Xylose induction transcription factor component and application thereof

Abstract

The invention relates to a xylose sensing transcription factor component and application thereof. The xylose induction transcription factor component comprises a xylose induction transcription factor XlnR derived from filamentous fungi and a binding consistency sequence of the xylose induction transcription factor XlnR. According to a xylose induction system based on the transcription factor XlnR, in the presence of 2% glucose, xylose can reach 50% of the maximum activation level after being induced for 3 hours, and can reach the maximum activation level after being induced for 6 hours, namely, the maximum activation level is greatly improved, and the induction speed is high. By compounding the transcription factor XlnR and a prokaryote xylose induction transcription inhibition factor XylR, the leakage level of the obtained dual-regulation induction system is extremely low, but the maximum activation level is equivalent to that of an XlnR single-regulation xylose induction system, so that the dynamic range is greatly expanded and can reach 4,000 times. The invention solves the problems of low induction intensity and slow response of a xylose transcription induction system dependent on transcription factors in the field of synthetic biology.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a xylose sensing transcription factor component and an application thereof. Background Art

[0002] The transcription induction system is an induction system that depends on transcription factors. It has irreplaceable advantages for the dynamic regulation of metabolic engineering and the design of synthetic gene circuits due to its strong specificity and high sensitivity. The transcription induction system generally consists of three parts: inducers, transcription factors, and promoters that can be bound by the corresponding transcription factors. According to whether the inducer turns off or turns on gene expression, the transcription induction system can be divided into transcription activation and transcription repression systems. For example, the tet system developed by Bujard in 1999, by mutating the transcription factor protein, transformed the tet-off system mediated by the transcription repressor protein TetR-VP16 into the tet-on system mediated by the transcription activator rtTA. In addition, even if they belong to the same shutdown or activation system, transcription factors have different modes of action. For example, the transcription activation system mediated by TetR and rtTA, the former dissociates from the promoter due to binding to inducers such as tetracycline, and transcription machinery such as RNA polymerase is able to bind to the promoter to turn on gene expression; while the latter binds to the promoter due to binding to the inducer, thereby recruiting the transcription machinery to turn on expression. The difference between the two is that the activation level of the former depends only on the promoter, while that of the latter depends not only on the promoter but also on the strength of the transcription activation domain of the transcription activator. Therefore, the activation transcription level of the latter is often higher than that of the former, but the leakage level of this system is often not to be ignored.

[0003] The transcription induction systems of eukaryotes are often divided into three types: 1) Endogenous induction systems. For example, the most classic galactose induction system in Saccharomyces cerevisiae; this endogenous induction system has complex signal transduction and regulatory networks, which is not conducive to migration to other hosts, and because of its non-orthogonality with its own endogenous system, it cannot be used in the design of synthetic biology; 2) Induction systems based on bacterial transcription repressors. This type of system cannot be widely used in application scenarios with output intensity requirements due to the generally low level of transcriptional activation, such as overexpression of key metabolic pathway enzymes in metabolic engineering; 3) Synthetic transcription induction systems assembled by modules. The transcription factors obtained by this strategy usually consist of three parts, the DNA binding domain (DBD), the ligand binding domain (LBD) and the transcription activation domain (AD). Each module has a developed library, and then the modules are assembled to obtain a synthetic induction system with the desired performance. This development method has greatly expanded the induction system with specific performance, but the limitations of LBD and the fact that most successful examples are developed based on bioactive substances as inducers (such as hormone inducers) limit their wide application. In summary, universal eukaryotic transcriptional induction systems are still scarce and cannot meet the needs of the booming field of synthetic biology.

[0004] Xylose is the second most abundant monosaccharide in nature and the main pentose. It is low-cost and has food-grade safety and stability, making it a promising inducer. In particular, as a eukaryotic cell factory, brewer's yeast cannot naturally utilize xylose and arabinose, making it a special inducer rather than a nutrient. Compared with the commonly used galactose, xylose as an inducer has significant advantages in large-scale fermentation and cost reduction. In addition, the importance of xylose is due to its role as an energy source (producing H 2 , ethanol, energy precursors, etc.), food, and the production of high-value compounds are becoming increasingly prominent.

