harvesting the product.

Preferably, after culturing the above yeast engineering bacteria, the strain is directly cultured and the production of the product is performed without performing the step of cell collection and/or concentration.

Preferably, the expression cassette further comprises a promoter, including but not limited to: a constitutive promoter or a methanol inducible promoter; such constitutive promoters include, but are not limited to, GAP promoters; such methanol inducible promoters include, but are not limited to, the AOX1 promoter.

Preferably, the culture medium used for culturing the yeast engineering bacteria comprises (but is not limited to): YPD, YPG, YPM, YPE medium. YPD or YEPD (Yeast Extract Peptone Dextrose Medium), also known as yeast extract peptone glucose medium, was added to agar, also known as yeast extract peptone glucose (YPD) agar medium. YPG medium is a commonly used Yeast medium, and its components mainly include Yeast Extract (Yeast Extract), peptone (Peptone) and glycerol (glycerol). YPE medium is a commonly used Yeast medium, and its components mainly include Yeast Extract (Yeast Extract), peptone (Peptone) and ethanol (ethanol). YPM medium is a commonly used Yeast medium, and its components mainly include Yeast Extract (Yeast Extract), peptone (Peptone) and methanol (methanol).

Preferably, glucose is supplemented during the culturing process; preferably, glucose is added at intervals or fed-batch; more preferably, the interval supplement includes: glucose (m/v) is added to the liquid medium every 12-36 h, more preferably 16-30 h (e.g. 15, 18, 20, 22, 24, 28, 32 h), more preferably 1.8-3.5% (e.g. 1.5%, 2%, 2.5%, 3%, 4%) during fermentation.

Preferably, the pH of the medium is from 5.8 to 7.6, preferably from 6.5 to 7.5, more preferably from 6.8 to 7.3. In a preferred embodiment of the present invention, pH7.0 is used.

Preferably, the cultivation temperature is 25 to 37 ℃, more preferably 30.+ -. 2 ℃ (e.g, 28, 29, 30, 31, 32 ℃).

Preferably, the rotation speed during cultivation is 150 r/min-250 r/min, more preferably 200+ -50 r/min (such as 200+ -40 r/min, 200+ -30 r/min, 200+ -20 r/min or 200+ -10 r/min).

Preferably, the incubation time is 1.5 to 10 days, more preferably 3 to 7 days (e.g., 2, 3, 4,5, 6, 8 days).

In a second aspect, the invention provides a yeast engineering bacterium for synthesizing nicotinamide mononucleotide from scratch, wherein PncA, pncC, nadR, ushA genes are knocked out from the yeast engineering bacterium. Further, the yeast engineering bacteria for synthesizing nicotinamide mononucleotide from the head comprises the expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3.

In one or more embodiments, the PhzE gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO.1, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more) to the sequence shown as SEQ ID NO. 1.

In one or more embodiments, the PhzD gene has a nucleotide sequence corresponding to the polypeptide shown in SEQ ID NO.2, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more) to the sequence shown in SEQ ID NO. 1.

In one or more embodiments, the PnuC gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO.3, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, yet more preferably 95% or more, yet more preferably 97% or more) homology to the sequence shown as SEQ ID NO.3.

In one or more embodiments, the NadC gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO.4, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, yet more preferably 95% or more, yet more preferably 97% or more) homology to the sequence shown as SEQ ID NO. 4.

In one or more embodiments, the NbaC gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO.5, or a degenerate sequence thereof, or a nucleotide coding sequence of a functional protein having greater than 70% (preferably greater than 80%, more preferably greater than 90%, more preferably greater than 93%, more preferably greater than 95%, more preferably greater than 97%) homology to the sequence shown as SEQ ID NO. 5.

In one or more embodiments, the DhbX gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO. 6, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, yet more preferably 95% or more, yet more preferably 97% or more) homology to the sequence shown as SEQ ID NO. 6.

In one or more embodiments, the NadE gene has a nucleotide sequence corresponding to the polypeptide shown as SEQ ID NO. 7, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, yet more preferably 95% or more, yet more preferably 97% or more) homology to the sequence shown as SEQ ID NO. 7.

In one or more embodiments, the PRS gene has a nucleotide sequence corresponding to the polypeptide shown in SEQ ID NO. 8, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more) to the sequence shown in SEQ ID NO. 8.

In one or more embodiments, the ARO3 gene has a nucleotide sequence corresponding to the polypeptide shown in SEQ ID NO. 9, or a degenerate sequence thereof, or a nucleotide coding sequence of a homologous functional protein having a homology of 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more) to the sequence shown in SEQ ID NO. 9.

