CN114774421B

CN114774421B - Mutant of endogenous promoter of zymomonas mobilis

Info

Publication number: CN114774421B
Application number: CN202210493106.4A
Authority: CN
Inventors: 杨世辉; 宋皓月; 杨永富; 李涵; 杜军
Original assignee: Hubei University
Current assignee: Wuhan Ruijiakang Biotechnology Co ltd
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2023-04-28
Anticipated expiration: 2042-05-07
Also published as: CN114774421A

Abstract

The present application discloses zymomonas mobilis endogenous promoter mutants directing expression of chimeric linked heterologous nucleic acids, promoting expression of the heterologous nucleic acids, while these promoter mutants are highly expressed promoters with significantly enhanced function relative to wild type promoters for expression of chimeric genes in zymomonas mobilis and/or escherichia cells.

Description

Mutant of endogenous promoter of zymomonas mobilis

Technical Field

The application relates to the technical fields of synthetic biology and genetic engineering, in particular to a zymomonas mobilis endogenous promoter mutant.

Background

Synthetic biology is an emerging interdisciplinary science and is being developed in the fields of agriculture, industry, intelligent materials, biomedicine and the like, and has wide application. The basis of synthetic biology is a biological element, and sufficient biological elements (including promoters, terminators, ribosome binding sites, riboswitches and the like) compatible with a chassis are key to effectively designing and assembling gene lines and realizing engineering of synthetic biology. Promoters, which are the core biological elements, are a DNA sequence required for transcription initiation, typically located upstream of the 5' end of a functional gene, and contain conserved sequences that specifically bind RNA polymerase and transcriptional regulators (e.g., regulatory proteins and small RNA). The gene sequence of the promoter determines the expression intensity and expression mode (constitutive or inducible) of the downstream gene, so that the promoter becomes a key component for driving gene expression, regulating a gene loop and obtaining a high-efficiency cell factory, and the high-efficiency and accurate expression and regulation of the target gene can be realized through the promoters with different intensities at the present stage.

However, as the requirements for efficient expression and precise regulation of target genes continue to increase, so do the requirements for strength of promoters and compatibility in different chassis cells, for example, often when promoters endogenous to one host are used in another, the strength of the promoters will often change, thereby affecting expression of downstream genes.

Disclosure of Invention

Zymomonas mobilis is a non-model strain of naturally occurring ethanol production, the only microorganism currently known to be anaerobic fermented using the Entner-Doudoroff (ED) pathway. It has many excellent properties developed as an industrial chassis, for example: rapid and efficient sugar utilization, high ethanol yield and tolerance, few anaerobic fermentation byproducts, and excellent fermentation performance in an acidic pH range (pH 4-8). In addition, the genome of Z.mobilis is small and various genomic engineering tools have been developed, including endogenous and exogenous CRISPR-cas editing systems; the development of whole genome metabolic network models and the availability of accumulated histology data also make Z.mobilis an ideal chassis for further development.

In Z.mobilis, the promoters that drive the expression of the genes encoding enzymes involved in glycolysis (e.g., pgap, ppdc, peno and PadhB) are typically strong promoters. Wherein the Pgap promoter is a constitutive tandem promoter driving expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (Gap). It is one of the strongest promoters in Z.mobilis, 305bp in length, containing 9 predicted Transcription Start Sites (TSS). Two transcription initiation sites (TSS) have been identified for Pgap by biochemical means, one starting from thymine and the other starting from adenine. The-35 region of the proximal promoter (Pgap 2) overlaps the-10 region of the distal promoter (Pgap 1). Pgap is widely used in metabolic engineering, and has been constructed in functional circuits of various strains such as zymomonas mobilis and pseudomonas putida to achieve the purpose of high yield. Based on the research background, the application is based on the endogenous promoters of the zymomonas mobilis, and a series of strong promoter mutants are obtained, and the promoters not only have strong promoter activity in the zymomonas mobilis, but also have strong promoter activity in heterologous escherichia coli, so that the application provides assistance for providing some strong promoters and/or improving the compatibility of chassis cells.

In a first aspect, embodiments herein disclose a nucleic acid molecule comprising a zymomonas mobilis endogenous promoter mutant, wherein the promoter mutant comprises at least one of a glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant and a ZMO1609 gene promoter, the glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant being formed by substitution of an UP element of a glyceraldehyde-3-phosphate dehydrogenase gene promoter comprising at least one of positions 28, 30, 86, 107, 109, 136, 142, 164 and 197 with a base substitution, or being a glyceraldehyde-3-phosphate dehydrogenase gene promoter; wherein the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene promoter is shown in SEQ ID NO:1 is shown in the specification; the ZMO1609 gene promoter mutant is formed by replacing an UP element of a ZMO1609 gene promoter, wherein the nucleotide sequence of the ZMO1609 gene promoter is shown in SEQ ID NO: 2.

In embodiments of the present application, the base substitution includes: 28T > A, 30A > G, 86G > T, 107C > T, 109G > C, 109G > A, 136T > C, 142T > A, 164C > T, 197A > T.

In the embodiment of the application, the UP element is replaced by 1-2 UP elements from E.coli.

In embodiments of the present application, the nucleic acid molecule comprises a sequence selected from any one of SEQ ID NOS.3-20.