[0005] So far, international and domestic researchers have independently developed some xylose-induced systems in yeast. For example, in 2014, Matthew Wook Chang et al. in South Korea took the lead in heterologously expressing the xylose-sensing transcriptional repressor XylR from Tetragenococcus halophile, Clostridium difficile and Lactobacillus pentosus in Saccharomyces cerevisiae, and used promoters of different strengths to express XylR from different sources. Xylose can successfully activate the expression of downstream target genes; then, Zhao Huimin et al. expressed the transcriptional repressor protein XylR from Staphylococcus xylosus, Bacillus subtilis, and Bacillus licheniformis in Saccharomyces cerevisiae in 2016. 3 , these transcription factors are also regulated by xylose to turn on gene expression. The above studies are all based on the xylose induction system developed by the transcriptional repressor protein XylR. Its working principle is as follows Figure 1 As shown in A. In simple terms, in the absence of xylose, XylR R Binds to the operator sequence xylO on the promoter, preventing the RNA polII transcription machinery from binding to the promoter through steric hindrance and shutting down gene expression; conversely, when xylose is added, XylR R Dissociate from the promoter, transcription starts, and gene expression begins. The upper limit of the response intensity of biosensors based on transcription factors is also related to the activation domain (AD) of the transcription activator. Therefore, some studies have used XylR R The chimeric transcription factor was obtained by fusion expression with the activation domain. However, the sensor designed in this way is a switch that controls the gene to be turned off, which does not meet the needs of direct induced expression. Therefore, in 2020, Ye Bangce and others developed a gene-based transcription activator XylR A Turn-on xylose induction system. To increase the level of transcriptional activation, XylR AIt is designed to be fused with a strong transcriptional activation domain (VPR+HSF), and its working principle is as follows Figure 1 As shown in B. The sensor based on the TEF core promoter in this system has a strong response to xylose, high selectivity for xylose (14.82 times higher than that for glucose) and a wide operating range (0-20 mM, 48-72 h).

[0006] At present, most of the xylose-induced transcription systems developed in yeast are based on prokaryotic transcription factors, which have low maximum transcriptional activation intensity, narrow dynamic range, and too narrow xylose concentration response range (operable range) to be widely used. For xylose sensors that rely on prokaryotic transcription factors, the expression level of their transcription factors needs to be precisely regulated, otherwise it will cause host cell toxicity; xylose sensors based on the fusion expression strategy of fusion strong activation domains (such as VP16) also increase the risk of inhibiting host cell growth. Existing xylose-induced transcription systems lack universal high-performance modular synthetic promoters. Due to the different transcriptional activation mechanisms of prokaryotes and eukaryotes, there is no definite optimization strategy to follow, and further optimization is difficult. Summary of the invention

[0007] Based on this, in order to solve the problem of low induction intensity and slow response of the xylose transcription induction system that depends on transcription factors in the field of synthetic biology, it is necessary to provide a xylose-sensing transcription factor component with high induction intensity and rapid response and its application.

[0008] A xylose sensing transcription factor component comprises: a xylose sensing transcription factor XlnR and a binding consensus sequence of the xylose sensing transcription factor XlnR, wherein the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi.

[0009] Among the xylose sensing transcription factor components, the xylose response activation transcription factor XlnR derived from eukaryotic filamentous fungi significantly increases the maximum activation level and the induction speed is very fast. It has been verified by experiments that the induction of the xylose response activation transcription factor XlnR derived from eukaryotic filamentous fungi can reach 50% of the maximum activation level within 3 hours.

[0010] A promoter component capable of binding to the xylose sensing transcription factor component described above, the promoter component comprising: a first promoter capable of binding to the xylose sensing transcription factor XlnR, the first promoter being obtained by modifying the ADH2 promoter as a chassis promoter;

[0011] Furthermore, the modification includes modifying at least one of the core promoter sequence of the chassis promoter, the spacer sequence between the TATA box and the nucleosome deletion region, the spacer sequence between the TATA box and the transcription start site, the transcription factor binding site motif, and the prokaryotic operator site sequence.

