Biosensor and its application and method for high-throughput screening of zingerone-producing bacteria

Abstract

The present invention relates to biological detection technology, and discloses a biosensor and its application and a method for high-throughput screening of zingerone-producing bacteria. The biosensor contains a first sensing element and a second sensing element, wherein the first sensing element contains α-tubulin and a first signal protein expressed by fusion with the α-tubulin, and the second sensing element contains β-tubulin and a second signal protein expressed by fusion with the β-tubulin; the end of one of the first signal protein and the second signal protein is the N-terminus and the end of the other is the C-terminus, and the first signal protein and the second signal protein can be combined to form a reporter signal. The biosensor can couple zingerone with a fluorescent signal, efficiently and accurately respond to the concentration of zingerone, and realize high-throughput screening of the intracellular product zingerone in microorganisms.

Description

Technical Field

[0001] The present invention relates to biological detection technology, in particular to a biosensor and its application and a method for high-throughput screening of zingerone-producing bacteria. Background Art

[0002] Zerumbone is an important natural plant product, widely found in plants such as ginger and zingiber officinale. It possesses a variety of biological activities, such as antioxidant, anti-inflammatory, and antibacterial properties, and therefore has broad application potential in drug development, food additives, and flavorings. Currently, zerumbone is primarily produced industrially through chemical synthesis or plant extraction. Due to the low natural yield of zerumbone, traditional plant extraction methods are time-consuming and costly, failing to meet market demand. Chemical synthesis methods are cumbersome, inefficient, and difficult to purify. Microbial methods have emerged to produce zerumbone. For example, metabolic engineering strategies have achieved 40 mg / L of zerumbone in Saccharomyces cerevisiae, but the yield is too low. Rational metabolic engineering efforts struggle to overcome existing bottlenecks and reach new production levels.

[0003] In recent years, high-throughput screening breeding technology has become a research hotspot in industrial microbial breeding. This technology uses strategies such as mutagenesis, directed evolution, and adaptive evolution to construct a mutant library with genetic diversity, and through the design and construction of specific screening methods, the target product concentration is converted into parameters that can be detected with high throughput, including fluorescence intensity, cell density, color depth, etc., in order to screen mutant strains with improved performance. On the one hand, the constructed mutant library has the characteristics of globality and non-specificity, which enables researchers to carry out targeted optimization of the host when they have little understanding of intracellular metabolic regulation, thereby broadening the design scope of metabolic engineering. On the other hand, high-throughput screening breeding technology has the characteristics of faster detection speed, lower cost, and higher throughput. Combined with flow cytometry, droplet microfluidics and other technologies, 10 6 These mutants offer significant advantages in constructing cell factories, facilitating rapid product development. Using synthetic biology techniques to build efficient production platforms in microorganisms is expected to become an important approach for producing zingerone.

[0004] However, there are currently no reports of high-throughput screening methods for zingerone mutants. Because zingerone is an intracellular product of microorganisms, existing gas phase detection methods, such as those used for gas phase analysis, are unable to rapidly and efficiently detect intracellular zingerone. Therefore, the bottleneck in constructing zingerone cell factories lies in the successful development of high-throughput screening technologies that can efficiently and accurately detect zingerone concentrations within microorganisms. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of low zingerone production by microorganisms in the prior art and the inability to achieve high-throughput screening, and to provide a biosensor and its application and a method for high-throughput screening of zingerone-producing bacteria. The biosensor can couple zingerone with a fluorescent signal, efficiently and accurately respond to the zingerone concentration, and achieve high-throughput screening of the intracellular product zingerone in microorganisms.

[0006] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides a biosensor, which comprises a first sensing element and a second sensing element, wherein the first sensing element comprises α-tubulin and a first signal protein expressed by fusion with the α-tubulin, and the second sensing element comprises β-tubulin and a second signal protein expressed by fusion with the β-tubulin; one of the first signal protein and the second signal protein has an N-terminus and the other has a C-terminus, and the first signal protein and the second signal protein can combine to form a reporter signal.

[0007] Preferably, the amino acid sequences of the α-tubulin and the β-tubulin are as shown in SEQ ID NO.1, SEQ ID NO.2, or as shown in SEQ ID NO.3, SEQ ID NO.4, or as shown in SEQ ID NO.5, SEQ ID NO.6, or as shown in SEQ ID NO.7, SEQ ID NO.8.

[0008] Preferably, the amino acid sequence of the α-tubulin is shown as SEQ ID NO.1, and the amino acid sequence of the β-tubulin is shown as SEQ ID NO.2.

[0009] Preferably, the first signal protein and the second signal protein are obtained by cleavage of a reporter protein, and the reporter protein is selected from at least one of GFP, RFP, MeCherry and URA3; and / or,

[0010] The α-tubulin is fused with the first signal protein via a first connecting peptide and expressed, and the β-tubulin is fused with the second signal protein via a second connecting peptide and expressed.