In an embodiment of the present application, the nucleic acid molecule comprises the sequence shown in SEQ ID NO. 21.

In a second aspect, embodiments of the present application disclose chimeric genes comprising the nucleic acid molecules of the first aspect, which are chimeric linked to a heterologous nucleic acid molecule.

In embodiments of the present application, the heterologous nucleic acid molecule encodes a protein or peptide.

In a third aspect, embodiments of the present application disclose a vector comprising the nucleic acid molecule of the first aspect.

In a fourth aspect, embodiments of the present application disclose a host cell comprising the vector of the third aspect.

In embodiments of the present application, wherein the host cell comprises a zymomonas mobilis cell and/or an escherichia cell.

Drawings

FIG. 1 shows the results of strength tests of the existing endogenous strong promoters of Zymomonas mobilis (Pgap, ppdc, peno) provided in the examples of the present application in E.coli and Zymomonas mobilis, respectively; FIG. 1A is a flow two-dimensional scattergram of a promoter (Pgap, ppdc, peno) tested in E.coli; FIG. 1B is a flow two-dimensional scatter plot of promoters (Pgap, ppdc, peno) tested in Zymomonas mobilis; FIG. 1C is the green fluorescence intensity of promoter (Pgap, ppdc, peno) in E.coli and Z.mobilis; FIG. 1D is a ratio of EGFP/opmCherry at exponential phase to stationary phase in E.coli and Z.mobilis; pEZ15A represents a control with only the vector backbone.

FIG. 2 shows the results of DNA gel electrophoresis detection of the wild-type Pgap promoter and the mutant library fragment of the Pgap promoter provided in the examples of the present application, wherein lane 1 is the target fragment of the wild-type Pgap promoter, lane 2 is the mutant library fragment of the Pgap promoter, and lane 3 is Marker.

FIG. 3 shows the result of DNA gel electrophoresis detection of the amplified product of the dual-fluorescence reporter vector provided in the embodiment of the present application, wherein lane 1 and lane 2 are both amplified products of the dual-fluorescence reporter vector, and lane 3 is a Marker.

FIG. 4 shows the ratio of exponential phase to stationary phase EGFP/opmCherry in E.coli and Z.mobilis for 7 Pgap mutants based on sanger sequencing as provided in the examples of the present application.

FIG. 5 shows the green fluorescence intensity fold difference between the exponential phase and the stationary phase of E.coli for the Pgap mutant obtained by site-directed mutagenesis in the examples of the present application.

FIG. 6 is an illustration of the expression of Pgap and Ptt1609 mutants provided in the examples of the present application in E.coli; FIG. 6A is a schematic diagram of a Pgap promoter comprising two UP elements, -10 region and-35 region; FIG. 6B is a graph showing the fold difference in green fluorescence intensity between the Pgap and Ptt1609 mutants at the exponential phase of E.coli and at the stationary phase.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Described herein are novel promoters useful for expressing chimeric genes in host cells. The inventors of the present application creatively found that zymomonas mobilis endogenous promoter: different types of mutants of glyceraldehyde-3-phosphate dehydrogenase gene promoter and ZMO1609 gene promoter may increase the level of expression directed by the promoters.

The glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant is formed by substitution of at least one of positions 28, 30, 86, 107, 109, 136, 142, 164 and 197 of the glyceraldehyde-3-phosphate dehydrogenase gene promoter with a base, or substitution of an UP element of the glyceraldehyde-3-phosphate dehydrogenase gene promoter; wherein the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene promoter is shown in SEQ ID NO. 1.

The ZMO1609 gene promoter mutant is formed by replacing an UP element of the ZMO1609 gene promoter, wherein the nucleotide sequence of the ZMO1609 gene promoter is shown as SEQ ID NO. 2.

The zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter comprises any of these mutations, which can be used to express heterologous, chimeric linked DNA sequences in a host cell.

The following abbreviations and definitions will be used for the interpretation of the specification and claims.

In this application, the term "gene" refers to a nucleic acid fragment expressing a particular protein or functional RNA molecule, which may comprise a regulatory sequence preceding (5 'non-coding region) and a regulatory sequence following (3' non-coding region) the coding sequence. "native gene" or "wild-type gene" refers to a naturally occurring gene having its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory sequences and coding sequences that are not naturally present together. Thus, a chimeric gene may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature. "endogenous gene" refers to a native gene located in its natural location within the genome of an organism. "exogenous gene" refers to a gene that is not normally present in the host organism and that is introduced into the host organism by gene transfer. The foreign gene may comprise a native gene inserted into a non-native organism, or a chimeric gene. "endogenous" refers to a source located at a natural location within the genome of an organism. The "UP element" (upstream promoter elements) is called an upstream promoter element, and is a sequence located in the region-35 upstream of the core promoter element-40 to-60, which is recognized by RNA polymerase itself, and can increase transcription efficiency of rrmB P1 gene of E.coli by 30-fold only in the presence of RNA polymerase.

In this application, the term "genetic construct" refers to a nucleic acid fragment encoding a molecule that expresses one or more specific proteins or functional RNAs. A gene may be native, chimeric, or foreign in a genetic construct. Typically the genetic construct will comprise a "coding sequence". "coding sequence" refers to a DNA sequence that encodes a particular amino acid sequence.