In a third aspect, the invention provides an application of the yeast engineering bacteria in producing nicotinamide mononucleotide by taking a basic carbon source as a substrate.

The invention provides an application of a gene combination, wherein the gene combination comprises the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS, ARO3; knocking out PncA, pncC, nadR, ushA genes;

The gene combination is used for transforming yeast engineering bacteria and producing nicotinamide mononucleotide by taking a basic carbon source as a substrate. The invention surprisingly discovers that compared with the fermentation engineering strain, the fermentation yield of the NMN of the delta 4 strain is obviously improved by knocking PncA, pncC, nadR, ushA out the delta 4 strain. The Δ4-1 strain includes the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS, ARO3 and the like; and knockout PncA, pncC, nadR, ushA, namely, phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS, ARO3 gene is increased on the basis of delta 4 strain, and the yield is further improved.

In a fourth aspect, the invention provides a kit for producing nicotinamide mononucleotide, wherein the kit comprises the yeast engineering bacteria for de novo synthesis of nicotinamide mononucleotide.

Preferably, a medium for culturing the yeast engineering bacteria; preferred include (but are not limited to): YPD, YPG, YPM, YPE medium; glucose; or ethanol.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Terminology

As used herein, the term "whole cell de novo synthesis of an end product" refers to the production process of the product being performed simultaneously with the fermentation process, or being closely linked back and forth; it is different from the process of cell collection and/or concentration (fermentation broth) followed by enzyme-catalyzed substrate and product production (a combined fermentation and enzyme-catalyzed production method) after fermentation.

As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system comprising all the necessary elements necessary for expression of a polypeptide of interest, typically including the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; optionally signal peptide coding sequences and the like; these elements are operatively connected.

As used herein, the terms "operably linked" or "operably linked" refer to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "expression construct" refers to a recombinant DNA molecule that comprises the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector.

As used herein, the term "exogenous" or "heterologous" refers to a relationship between two or more nucleic acid or protein sequences from different sources, or a relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, the nucleic acid is exogenous to the host cell. The particular sequence is "exogenous" to the cell or organism into which it is inserted.

Gene and expression system thereof

In the invention, the efficient production of NMN in the yeast engineering bacteria by utilizing the basic carbon source is realized by introducing exogenous genes into the yeast engineering bacteria and combining an intracellular system of the yeast cells. In a preferred mode, pichia pastoris is used as a chassis cell, and the efficient fermentation synthesis of NMN by the yeast cell is realized through metabolic engineering of the strain.

In the present invention, the introduced foreign gene includes ：PhzE(SEQ ID NO:1),PhzD(SEQ ID NO:2),PnuC(SEQ ID NO:3),NadC(SEQ ID NO:4),NbaC(SEQ ID NO:5),DhbX(SEQ ID NO:6),NadE(SEQ ID NO:7),PRS(SEQ ID NO:8),ARO3(SEQ ID NO:9).

In the present invention, the above-mentioned gene may be naturally occurring, for example, it may be isolated or purified from an autotrophic plant or a microorganism. In addition, the gene may be artificially prepared, for example, the gene may be obtained according to a conventional genetic engineering recombination technique, or the gene may be obtained by an artificial synthesis method.

The nucleotide sequence of the above gene may be identical to the corresponding sequences shown in SEQ ID NOS.1 to 9, or may be degenerate variants thereof. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes a protein having the same function, but differs from the corresponding sequence set forth in SEQ ID NOs 1-9.

The gene may include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The invention also relates to variants of the gene, which encode polypeptides differing in amino acid sequence from their corresponding wild-type polypeptides, and which are fragments, analogs or derivatives of the wild-type polypeptides. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the corresponding wild-type polypeptide.

It should be understood that while each gene of the present invention is preferably obtained from Saccharomyces cerevisiae and Pichia pastoris, other genes obtained from other microorganisms that are highly homologous (e.g., have more than 70%, such as 80%, 90%, 95%, or even 98% sequence identity) to the corresponding genes in Saccharomyces cerevisiae and Pichia pastoris are also within the contemplation of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

The full-length sequence of each gene of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences. When the sequence is longer, two or more PCR amplifications can be performed, and then the amplified fragments are spliced together in the correct order.

The invention also relates to vectors comprising said polynucleotides, and host cells genetically engineered with said vectors.