[0011] Preferably, the reporter protein is GFP and its amino acid sequence is as shown in SEQ ID NO.9; the amino acid sequence of GFP is broken at any one of positions 140-160 at the N-terminus to form the first signal protein and the second signal protein; and / or,

[0012] The amino acid sequences of the first connecting peptide and the second connecting peptide are shown in SEQ ID NO.10 respectively.

[0013] Preferably, the first sensor element further comprises a first promoter linked to the α-tubulin, and the second sensor element further comprises a second promoter linked to the β-tubulin;

[0014] The first promoter is AAT2 and / or FAD2, and the second promoter is TEFIN and / or TDH.

[0015] The second aspect of the present invention provides the use of the aforementioned biosensor in detecting the concentration of zingerone and / or screening zingerone-producing bacteria.

[0016] A third aspect of the present invention provides a method for high-throughput screening of zingerone-producing bacteria, the method comprising the following steps:

[0017] S1. Construction of zingerone-producing bacteria containing the aforementioned biosensor as chassis cells;

[0018] S2. Establish a strain mutation library based on the chassis cells, and use a cell screening device to culture the strains in the strain mutation library and sort them based on the reporter signal intensity.

[0019] Preferably, the zingiberone-producing bacteria is a recombinant strain into which a multi-gene co-expression plasmid of P450 hydroxylase-zingiberone synthase-P450 reductase is introduced, and the starting strain of the recombinant strain is an engineered strain of Yarrowia lipolytica with a deposit number of CGMCC No. 24525. The starting strain is constructed by the inventors. Yarrowia lipolyticaGQ3007 was deposited on March 14, 2022 at the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101). The specific preparation process and information have been disclosed in CN114525215A. The vector plasmid for the multigene co-expression plasmid encoding P450 hydroxylase, zingiberenone synthase, and P450 reductase was pGGYL3. For the construction of pGGYL3, see Li YW, Yang CL, Shen Q, Peng QQ, Guo Q, Nie ZK, Sun XM, Shi TQ, Ji XJ and Huang H (2022). YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of MultigenePathway and Terpenoid Production in Yarrowia lipolytica. Front. Bioeng. Biotechnol. 9:816980. doi: 10.3389 / fbioe.2021.816980.

[0020] Preferably, the establishment of the strain mutation library adopts at least one of UV mutagenesis, ARTP mutagenesis, CRISPR-guided whole genome mutagenesis and random integration mutagenesis; and / or,

[0021] The cell screening device adopts at least one of a high-throughput microliter droplet culture genomics system, a flow cytometer, a microplate screening instrument and a microfluidic chip; and / or,

[0022] The isolation and culture conditions include: a temperature of 25-35° C. and a time of 48-120 h; and / or,

[0023] The reporter signal intensity sorting process includes: measuring the reporter signal intensity of the strains in the strain mutation library after separation and culture, obtaining the strain with the lowest reporter signal intensity or obtaining a strain with a reporter signal intensity lower than that of the chassis strain.

[0024] Through the above technical solution, the beneficial effects of the present invention are:

[0025] The biosensor provided by the present invention can couple the zingerone content with the reporter signal intensity to respond to the zingerone concentration efficiently and accurately. By detecting the reporter signal emitted by the microbial strain, the zingerone production can be quickly measured, thereby realizing high-throughput screening of microorganisms that synthesize the intracellular product zingerone. Combined with cell screening devices such as droplet microfluidics or flow cytometers, the intracellular zingerone concentration of the microbial strain is converted into the strength of the reporter signal that can be detected with high throughput. High-throughput screening technology suitable for zingerone-producing bacteria can be developed. This high-throughput screening technology can expand the design scope of metabolic engineering and can carry out targeted optimization of chassis cells when little is known about intracellular metabolic regulation. It has the advantages of high throughput, short time, and low cost.

[0026] Furthermore, the present invention uses an engineered strain of Yarrowia lipolytica as a chassis cell, and the obtained zingerone-producing bacteria have the advantages of raw material regeneration, mild conditions, green production, and no time or location constraints. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the principle of the biosensor for detecting zingerone in the present invention;

[0028] Figure 2 is a graph showing the effect of tubulin from different sources on the fluorescence intensity of the biosensor in Example 2;

[0029] Figure 3 is a graph showing the effect of different connecting peptides on the fluorescence intensity of the biosensor in Example 2;

[0030] Figure 4 2. The effect of different truncation positions of the fluorescent protein on the fluorescence intensity of the biosensor in Example 2;

[0031] Figure 5 is a graph showing the effect of different promoter strengths on the fluorescence intensity of the biosensor in Example 2;

[0032] Figure 6 This is a graph showing the relative fluorescence intensity and zingerone yield of the zingerone-enhanced evolved strains screened by high-throughput biosensor screening in Example 3. DETAILED DESCRIPTION

[0033] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0034] In a first aspect, the present invention provides a biosensor comprising a first sensing element and a second sensing element, wherein the first sensing element comprises α-tubulin and a first signal protein expressed by fusion with the α-tubulin, and the second sensing element comprises β-tubulin and a second signal protein expressed by fusion with the β-tubulin; one of the first signal protein and the second signal protein has an N-terminus and the other has a C-terminus, and the first signal protein and the second signal protein can combine to form a reporter signal.