In this application, a "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters may be derived entirely from a native gene, or consist of different elements derived from different naturally occurring promoters, or even comprise synthetic DNA fragments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause expression of genes in most cell types in most cases are commonly referred to as "constitutive promoters".

In this application, the term "expression" refers to the transcription and stable accumulation of coding RNA (mRNA) or functional RNA derived from a gene, and may also refer to translation of mRNA into a polypeptide or protein. "overexpression" refers to the production of a gene product in a transgenic organism that exceeds the level of the gene product produced in a normal organism or an untransformed organism.

In this application, the term "messenger RNA (mRNA)" refers to RNA that is intronless and can be translated into protein by a cell.

In the present application, the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in a genetically stable inheritance. The transformed nucleic acid may be in the form of a plasmid that remains in the host cell, or some of the transformed nucleic acid may be integrated into the host cell genome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

In the present application, the terms "plasmid" and "vector" refer to an extrachromosomal element that normally carries a gene that is not part of the central metabolism of the cell, and is often in the form of a circular double stranded DNA molecule. Such elements may be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences (linear or circular) of single-or double-stranded DNA or RNA derived from any source, wherein multiple nucleotide sequences have been joined or recombined into a unique construct capable of introducing a promoter fragment of a selected gene product and the DNA sequence into a cell along with the corresponding 3' -terminal untranslated sequence.

In the present application, the term "chimeric ligation" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one nucleic acid sequence is affected by the other. For example, a promoter is chimeric to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be chimeric linked to the regulatory sequence in either sense or antisense orientation. Correspondingly, a "chimeric gene" refers to nucleic acid sequences that are "chimeric linked" together, for example, by "chimeric linking" a promoter to a coding sequence that it can affect.

In this application, the term "heterologous" refers to a site that does not naturally occur at the site of interest. For example, a "heterologous gene" refers to a gene that does not naturally occur in a host organism and that is introduced into the host organism by gene transfer. For example, a heterologous nucleic acid molecule present in a chimeric gene is a nucleic acid molecule that does not naturally occur with other fragments in the chimeric gene, such as a nucleic acid molecule having coding regions and promoter fragments that do not naturally associate with each other.

In this application, a "nucleic acid molecule" is a polymer of RNA or DNA that is single-stranded or double-stranded, optionally comprising synthetic, non-natural or altered nucleotide bases. The nucleic acid molecule in the form of a DNA polymer may be composed of one or more segments of cDNA, genomic DNA, or synthetic DNA.

In the present application, the terms "zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter" and "ZMO1609 gene promoter" both refer to nucleic acid molecules having promoter activity. The "ZMO1609 Gene promoter" has a nucleotide sequence that is naturally present in the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase Gene (Gene ID: 58026057) upstream of the coding region, and the "ZMO1609 Gene promoter" has a nucleotide sequence that is naturally present in the ZMO1609 Gene (Gene ID: 58027328) upstream of the coding region. These terms also include reference to promoters of zymomonas mobilis strains such as the ZM4 strain (ATCC 31821) and to variants in sequence and/or length that direct expression, e.g., the variants do not direct significant differences in the level of expression.

Discovery of endogenous promoter mutants of Zymomonas mobilis

The zymomonas mobilis endogenous promoters (glyceraldehyde-3-phosphate dehydrogenase promoter Pgap, pyruvate decarboxylase promoter Ppdc, enolase promoter Peno) have been used to express chimeric genes in zymomonas mobilis, palm fermentation bacteria and escherichia coli. However, when used alone to express and initiate expression of certain genes, the expression was not as effective as desired, as shown, for example, in FIG. 1, the three strong promoters previously identified in Zymomonas mobilis (Pgap, ppdc, peno) were tested for strength in E.coli and the strong promoters in Zymomonas mobilis were significantly reduced in strength in E.coli.

For this reason, the inventor of the application starts from a zymomonas mobilis endogenous promoter Pgap, utilizes an error-prone PCR technology to construct a Pgap mutation library, constructs the Pgap mutation library onto a double-report system carrier, then converts escherichia coli T1 in a plasmid form to obtain a mutant library in escherichia coli, and utilizes a flow cytometer to sort out cell subsets with high EGFP fluorescence intensity. Multiple mutation sites are determined by combining Sanger sequencing and a new generation sequencing (Next-generation sequencing, NGS) method, and positive and negative experimental design shows that the mutation sites can indeed influence Pgap promoter compatibility, and the results are shown in figures 4-5. The structural characteristics of the mutation site and the promoter are linked, the effect of the UP element of the promoter on the compatibility of the promoter in the zymomonas mobilis and the escherichia coli is analyzed, the UP element is verified in another strong promoter Ptt1609 of the zymomonas mobilis, and the molecular mechanism for restricting the universality of the promoter is further analyzed, and the result is shown in figure 6.

