Biosensor, application thereof and method for high-throughput screening of zingiberone producing strains

Abstract

The invention relates to a biological detection technology, and discloses a biosensor, application thereof and a method for high-throughput screening of zingiberone producing strains. The biosensor comprises a first sensing element and a second sensing element, the first sensing element comprises alpha-tubulin and a first signal protein which is in fusion expression with the alpha-tubulin, and the second sensing element comprises beta-tubulin and a second signal protein which is in fusion expression with the beta-tubulin; the tail end where one of the first signal protein and the second signal protein is located is an N end, the tail end where the other one of the first signal protein and the second signal protein is located is a C end, and the first signal protein and the second signal protein can be combined to form a report signal. The biosensor can couple zingiberone with a fluorescence signal, efficiently and accurately respond to the concentration of zingiberone, and realize high-throughput screening of an intracellular product zingiberone in microorganisms.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and in particular, to a biosensor and its applications, as well as a method for high-throughput screening of zerumbone-producing bacteria. Background Art

[0002] Zerumbone is an important natural plant product, widely present in plants such as ginger and Zingiber zerumbet, and has various biological activities, such as antioxidant, anti-inflammatory, antibacterial, etc. Therefore, it has broad application potential in the fields of drug development, food additives, and spices. Currently, zerumbone is mainly industrially produced by chemical synthesis or plant extraction methods; due to the low natural yield of zerumbone, the traditional extraction method from plants is both time-consuming and costly, unable to meet market demand. The chemical synthesis method has cumbersome steps, low efficiency, and difficult purification. Currently, there has emerged a method of producing zerumbone using microorganisms. For example, 40 mg / L of zerumbone has been achieved in Saccharomyces cerevisiae through metabolic engineering strategies, but the yield is too low; rational metabolic engineering is difficult to break through the existing bottleneck to reach a new yield level.

[0003] In recent years, high-throughput screening and breeding technologies have 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 by designing and constructing specific screening methods, converts the target product concentration into parameters that can be detected by high-throughput, including fluorescence intensity, cell density, color depth, etc., so as to screen mutant strains with improved performance. On the one hand, the constructed mutant library has characteristics such as global and non-specific, enabling researchers to perform directional optimization of the host with less understanding of intracellular metabolic regulation, broadening the design scope of metabolic engineering. On the other hand, high-throughput screening and breeding technologies have characteristics such as faster detection speed, lower cost, and larger throughput. Combining technologies such as flow cytometry and droplet microfluidics, more than 10 6 above mutants can be detected in one day, having great advantages in constructing cell factories and promoting the rapid development of products. Using synthetic biology technology to construct an efficient production platform in microorganisms is expected to become an important way to produce zerumbone.

[0004] However, there is currently no report on a high-throughput screening method for zerumbone mutant strains. Since zerumbone is an intracellular product of microorganisms, existing gas detection methods and the like cannot rapidly and high-throughput detect intracellular zerumbone. Therefore, the "bottleneck" problem in realizing the construction of a zerumbone cell factory lies in whether an efficient and accurate high-throughput screening technology that responds to the intracellular zerumbone concentration of microorganisms can be successfully developed. Summary of the Invention

[0005] The object of the present invention is to overcome the problems in the prior art that the yield of zerumbone produced by microorganisms is low and high-throughput screening cannot be achieved, and to provide a biosensor, its application and a method for high-throughput screening of zerumbone-producing bacteria. This biosensor can couple zerumbone with a fluorescence signal, efficiently and accurately respond to the concentration of zerumbone, and achieve high-throughput screening of intracellular product zerumbone in microorganisms.

[0006] To achieve the above object, the first aspect of the present invention provides a biosensor, which contains a first sensing element and a second sensing element. The first sensing element contains α-tubulin and a first signal protein fused with the α-tubulin. The second sensing element contains β-tubulin and a second signal protein fused with the β-tubulin. One of the first signal protein and the second signal protein has an N-terminal end, and the other has a C-terminal end. 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 and SEQ ID NO.2, or as shown in SEQ ID NO.3 and SEQ ID NO.4, or as shown in SEQ ID NO.5 and SEQ ID NO.6, or as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0008] Preferably, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.1, and the amino acid sequence of the β-tubulin is as shown in 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, The α-tubulin is fused with the first signal protein through a first linker peptide, and the β-tubulin is fused with the second signal protein through a second linker peptide.

[0010] 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 cleaved at any position between the 140th and 160th positions at the N-terminal to form the first signal protein and the second signal protein; and / or, The amino acid sequences of the first linker peptide and the second linker peptide are respectively as shown in SEQ ID NO.10.