[0012] A dual-regulatory promoter, which can bind to a xylose sensing transcription factor XlnR and a xylose sensing transcription factor XylR. The xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi, and the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria.

[0013] A transcription factor expression plasmid is an expression skeleton plasmid into which the above-mentioned xylose sensing transcription factor component is inserted.

[0014] A promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with an inducible promoter inserted therein, wherein the inducible promoter is selected from any one of the promoter components and the dual-regulatory promoters described above.

[0015] A recombinant bacterium is a host bacterium carrying the above-mentioned transcription factor expression plasmid, and the host bacterium is Saccharomyces cerevisiae carrying a TetR expression frame.

[0016] A recombinant bacterium is a host bacterium with a recombinant plasmid integrated into its chromosome, the recombinant plasmid is a backbone plasmid containing a xylose sensing transcription factor XlnR expression cassette and a reporter gene expression cassette driven by a response promoter, and the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The schematic diagram of the design of the existing xylose biosensor, wherein: A is the schematic diagram of the xylose induction system based on the transcription inhibitor XylR, and B is the schematic diagram of the xylose induction system designed based on the transcription activator;

[0018] Figure 2 Schematic diagram of the TF-based biosensor screening strategy;

[0019] Figure 3 Based on the natural inducible promoter P ADH2 Schematic diagram of the transformation strategy;

[0020] Figure 4 Schematic diagram of the construction principle of a high-performance xylose-inducible system based on bacterial XylR;

[0021] Figure 5 This is a schematic diagram of the construction principle of the yeast strain carrying the xylose-responsive transcription factor array in Example 3;

[0022] Figure 6 This is a schematic diagram of the construction principle of the single-regulatory xylose-induced transcription system in Example 3;

[0023] Figure 7 It is a schematic diagram of the structure of the functional dual transcription factor regulatory topology in Example 4;

[0024] Figure 8 A schematic diagram of the structure of the preferred dual transcription factor regulatory topology design in Example 4;

[0025] Fig. 9 This is a schematic diagram of the structure of the commonly used induction system reconstructed in Saccharomyces cerevisiae;

[0026] Fig.10 This is a schematic diagram of the preparation process of corncob hydrolyzate in Example 5;

[0027] Fig.11 Schematic diagram of the design of the XlnR-based xylose transcription system and genome recombination strategy in Pichia pastoris;

[0028] Fig.12 Schematic diagram of the design of the XlnR-based xylose transcription system and genome recombination strategy in Candida glabrata;

[0029] Fig.13 Schematic diagram of the construction of a universal plasmid adapted to the GOLDEN-GATE technology in Candida albicans;

[0030] Fig.14 Schematic diagram of the design of the XlnR-based xylose transcription system and genome recombination strategy in Candida albicans;

[0031] Fig.15 It is a statistical graph of the activation / inhibition fold detection results of different xylose-responsive transcription factor screening systems in Example 1 under xylose induction and non-induction conditions;

[0032] Fig.16 Figure 1 is a test result diagram based on the optimization of the XlnR xylose transcription induction system, where: a is a test result diagram based on the optimization of the synthetic promoter; b is a test result diagram based on the optimization of the transcription factor concentration

[0033] Fig.17 a is a schematic diagram of the substrate response curve of the xylose single regulation induction system in Example 3, b is a schematic diagram of the induced heterogeneity, and c is a schematic diagram of the kinetic behavior measurement and fitting;

[0034] Fig.18Schematic diagram of the construction and design of the xylose dual-regulation induction system in Example 4, wherein: a is a fitting curve diagram of 6 topologies, and b is a structural statistical diagram of the simulated calculation of τ1 / 2 (response time at half-activation level) of 6 topologies;

[0035] Fig.19 a is a schematic diagram of the topological structure and substrate response curve of the galactose induction system and the xylose dual-regulation induction system of Saccharomyces cerevisiae in Example 4, and b is a schematic diagram of the kinetic behavior measurement and fitting;

[0036] Fig. 20 a is a schematic diagram of the polysaccharide composition of corncob hydrolysate in Example 5, and b is a schematic diagram of the substrate response curve for activating the xylose dual-regulation induction system;