The inventors have further found that having single or multiple nucleotide changes in the wild-type Pgap promoter (shown in SEQ ID No. 1) results in enhanced expression of the coding sequence of the mutant chimeric linkage of the promoter. And this nucleotide change is a new change relative to the Pgap promoter of the strain, compared to the sequence of the wild-type Pgap promoter of the ZM4 strain, ATCC 31821 strain, as shown in SEQ ID NO. 1. The glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant is formed by substitution of at least one of positions 28, 30, 86, 107, 109, 136, 142, 164 and 197 of the glyceraldehyde-3-phosphate dehydrogenase gene promoter with a base, or substitution of an UP element of the glyceraldehyde-3-phosphate dehydrogenase gene promoter.

Preparation of Pgap mutant and Ptt1609 mutant

The mutations at positions 28, 30, 86, 107, 109, 136, 142, 164 and 197 may be introduced into the nucleic acid molecule of the wild-type Pgap promoter (shown in SEQ ID NO. 1) by any method known to those skilled in the art, or may result in a Pgap promoter in which the UP element is replaced by an E.coli UP element, or may result in a Ptt1609 promoter mutant in which the UP element in the wild-type Ptt1609 promoter (shown in SEQ ID NO. 2) is replaced. For example, oligonucleotides having mutations and surrounding DNA sequences can be synthesized and cloned into larger promoter DNA fragments to replace non-mutated fragments. Primers containing the mutation and some adjacent promoter sequences can be synthesized and used in PCR to prepare promoter fragments. Full-length promoter DNA fragments can be synthesized as multiple oligonucleotides that are ligated together. Site-directed mutagenesis may be used to introduce mutations. In addition, genomic DNA from ZM4 (ATCC 3182) can be used as a template to prepare mutant promoters as PCR-amplified DNA fragments.

Table 1 shows Pgap mutants and Ptt1609 mutants prepared in the examples of the present application, and mutation sites of each mutant. Wherein, "Pgap-WT" represents a wild type glyceraldehyde-3-phosphate dehydrogenase gene promoter shown in SEQ ID NO.1, "Ptt1609-WT" represents a wild type ZMO1609 gene promoter shown in SEQ ID NO.2, "28T > A" represents substitution of the 28 th base T of "Pgap-WT" with A, and other base substitution forms are analogized. The nucleotide sequence of the UP element of the E.coli is shown in any one of SEQ ID NO. 36-38. "+" indicates that the plasmid has been transformed into Zymomonas or Escherichia.

TABLE 1 Pgap mutant and Ptt1609 mutant

Chimeric genes and vectors comprising Pgap mutants and Ptt1609 mutants into host cells

The Pgap mutants and Ptt1609 mutants disclosed herein may be chimeric linked as promoters to a heterologous nucleic acid molecule intended for expression in a host cell, forming a chimeric nucleic acid molecule or chimeric gene of the present application. The design and construction of chimeric genes is well known to those skilled in the art. Chimeric genes typically include a promoter, a heterologous nucleic acid molecule intended for expression, and 3' termination control regions. The termination control region may be derived from a different gene and is often derived from a gene naturally found in the target host cell. The chimeric linked heterologous nucleic acid molecule may be any nucleic acid molecule desired to be expressed in a host cell, including, for example, a coding region for a protein or peptide. Furthermore, an operon comprising the promoters described herein and a plurality of coding regions expressed from the promoters may be constructed.

The promoters described herein may be used in chimeric genes to express the chimeric genes in bacteria belonging to the genus Zymomonas and/or the genus Escherichia. The chimeric genes may be used to express any protein involved in the production of zymomonas and/or escherichia products. For example, one or more enzymes involved in the synthesis of bioenergy substances such as isobutanol, in the production of bioplastic such as PHB can be expressed starting from chimeric genes with these promoters. The chimeric gene may be expressed in a strain of natural zymomonas and/or escherichia that does not utilize xylose, or in a strain that utilizes xylose. The promoters described herein may also be used to express enzymes involved in isobutanol metabolism or another metabolic pathway.

The chimeric genes described herein are typically constructed in a vector or transferred to a vector for further manipulation. Vectors are well known. Some vectors are capable of replication in a wide range of host bacteria and can be transferred by conjugation, for example, pET series vectors are one of the most widely used vectors at present, have a powerful function in E.coli, and are capable of expressing recombinant proteins; pEZ-Dual Dual reporter vectors have identified promoters, terminators, RBS and alcohol inducible promoters of varying strengths in Zymomonas mobilis. Vectors may include plasmids for autonomous replication in cells, as well as plasmids for transporting constructs for integration into the bacterial genome. Plasmids used for DNA integration may include a transposon, a region of nucleic acid sequence homologous to the target bacterial genome, or other sequences involved in integration.

The promoters described herein may also be constructed for expression in vectors without chimeric linked nucleic acid molecules and integrated into the vicinity of the endogenous coding region to replace the endogenous promoter in the bacterial genome or to add at least one promoter to the coding region within, for example, an operon. Vectors comprising the promoters described herein may be introduced into bacterial cells by well known methods, such as using freeze-thaw transformation, calcium-mediated transformation, electroporation, or conjugation.