[0011] Preferably, the first sensing element further contains a first promoter linked to the α-tubulin, and the second sensing element further contains 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.

[0012] The second aspect of the present invention provides the application of the biosensor as described above in detecting the concentration of zerumbone and / or screening zerumbone-producing bacteria.

[0013] The third aspect of the present invention provides a method for high-throughput screening of zerumbone-producing bacteria, which includes the following steps: S1. Construct a zerumbone-producing bacterium containing the biosensor as described above as a chassis cell; S2. Establish a strain mutant library based on the chassis cell, and use a cell screening device to culture and sort the strains in the strain mutant library according to the signal intensity of the reporter.

[0014] Preferably, the zerumbone-producing bacterium is a recombinant strain introduced with a multi-gene co-expression plasmid of P450 hydroxylase-zerumbone synthase-P450 reductase, and the starting strain of this recombinant strain is the Yarrowia lipolytica engineering strain with the preservation number of CGMCC No. 24525. This starting strain was constructed by the inventor Yarrowia lipolytica GQ3007, which was deposited on March 14, 2022 at the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101). The specific preparation process and information have been disclosed in CN114525215A. Among them, the vector plasmid of the multi-gene co-expression plasmid of P450 hydroxylase-zerumbone synthase-P450 reductase is pGGYL3, and the construction method of pGGYL3 can be referred to Li Y-W, Yang C-L, Shen Q, Peng Q-Q, Guo Q, Nie Z-K, Sun X-M, Shi T-Q, Ji X-J and Huang H (2022). YALIclone NHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica. Front. Bioeng. Biotechnol. 9:816980. doi: 10.3389 / fbioe.2021.816980.

[0015] Preferably, at least one of ultraviolet mutagenesis, ARTP mutagenesis, CRISPR-guided genome-wide mutagenesis, and random integration mutagenesis is used to establish the strain mutant library; and / or, at least one of a high-throughput microliter-scale droplet culture omics system, a flow cytometer, a microplate screening instrument, and a microfluidic chip is used as the cell screening device; and / or, the conditions for the separation and culture include: a temperature of 25-35°C and a time of 48-120 h; and / or, the process of sorting by the reporter signal intensity includes: measuring the reporter signal intensity of the strains in the strain mutant library after separation and culture, and obtaining the strain with the lowest reporter signal intensity or the strain with a reporter signal intensity lower than that of the chassis strain.

[0016] Through the above technical solutions, the beneficial effects of the present invention are as follows: The biosensor provided by the present invention can couple the content of zerumbone with the reporter signal intensity to efficiently and accurately respond to the zerumbone concentration. By detecting the reporter signal emitted by the microbial strain, the yield of zerumbone can be quickly measured, realizing the high-throughput screening of microorganisms synthesizing intracellular product zerumbone; combined with cell screening devices such as droplet microfluidics or flow cytometers, the intracellular zerumbone concentration of microbial strains is converted into the strength of a report signal that can be detected by high throughput, and a high-throughput screening technology applicable to zerumbone-producing bacteria can be developed. Moreover, this high-throughput screening technology can expand the design scope of metabolic engineering, and can perform directional optimization on chassis cells with less understanding of intracellular metabolic regulation, having advantages such as high throughput, short time, and low cost.

[0017] Furthermore, the present invention uses an engineered strain of Yarrowia lipolytica as the chassis cell, and the obtained zerumbone-producing bacteria have the advantages of raw material regeneration, mild conditions, green production, and being unrestricted by time and location. Description of the Drawings

[0018] Figure 1 is the schematic diagram of the biosensor detecting zerumbone in the present invention; Figure 2 is the graph showing the influence of tubulin from different sources on the fluorescence intensity of the biosensor in Example 2; Figure 3 is the graph showing the influence of different linkers on the fluorescence intensity of the biosensor in Example 2; Figure 4 is the graph showing the influence of different truncation positions of the fluorescent protein on the fluorescence intensity of the biosensor in Example 2; Figure 5 is the graph showing the influence of different promoter strengths on the fluorescence intensity of the biosensor in Example 2; Figure 6It is a graph showing the relative fluorescence intensity and zerumbone yield of the evolved strain of zerumbone with improved high-throughput screening yield of the biosensor in Example 3. Detailed implementation mode

[0019] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0020] The first aspect of the present invention provides a biosensor, which contains a first sensing element and a second sensing element. The first sensing element contains α-tubulin and a first signal protein fused and expressed with the α-tubulin. The second sensing element contains β-tubulin and a second signal protein fused and expressed with the β-tubulin. One of the first signal protein and the second signal protein has an N-terminal end and the other has a C-terminal end, and the first signal protein and the second signal protein can combine to form a reporting signal.