In the present invention, the sequence of each gene may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

The sequences of the genes can be respectively inserted into recombinant expression vectors, and a plurality of recombinant expression vectors co-transform host cells; the expression cassettes of multiple genes can also be inserted into the same recombinant expression vector in a tandem manner and transferred into host cells. The recombinant expression vector may further comprise an expression control sequence operably linked to the sequence of the gene to facilitate expression of the protein. It will be appreciated that recombinant expression vectors may be conveniently constructed after the technical context of the present invention is understood by those skilled in the art. The recombinant expression vectors obtained are also encompassed by the present invention.

In the expression control sequence or the expression cassette, inducible or constitutive promoters can be used according to different needs, and the inducible promoters can realize more controllable protein expression and compound production, thereby being beneficial to industrial application.

As a preferred mode of the present invention, there is provided an expression vector (expression construct) comprising an expression cassette of the following genes: phzE, phzD, dhbX; also provided is an expression vector (expression construct) comprising an expression cassette for the following genes: nabC, nadC; and also provides an expression vector (expression construct) comprising an expression cassette of the following genes: nadE, pnuC, PRS; also provided is an expression vector (expression construct) comprising an expression cassette for the following genes: ARO3.

The construction of expression vectors (expression constructs) is now a technique familiar to the person skilled in the art. Thus, after knowing the desired selected gene, the person skilled in the art is readily able to make the establishment of expression constructs. The gene sequences may be inserted into different expression constructs (e.g., expression vectors) or into the same expression construct, provided that the polypeptide encoded by the gene is efficiently expressed and active upon transfer into a cell. As a preferred mode of the invention, the expression vectors are pGAPZB, pPIC 3.5K and pAG32.

Vectors comprising the appropriate gene sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein. In the present invention, the host cell is preferably a yeast engineering bacterium, more preferably pichia pastoris, such as pichia pastoris Δku70, GS115. Of these, the most preferred Pichia is Δku70.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained may be cultured by a conventional method, and the medium used in the culture may be a yeast medium well known in the art. The culture is carried out under conditions suitable for the growth of yeast cells.

The recombinant pichia pastoris strain obtained by the gene recombination technology has the characteristics of low metabolic background, strong heterologous expression capability, capability of synthesizing the end product by whole cells, easiness in separating the end product, few byproducts and the like, and can solve the problems existing in the synthesis of the traditional biological and chemical methods to a great extent, thereby providing a new idea for industrially producing NMN medicaments.

Method for synthesizing NMN

The invention discloses a method for synthesizing and producing NMN by using pichia pastoris. The method comprises the following steps: the 9 genes (PhzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO 3) were transformed into yeast engineering bacteria to produce NMN. The specific method is shown in figure 3, a carbon source and the like generate precursor chorismate of an NMN synthesis pathway through metabolism, then the chorismate synthesizes NMN through catalysis of enzymes in a heterologous pathway, and finally the generated NMN is transported to the outside of cells through PnuC proteins; the whole reaction process is that whole cells synthesize the final product from the head.

The fermentation culture of recombinant yeast engineering bacteria can be carried out by adopting a yeast fermentation method known in the art, and one preferable method is as follows: in liquid YPD culture at 30deg.C at 200r/min, recombinant bacteria were cultured to logarithmic phase, and the collected cells 1OD were inoculated into YPD medium containing nicotinamide, and cultured at 30deg.C at 200r/min for 48h. 2% glucose was added to the liquid medium every 24h during fermentation.

After the fermentation product is obtained, NMN may be extracted from the fermentation product using techniques known in the art. Fluorescence can be used to analyze the product to determine the amount of compound and product desired.

The invention uses recombinant pichia pastoris to produce NMN, which not only solves the problems of difficult transfer of substrate into cells, unstable enzyme method and the like in the biological fermentation method, but also avoids the adverse factors of more byproducts, difficult purification, large environmental pollution and the like in the chemical synthesis method, and opens up a new way for producing NMN. Therefore, the recombinant pichia pastoris strain provided by the invention has the capacity of producing NMN at low cost.

The invention realizes the technical breakthrough of synthesizing NMN from whole cells by taking the recombinant yeast as a substrate and provides a new way for producing NMN.

In the description of the present invention, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element. It should be noted that in this document, relational terms such as "first," "second," and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

The recombinant pichia pastoris strain obtained by the gene recombination technology has the characteristics of low metabolic background, strong heterologous expression capability, capability of synthesizing the end product by whole cells, easiness in separating the end product, few byproducts and the like, and can solve the problems existing in the synthesis of the traditional biological and chemical methods to a great extent, thereby providing a new idea for industrially producing NMN medicaments.

Drawings

In order to more clearly illustrate the technical solutions of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that, for some embodiments of the present application, each drawing in the following description can be further obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 NMN production by fermentation of different metabolically engineered strains. _Δ 4 is a knockout catabolic pathway strain, _Δ 4-1 is a heterologous de novo metabolic pathway expression strain, and the fermentation experiments were performed in shake flasks.