[0037] Fig.21 The diagram is a test result of substrate induction curve and fluorescence distribution of the xylose-induced transcription system in Example 6 and the existing induction system in Saccharomyces cerevisiae;

[0038] Fig. 22 It is a multi-dimensional comparison diagram of the xylose-induced transcription system and the existing induction system in Saccharomyces cerevisiae, wherein: a is a schematic diagram of the maximum activation level, b is a schematic diagram of the toxic effect on the growth of the host, c is a schematic diagram of the induction speed, and d is a schematic diagram of the radar diagram (visualized multi-dimensional comparison);

[0039] Fig.23 The xylose induction system based on XlnR in Example 8 was expressed in Pichia pastoris ( Fig.23 a), Candida glabrata ( Fig.23 b) and Candida albicans ( Fig.23 c) Substrate induction curve determination and Hill fitting diagram;

[0040] Fig.24 This is a comparison of the ability of the xylose system and the methanol system in Pichia pastoris to induce secretion of msfGFP in Example 8. DETAILED DESCRIPTION

[0041] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described in the application, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used in this application and in the specification of this application are only for the purpose of describing the implementation methods and embodiments and are not intended to limit this application.

[0043] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0044] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0045] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0046] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0047] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0048] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0049] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0050] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In the present application, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges included therein.

[0051] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0052] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0053] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0054] In a first aspect of an embodiment of the present application, a xylose-sensing transcription factor component is provided, comprising: a xylose-sensing transcription factor XlnR and a binding consensus sequence of the xylose-sensing transcription factor XlnR, wherein the xylose-sensing transcription factor XlnR is a xylose-sensing transcription factor XlnR derived from filamentous fungi.

[0055] Among the xylose sensing transcription factor components, the xylose response activation transcription factor XlnR derived from eukaryotic filamentous fungi significantly increases the maximum activation level and the induction speed is very fast. It has been verified by experiments that the induction of the xylose response activation transcription factor XlnR derived from eukaryotic filamentous fungi can reach 50% of the maximum activation level within 3 hours.

[0056] In some embodiments, the filamentous fungus includes Aspergillus nidulans. Aspergillus nidulans is a fungus belonging to the family Aspergillus and the genus Aspergillus. It should be noted that the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from Aspergillus nidulans, and can also be a sequence homolog (orthologs) of the xylose sensing transcription factor XlnR derived from Aspergillus nidulans, and the binding consensus sequence can replace the system and play a similar function.

[0057] Furthermore, the amino acid sequence of the xylose sensing transcription factor XlnR is shown in SEQ ID NO.1; and / or, the binding consensus sequence nucleotide sequence is shown in SEQ ID NO.50. The above xylose sensing transcription factor XlnR and / or its binding consensus sequence can increase the maximum activation level and induction rate. Specifically, the sequence shown in SEQ ID NO.50 is: 5'-GGCTAAW-3', wherein W is a mixture of adenine (A) and thymine (T).

[0058] In some embodiments, the xylose sensing transcription factor assembly further comprises a xylose sensing transcription factor XylR, wherein the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria. The combination of the xylose sensing transcription factor XylR derived from bacteria can reduce the leakage level of the transcription induction system.

[0059] The bacterium is Bacillus licheniformis. Furthermore, the amino acid sequence of the xylose sensing transcription factor XylR is shown in SEQ ID NO. 2. The dual regulation induction system combined with the Bacillus licheniformis transcription factor XylR greatly reduces the leakage level, and the final induction dynamic range is as high as 4000 times, and the maximum activation intensity and response speed are not affected relative to the single XlnR. It should be noted that the above-mentioned xylose-sensing transcription factor XylR is not limited to that derived from Bacillus licheniformis, but may also be sequence homologs (orthologs) of the xylose-sensing transcription factor XylR derived from Bacillus licheniformis and the binding consensus sequence can replace this system and play a similar function. It may also be derived from other bacteria, for example, it may be Lactobacillus pentosus, Clostridium difficile, Tetragenococcus halophile, Bacillus subtilis, Caulobacter crescentus and other bacteria.