[0021] The biorecognition element in the biosensor can recognize the target substance and convert it into observable signals with different intensities. Therefore, constructing a zerumbone biosensor suitable for high-throughput screening methods is an opportunity to solve the difficulties of high-throughput screening and breeding of zerumbone cell factories. During the research process, the inventor unexpectedly found that zerumbone inhibits the formation of tubulin dimers by embedding into the active site in the binding interface between α-tubulin and β-tubulin. Furthermore, α-tubulin and β-tubulin are used as biorecognition elements, and the reporter protein is split into two parts to form a first signal protein and a second signal protein, which are used as signal conversion elements. α-tubulin and β-tubulin are respectively fused and expressed with the C-terminal and N-terminal ends of the split reporter protein to form a first sensing element and a second sensing element. When the concentration of zerumbone is low, α-tubulin and β-tubulin form dimers, causing the first sensing element and the second sensing element to combine, and the first signal protein and the second signal protein can combine to form a reporter protein with high expression activity and emit a strong reporting signal. When the concentration of zerumbone increases, α-tubulin and β-tubulin depolymerize, the combination of the first sensing element and the second sensing element decreases, the activity of the reporter protein decreases, and the reduction of protein expression weakens the intensity of the reporting signal, so as to couple the content of zerumbone with the intensity of the reporting signal (taking the reporting signal as the GFP fluorescence signal as an example, the principle of the specific detection process is shown in Figure 1), efficiently and accurately respond to the concentration of zerumbone, quickly measure the yield of zerumbone by detecting the reporter signal emitted by the microbial strain, and achieve high-throughput screening of the intracellular product zerumbone in microorganisms.

[0022] In the present invention, one of the first signal protein and the second signal protein has an N-terminal end and the other has a C-terminal end. Specifically, when the C-terminal end of the first signal protein is fused and expressed with α-tubulin (i.e., the end where the first signal protein is located is the N-terminal end), the N-terminal end of the second signal protein is fused and expressed with β-tubulin (i.e., the end where the second signal protein is located is the C-terminal end); when the N-terminal end of the first signal protein is fused and expressed with α-tubulin (i.e., the end where the first signal protein is located is the C-terminal end), the C-terminal end of the second signal protein is fused and expressed with β-tubulin (i.e., the end where the second signal protein is located is the N-terminal end).

[0023] The preparation process of the biosensor provided by the present invention can adopt any method capable of fusing and expressing two proteins to fuse and express α-tubulin with the N-terminal or C-terminal end of the first signal protein, and fuse and express β-tubulin with the C-terminal or N-terminal end of the second signal protein; for example, Gibson assembly is adopted.

[0024] In the present invention, preferably, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.1, and the amino acid sequence of the β-tubulin is as shown in SEQ ID NO.2; Or, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.3, and the amino acid sequence of the β-tubulin is as shown in SEQ ID NO.4; Or, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.5, and the amino acid sequence of the β-tubulin is as shown in SEQ ID NO.6; Or, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.7, and the amino acid sequence of the β-tubulin is as shown in SEQ ID NO.8.

[0025] More preferably, the amino acid sequence of the α-tubulin is as shown in SEQ ID NO.1, and the amino acid sequence of the β-tubulin is as shown in SEQ ID NO.2. The inventors found that under this preferred specific embodiment, the biosensor has higher detection sensitivity and accuracy for zerumbone. Especially after the biosensor is transformed into the zerumbone-producing bacteria, when there is no zerumbone present, the intensity of its reporter signal is closer to that of the control strain, and the adaptability and expression of tubulin and zerumbone are better and more excellent.

[0026] In the present invention, the nucleotide sequences of α-tubulin and β-tubulin can be obtained by amino acid sequence conversion. As is well known in the art, among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) is encoded by a single codon respectively, the other 18 amino acids are each encoded by 2 - 6 codons. That is, due to the degeneracy of the genetic code, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different.

[0027] According to the present invention, preferably, the nucleotide sequence of the α-tubulin is as shown in SEQ ID NO.11, and the nucleotide sequence of the β-tubulin is as shown in SEQ ID NO.12. Under this preferred embodiment, the nucleotide sequences of α-tubulin and β-tubulin are optimized by specific codons, which can make the biosensor have higher detection sensitivity and accuracy for zerumbone.