FIG. 2, Δ4-1, NMN production by fermentation in a 3L reactor using a basal carbon source as a substrate.

FIG. 3, schematic representation of the synthetic pathway for NMN production by carbon source metabolism. Carbon sources and the like are metabolized to generate precursor branched acid of an NMN synthesis pathway, then the branched acid synthesizes NMN through catalysis of enzymes in a heterologous pathway, and finally the generated NMN is transported to the outside of cells through PnuC proteins; the whole reaction process is that whole cells synthesize the final product from the head.

FIGS. 4, Δ4 and Δ4-1 are schematic diagrams of the preparation process. Fig. 4 (a) shows the preparation process of Δ4, and fig. 4 (B) shows the preparation process of Δ4-1.

Detailed Description

Through intensive research, the inventor introduces exogenous genes into yeast engineering strains through a gene recombination technology to obtain yeast engineering strains capable of producing NMN by using a basic carbon source as a substrate for fermentation. The yeast engineering bacteria prepared by the invention has the characteristics of low metabolic background, strong heterologous expression capability, capability of synthesizing a final product from the head of whole cells, easiness in separating the final product, few byproducts and the like, and provides a new idea for industrial production of NMN.

Pichia pastoris is used as an FDA approved GRAS strain, can be applied to the fields of medicines, foods and the like, and has the advantage of high safety. The recombinant yeast engineering bacteria established by the invention have the advantages of high fermentation density, high expression quantity and strict regulation and control; meanwhile, the production method of the invention has simple operation, high efficiency and high enzyme expression, and the exogenous gene is correctly translated and processed and modified after translation.

The invention will be further illustrated with reference to specific examples. It is to 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 methods, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.

Material

The plasmid construction method uses a seamless cloning kit from Norwegian biotechnology company.

The tool enzymes used were all purchased from TaKaRa Biolabs (Dai, china), and specific reaction conditions and methods used were referred to the commercial specifications.

The following commercial plasmids and strains were used for gene cloning and protein expression: plasmid pGAPZB, E.coli Top10, were purchased from Invitrogen. The (PhzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO 3) genes were synthesized by Jin Weizhi Biotechnology Inc. and constructed into pGAPZB vectors, respectively, to give plasmid pGAP-PnuC,pGAP-PhzE,pGAP-PhzD,pGAP-NbaC,pGAP-NadC,pGAP-DhbX,pGAP-PRS,pGAP-ARO3.Δku70、pDPFg1、pDAg2、pPIC3.5K-PFLDup-gRNA1、pPIC3.5K-PAOX1up-gRNA2, see Liu Q,Shi X,Song L,Liu H,Zhou X,Wang Q,Zhang Y,Cai M.CRISPR-Cas9-mediated genomic multilociintegration in Pichia pastoris.Microbial Cell Factories.2019,18:144.

Culture medium

YPD liquid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract 10.0g/L;

YPD solid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract 10.0g/L, agar 20.0g/L;

YPE liquid medium: ethanol 10.0g/L, peptone 20.0g/L, yeast extract 10.0g/L;

YND medium: 20.0g/L glucose, 0.67% YNB, and 20.0g/L agar.

When preparing the culture medium, glucose is autoclaved for 20min at 115 ℃, and methanol is added when in use. The other components were autoclaved at 121℃for 20min.

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 construction of expression plasmid

1. Construction of PhzE, phzD, dhbX Gene expression plasmid

The recombinant plasmid obtained by ligating PhzE expression cassette (amplification product contains GAP promoter) to pDAg vector fragment was Ag2-PhzE.

The recombinant plasmid obtained by inserting the PhzD expression cassette (amplified product contains GAP promoter) into SpeI cleavage plasmid Ag2-PhzE is Ag2-PhzE-PhzD.

The recombinant plasmid obtained by inserting DhbX expression cassette (amplified product contains GAP promoter) into BamHI enzyme cut plasmid Ag2-PhzE-PhzD is Ag2-PhzE-PhzD-DhbX.

2. Construction of NadE, PRS Gene expression plasmid

NadE expression cassette (amplification product contains GAP promoter); the recombinant plasmid obtained by ligating pDFg to the vector fragment was Fg1-NadE.

The recombinant plasmid obtained by inserting the PRS expression cassette (amplification product contains GAP promoter) into SpeI cleavage plasmid Fg1-NadE was Fg1-NadE-PRS.