[0035] The biological sensitive elements in the biosensor can identify the target substance and convert it into observable signals of different intensities. Therefore, the construction of a zingerone biosensor suitable for high-throughput screening methods is an opportunity to solve the difficulties in high-throughput screening and breeding of zingerone cell factories. During the research process, the inventors unexpectedly discovered that zingerone can inhibit the formation of tubulin dimers by embedding into the active site in the binding interface of α-tubulin and β-tubulin. Then, α-tubulin and β-tubulin are used as biological sensitive elements, and the reporter protein is split into two parts to form the first signal protein and the second signal protein. As signal conversion elements, α-tubulin and β-tubulin are fused with the C-terminus and N-terminus of the split reporter protein to form the first sensor element and the second sensor element; when the zingerone concentration is high, the α-tubulin and β-tubulin are fused with the C-terminus and N-terminus of the split reporter protein to form the first sensor element and the second sensor element. When the concentration is low, α-tubulin and β-tubulin form a dimer, allowing the first sensor element to bind to the second sensor element, and the first signal protein and the second signal protein can combine to form a reporter protein. The expression activity of the reporter protein is high, and a strong reporter signal is emitted. When the concentration of zingerone increases, α-tubulin and β-tubulin disaggregate, the binding of the first sensor element and the second sensor element decreases, the activity of the reporter protein decreases, and the reduced protein expression weakens the intensity of the reporter signal. The zingerone content is coupled with the reporter signal intensity (taking the reporter signal as GFP fluorescence signal as an example, the specific detection process principle is shown in FIG). Figure 1 ), it can respond efficiently and accurately to the concentration of zingerone, and quickly measure the production of zingerone by detecting the reporter signal emitted by the microbial strain, thereby realizing high-throughput screening of zingerone, an intracellular product in microorganisms.

[0036] In the present invention, the terminus of one of the first signal protein and the second signal protein is the N-terminus and the terminus of the other is the C-terminus, which specifically refers to when the C-terminus of the first signal protein is expressed in fusion with α-tubulin (that is, the terminus of the first signal protein is the N-terminus), the N-terminus of the second signal protein is expressed in fusion with β-tubulin (that is, the terminus of the second signal protein is the C-terminus); when the N-terminus of the first signal protein is expressed in fusion with α-tubulin (that is, the terminus of the first signal protein is the C-terminus), the C-terminus of the second signal protein is expressed in fusion with β-tubulin (that is, the terminus of the second signal protein is the N-terminus).

[0037] The preparation process of the biosensor provided by the present invention can adopt any method that can fuse two proteins, so as to fuse α-tubulin with the N-terminus or C-terminus of the first signal protein, and fuse β-tubulin with the C-terminus or N-terminus of the second signal protein; for example, using Gibson assembly.

[0038] In the present invention, preferably, the amino acid sequence of the α-tubulin is shown as SEQ ID NO.1, and the amino acid sequence of the β-tubulin is shown as SEQ ID NO.2;

[0039] Alternatively, the amino acid sequence of the α-tubulin is shown in SEQ ID NO.3, and the amino acid sequence of the β-tubulin is shown in SEQ ID NO.4;

[0040] Alternatively, the amino acid sequence of the α-tubulin is shown in SEQ ID NO.5, and the amino acid sequence of the β-tubulin is shown in SEQ ID NO.6;

[0041] Alternatively, the amino acid sequence of the α-tubulin is shown as SEQ ID NO.7, and the amino acid sequence of the β-tubulin is shown as SEQ ID NO.8.

[0042] Further preferably, the amino acid sequence of the α-tubulin is shown in SEQ ID NO. 1, and the amino acid sequence of the β-tubulin is shown in SEQ ID NO. 2. The inventors have found that under this preferred embodiment, the biosensor has higher sensitivity and accuracy in detecting zingerone. In particular, after the biosensor is transformed into a zingerone-producing strain, when zingerone is absent, its report signal intensity is closer to that of the control strain, indicating better compatibility between tubulin and zingerone and better expression.

[0043] In the present invention, the nucleotide sequences of α-tubulin and β-tubulin can be obtained by amino acid sequence conversion. It is well known in the art that of the 20 different amino acids that make up proteins, with the exception of Met (ATG) and Trp (TGG), which are each encoded by a single codon, the other 18 amino acids are each encoded by 2-6 codons. That is, due to the degeneracy of the genetic code, there is often more than one codon that specifies an amino acid. Substitution of the third nucleotide in a triplet codon generally does not change the amino acid composition, so the nucleotide sequences of genes encoding the same protein can differ.

[0044] According to the present invention, preferably, the nucleotide sequence of the α-tubulin is shown as SEQ ID NO. 11, and the nucleotide sequence of the β-tubulin is shown as SEQ ID NO. 12. In this preferred embodiment, the nucleotide sequences of the α-tubulin and β-tubulin undergo specific codon optimization, which can enable the biosensor to detect zingerone with higher sensitivity and accuracy.