[0028] In the present invention, the nucleotide sequences of α-tubulin and β-tubulin can be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. For example, those skilled in the art can easily obtain the template and primers according to the nucleotide sequences provided by the present invention, and use PCR to amplify the relevant sequences. Once the relevant nucleotide sequences are obtained, the relevant amino acid sequences can be obtained in large quantities by recombination. Usually, the obtained nucleotide sequences are cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequences are separated from the proliferated host cells by conventional methods.

[0029] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0030] In the present invention, a certain reporter protein is cleaved at a certain site to form two parts, one part of the sequence serves as the first signal protein, and the other part of the sequence serves as the second signal protein; the reporter protein can be any protein sequence capable of generating a specific reporter signal, for example, a fluorescent protein that generates a fluorescent signal. Preferably, the first signal protein and the second signal protein are obtained by cleaving 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 its amino acid sequence is as shown in SEQ ID NO.9; the amino acid sequence of GFP is cleaved at any position between the 140th and 160th positions at the N-terminus to form the first signal protein and the second signal protein. In this preferred embodiment, when α-tubulin and β-tubulin form a dimer, the binding effect between the first signal protein and the second signal protein can be further improved, the signal of the reporter protein can be enhanced, and the detection sensitivity and accuracy of the biosensor for zerumbone can be higher.

[0031] Further preferably, the nucleotide sequence of the reporter protein GFP is as shown in SEQ ID NO.13.

[0032] In the present invention, the α-tubulin can be directly connected to the first signal protein, or can be formed with the first signal protein through a linker peptide to form a first sensing element; similarly, the β-tubulin can be directly connected to the second signal protein, or can be formed with the second signal protein through a linker peptide to form a second sensing element. Preferably, the α-tubulin is fused and expressed with the first signal protein through a first linker peptide, and the β-tubulin is fused and expressed with the second signal protein through a second linker peptide. Further preferably, the amino acid sequences of the first linker peptide and the second linker peptide are respectively as shown in SEQ ID NO.10. In this preferred embodiment, when α-tubulin and β-tubulin form a dimer, the binding effect between the first signal protein and the second signal protein can be further improved, the reporter signal can be enhanced, and the detection sensitivity and accuracy of the biosensor for zerumbone can be higher.

[0033] According to the present invention, preferably, the first sensing element further contains a first promoter linked to the α-tubulin, and the second sensing element further contains a second promoter linked to the β-tubulin; wherein, 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. In this preferred embodiment, when α-tubulin and β-tubulin form a dimer, the binding effect between the first signal protein and the second signal protein can be further improved, the reporting signal can be enhanced, and the detection sensitivity and accuracy of the biosensor for zerumbone are higher.

[0034] Based on the biosensor provided by the present invention, the coupling of the zerumbone concentration and the fluorescence intensity can be realized, which can be used to detect the zerumbone concentration in a sample, and can also be used to convert the intracellular zerumbone concentration of microorganisms into the fluorescence intensity that can be detected by high-throughput, quickly measure and judge the yield of zerumbone synthesis. Therefore, a high-throughput microbial strain screening technology suitable for zerumbone is developed, and then a cell factory with high-yield zerumbone is obtained through this high-throughput screening and breeding technology.

[0035] The second aspect of the present invention provides the application of the biosensor as described above in detecting the zerumbone concentration and / or screening zerumbone-producing bacteria.

[0036] The third aspect of the present invention provides a method for high-throughput screening of zerumbone-producing bacteria, which includes the following steps: S1. Construct a zerumbone-producing bacterium containing the biosensor as described above as a chassis cell; S2. Based on the chassis cell, establish a strain mutant library, and use a cell screening device to culture and sort the strains in the strain mutant library according to the reporting signal intensity.

[0037] The method for high-throughput screening of zerumbone-producing bacteria provided by the present invention has a simple process, can efficiently and accurately screen out the strains of zerumbone-producing bacteria with high yield, and this process is easy to cooperate with various cell screening devices in the prior art, and has strong operability.

[0038] In the present invention, the zerumbone-producing bacterium can be any strain capable of synthesizing zerumbone, for example, Yarrowia lipolytica, Saccharomyces cerevisiae, etc. Preferably, the zerumbone-producing bacterium is a recombinant strain introduced with a multi-gene co-expression plasmid of P450 hydroxylase-zerumbone synthase-P450 reductase, and the starting strain of this recombinant strain is the Yarrowia lipolytica engineering strain with the preservation number of CGMCC No. 24525.