Expression Using Pgap mutant and Ptt1609 mutant as promoters

The Pgap mutants and Ptt1609 mutants disclosed herein can be used as promoters to increase expression of luciferase genes. For example, as shown in FIGS. 2 and 3, the Pgap mutant was chimeric into pEZ-Dual (refer to the method disclosed in CN 109913487A), and transformed into Zymomonas mobilis and E.coli by constituting chimeric genes with EGFP in pEZ-Dual, positive clones were selected, transformants were identified, and PCR products of the positive clones were subjected to fluorescence detection, as shown in Table 1, and it was found that 16 Pgap mutants and Pgap-COUP (SEQ ID NO. 3) were increased in the promoter strength of Pgap-2COUP (SEQ ID NO. 4) by at least 1.5 times, particularly by 20 times, as compared with wild type Pgap in the promoter strength of 6M, UP-3M. And these mutants still have high promoter strength in Z.mobilis. The results are shown in FIGS. 4 to 6.

For example, as shown in Table 1 and FIG. 6, ptt1609 mutant (SEQ ID NO. 5) was chimeric into pEZ-Dual (see methods disclosed in CN 109913487A), constituted chimeric genes with EGFP in pEZ-Dual, transformed into Zymomonas mobilis and E.coli, positive clones were selected, transformants were identified, PCR products of the positive clones were subjected to fluorescent detection, and Ptt1609 mutant Ptt1609-COUP promoter strength was found to be more than 5-fold higher than that of wild-type Ptt1609, and still maintained high in original host Zymomonas mobilis.

The following is a description of more specific examples.

1. Random mutagenesis to produce Pgap promoter

1. Obtaining a fragment of the order wild-type Pgap promoter

The genome DNA of ZM4 (ATCC 31821) of Zymomonas mobilis is used as a template to amplify to obtain a Pgap promoter (SEQ ID NO. 1) as a target fragment, and the primers used for amplifying the Pgap are Pgap-fra-F (SEQ ID NO. 6) and Pgap-fra-R (SEQ ID NO. 7) and are synthesized by the company of the Wuhan engine family. The amplification results of the target fragment are shown in FIG. 2.

2. Random mutation

The step employs random mutagenesis to obtain Pgap promoter mutants, particularly by error-prone PCR, wherein a low fidelity DNA polymerase is used and Mg is adjusted ²⁺ The frequency of introducing mismatched bases in the amplification process is increased by three factors of template concentration and cycle number, so that the amplification product of the target gene Pgap generates proper random mutation, and a random mutation library is constructed. The error-prone PCR was performed 4 times using Pgap-fra-F, pgap-fra-R as a primer, and the products after the 4 error-prone PCR were mixed and purified for recovery. The PCR reaction (50. Mu.L) and the procedure are shown in Table 2. The amplification results of the target fragment are shown in FIG. 2.

TABLE 2 error-prone PCR reaction System and reaction procedure

3. Amplification dual fluorescent reporting system carrier

Plasmid pEZ-Dual (prepared by the method disclosed in CN109913487A, p15A_ori, zymo_ori, ptet, speR, placUV5:: opmCherry, ptet::: EGFP) is used as a DNA amplification template, and primers mDual-Rev-F (SEQ ID NO. 8) and mDual-Rev-R (SEQ ID NO. 9) are designed for amplification to give a double-fluorescence report vector (primers were synthesized by Wohanoaceae). The PCR reaction system was as follows using mDual-F, mDual-R reverse-amplified plasmid pEZ-Dual, 20. Mu.L, and the PCR amplification procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 53℃for 10s, extension at 72℃for 50s for 29 cycles; and after the circulation is finished, the temperature is kept at 72 ℃ for 5min to obtain a double-fluorescence report system carrier, and a section of fragment with the size of 4871bp is obtained through purification and recovery. The purified fragments were subjected to DNA gel electrophoresis.

TABLE 3 reaction System for amplifying double fluorescent reporter System Carrier

4. Construction of recombinant E.coli library

Adding the purified Pgap promoter mixed fragment subjected to random mutation with a double-fluorescence reporting system carrier, water and T5 enzyme respectively, adding Buffer solution according to a reaction system shown in table 4, standing on ice for 5min, adding 50 mu L of escherichia coli competent T1 into a sterile operation table, and standing on ice for 30min; standing on ice for 2min after heat shock at 42 ℃ for 45 s; then 500. Mu.L of LB liquid culture is added and cultured for 1 hour in a shaking table at 37 ℃ and 250 rpm; using 5000rpm in a bench-type high-speed centrifuge, 2min centrifugation was performed, 400. Mu.L of the supernatant was removed, and the remaining bacterial liquid was blown and mixed uniformly, and spread on a solid plate of LB+Spectinomycin (Spe, 100. Mu.g/mL) and incubated overnight at 37℃in an inverted state. All colonies on the plates were collected the next day in EP tubes and frozen by adding 60% glycerol.

TABLE 4 Table 4

5. Fluorescence activated cell sorting and detection of recombinant E.coli library

Sorting the Pgap mutant library by fluorescence activated cells to recombine cell subsets with different intensities in the E.coli cell library. Using phosphate buffer(PBS) the collected cells were washed 3 times and finally resuspended to 10 ⁷ Each cell/mL was prepared as a sample solution. The ultrasonic cleaning experiment was performed using a nozzle to expel air bubbles. And (5) sterilizing the working environment and instruments by using 75% alcohol. And (3) using sorting quality control microspheres to debug to an optimal value. The 488nm excitation wavelength and FITC channel were set. And the deflection voltage plate voltage of the side liquid flow window is opened, and a proper deflection angle is set, so that liquid flow beam splitting is clear, and sorting is facilitated. After washing the tube with 0.5% sodium hypochlorite solution for 5min, the sample was placed in a loading rack, cells expressing EGFP protein were detected, and the cells were collected by setting a door. The cells obtained after sorting were subjected to aseptic culture. 200 mu L of the cultured bacterial liquid is absorbed and evenly coated on an LB+Spe solid plate, and the bacterial liquid is inversely cultured in a biochemical incubator at the constant temperature of 37 ℃ for overnight.