[0045] In the present invention, the nucleotide sequences of α-tubulin and β-tubulin can be obtained using polymerase chain reaction (PCR) amplification, recombinant methods, or artificial synthesis methods. For example, based on the nucleotide sequences provided by the present invention, those skilled in the art can easily obtain templates and primers and amplify the relevant sequences using PCR. Once the relevant nucleotide sequences are obtained, the relevant amino acid sequences can be obtained in large quantities using recombinant methods. The obtained nucleotide sequences are typically cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequences are isolated from the propagated host cells using conventional methods.

[0046] In addition, the relevant nucleotide sequences can also be synthesized using known artificial chemical synthesis methods.

[0047] In the present invention, a certain reporter protein is used to break from a certain place to form two parts, one part of the sequence is used as the first signal protein, and the other part of the sequence is used as the second signal protein; the reporter protein can use any protein sequence that can produce a specific reporter signal, for example, a fluorescent protein that produces a fluorescent signal. Preferably, the first signal protein and the second signal protein are obtained by breaking the reporter protein, and the reporter protein is selected from at least one of GFP, RFP, MeCherry and URA3. Further preferably, the reporter protein is GFP, and the amino acid sequence is as shown in SEQ ID NO.9; the amino acid sequence of GFP is broken at any one of positions 140-160 at the N-terminus to form the first signal protein and the second signal protein. Under this preferred embodiment, the binding effect of the first signal protein and the second signal protein when α-tubulin and β-tubulin form a dimer can be further improved, the signal of the reporter protein can be enhanced, and the detection sensitivity and accuracy of the biosensor for zingerone can be higher.

[0048] More preferably, the nucleotide sequence of the reporter protein GFP is shown as SEQ ID NO.13.

[0049] In the present invention, the α-tubulin can be directly connected to the first signal protein, or it can be formed with the first signal protein through a connecting peptide to form a first sensing element; similarly, the β-tubulin can be directly connected to the second signal protein, or it can be formed with the second signal protein through a connecting peptide to form a second sensing element. Preferably, the α-tubulin is expressed by fusion with the first signal protein through a first connecting peptide, and the β-tubulin is expressed by fusion with the second signal protein through a second connecting peptide. Further preferably, the amino acid sequences of the first connecting peptide and the second connecting peptide are respectively as shown in SEQ ID NO.10. Under this preferred embodiment, the binding effect of the first signal protein and the second signal protein when α-tubulin and β-tubulin form a dimer can be further improved, the reporter signal can be enhanced, and the detection sensitivity and accuracy of the biosensor for zingerone can be higher.

[0050] According to the present invention, preferably, the first sensing element further comprises a first promoter linked to the α-tubulin protein, and the second sensing element further comprises a second promoter linked to the β-tubulin protein; the first promoter is a medium-strength promoter, and the second promoter is a strong promoter. Further preferably, the first promoter is AAT2 and / or FAD2, and the second promoter is TEFIN and / or TDH. This preferred embodiment further enhances the binding of the first and second signal proteins when α-tubulin and β-tubulin form dimers, amplifying the reporter signal and improving the biosensor's sensitivity and accuracy in detecting zingerone.

[0051] The biosensor provided by the present invention can achieve the coupling of zingerone concentration and fluorescence intensity, and can be used to detect the concentration of zingerone in a sample. It can also be used to convert the intracellular zingerone concentration of microorganisms into fluorescence intensity that can be detected with high throughput, and quickly measure and judge the yield of zingerone synthesis. For this purpose, a high-throughput microbial strain screening technology suitable for zingerone is developed, and then a cell factory with high zingerone production is obtained through this high-throughput screening and breeding technology.

[0052] The second aspect of the present invention provides the use of the aforementioned biosensor in detecting the concentration of zingerone and / or screening zingerone-producing bacteria.

[0053] A third aspect of the present invention provides a method for high-throughput screening of zingerone-producing bacteria, the method comprising the following steps:

[0054] S1. Construction of zingiberone-producing bacteria containing the aforementioned biosensor as chassis cells;

[0055] S2. Establish a strain mutation library based on the chassis cells, and use a cell screening device to culture the strains in the strain mutation library and sort them based on the reporter signal intensity.

[0056] The method for high-throughput screening of zingerone-producing bacteria provided by the present invention has a simple process and can efficiently and accurately screen out high-yielding zingerone-producing bacterial strains. The process is easy to cooperate with various cell screening devices in the prior art and has strong operability.

[0057] In the present invention, the zingerone-producing bacteria can be any strain capable of synthesizing zingerone, for example, Yarrowia lipolytica, Saccharomyces cerevisiae, etc. Preferably, the zingerone-producing bacteria is a recombinant strain into which a multi-gene co-expression plasmid for P450 hydroxylase-zingerone synthase-P450 reductase has been introduced, and the starting strain of the recombinant strain is an engineered strain of Yarrowia lipolytica with a deposit number of CGMCC No. 24525.