3. Construction of PnuC, ARO3 Gene expression plasmid

The 5 'and 3' flanking regions (about 1000 bp) of PNSI4 were amplified from the GS115 genome and then the two fragments were cloned into the pUC18 vector to generate the PNSI plasmid.

The recombinant plasmid obtained by inserting PnuC expression cassette (amplification product contains GAP promoter) into PNSI plasmid was PNSI-PnuC.

The recombinant plasmid obtained by inserting the ARO3 expression cassette (amplified product contains GAP promoter) into BamHI cleavage plasmid PNSI4-PnuC was PNSI4-PnuC-ARO3.

4. Construction of pUC18-PncA plasmid

Fragments of the 5 'and 3' flanking regions (about 1000 bp) of PncA were amplified using the GS115 genome as a template, and then cloned into the pUC18 vector to generate the pUC18-PncA plasmid.

5. Construction of pUC18-PncC plasmid

Fragments of the 5 'and 3' flanking regions (about 1000 bp) of PncC were amplified using the GS115 genome as a template, and then cloned into the pUC18 vector to generate the pUC18-PncC plasmid.

6. PUC18-NadR plasmid construction

Fragments of 5 'and 3' flanking regions (about 1000 bp) of NadR were amplified using the GS115 genome as a template, and then cloned into pUC18 vector to generate pUC18-NadR plasmid.

7. Construction of UshA-NbaC-NadC plasmid

Fragments of 5 'and 3' flanking regions (about 1000 bp) of UshA were amplified using the GS115 genome as a template, and then cloned into pUC18 vector to generate pUC18-UshA plasmid.

The recombinant plasmid obtained by inserting NbaC expression cassette (amplification product contains GAP promoter) into pUC18-UshA plasmid was UshA-NbaC.

The recombinant plasmid obtained by inserting NadC expression cassette (amplification product contains GAP promoter) into BamHI cleavage plasmid UshA-NbaC was UshA-NbaC-NadC.

EXAMPLE 2 construction of Pichia electrotransport and Strain

1. Delta 4 Strain construction

The pUC18-PncA plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, the fragments are transformed into delta ku70 together with plasmid PncA-gRNA1, YND plates are coated for screening transformants, and correct transformants with gene deletion are screened to obtain the delta ku70 delta PncA strain. The pUC18-PncC plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, the fragments are transformed into delta ku70Delta PncA together with plasmid PncC-gRNA1, YND plates are coated for screening transformants, and correct transformants with the genes deleted are screened to obtain the delta ku70Delta PncA Delta PncC strain. The pUC18-NadR plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, the fragments are transformed into delta ku70Delta PncA Delta PncC together with the plasmid NadR-gRNA1, YND plates are coated for screening transformants, and correct transformants with the genes deleted are screened to obtain the delta ku70Delta PncA Delta PncC DeltaNadR strain. The pUC18-UshA plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, and the fragments are transformed into delta ku70Delta PncA Delta PncC DeltaNadR together with plasmid UshA-gRNA1, and YND plates are coated for screening transformants, so that correct transformants with the genes deleted are screened to obtain delta 4 strains.

2. Construction of Δ4-1 Strain

The UshA-NbaC-NadC plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, and the fragments are transformed into delta ku70delta PncA delta PncC delta NadR together with plasmid UshA-gRNA1, and YND plates are coated for screening transformants, so that correct transformants with gene deletion are screened to obtain the delta 4-bd strain.

The Ag2-PhzE-PhzD-DhbX plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain a fragment, the fragment is transformed into delta 4-bd together with a plasmid pPICC 3.5K-AOX1up-gRNA2, YND plates are coated for screening transformants, and correct transformants with gene deletion are screened to obtain the delta 4-bd-edx strain.

Fg1-NadE-PRS plasmid template, using primer pair pPUP-F/pPDO-R to amplify to obtain fragment, converting delta 4-bd-edx together with plasmid pPICC 3.5K-PFLDup-gRNA1, coating YND plate to make screening of transformant, screening correct transformant with gene deletion so as to obtain delta 4-bd-edx-ep strain.

The PNSI-PnuC-ARO 3 plasmid template is amplified by using a primer pair pPUP-F/pPDO-R to obtain fragments, the fragments are transformed into delta 4-bd-edx-ep together with plasmid PNSI-gRNA, YND plates are coated for screening transformants, and correct transformants with gene deletion are screened to obtain the delta 4-1 strain.