6. Pgap mutant obtained by screening random mutation through Sanger sequencing

Single colonies growing on the plates after sorting are used as DNA amplification templates, and primers Pseq-F (SEQ ID NO. 10) and Pseq-R (SEQ ID NO. 11) are designed for colony PCR detection (primers are synthesized by the company of the Gramineae) as follows:

a plurality of monoclonals are randomly selected from the flat plate, colony PCR is used for identifying transformants, the used primers are Pseq-F, pseq-R, and the theoretical size of the positive clone PCR product band is 815bp. From the solid plates after overnight incubation, 15 single colonies were randomly picked with a 10. Mu.L sterile small gun head in a 200. Mu.L centrifuge tube, added with 10. Mu.L ultrapure water, thoroughly mixed, and 1. Mu.L was aspirated therefrom as DNA templates for colony PCR amplification. The PCR reaction system using 10. Mu.L was as follows, and the PCR amplification procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 53℃for 10s, extension at 72℃for 10s for 27 cycles; after the cycle was completed, the temperature was maintained at 72℃for 5 minutes. The PCR products were detected by agarose gel electrophoresis and the results were observed using an ultraviolet gel imaging system. And selecting bacteria with correct band sizes from the electrophoresis results for culturing.

TABLE 5

Reagent(s)	Volume (mu L)	Concentration of
			Primer F	0.4	10μM
Primer R	0.4	10μM
			Template
	1
			ddH ₂ O	3.2
T5 Super PCR Mix	5	2×

The four consecutive sorted Pgap promoter library was sent to GENEWIZ company, su for NGS sequencing. The paired end read quality was checked using the FastQC program (http:// www.bioinformatics.babraham.ac.uk/subjects/FastQC /), and the data was mapped to Pgap wild-type DNA sequences to identify variations. NGS result data is shown in table 6.

TABLE 6 NGS-based read count and mutation type data

Total reading number	18402322
		Reading number (unmutated)	6251443
Reading number (mutation)	12150879
		Type of mutation	593745
Number of reads>1,000 mutation	356

7. Flow cytometry detection and fluorescence intensity calculation

EGFP fluorescence was detected using a Beckman CytoFLEX FCM flow cytometer, with FITC channel, opmCherry was detected with PC5.5 channel, and fluorescence compensation was set. The parameters recommended by FlowJo software are used for processing, the minimum value of Events is set to 20000, the error in analysis is effectively reduced, and the flow cytometry analysis time is shortened. The average fluorescence intensity of each sample was calculated, the intensity of each promoter was quantified using the average EGFP/opmCherry ratio of the three parallel groups, and the standard deviation (STDEV) was set as the error bar. The resulting EGFP/opmCherry ratio was imported into GraphPad Prism 8.0 software and subjected to significance analysis.

The result is shown in FIG. 4, compared with the wild type promoter, the EGFP/opmC herry values of the Pgap promoter mutants obtained by random mutation screening are obviously enhanced within the same growth time of escherichia coli, and the Pgap promoter mutants provided by the embodiment of the application have the expression enhancement function compared with the wild type Pgap promoter.

8. Intensity detection of mutants in Zymomonas mobilis

(1) Preparation of the competent Zymomonas mobilis Strain of interest

A suitable amount of ZM4 glycerol bacteria of Zymomonas mobilis was selected with an inoculating loop in RMG5 solid medium (RMG 5:50g/L glucose, 10g/L yeast extract, 2g/L KH) ₂ PO ₄ 3g/L agar) plate is streaked, and is cultured for 2 to 3 days at the temperature of 30 ℃ in an inverted way for activation; the activated single colonies were picked and transferred to a strain containing about 10mL of RMG5 (RMG 5:50g/L glucose, 10g/L yeast extract, 2g/L KH) ₂ PO ₄ ) In a liquid culture medium, standing and culturing at 30 ℃ until mid-log phase is used as seed liquid; transferring the seed liquid into a 250mL blue cap bottle containing 200mL of RMG5 liquid culture medium, and controlling the initial OD to be between 0.025 and 0.03. Standing and culturing at 30 ℃ until OD=0.4-0.6; after a blue cap bottle filled with bacterial liquid is placed on ice and cooled for 30min, a precooled 50mL centrifuge tube is used for centrifuging at 4000rpm for 10min to collect bacterial bodies, and the supernatant is discarded; adding 30mL of pre-cooled sterile water into the centrifuge tube, re-suspending and washing thalli, uniformly mixing, centrifuging at 4000rpm for 10min, and discarding the supernatant; adding 30mL of pre-cooled 10% glycerol to the centrifuge tube to resuspend and wash the thalli, uniformly mixing, centrifuging at 4000rpm for 10min, discarding the supernatant, and repeating the steps once; adding 1% (volume ratio) pre-cooled 10% glycerol re-suspended thallus, slowly mixing, packaging on ice, packaging every 50 μl into sterile 1.5mL centrifuge tube, quick freezing in liquid nitrogen, and storing at-80deg.C.