[0058] In the present invention, the zingerone-producing bacteria containing the biosensor can be constructed by using conventional gene transformation methods in the art to transfer the biosensor into the zingerone-producing bacteria, for example, by transferring the recombinant plasmid containing the biosensor into the competent cells of the zingerone-producing bacteria.

[0059] According to the present invention, the strain mutation library can be established by mutagenizing the chassis cells using conventional microbial mutagenesis methods to obtain a mutant library comprising multiple chassis cell strains. Preferably, the strain mutation library is established using at least one of UV mutagenesis, ARTP mutagenesis, CRISPR-guided whole-genome mutagenesis, and random integration mutagenesis.

[0060] Exemplarily, the process of establishing a strain mutation library includes: first constructing a pUC-B2-HUH-Cas9 integration plasmid containing a URA3 selection marker and carrying a Cas9 protein controlled by a strong TEFin promoter and an XPR2t terminator. Using homologous recombination technology, the URA3 and Cas9 expression cassettes are integrated into a zingiberone-producing bacterium containing a biosensor and producing zingiberone as a chassis cell; subsequently, based on the genomic information of the chassis cells, a sgRNA library targeting each gene of the genome is synthesized and cloned into a replicating plasmid containing a LEU selection marker through high-throughput Gibson assembly. To ensure editing efficiency, three sgRNAs targeting different sites are designed for each gene; the constructed sgRNA plasmid library is transformed into the above-mentioned chassis cells, and the transformants are cultured at 30°C for 96 hours in a droplet containing a leucine nutrient-deficient medium to obtain a strain mutation library.

[0061] According to the present invention, preferably, the cell screening device adopts at least one of a high-throughput microliter droplet culture omics system, a flow cytometer, a microplate screening instrument and a microfluidic chip, and more preferably a high-throughput microliter droplet culture omics system (MISS cell omics system).

[0062] In the present invention, the culturing may be to first mix and culture the strains in the strain mutation library and then directly sort them for reporter signal intensity, or to separate the strains and then culture them and then sort them for reporter signal intensity.

[0063] Taking the high-throughput microliter droplet culture omics system as an example, the strain culture conditions in the strain mutation library include: the internal phase is the strain culture medium, and the external phase is mineral oil, fluorinated oil, soybean oil, silicone oil, tetradecane, etc., preferably mineral oil; the surfactants are Span 80 (added amount is 0.5-5%), Tween 80 (added amount is 0.3-3%), Pluronic F-68 (added amount is 0.1-1%), preferably Span 80; the internal phase flow rate is usually set between 0.23 and 1.3 μL / min, while the external phase flow rate is between 8.1 and 50 μL / min.

[0064] According to the present invention, the zingerone-producing bacteria is an engineered strain of Yarrowia lipolytica, and when the deposit number is CGMCC No. 24525, preferably, the culture conditions include: a temperature of 25-35°C, specifically 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the above two numbers; a time of 48-120h, specifically 48h, 60h, 72h, 84h, 96h, 108h, 120h, or any value between the above two numbers. Under this preferred embodiment, it is beneficial to promote the growth and reproduction of cells in the strain mutant library and improve the sensitivity and accuracy of detection and screening.

[0065] According to the present invention, preferably, the reporter signal intensity sorting process includes: measuring the reporter signal intensity of the strains in the strain mutation library after the culture, obtaining a strain with the lowest reporter signal intensity or obtaining a strain with a reporter signal intensity lower than that of the chassis strain. The higher the concentration of intracellular zingerone in the strain, the weaker the reporter signal intensity generated by the biosensor, so as to couple the zingerone content with the reporter signal intensity, efficiently and accurately respond to the zingerone production synthesized in the strain, and quickly measure the zingerone production by detecting the fluorescence emitted by the microbial strain, thereby realizing high-throughput screening of the intracellular product zingerone in the microorganism.

[0066] The present invention will be described in detail below through examples.

[0067] In the following examples, unless otherwise specified, the remaining raw materials or reagents are conventional commercially available products.

[0068] YPD liquid medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L;

[0069] YPD solid medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, agar powder 20 g / L;

[0070] YPD 60 Fermentation medium: peptone 20g / L, yeast powder 10g / L, glucose 60g / L,

[0071] The content of zingerone was detected by gas chromatography (GC). The specific process was as follows:

[0072] Gas chromatograph model: Shimadzu QP2020NX, Japan, HP-5MS chromatographic column (30m×320μm×0.5μm); detection conditions of zingerone: injection port temperature 250°C, injection volume 1μL, split ratio 20:1; chromatographic column: HP-5ms (30m×0.25mM); chromatographic conditions: initial temperature 60°C, increased to 150°C at a rate of 10°C / min, then increased to 280°C at 20°C / min, and maintained for 2min; zingerone standards were used for qualitative and quantitative analysis.