Example 3 recombinant engineering bacteria fermentation and product identification

1. Recombinant engineering bacterium shake flask fermentation process and quantitative product analysis

In liquid YPD culture at 30deg.C and rotation speed of 200r/min, recombinant bacteria were cultured to logarithmic phase, and after the collected bacteria were resuspended 1OD/mL, they were inoculated into 250mL conical flasks (OD value was yeast concentrate unit, 1OD was about 5X10 ⁷ yeast cells) containing 100mL of YPD liquid medium pH 7.0 phosphate buffer pair. OD value was measured by ultraviolet spectrophotometer at 600nm wavelength, and cultured at 30℃and rotation speed 200r/min for 48 hours. 2% (m/v) glucose was added to the liquid medium every 24h during fermentation.

2. Identification of recombinant strain fermentation products

After fermentation culture, 1mL of the fermentation broth was centrifuged at 12000rpm for 5min, and the supernatant was filtered with a 0.22 μm filter membrane and analyzed by High Performance Liquid Chromatography (HPLC). The HPLC method is described in patent CN114836495B.

Example 4 production of products by engineering Strain fermentation Using glucose as substrate in shake flask

Fermentation was performed according to the method of example 3. From FIG. 1, it is understood that the Δ4 strain has a significantly increased yield relative to the Δku70 strain, and that knockout of the catabolic pathway-related gene PncA, pncC, nadR, ushA in the Δku70 strain has a promoting effect on the accumulation of NMN product. In addition, a heterologous NMN de novo synthesis pathway was introduced into the knocked-out strain, resulting in strain Δ4-1. And the delta 4-1 strain has further improved yield relative to the delta 4 strain, and the heterologous NMN is proved to have better expression suitability in Pichia pastoris from the de novo pathway.

Example 5 production of products produced by fermentation of engineering Strain Using glucose as substrate in reactor

In fed-batch fermentation, the strain is cultured in 1L shake flasks containing 200mL YPD medium for seed preparation. After 12-16 hours of incubation, the seed culture was transferred to a 3L fermenter containing 2L of fermentation medium. The pH was maintained at 4.5 during the batch phase and adjusted to 7.0 during the feed phase by automatic control of NH ₄ OH (50%, v/v) addition. The Dissolved Oxygen (DO) was maintained at 30% or more by automatically adjusting the stirring speed to 200-1000 rpm. As shown in FIG. 2, when the strain delta 4-1 is fermented by taking glucose as a substrate, the concentration of NMN products is greatly improved, the maximum production value is reached at 91h, and the yield is about 1g/L. The study was the highest yield of NMN from de novo synthetic fermentation.

The Δ4-1 strain expresses genes related to the synthetic pathway from chorismate to NMN as compared to the Δ4 strain. As particularly shown in fig. 3.

Sequence information

SEQ ID NO:1(PhzE)

MNALPTSLLQRLLERPAPFALLYRPESNGPGLLDVIRGEALELHGLADLPLDEPGPGLPRHDLLALIPYRQIAERGFEALDDGTPLLALKVLEQELLPLEQALALLPNQALELSEEGFDLDDEAYAEVVGRVIADEIGRGEGANFVIKRRFQARIDGYATASALSFFRQLLLREKGAYWTFIVHTGERTLVGASPERHISVRDGLAVMNPISGTYRYPPAGPNLAEVMEFLDNRKEADELYMVVDEELKMMARICEDGGRVLGPYLKEMAHLAHTEYFIEGQTSRDVREVLRETLFAPTVTGSPLESACRVIRRYEPQGRGYYSGVAALIGGDGQGGRTLDSAILIRTAEIEGDGRLRIGVGSTIVRHSDPLGEAAESRAKASGLIAALKSQAPQRLGSHPHVVAALASRNAPIADFWLRGASERQQLQADLSGREVLIVDAEDTFTSMIAKQLKSLGLTVTVRGFQEPYSFDGYDLVIMGPGPGNPTEIGQPKIGHLHLAIRSLLSERRPFLAVCLSHQVLSLCLGLDLQRRQEPNQGVQKQIDLFGAAERVGFYNTFAARALQDRIEIPEVGPIEISRDRETGEVHALRGPRFASMQFHPESVLTREGPRIIADLLRHALVERRPVD

SEQ ID NO:2(PhzD)

MSGIPEITAYPLPTAQQLPANLARWSLEPRRAVLLVHDMQRYFLRPLPESLRAGLVANAARLRRWCVEQGVQIAYTAQPGSMTEEQRGLLKDFWGPGMRASPADREVVEELAPGPDDWLLTKWRYSAFFHSDLLQRMRAAGRDQLVLCGVYAHVGVLISTVDAYSNDIQPFLVADAIADFSEAHHRMALEYAASRCAMVVTTDEVLE