[0039] In the present invention, the zingerone-producing bacterium containing the biosensor can be constructed by transferring the biosensor into the zingerone-producing bacterium using conventional gene transformation methods in the art. For example, it can be obtained by transferring a recombinant plasmid containing the biosensor into the competent cells of the zingerone-producing bacterium.

[0040] According to the present invention, the establishment of the strain mutant library can be carried out by mutating the chassis cells using conventional microbial mutagenesis methods to obtain a mutant library containing multiple strains of the chassis cells. Preferably, the establishment of the strain mutant library uses at least one of ultraviolet mutagenesis, ARTP mutagenesis, CRISPR-guided genome-wide mutagenesis, and random integration mutagenesis.

[0041] Exemplarily, the process of establishing the strain mutant library includes: First, construct the pUC-B2-HUH-Cas9 integration plasmid, which contains the URA3 selection marker and carries the Cas9 protein controlled by the strong promoter TEFin and the XPR2t terminator. Using homologous recombination technology, integrate URA3 and the Cas9 expression cassette into the zingerone-producing bacterium containing the biosensor and producing zingerone as the chassis cells; Subsequently, according to the genomic information of the chassis cells, synthesize an sgRNA library targeting each gene of the genome and clone it onto a replicative plasmid containing the LEU selection marker through high-throughput Gibson assembly. To ensure the editing efficiency, 3 sgRNAs targeting different sites are designed for each gene; Transform the constructed sgRNA plasmid library into the above-mentioned chassis cells, and the transformants are cultured at 30 °C for 96 hours in droplets containing leucine auxotrophic medium to obtain the strain mutant library.

[0042] According to the present invention, preferably, the cell screening device uses 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).

[0043] In the present invention, the cultivation can be to first mix and culture the strains in the strain mutant library and then directly perform sorting based on the reporter signal intensity, or to separate the strains for cultivation and then perform sorting based on the reporter signal intensity.

[0044] Taking the high-throughput microliter droplet culture omics system as an example, the culture conditions of the strains in the strain mutant library include: the internal phase is the strain culture solution, and the external phase is mineral oil, fluorinated oil, soybean oil, silicone oil, tetradecane, etc., preferably mineral oil; the surfactants are Span 80 (addition amount is 0.5-5%), Tween 80 (addition amount is 0.3-3%), Pluronic F-68 (addition 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.

[0045] According to the present invention, the zingerone-producing bacterium is an engineered strain of Yarrowia lipolytica, and when the preservation number is CGMCC No. 24525, preferably, the culture conditions include: the temperature is 25-35°C, specifically it can be 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the above two numbers; the time is 48-120 h, specifically it can be 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h, 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.

[0046] According to the present invention, preferably, the process of sorting the reporter signal intensity includes: measuring the reporter signal intensity of the strains in the strain mutant library after the culture, and obtaining the strain with the lowest reporter signal intensity or obtaining the 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 yield of zingerone synthesized in the strain, and quickly measure the yield of zingerone by detecting the fluorescence emitted by the microbial strain, realizing the high-throughput screening of intracellular product zingerone in microorganisms.

[0047] The present invention will be described in detail below through examples.

[0048] In the following examples, unless otherwise specified, the remaining raw materials or reagents are all conventional commercially available products.

[0049] YPD liquid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L; YPD solid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar powder 20 g / L; YPD 60 Fermentation medium: peptone 20 g / L, yeast extract 10 g / L, glucose 60 g / L, The content of zerumbone was detected by gas chromatography (GC). The specific process is as follows: Model of the gas chromatograph: Shimadzu QP2020NX of Japan, HP-5MS chromatographic column (30m×320μm×0.5μm); Detection conditions for zerumbone: inlet temperature 250°C, injection volume 1μL, split ratio 20:1; Chromatographic column: HP-5ms (30m×0.25mM); Chromatographic conditions: initial temperature 60°C, rising to 150°C at a rate of 10°C / min, then rising to 280°C at 20°C / min and holding for 2 min; Qualitative and quantitative analysis was carried out using a standard product of zerumbone.

[0050] Example 1 1. Construction of component plasmids According to the CYP71BA1, ZSD1S144A, and AtCPR nucleotide sequences provided on NCBI, after specific codon optimization, they were synthesized by Tsingke Biotechnology Co., Ltd. and inserted into plasmid pUC57 to obtain plasmids pUC57-34-CYP71BA1, pUC57-910-ZSD1S144A, and pUC57-67-AtCPR respectively.