[0073] Example 1

[0074] 1. Construction of component plasmids

[0075] According to the CYP71BA1, ZSD1S144A, and AtCPR nucleotide sequences provided by NCBI, after specific codon optimization, they were commissioned to Qingke Biotechnology Co., Ltd. for synthesis and inserted into plasmid pUC57 to obtain plasmids pUC57-34-CYP71BA1, pUC57-910-ZSD1S144A, and pUC57-67-AtCPR, respectively.

[0076] The constructed element plasmids pUC57-34-CYP71BA1, pUC57-910-ZSD1S144A, and pUC57-67-AtCPR were digested with restriction endonuclease BsaI and the target fragments were recovered by gel running.

[0077] Among them, P450 hydroxylase CYP71BA1 (the amino acid sequence of P450 hydroxylase is shown in SEQ ID NO.14, and the nucleotide sequence encoding the gene CYP71BA1 is shown in SEQ ID NO.15),

[0078] Zingiberone synthase ZSD1S144A (the amino acid sequence of zingiberone synthase is shown in SEQ ID NO.16, and the nucleotide sequence encoding the gene ZSD1S144A is shown in SEQ ID NO.17), P450 reductase AtCPR (NCBI No.: BT008426).

[0079] The above coding genes were all codon-optimized and then commissioned to Qingke Biotechnology Co., Ltd. for synthesis.

[0080] 2. Construction of multi-gene co-expression plasmid

[0081] The target fragments in the element plasmid were connected with pGGYL3 as the backbone and T4 ligase. The multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR of the zingiberenone biosynthesis pathway was constructed using Golden Gate modular assembly technology, and then amplified using the Yarrowia lipolytica genome as a template.

[0082] 3. Construction of recombinant strains

[0083] The Yarrowia lipolytica engineered strain GQ3007, which produces high α-humulene, was used as the starting strain. The specific construction process was based on the method disclosed in CN114525215A, with a deposit number of CGMCC No. 24525.

[0084] The starting strain Yarrowia lipolytica engineered strain GQ3007 was cultured in YPD liquid medium for 12 h until OD 600 The p-value of the yeast strain was 0.8. The multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR obtained in Example 1 was transformed into the starting strain using the Zymogen Frozen EZ Yeast Transformation Kit II kit produced by Zymo Research Corporation for homologous recombination to prepare competent cells. After each multi-gene co-expression plasmid was transformed, it was screened using screening medium, and the correct positive clones were identified by PCR to obtain the recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR.

[0085] The PCR enzyme used in PCR amplification was TAKARA's PrimeSTAR Max DNA polymerase; the PCR amplification system is shown in Table 1.

[0086] Table 1

[0087]

[0088] The PCR amplification process was as follows: denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C. After 35 cycles, each fragment was purified and recovered using the AxyPrep™ DNA Gel Extraction Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd.). The extension time was calculated as the target fragment length / 1 kb (in minutes). One-step cloning was then achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The reaction system is shown in Table 2.

[0089] Table 2

[0090]

[0091] 4. Fermentation to produce zingerone

[0092] The recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR was streaked on a YPD solid medium plate. After culturing for 2-3 days, a single colony was picked and transferred to YPD liquid medium. The culture was carried out at 30°C and 220 rpm to obtain the recombinant strain I seed solution. The recombinant strain I seed solution was then inoculated into 50 mL of YPD medium at a volume ratio of 1%. 60 In the fermentation medium, after shaking culture at 30°C and 220 rpm for 24 h, YPD was added. 60 The fermentation medium was filled with 25% n-dodecane by volume, and the shaking culture was continued for 72 h to obtain the fermentation broth of the recombinant strain I.

[0093] The fermentation broth of the recombinant strain I was transferred to 50 mL centrifuge tubes, centrifuged at 8000 rpm for 5 min, and the upper organic phase was collected and filtered. The yield of zingerone was detected to be 6.3 mg / L.

[0094] Example 2 Construction of a biosensor based on α-tubulin and β-tubulin

[0095] Based on the nucleotide sequences of α-tubulin and β-tubulin provided by NCBI, after specific codon optimization, they were commissioned to Qingke Biotechnology Co., Ltd. for synthesis and inserted into plasmid pUC57 to obtain plasmid pUC57-α-tubulin and plasmid pUC57-β-tubulin, respectively.

[0096] The promoter and terminator used were endogenous to Yarrowia lipolytica , so the Yarrowia lipolytica genome was used as a template for amplification;

[0097] Using pUC-intB2-HUH as the backbone, the target fragments α-tubulin and the first signal protein were inserted into the recombinant plasmid pUC-HUH-intB2-(α-tubulin-N first signal protein) by Gibson assembly. The target fragments β-tubulin and the second signal protein were then inserted into the recombinant plasmid pUC-HUH-intB2-(α-tubulin-N first signal protein)-(β-tubulin-C second signal protein). The PCR enzyme used in the PCR amplification was TAKARA PrimeSTAR Max DNA polymerase. The PCR amplification system is shown in Table 1.