SEQ ID NO:3(PnuC)

MVRSPLFLLISSIICILVGFYIRSSYIEIFASVMGIINVWLLAREKVSNFLFGMITVAVFLYIFTTQGLYAMAVLAAFQFIFNVYGWYYWIARSGEEKVKPTVRLDLKGWIIYILFILVAWIGWGYYQVRYLESTNPYLDALNAVLGLVAQFMLSRKILENWHLWILYNIVSIVIYISTGLYVMLVLAIINLFLCIDGLLEWKKNHKERERVNNY

SEQ ID NO:4(NadC)

MSFSNPNPEFAHLLPVDGKWKKDITSWLDEDTPSFDYGGYVVGENLQSATLWIKSNGVISGVPFAQEVYKQCGLEVEWFLKEGDYICGGDEGKVKVALVKGPVRNILLAERLSLNILARCSGVATQSYITIKRAREAGYKGIIAGTRKTTPGLRLLEKYSMLVGGCDSHRYDLSSMIMLKDNHIWSTGSITKAIESAQKVIGFSTKIEVEVQDEAEANEAIAAGADIIMLDNFTGEGLKVAAINLKNKWANTNKKFYLECSGGLTLDNIKDYLCNEIDIYSTSSIHQGVGIVDFSLKINK

SEQ ID NO:5(NbaC)

MSKTLQSFNLLKWIDENKELLKPPVNNKVIWQDSEFIAMILGGPNRRRDFHVDPSDEFFYQIKGECYVECITE EGKREVVTVKEGDVFMLPAMVPHSPHRVANTYGLVIERKRNQGELEDFVWFCDECNHEMHRVRVQLTDIEKQVKEAIHSFNSNKEIRACKNCGHIMPEEVEEWKCE

SEQ ID NO:6(DhbX)

MTFDKDYIAVITGACGGIGESVAHALAKEGLSLALLDNNATQLATLVATLQDNHPQPIAGFTVDVADDRCVAEAFTAVGHQLGPVGYLVNGAGVLCHASVAETQPQDWAKTFAVNATGVFNTSRHAANLMMAQRKGSIVTIASNAARVPRATMAAYCASKAAAQAFTYALGLEVAPYGIRCNVVAPGSTDTPMLRGMWHSESDKQNTLNGNPQQFRIGIPLNKVATAEEIAAAVCFYLCEESGQTTLSTLLVDGGAALGSC

SEQ ID NO:7(NadE)

MGMKIVKDFSPKEYSQKLVNWLSDSCMNYPAEGFVIGLSGGIDSAVAASLAVKTGLPTTALILPSDNNQHQDMQDALELIEMLNIEHYTISIQPAYEAFLASTQSFTNLQNNRQLVIKGNAQARLRMMYLYAYAQQYNRIVIGTDNACEWYMGYFTKFGDGAADILPLVNLKKSQVFELGKYLDVPKNILDKAPSAGLWQGQTDEDEMGVTYQEIDDFLDGKQVSAKALERINFWHNRSHHKRKLALTPNFVD

SEQ ID NO:8(PRS)

MSNQYGDKNLKIFSLNSNPELAKEIADIVGVQLGKCSVTRFSDGEVQINIEESIRGCDCYIIQSTSDPVNEHIMELLIMVDALKRASAKTINIVIPYYGYARQDRKARSREPITAKLFASLLETAGATRVIALDIHAPQIQGFFDIPIDHLMGVPILGEYFEGKNLEDIVIVSPDHGGVTRARKLADRLKAPIAIIDKRRPRPNVAEVMNIVGNIEGKTAILIDDIIDTAGTITLAANALVENGAKEVYACCTHPVLSGPAVERINNSTIKELVVTNSIKLPEEKKIERFKQLSVGPLLAEAIIRVHEQQSVSYLFS

SEQ ID NO:9(ARO3)

MFIKNDHAGDRKRLEDWRIKGYDPLTPPDLLQHEFPISAKGEENIIKARDSVCDILNGKDDRLVIVIGPCSLHDPKAAYDYADRLAKISEKLSKDLLIIMRAYLEKPRTTVGWKGLINDPDMNNSFQINKGLRISREMFIKLVEKLPIAGEMLDTISPQFLSDCFSLGAIGARTTESQLHRELASGLSFPIGFKNGTDGGLQVAIDAMRAAAHEHYFLSVTLPGVTAIVGTEGNKDTFLILRGGKNGTNFDKESVQNTKKQLEKAGLTDDSQKRIMIDCSHGNSNKDFKNQPKVAKCIYDQLTEGENSLCGVMIESNINEGRQDIPKEGGREGLKYGCSVTDACIGWESTEQVLELLAEGVRNRRKALKK

The above-described embodiments are merely specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be suggested to one skilled in the art without inventive effort are intended to be included in the scope of the present application. Therefore, the protection scope of the application is subject to the protection scope of the claims in the present application.