[0051] The constructed component plasmids pUC57-34-CYP71BA1, pUC57-910-ZSD1S144A, and pUC57-67-AtCPR were digested with the restriction endonuclease BsaI, and the target fragments were obtained by gel electrophoresis and recovery.

[0052] Among them, P450 hydroxylase CYP71BA1 (the amino acid sequence of P450 hydroxylase is shown in SEQ ID NO.14, and the nucleotide sequence of the encoding gene CYP71BA1 is shown in SEQ ID NO.15), Zerumbone synthase ZSD1S144A (the amino acid sequence of zerumbone synthase is shown in SEQ ID NO.16, and the nucleotide sequence of the encoding gene ZSD1S144A is shown in SEQ ID NO.17), P450 reductase AtCPR (NCBI number: BT008426).

[0053] The above encoding genes were all synthesized by Tsingke Biotechnology Co., Ltd. after codon optimization.

[0054] 2. Construction of multi-gene co-expression plasmids Using pGGYL3 as the backbone, ligate the target fragment in the component plasmid with T4 ligase. Utilize the Golden Gate modular assembly technology to construct the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR for the curcumenone biosynthetic pathway, and then amplify it using the Yarrowia lipolytica genome as a template.

[0055] 3. Construction of recombinant strains Using the Yarrowia lipolytica engineering strain GQ3007 with high-yield α-humulene as the starting strain, the specific construction process refers to the method disclosed in CN114525215A, and the preservation number is CGMCC No. 24525; Cultivate the starting strain Yarrowia lipolytica engineering strain GQ3007 in YPD liquid medium for 12 h until the OD 600 reaches 0.8. Use the Zymogen Frozen EZ Yeast TransformationKit II kit produced by Zymo Research Corporation to transfer the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR obtained in Example 1 into the starting strain for homologous recombination to prepare competent cells. After each transfer of the multi-gene co-expression plasmid, screening is carried out using a screening medium, and correct positive clones are identified by PCR to obtain the recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR.

[0056] The PCR enzyme used in PCR amplification is PrimeSTAR Max DNA polymerase from TAKARA; the PCR amplification system is shown in Table 1. Table 1

[0057] Among them, the process of PCR amplification is: denaturation treatment at 98 °C for 10 s, annealing at 55 °C for 10 s, and then extension at 72 °C. After repeating 35 cycles, each fragment is purified and recovered using the AxyPrepTM DNA Gel Extraction Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd.). Among them, the extension time = target fragment length / 1 kb, unit min; then use the ClonExpress MultiS One Step Cloning Kit from Nanjing Novozymes Biotech Co., Ltd. to achieve one-step cloning, and the reaction system is shown in Table 2.

[0058] Table 2

[0059] 4. Fermentation production of zerumbone The recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR was streaked on the plate of YPD solid medium. After culturing for 2 - 3 days, single colonies were picked and transferred into YPD liquid medium, and cultured at 30 °C and 220 rpm to obtain the seed liquid of recombinant strain I. Then, the seed liquid of recombinant strain I was inoculated into 50 mL of YPD 60 fermentation medium at an inoculation amount of 1% (v / v), and cultured with shaking at 30 °C and 220 rpm for 24 h, then n-dodecane accounting for 25% of the volume of YPD 60 fermentation medium was added, and the culture was continued with shaking for 72 h to obtain the fermentation broth of recombinant strain I.

[0060] The fermentation broth of recombinant strain I was transferred into 50 mL centrifuge tubes respectively, centrifuged at 8000 rpm for 5 min, the upper organic phase was collected, filtered through a membrane, and the yield of zerumbone was detected to be 6.3 mg / L.

[0061] Example 2 Construction of a biosensor based on α-tubulin and β-tubulin According to the nucleotide sequences of α-tubulin and β-tubulin provided on NCBI, after specific codon optimization, they were entrusted to Tsingke Biotechnology Co., Ltd. for synthesis and inserted into the plasmid pUC57 to obtain the plasmids pUC57-α-tubulin and pUC57-β-tubulin respectively.

[0062] Both the promoter and the terminator were endogenous to Yarrowia lipolytica, so the genome of Yarrowia lipolytica was used as a template for amplification; Using puc-intB2-HUH as the backbone, through Gibson assembly, the target fragments α-tubulin and the first signal protein were inserted to obtain the recombinant plasmid pUC-HUH-intB2-(α-tubulin-N the first signal protein), and then the target fragments β-tubulin and the second signal protein were inserted to obtain the recombinant plasmid pUC-HUH-intB2-(α-tubulin-N the first signal protein)-(β-tubulin-C the second signal protein); the PCR enzyme used in PCR amplification was PrimeSTAR Max DNA polymerase from TAKARA; the PCR amplification system is shown in Table 1.