[0098] The PCR amplification process was as follows: denaturation at 98°C for 10 seconds, annealing at 55°C for 10 seconds, and extension at 72°C. After 35 cycles, each fragment was purified and recovered using the AxyPrepTM DNA Gel Extraction Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd.).

[0099] Where, extension time = target fragment length / 1kb, unit min

[0100] Fragment ligation (one-step cloning):

[0101] One-step cloning was achieved using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotechnology Co., Ltd. The reaction system is shown in Table 2. The reaction system was incubated at 50°C for 15 minutes to obtain a circular recombinant vector.

[0102] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0103] Yarrowia lipolytica po1f (Yarrowia lipolytica strain MYA2613 purchased from the American Type Culture Collection ATCC) was cultured in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) for 12 h until OD 600 was 0.8; the linearized plasmid pUC-HUH-intB2-(α-tubulin-N first signal protein)-(β-tubulin-C second signal protein) was transformed into Yarrowia lipolytica using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation for homologous recombination to prepare competent cells (Kit: Zymogen Frozen EZ Yeast Transformation Kit II, Manufacturer: Zymo Research Corporation); the above plasmid was transformed into competent cells, and screening was performed using screening medium. The positive clones identified correctly by PCR were obtained to obtain the test strains.

[0104] The recombinant plasmid pUC-HUH-intB2-reporter protein GFP expressing the reporter protein alone was constructed and integrated into the Yarrowia lipolytica po1f strain, which was used as a control strain.

[0105] The strain to be tested was picked from the streaked plate and cultured overnight in YPD medium. It was then transferred to the fermentation medium at a 10% inoculation ratio, with two conditions: the absence of zingerone and the presence of zingerone (concentration of 100 mg / 100 mL). After culturing for 24 hours, samples were taken and the excitation and absorption wavelengths of the test were set to 488 nm and 520 nm, respectively, using a microplate reader (no brand requirements). The OD of the strain growth was measured at a wavelength of 600 nm. The reported signal intensity = fluorescence value / OD 600 , the relative fluorescence intensity was calculated based on the reporter signal intensity of the control strain, and comparative analysis was performed.

[0106] The above experimental process was conducted on multiple groups of tests. The types of α-tubulin, β-tubulin, first signal protein, second signal protein, first connecting peptide, first promoter, and second promoter in each group are specifically shown in Table 3. Among them, the amino acid sequence of the reporter protein GFP is shown in SEQ ID NO.9, the nucleotide sequence is shown in SEQ ID NO.13, and the second connecting peptide is the remaining sequence of GFP after removing the first signal protein.

[0107] Table 3

[0108]

[0109] The test results of groups 1, 2, 3, and 4 are as follows Figures 2 to 5 As shown in the figure, after comparative analysis, the optimal preparation method of the biosensor was finally obtained as group 4-4, specifically: Euplotes octocarinatus The microtubule protein α-tubulin from the plant was fused with the N-terminal 450bp domain of green fluorescent protein GFP, connected by a GGGGS linker in the middle, and the medium-strength promoter AAT2 was used to express the fusion of α-tubulin and GFP as the first sensor element; β-tubulin was fused with the C-terminal 270bp domain of green fluorescent protein GFP, connected by a GGGGS linker in the middle, and expressed by the strong promoter TEFin as the second sensor element; both were cloned and integrated into the plasmid pUC-HUH-intB2, and transformed into the above-mentioned zingerone-producing bacteria (the recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR obtained in Example 1) to obtain a recombinant strain II containing a biosensor and producing zingerone, and the zingerone content was characterized by fluorescence. The fluorescence signal results showed that when there was no zingerone, the fluorescence intensity of the recombinant strain II was close to that of the control strain, and the relative fluorescence reached more than 90% of that of the control strain, indicating that the first sensor element and the second sensor element were tightly bound to form a dimer.

[0110] Example 3 High-throughput screening of zingerone-producing evolved strains based on the constructed biosensor

[0111] The recombinant strain II obtained in Example 2 was used to establish a strain mutation library. The strains with weakened fluorescence intensity were sorted using the MISS cell high-throughput microliter droplet culture omics system, and shake flask fermentation was used for verification. Specifically:

[0112] First, a pUC-B2-HUH-Cas9 integration plasmid was constructed. This plasmid contains the URA3 selection marker and carries the Cas9 protein controlled by the TEFin strong promoter and the XPR2t terminator. Using homologous recombination technology, the URA3 and Cas9 expression cassettes were integrated into the chassis cells containing the biosensor and producing zingerone (recombinant strain II in Example 2).

[0113] Subsequently, based on the genomic information of the chassis cells, a sgRNA library targeting each gene of the genome was synthesized and cloned into a replicative plasmid containing the LEU screening marker through high-throughput Gibson assembly. In order to ensure the editing efficiency, three sgRNAs targeting different sites were designed for each gene; the constructed sgRNA plasmid library was transformed into the above-mentioned chassis cells to obtain a strain mutation library; each transformant in the strain mutation library was cultured at 30°C in a droplet containing leucine nutrient-deficient medium using the MISS cell high-throughput microliter droplet culture omics system for 96 hours; specific conditions: the external phase was mineral oil containing 1% Span 80, the internal phase flow rate was 0.4 μL / min, and the external phase flow rate was 38 μL / min. The droplets of each strain in the strain mutation library were then tested for fluorescence intensity, and 40 single cells with relatively different fluorescence were sorted out. The relative fluorescence intensities are shown in Tables 4 and Figure 6 Finally, the sorted single cells were preserved and fermented in shake flasks for verification. The yield of zingerone is shown in Table 4 and Figure 6 shown.