[0060] In the above-mentioned xylose-sensing transcription factor component, the xylose-responsive activation transcription factor XlnR derived from eukaryotic filamentous fungi is used, which greatly improves the maximum activation level and makes the induction speed very rapid (50% of the maximum activation level can be reached after 3 hours of induction); on the basis of the XlnR system, the dual-regulation induction system of the Bacillus licheniformis transcription factor XylR is combined to greatly reduce the leakage level, and the final induction dynamic range is as high as 4000 times, and the maximum activation intensity and response speed are not affected relative to the single XlnR.

[0061] In a second aspect of the present application, a promoter component is provided, which can be combined with the above-mentioned xylose sensing transcription factor component to regulate xylose induction of the host bacteria. The promoter component includes: a first promoter, which can be combined with the xylose sensing transcription factor XlnR.

[0062] Since natural promoters such as P xylPToo long, unstructured frameworks increase the difficulty of further modification, and may also be subject to unknown endogenous regulation, so it is necessary to rationally design a structured synthetic promoter. Therefore, in some embodiments, a well-defined inducible ADH2 natural promoter is first selected, and the first promoter is obtained by modifying the Saccharomyces cerevisiae-derived ADH2 promoter as a chassis promoter. Among them, the Saccharomyces cerevisiae-derived ADH2 promoter is the ADH2 promoter derived from Saccharomyces cerevisiae.

[0063] Due to the structured sequence characteristics, the natural inducible ADH2 promoter of Saccharomyces cerevisiae was selected as the chassis for the design of a fully synthetic promoter. The principle is to ensure that the characteristic sequence remains unchanged: nucleosome depletion region (NDR)-poly (A) sequence, TATA-box, transcription start site (TSS), and change the spacer sequence between the characteristic sequences to obtain a fully synthetic promoter that meets the required performance; in addition, depending on the induction system, the upstream activation sequence (UAS) can be replaced. Therefore, the transformation described in the present application includes transforming at least one of the core promoter sequence of the chassis promoter, the spacer sequence between the TATA box and the nucleosome depletion region, the spacer sequence between the TATA box and the transcription start site, the transcription factor binding site motif, and the prokaryotic operator site sequence.

[0064] In some embodiments, the first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the Saccharomyces cerevisiae ADH2 promoter with the binding sequence of the xylose sensing transcription factor XlnR. The promoter obtained by this step is a semi-synthetic promoter. Further, the base sequence of the first promoter is shown in SEQ ID NO.4. However, the leakage level of the semi-synthetic promoter cannot be ignored.

[0065] Since the leakage level of the semi-synthetic promoter cannot be ignored, in some embodiments, the first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the Saccharomyces cerevisiae ADH2 promoter with the binding sequence of the xylose sensing transcription factor XlnR, and then changing the spacer sequence between the TATA box and the nucleosome-deficient region and / or the spacer sequence between the TATA box and the transcription initiation site. The leakage level of the promoter is improved by the above transformation.

[0066] Furthermore, the first promoter is selected from any one of the promoters with base sequences as shown in SEQ ID NO.5-SEQ ID NO.11 and SEQ ID NO.18-SEQ ID NO.20.

[0067] Specifically, the sequence between NDR and TATA-box was screened by synthesizing random primers (hereinafter referred to as random primer 1, with the base sequence: 5'GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAGCA-3'), and finally a P with low background and strong activation was obtained. xln.1b (The base sequence is shown in SEQ ID NO.5). Previously, it was reported in the literature that the core promoter determines the maximum activation strength. Therefore, the core promoter region of ADH2 was replaced with other highly active promoter core regions in the present application, and 6 synthetic promoters were obtained (the base sequences are shown in SEQ ID NO.6-SEQ ID NO.11). Although all of these can support xylose-induced activation, the maximum activation level is not as good as the original ADH2 core promoter. These results also reveal that the core promoter part has programmability. In order to obtain a fully synthetic promoter, the TATA-BOX and TSS region sequences were subjected to high-throughput screening by superimposing synthesis of multiple random primers. Finally, a series of fully synthetic inducible promoters were obtained, especially three fully synthetic promoters with base sequences shown in SEQ ID NO.18-SEQ ID NO.20.