[0063] The process of PCR amplification is as follows: denaturation treatment is carried out at 98°C for 10 s, annealing is carried out at 55°C for 10 s, and then extension is carried out at 72°C. After repeating 35 cycles, each fragment is purified and recovered using the AxyPrepTM DNA Gel Extraction Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd). Among them, the extension time = target fragment length / 1 kb, unit: min Ligation of fragments (one-step cloning): One-step cloning is achieved using the ClonExpress MultiS One Step CloningKit from Nanjing Novoprotein Scientific Inc. The reaction system is shown in Table 2. After incubating the reaction system at 50°C for 15 min, a circular recombinant vector is obtained; The circular recombinant vector is transformed into Escherichia coli DH5α competent cells. After screening through an ampicillin-resistant plate and verifying by colony PCR and sequencing, a positive recombinant plasmid is obtained.

[0064] Yarrowia lipolytica po1f (Yarrowia lipolytica strain MYA2613 purchased from the American Type Culture Collection ATCC) is cultured in YPD liquid medium (containing 2% peptone, 1% yeast extract, and 2% glucose) for 12 h until the OD 600 is 0.8; the linearized plasmid pUC-HUH-intB2-(α-tubulin-N first signal protein)-(β-tubulin-C second signal protein) is transferred 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 is transferred into the competent cells, and screening is carried out using a screening medium. The positive clones identified correctly by PCR are used to obtain the strains to be tested.

[0065] Construct a recombinant plasmid pUC-HUH-intB2-report protein GFP that expresses the reporter protein alone and integrate it into Yarrowia lipolytica po1f. This strain is used as a control strain.

[0066] Pick the test strain streaked on the plate and culture it overnight in YPD medium. Then, transfer it to the fermentation medium at an inoculation ratio of 10%, and divide it into two conditions: the absence and presence of paradol (concentration: 100 mg / 100 mL). After culturing for 24 hours, take samples and use an ELISA reader (no brand requirement). Set the excitation wavelength and absorption wavelength for detection at 488 nm and 520 nm respectively, and set the wavelength for detecting the OD of the strain growth at 600 nm. Report signal intensity = fluorescence value / OD 600 , taking the report signal intensity of the control strain as the benchmark, calculate the relative fluorescence intensity, and conduct comparative analysis.

[0067] The above test process is carried out for multiple group tests. The categories of α-tubulin, β-tubulin, the first signal protein, the second signal protein, the first linker peptide, the first promoter, and the second promoter in each group are shown in Table 3 specifically; among them, the amino acid sequence of the reporter protein GFP is as shown in SEQ ID NO.9, and the nucleotide sequence is as shown in SEQ ID NO.13. The second linker peptide is the remaining sequence after removing the first signal protein from GFP.

[0068] Table 3

[0069] The test results of groups 1, 2, 3, and 4 are as Figures 2 to 5 shown. Through comparative analysis, the optimal preparation method of the biosensor is finally obtained as group 4-4, specifically: fusing the α-tubulin from Euplotes octocarinatus with the N-terminal 450 bp domain of green fluorescent protein GFP for fusion expression, connecting them with a GGGGS linker in the middle, and using the medium-strength promoter AAT2 to express the fusion of α-tubulin and GFP as the first sensing element; fusing β-tubulin with the C-terminal 270 bp domain of green fluorescent protein GFP for fusion expression, connecting them with a GGGGS linker in the middle, and using the strong promoter TEFin for expression as the second sensing element; cloning both of them and integrating them separately into the plasmid pUC-HUH-intB2, and transforming them into the above paradol-producing bacteria (the recombinant strain I containing the multi-gene co-expression plasmid pGGYL3-CYP71BA1-ZSD1S144A-AtCPR obtained in Example 1) to obtain the recombinant strain II containing the biosensor and producing paradol, and performing fluorescence characterization on the content of paradol. The fluorescence signal results show that when there is no paradol, the fluorescence intensity of the recombinant strain II is close to that of the control strain, and the relative fluorescence reaches more than 90% of the control strain; indicating that the first sensing element and the second sensing element are tightly combined to form a dimer.