[0114] The process steps of shake flask fermentation verification are as follows: the sorted single cells are streaked on a plate of YPD solid medium, cultured for 2.5 days, and then a single colony is picked and transferred to YPD liquid medium. The culture is carried out at 30°C and 220 rpm to obtain a single cell seed solution. The single cell seed solution is then inoculated into 50 mL of YPD at a volume ratio of 1%. 60 In the fermentation medium, after shaking culture at 30°C and 220 rpm for 24 h, YPD was added. 60 The fermentation medium was filled with 25% n-dodecane by volume, and the culture was continued with shaking for 72 h to obtain a single-cell fermentation broth.

[0115] The fermentation broth of the single cells was transferred to 50 mL centrifuge tubes, centrifuged at 8000 rpm for 5 min, and the upper organic phase was collected and filtered to detect the yield of zingerone.

[0116] Table 4

[0117]

[0118] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A biosensor, characterized in that: The biosensor comprises a first sensing element and a second sensing element, wherein the first sensing element comprises α-tubulin and a first signal protein expressed by fusion with the α-tubulin, and the second sensing element comprises β-tubulin and a second signal protein expressed by fusion with the β-tubulin; one of the first signal protein and the second signal protein has an N-terminus and the other has a C-terminus, and the first signal protein and the second signal protein can combine to form a reporter signal; The first signal protein and the second signal protein are obtained by cleavage of a reporter protein, the reporter protein is GFP and the amino acid sequence is shown in SEQ ID NO.9; the amino acid sequence of GFP is cleaved at any one of positions 83, 117, 150, and 183 at the N-terminus to form the first signal protein and the second signal protein.

2. The biosensor according to claim 1, wherein The amino acid sequences of the α-tubulin and the β-tubulin are shown in SEQ ID NO.1 and SEQ ID NO.2, or in SEQ ID NO.3 and SEQ ID NO.4, or in SEQ ID NO.5 and SEQ ID NO.6, or in SEQ ID NO.7 and SEQ ID NO.

8.

3. The biosensor according to claim 2, wherein The amino acid sequence of the α-tubulin is shown in SEQ ID NO.1, and the amino acid sequence of the β-tubulin is shown in SEQ ID NO.

2.

4. The biosensor according to any one of claims 1 to 3, characterized in that The α-tubulin is fused with the first signal protein via a first connecting peptide and expressed, and the β-tubulin is fused with the second signal protein via a second connecting peptide and expressed.

5. The biosensor according to claim 4, wherein The amino acid sequences of the first connecting peptide and the second connecting peptide are shown in SEQ ID NO.10 respectively.

6. The biosensor according to any one of claims 1 to 3, characterized in that The first sensor element further comprises a first promoter linked to the α-tubulin, and the second sensor element further comprises a second promoter linked to the β-tubulin; The first promoter is AAT2 and / or FAD2, and the second promoter is TEFIN and / or TDH.

7. Use of the biosensor according to any one of claims 1 to 6 in detecting the concentration of zingerone and / or screening zingerone-producing bacteria.

8. A method for high-throughput screening of zingerone-producing bacteria, characterized in that: The method comprises the following steps: S1. Constructing a zingiberone-producing bacterium containing the biosensor according to any one of claims 1 to 6 as a chassis cell; S2. Establish a strain mutation library based on the chassis cells, and use a cell screening device to culture the strains in the strain mutation library and sort them based on the reporter signal intensity.

9. The method according to claim 8, characterized in that The zingiberone-producing bacteria is a recombinant strain into which a multi-gene co-expression plasmid of P450 hydroxylase-zingiberone synthase-P450 reductase is introduced. The starting strain of the recombinant strain is an engineered strain of Yarrowia lipolytica with a deposit number of CGMCC No. 24525.

10. The method according to claim 8 or 9, characterized in that The establishment of the strain mutation library adopts at least one of UV mutagenesis, ARTP mutagenesis, CRISPR-guided whole genome mutagenesis and random integration mutagenesis; and / or, The cell screening device adopts at least one of a high-throughput microliter droplet culture genomics system, a flow cytometer, a microplate screening instrument and a microfluidic chip; and / or, The culture conditions include: a temperature of 25-35°C and a time of 48-120 hours; and / or, The reporter signal intensity sorting process includes: measuring the reporter signal intensity of the strains in the strain mutation library after the culture, obtaining the strain with the lowest reporter signal intensity or obtaining a strain with a reporter signal intensity lower than that of the chassis strain.

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