Claims (10)

1. A method of producing nicotinamide mononucleotide, said method comprising:

providing yeast engineering bacteria for synthesizing nicotinamide mononucleotide from the head, wherein PncA, pncC, nadR, ushA genes are knocked out from the yeast engineering bacteria; and

The nicotinamide mononucleotide product is produced without additional substrate addition using a base carbon source and a nitrogen source.

2. The method of claim 1, wherein the yeast engineering bacteria are transformed with an expression cassette comprising the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3;

After the yeast engineering bacteria are obtained, the bacterial strain is directly cultured and the production of the product is carried out without cell collection and/or concentration.

3. The method of claim 2, wherein the method comprises one or more of the following steps:

(1) Obtaining a yeast engineering bacterium I, wherein the yeast engineering bacterium I is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3;

(2) Obtaining yeast engineering bacteria II, wherein the yeast engineering bacteria II are transformed with expression cassettes of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA gene in the strain;

(3) Obtaining a yeast engineering bacterium III, wherein the yeast engineering bacterium III is transformed with an expression cassette of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock out the PncA, pncC genes in the strain;

(4) Obtaining yeast engineering bacteria IV, wherein the yeast engineering bacteria IV are transformed with expression cassettes of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3, and knock-out PncA, pncC, nadR gene in the strain;

the yeast engineering bacteria are pichia pastoris.

4. A method according to any one of claims 1 to 3, wherein the yeast engineering bacterium is pichia pastoris GS115 or KU70 deleted strain (Δku70).

5. A method according to any one of claims 1 to 3, wherein the expression cassette further comprises a promoter, including but not limited to: a constitutive promoter or a methanol inducible promoter;

Such constitutive promoters include, but are not limited to, GAP promoters;

Such methanol inducible promoters include, but are not limited to, the AOX1 promoter.

6. A method according to any one of claims 1 to 3, wherein the medium used for culturing the yeast engineering bacteria includes, but is not limited to: YPD, YPG, YPM, YPE medium; and/or

Glucose is supplemented during the culture process; preferably, glucose is added at intervals or fed-batch; more preferably, the interval supplement includes: glucose of 1% -5% (m/v) is added into the liquid culture medium every 12-36 h in the fermentation process; and/or

The pH of the culture medium is 5.8-7.6, preferably 6.5-7.5, more preferably 6.8-7.3;

culturing at 30+ -2deg.C and rotation speed of 200+ -50 r/min; preferably for 1.5 to 10 days;

The yeast engineering bacteria are Pichia pastoris GS115 or KU70 deletion strains (delta KU 70).

7. A yeast engineering bacterium for synthesizing nicotinamide mononucleotide from the head is characterized in that PncA, pncC, nadR, ushA genes are knocked out from the yeast engineering bacterium.

8. The yeast engineering bacterium for de novo synthesis of nicotinamide mononucleotide according to claim 7,

The yeast engineering bacteria comprise expression cassettes of the following genes: phzE, phzD, nabC, nadC, dhbX, nadE, pnuC, PRS and ARO3;

the PhzE gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO.1, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 1;

the PhzD gene has a nucleotide sequence for encoding an amino acid sequence shown as SEQ ID NO. 2, or a degenerate sequence thereof, or a nucleic acid encoding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 1;

the PnuC gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 3, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 3;

The NadC gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 4, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 4;

the NbaC gene has a nucleotide sequence of an amino acid sequence shown in SEQ ID NO. 5, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown in SEQ ID NO. 5;

the DhbX gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 6, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 6;

The NadE gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 7, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 7;

The PRS gene has a nucleic acid sequence for encoding an amino acid sequence shown in SEQ ID NO. 8, or a degenerate sequence thereof, or a nucleic acid encoding sequence of a homofunctional protein with more than 70% of identity with the sequence shown in SEQ ID NO. 8;

The ARO3 gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO. 9, or a degenerate sequence thereof, or a nucleic acid coding sequence of a homofunctional protein with more than 70% of identity with the sequence shown as SEQ ID NO. 9.

9. The use of the yeast engineering bacteria of claim 7 for producing nicotinamide mononucleotide using a basic carbon source as a substrate.

10. A kit for producing nicotinamide mononucleotide, the kit is characterized by comprising the following components: the yeast engineering strain of claim 7.