[0068] Studies have found that appropriately increasing the binding sites of transcription factors is a reliable strategy to increase the level of transcriptional activation. Therefore, in some embodiments, the first promoter is mainly obtained by the following steps: inserting the binding site xlno of the xylose sensing transcription factor XlnR into the ADH2 promoter. Furthermore, the number of inserted binding sites xlno is 1-4. Specifically, the first promoter is selected from at least one of the promoters with base sequences such as SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.12-SEQ ID NO.15. Among them, the promoter containing 2 xlno has significantly increased the activation level, especially the promoter containing 2 xlno. xln.2b (The base sequence is shown in SEQ ID NO.13), and its activation level even exceeds that of the promoter P containing 4 xlno xln.4 (base sequence as shown in SEQ ID NO.15), but its background leakage level is too high, so P xln.4 Has the largest dynamic range.

[0069] The ADH2 promoter chassis architecture of Saccharomyces cerevisiae and the optimization strategy for synthetic promoters used in this application specifically refer to the characteristic sequences (kozak, TSS, TATA-Box, NDR, UAS, see Table 1 Notes) contained in the architecture and the length of the intervals between the characteristic sequences (see Figure 3 ). For the spacer sequences between the characteristic sequences (TSS, TATA-Box, NDR) Figure 3 Spacer.a and Spacer.b) in the assay were subjected to high-throughput screening by designing random primers with fixed spacer sequences.

[0070] The first promoter is a series of short (<300bp) adjustable synthesized promoters designed and constructed with the natural ADH2 promoter from Saccharomyces cerevisiae as the modified chassis, which can achieve high-intensity activation and avoid the problems caused by the use of natural promoters: potential endogenous regulation, construction and transformation difficulties caused by repeated use, and genomic instability.

[0071] It should be noted that the Saccharomyces cerevisiae ADH2 promoter architecture used in this application has an intervening sequence ( Figure 3 The length and sequence of spacer.a and spacer.b) in the expression vector have certain compatibility within a certain range, and the UAS sequence can be replaced with other transcription factor binding sites, thereby being used in other transcription induction systems.

[0072] In some embodiments, the promoter component further includes a second promoter capable of binding to the xylose sensing transcription factor XylR.

[0073] Furthermore, the second promoter contains the binding site xylo of the xylose sensing transcription factor XylR, and the binding site xylo is located upstream of the adjacent transcription initiation site and near the TATA box. Furthermore, the binding sequence (operator) of XylR is placed downstream of the TATA-box and upstream of the TSS. In some specific examples, the base sequence of the binding site xylo is shown in SEQ ID NO.49; and / or, the base sequence of the second promoter is shown in SEQ ID NO.3.

[0074] In a third aspect of the embodiments of the present application, a dual-regulated promoter is provided, wherein the dual-regulated promoter can bind to a xylose sensing transcription factor XlnR and can bind to a xylose sensing transcription factor XylR, wherein the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi, and the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria. The dual-regulated promoter can dual-regulate xylose induction of the host bacteria.

[0075] In some embodiments, the dual-regulated promoter is selected from at least one of the promoters with base sequences as shown in SEQ ID NO.16-SEQ ID NO.17.

[0076] In a fourth aspect of the present application, a transcription factor expression plasmid is provided, wherein the transcription factor expression plasmid is an expression backbone plasmid into which the xylose-sensing transcription factor component described above is inserted. The xylose-sensing transcription factor component described above can be expressed by the transcription factor expression plasmid.

[0077] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are inserted simultaneously, that is, the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are inserted into the same backbone plasmid, which can be used to construct a transcription factor array yeast strain.

[0078] In other embodiments, the transcription factor expression plasmid includes a first expression plasmid and a second expression plasmid, wherein the first expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XlnR is inserted, and the second expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XylR is inserted. That is, the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are respectively inserted into different backbone plasmids, which can be used to construct a prototype response test platform for the xylose transcription induction system. Furthermore, the expression backbone plasmid corresponding to the xylose sensing transcription factor XlnR is a pGD137 plasmid having a base sequence as shown in SEQ ID NO.23, and the expression backbone plasmid corresponding to the xylose sensing transcription factor XylR is a pGS001 plasmid having a base sequence as shown in SEQ ID NO.24.