(2) Transformation of mutants into competent cells of Zymomonas mobilis of interest

ZM4 competent cells of Zymomonas mobilis were taken on ice, 50. Mu.L of competent cells were added to the electrorotor after thawing, and 1. Mu.g of plasmid was added to the electrorotor. The electrical switching conditions were 160V, 25. Mu.F, 200Ω. Resuscitates in an incubator at 30℃in RMG5 liquid medium after the completion of the electrotransformation. Resuscitates the cultures for 4-6 hours at 6000rpm,1min centrifugation and part of the supernatant removed. The suspension cells were plated at 100. Mu.L on 100. Mu.g/mL spectinomycin-resistant plates and incubated at 30℃for 2 days.

After colonies grow out, colony PCR detection is carried out on the recombinant strain, and the PCR amplification program is set as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 10s, extension at 72℃ (set according to fragment length of 10 s/kb) for 30 cycles; maintaining at 72 deg.c for 5min after the cyclic reaction; the reaction system is as follows:

TABLE 7

Reagent(s)	Volume of
		F-primer(10μM)	0.3μL
R-primer(10μM)	0.3μL
		2×T5 Super PCR Mix(Tsingke)	5μL
Template	XμL
		ddH ₂ O	To 10μL
Total volume	10μL

The correct positive clones obtained were glycerol-protected after activation in the medium with resistant liquid RMG 5.

The average EGFP/opmCherry ratio was calculated using flow cytometric detection and fluorescence intensity as described in the examples above to quantify the intensity of each promoter and set the standard deviation (STDEV) as the error bar. The results are shown in fig. 4, and compared with the wild type promoter, the EGFP/opmCherry value of the Pgap promoter mutant obtained by random mutation screening in the same growth time of the zymomonas mobilis is not significantly reduced, which indicates that the Pgap promoter mutant provided by the embodiment of the application has the expression enhancement function compared with the wild type Pgap promoter.

2. UP element substitution mutation

The sequence of the wild-type Pgap promoter and the sequence of the wild-type Ptt1609 gene were used as templates, the desired fragment was prepared by referring to the above examples, primers (shown in Table 5) were designed to construct recombinant plasmids, and after transformation into competent cells of E.coli T1, single colonies were selected by using a spectinomycin resistance (100. Mu.g/mL) plate, PCR verification was performed by using two primers of Pseq-F (SEQ ID NO. 10) and Pseq-R (SEQ ID NO. 11), and the primers were synthesized by Wohan's Optimaceae company as shown in Table 8. Verification System the fluorescent protein expression assays were performed on promoter mutants from which several UP elements were obtained, as described in the examples above.

As shown in FIG. 6, EGFP/opmCherry values were significantly enhanced during the same growth time, indicating that the promoter mutants of the UP element provided in the examples of the present application have an enhanced expression function relative to the wild-type Pgap promoter.

TABLE 8 Pgap-COUP, pgap-2COUP and Ptt1609-COUP primers used in construction

3. Combinatorial mutation

The four consecutive sorted Pgap promoter library was sent to GENEWIZ company, su for NGS sequencing. The paired end read quality was checked using the FastQC program (http:// www.bioinformatics.babraham.ac.uk/subjects/FastQC /), and the data was mapped to Pgap wild-type DNA sequences to identify variations. NGS data has been uploaded to the NCBI biological sample database under the number SAMN24913892. Compared to the wild-type Pgap sequence, 6 sites with significant mutation rates were identified, T28, G86, C107, G109, T136 and C164, respectively, in the Pgap sequence. The 6 sites were subjected to combinatorial mutation to construct plasmid Pgap-6M.

Taking Pgap-4M obtained by sanger sequencing as a DNA amplification template, designing a primer for amplification to obtain a target fragment (the primer is synthesized by Wuhan qingke company), and constructing a specific plasmid as follows: 4M+C107T-F (SEQ ID NO. 20) and DOWN-spe-R (SEQ ID NO. 13), respectively; 4M+G86T-R (SEQ ID NO. 21) and UP-spe-F (SEQ ID NO. 15) two sets of primers, performing DNA amplification by taking a Pgap-4M plasmid as a DNA amplification template to obtain two fragments with homology arms, transforming the fragments into competent cells of escherichia coli T1, screening by using a spectinomycin resistance (100 mug/mL) plate, picking a single colony, performing PCR verification by using the Pseq-F, pseq-R two primers, and sequencing to obtain a correct transformant Pgap-6M.

And the promoter mutants from which several UP elements were obtained were examined for fluorescent protein expression by the method described in the above examples.