[0070] Example 3: High-throughput screening of evolved Zerumbone-producing strains with increased yield based on the constructed biosensor A strain mutation library was established from the recombinant strain II obtained in Example 2 above. Then, the MISS cell high-throughput microliter droplet culture omics system was used to sort the strains with weakened fluorescence intensity, and shake flask fermentation was used for verification. Specifically: First, a pUC-B2-HUH-Cas9 integration plasmid was constructed. This plasmid contains a 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 (recombinant strain II in Example 2) containing the biosensor and producing zerumbone.

[0071] Subsequently, according to the genomic information of the chassis cells, an sgRNA library targeting each gene in the genome was synthesized and cloned onto a replicative plasmid containing a LEU selection marker through high-throughput Gibson assembly. To ensure the editing efficiency, 3 sgRNAs targeting different sites were designed for each gene. The constructed sgRNA plasmid library was transformed into the above chassis cells to obtain a strain mutation library. The MISS cell high-throughput microliter droplet culture omics system was used to culture each transformant in the strain mutation library in droplets containing leucine auxotrophic medium at 30 °C for 96 hours. Specific conditions: the outer phase was mineral oil containing 1% Span 80, the inner phase flow rate was 0.4 μL / min, and the outer 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. The relative fluorescence intensities are shown in Table 4 and Figure 6 as shown; finally, the sorted single cells were preserved and verified by shake flask fermentation. The yields of zerumbone are shown in Table 4 and Figure 6 as shown.

[0072] The process steps for shake flask fermentation verification were as follows: The sorted single cells were streaked on a plate of YPD solid medium. After culturing for 2.5 days, single colonies were picked into YPD liquid medium and cultured at 30 °C and 220 rpm to obtain a single cell seed solution. Then, the single cell seed solution was inoculated into 50 mL of YPD 60 fermentation medium at an inoculation amount of 1% by volume and shaken at 30 °C and 220 rpm for 24 h. Then, n-dodecane accounting for 25% of the volume of the YPD 60 fermentation medium was added, and shaking culture was continued for 72 h to obtain a single cell fermentation broth.

[0073] The fermentation broth of the single cells was transferred to 50 mL centrifuge tubes respectively, centrifuged at 8000 rpm for 5 min, the upper organic phase was collected, filtered through a membrane, and the yield of zerumbone was detected.

[0074] Table 4

[0075] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A biosensor, characterized in that, The biosensor contains a first sensing element and a second sensing element. The first sensing element contains α-tubulin and a first signal protein fused and expressed with the α-tubulin. The second sensing element contains β-tubulin and a second signal protein fused and expressed with the β-tubulin. One of the first signal protein and the second signal protein has an N-terminal end, and the other has a C-terminal end. The first signal protein and the second signal protein can combine to form a reporter signal.

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

8.

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

2.

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

5. The biosensor according to claim 4, characterized in that, 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 cleaved at any position from the 80th to the 190th at the N-terminal to form the first signal protein and the second signal protein; and / or, The amino acid sequences of the first linker peptide and the second linker peptide are as shown in SEQ ID NO.10, respectively.

6. The biosensor according to any one of claims 1 to 3, characterized in that, The first sensing element further contains a first promoter connected to the α-tubulin, and the second sensing element further contains a second promoter connected 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 zerumbone and / or screening zerumbone-producing bacteria.

8. A method for high-throughput screening of zerumbone-producing bacteria, characterized in that, This method includes the following steps: S1. Construct a zerumbone-producing bacterium containing the biosensor described in any one of claims 1 to 6 as a chassis cell; S2. Based on the chassis cell, establish a strain mutant library, and use a cell screening device to culture and sort the strains in the strain mutant library according to the intensity of the reporter signal.

9. The method according to claim 8, characterized in that, The zerumbone-producing bacterium is a recombinant strain introduced with a multi-gene co-expression plasmid of P450 hydroxylase-zerumbone synthase-P450 reductase. The starting strain of this recombinant strain is the Yarrowia lipolytica engineering strain with the preservation number of CGMCC No. 24525.

10. The method according to claim 8 or 9, characterized in that, The establishment of the strain mutant library adopts at least one of ultraviolet mutagenesis, ARTP mutagenesis, CRISPR-guided genome-wide mutagenesis, and random integration mutagenesis; and / or, The cell screening device employs at least one of a high-throughput microliter droplet culture omics 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 h; and / or, The process of sorting by the reporter signal intensity includes: measuring the reporter signal intensity of the strains in the strain mutant library after the culture, and obtaining the strain with the lowest reporter signal intensity or the strain with a reporter signal intensity lower than that of the chassis strain.

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