[0079] In a fifth aspect of the embodiments of the present application, a promoter-reporter gene plasmid is provided, which is a reporter gene backbone plasmid with an inducible promoter inserted therein, wherein the inducible promoter is selected from any one of the promoter components described above and the dual-regulatory promoter described above. The promoter-reporter gene plasmid can be used together with a transcription factor expression plasmid to construct a xylose transcription induction system.

[0080] In a sixth aspect of the embodiment of the present application, a recombinant bacterium is provided, which is a host bacterium carrying the above-mentioned transcription factor expression plasmid, and the host bacterium is Saccharomyces cerevisiae carrying the TetR expression frame. The transcription factor expression plasmid is transferred into the host bacterium to express the above-mentioned xylose sensing transcription factor XlnR and xylose sensing transcription factor XylR.

[0081] In some embodiments, the recombinant bacteria also carries a promoter-reporter gene plasmid, which is a reporter gene backbone plasmid into which the above-mentioned inducible promoter is inserted. The inducible promoter is selected from any one of the above-mentioned promoter components and the above-mentioned dual-regulatory promoters. The recombinant bacteria can be used to construct a transcription factor array yeast strain, or to construct a prototype response test platform for a xylose transcription induction system.

[0082] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are inserted at the same time, and the expression frames of the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are integrated into the NRT1 gene spacing site of the Saccharomyces cerevisiae. The recombinant bacteria can express both the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR.

[0083] Further, in some examples, based on the recombinant bacteria of the above embodiment, the recombinant bacteria are further introduced with a first promoter and a second promoter, the first promoter can bind to the xylose sensing transcription factor XlnR, and the second promoter can bind to the xylose sensing transcription factor XylR. The specific description of the first promoter and the second promoter is detailed above and will not be repeated here. The recombinant bacteria introduced with the first promoter and the second promoter can regulate xylose-induced expression alone.

[0084] In some examples, based on the recombinant bacteria of the above embodiment, the recombinant bacteria also carries a promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with an inducible promoter inserted, and the inducible promoter is selected from any one of the dual-regulatory promoters described above (see above for specific descriptions, which will not be repeated here). In the recombinant bacteria, the signals of the transcription activator and the transcription repressor are integrated into the same synthetic promoter, that is, the binding site of the transcription repressor is located in the core promoter region, and the binding site of the transcription activator is located in the UAS region, and the selected transcription activator and the transcription repressor do not have competitive binding, that is, they interact with the same synthetic promoter independently of each other. The above recombinant bacteria can dual-regulate xylose-induced expression.

[0085] The dual transcription factor regulation system can be used to further optimize the xylose transcription regulation system by integrating the advantages of both. However, there are many design schemes for the dual regulation mode (such as Fig.18 a), of which there are 6 functional topological structures (see Figure 7 ), that is, the dual-regulatory topology of the recombinant bacteria is selected from any one of topology 1 to topology 6. Topology 1 is the most effective dual-regulatory topology. The fitting equations of topology 1 to topology 6 are shown in Table 5 below. In the fitting equation, m ais the mRNA concentration of transcription activator a; m [yfp] is the mRNA concentration of the fluorescent reporter protein YFP; a is the protein concentration of the transcription activator a; [YFP] is the protein concentration of the fluorescent reporter protein YFP; β 1 is the rate of mRNA synthesis driven by a dual-regulated promoter; β 2 Is the rate of mRNA synthesis driven by transcriptional activators; β 3 is the rate of mRNA synthesis driven by transcriptional repressors; β p is the protein synthesis rate of the transcription factor protein and the fluorescent reporter protein YFP. Here, it is assumed that the protein synthesis rates of the two are the same; γ m is the degradation and dilution rate of mRNA; γ p is the rate of protein degradation and dilution; k 1 is the concentration of the transcriptional activator when the transcriptional output reaches half the activation intensity; n 1 is the Hill coefficient in the function of transcription activator concentration and output.