As shown in FIG. 5, the EGFP/opmCherry values of Pgap-6M were significantly enhanced during the same growth time, demonstrating that the Pgap-6M promoter mutants provided in the examples of the present application have an optimal expression enhancing function relative to the wild-type Pgap promoter

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Sequence listing

<110> university of Hubei

<120> Zymomonas mobilis endogenous promoter mutant

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 305

<212> DNA

<213> Artificial Sequence

<400> 1

gttcgatcaa caacccgaat cctatcgtaa tgatgttttg cccgatcagc ctcaatcgac 60

aattttacgc gtttcgatcg aagcagggac gacaattggc tgggaacggt atactggaat 120

aaatggtctt cgttatggta ttgatgtttt tggtgcatcg gccccggcga atgatctata 180

tgctcatttc ggcttgaccg cagtcggcat cacgaacaag gtgttggccg cgatcgccgg 240

taagtcggca cgttaaaaaa tagctatgga atatagtagc tacttaataa gttaggagaa 300

taaac 305

<210> 2

<211> 201

<212> DNA

<213> Artificial Sequence

<400> 2

atcgaaacct ttcttaaaaa atttttttcg aaaatctttt tgaactcagt ccgtcaatga 60

tctatccttc cttgacgcat aaggcaattc cactgttgca atgaatatat tgcttatggt 120

gaaacgttat cgcttctcat gcgattctat agttaggata aactgattat tgttacgtat 180

tgagtaactg gagtatagac a 201

<210> 3

<211> 305

<212> DNA

<213> Artificial Sequence

<400> 3

gttcgatcaa caacccgaat cctatcgtaa tgatgttttg cccgatcagc ctcaatcgac 60

aattttacgc gtttcgatcg aagcagggac gaaaaattat tttgaaaaat atactggaat 120

aaatggtctt cgttatggta ttgatgtttt tggtgcatcg gccccggcga atgatctata 180

tgctcatttc ggcttgaccg cagtcggcat cacgaacaag gtgttggccg cgatcgccgg 240

taagtcggca cgttaaaaaa tagctatgga atatagtagc tacttaataa gttaggagaa 300

taaac 305

<210> 4

<211> 305

<212> DNA

<213> Artificial Sequence

<400> 4

gttcgatcaa caacccgaat cctatcgtaa tgatgttttg cccgatcagc ctcaatcgac 60

aataaaatta ttttcgaaaa aagcagggac gaaaaattat tttgaaaaat atactggaat 120

aaatggtctt cgttatggta ttgatgtttt tggtgcatcg gccccggcga atgatctata 180

tgctcatttc ggcttgaccg cagtcggcat cacgaacaag gtgttggccg cgatcgccgg 240

taagtcggca cgttaaaaaa tagctatgga atatagtagc tacttaataa gttaggagaa 300

taaac 305

<210> 5

<211> 201

<212> DNA

<213> Artificial Sequence

<400> 5

atcgaaacct ttcttaaaaa atttttttcg aaaatctttt tgaactcagt ccgtcaatga 60

tctatccttc cttgacgcat aaggcaattc cactgttgca atgaatatat tgcttatggt 120

gaaacgttat cgcttctcat gcgattctat agttaggata aactgattat tgttacgtat 180

tgagtaactg gagtatagac a 201

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 6

gcggccgcta ctagtgttcg atcaacaacc cgaatc 36

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 7

gcccttgctc accatgttta ttctcctaac ttattaagta gc 42

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 8

atggtgagca agggcgag 18

<210> 9

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 9

actagtagcg gccgctg 17

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 10

gccattgacg ctacctt 17

<210> 11

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 11

tggtggcatc gccctcg 17

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 12

ggacgaaaaa ttattttgaa aaatatactg 30

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 13

gagaatctcg ctctctccag gg 22

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 14

aataattttt cgtccctgct tttttcgaaa ataattttat tgtcgattga ggctgatcgg 60

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 15

gagagcgaga ttctccgcgc 20

<210> 16

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 16

ggacgaaaaa ttattttgaa aaatatactg gaataaatgg tcttcgttat gg 52

<210> 17

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 17

tttcaaaata atttttcgtc cctgcttcga tcg 33

<210> 18

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 18

aaaatttttt tcgaaaatct ttttgaactc agtccgtcaa tg 42

<210> 19

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 19

gcccttgctc accattgtct atactccagt tactcaatac gtaac 45

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 20

cgacaattgg ctgggaatgc tatactggaa taaatggtct tcg 43

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 21

cccagccaat tgtcgtccat gcttcgatcg a 31

Claims

1. An endogenous promoter mutant of zymomonas mobilis, wherein the promoter mutant is glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant, the glyceraldehyde-3-phosphate dehydrogenase gene promoter mutant is obtained by 6 site mutations of a glyceraldehyde-3-phosphate dehydrogenase gene promoter, and the specific mutations are as follows:

28T > A, 86G > T, 107C > T, 109G > A, 136T > C and 164C > T;

the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene promoter is shown as SEQ ID NO. 1.

2. A nucleic acid molecule comprising the promoter mutant of claim 1.

3. A chimeric gene comprising the nucleic acid molecule of claim 2, said nucleic acid molecule being chimeric linked to a heterologous nucleic acid molecule.

4. The chimeric gene of claim 3, wherein the heterologous nucleic acid molecule encodes a protein or peptide.

5. A vector comprising the nucleic acid molecule of claim 2.

6. A host cell comprising the vector of claim 5, wherein the host cell comprises a zymomonas mobilis cell and/or an escherichia cell.