[0086] Based on the dual transcription factor regulation system, the kinetic model formula 1 of the substrate response curve of the recombinant bacteria is as follows:

[0087]

[0088] The meaning of each letter is: min is the output of the induction system when no inducer is added, y max is the output when the induction system reaches steady state after the inducer is added. x is the inducer concentration. k 1 ,k 2 ,n 1 , and n 2 They are the Hill parameters of the xylose single regulation system (XlnR-based and XylR-based induction system), respectively.

[0089] Agricultural waste such as corn, wheat, cotton straw, etc. are rich in xylose. If the xylose in their hydrolysates can be recycled, it will bring huge economic returns. In some embodiments, the induction substrate of the recombinant bacteria includes corn cob hydrolysate, and the corn cob hydrolysate is mainly prepared by the following steps: 3g of 20-mesh corn cob powder is mixed with 2% H 2 SO 4 The solution is treated at 120° C. for 45 minutes, then treated with calcium carbonate, then hydrolyzed by adding 1,800 U of cellulase and 1,500 U of hemicellulase, and then solid-liquid separation is performed, and the supernatant is collected to obtain the corncob hydrolyzate. The hydrolyzate of crop waste (such as corncobs) rich in xylose can replace pure xylose to activate the xylose induction system, thereby greatly reducing the cost of the system for the production of bulk industrial products.

[0090] In order to verify the portability of the XlnR-based xylose induction system in other yeast species, the single-regulatory XlnR-based xylose induction system was integrated into the genome of Pichia pastoris, Candida glabrata, and Candia albicans to detect the xylose response of the system. Therefore, in the seventh aspect of the embodiment of the present application, a recombinant bacterium is provided, wherein the recombinant bacterium is a host bacterium with a recombinant plasmid integrated on the chromosome, the recombinant plasmid is a backbone plasmid containing a xylose-sensing transcription factor XlnR expression cassette and a reporter gene expression cassette driven by a response promoter, and the xylose-sensing transcription factor XlnR is a xylose-sensing transcription factor XlnR derived from filamentous fungi. The specific description of the xylose-sensing transcription factor XlnR is detailed above and will not be repeated here.

[0091] In some embodiments, the host bacteria is Pichia pastoris or Candida glabrata, and the xylose sensing transcription factor XlnR expression cassette is located upstream of the reporter gene expression cassette;

[0092] Furthermore, the reporter gene is yEmCitrine; and / or the base sequence of the promoter is as shown in SEQ ID NO.15.

[0093] In some embodiments, the host bacteria is Candida albicans, and the xylose sensing transcription factor XlnR expression cassette is located downstream of the reporter gene expression cassette;

[0094] Furthermore, the reporter gene is mNeonGreen; and / or the base sequence of the promoter is shown in SEQ ID NO.21; and / or the backbone plasmid is pG S173 plasmid, and the base sequence of the backbone plasmid is shown in SEQ ID NO.22.

[0095] The above-mentioned recombinant bacteria can illustrate that the XlnR-mediated xylose-inducible transcription system is highly portable and can still respond to xylose according to doses in multiple unconventional yeasts with distant genetic relationships; the XlnR-mediated xylose-inducible transcription system is highly practical and can efficiently induce the secretion of the secretory protein msfGFP in Pichia pastoris, and the secretion level is higher than that of the endogenous methanol system.

[0096] The technical solution of this application has the following advantages:

[0097] The present application develops an efficient and low-cost transcription induction system: using the xylose-responsive activation transcription factor XlnR from eukaryotic filamentous fungi, the maximum activation level is greatly increased, and the induction speed is very rapid (50% of the maximum activation level can be reached within 3 hours of induction).

[0098] Based on the XlnR system, the present application combines the dual regulation induction system of Bacillus licheniformis transcription factor XylR to greatly reduce the leakage level, and the final induction dynamic range is as high as 4000 times, and the maximum activation intensity and response speed are not affected relative to the single XlnR.

[0099] The dual-regulation mode induction system designed in the present application has a substrate response function that can be predicted by the parameters of the single-regulation substrate response.

[0100] This application uses the natural ADH2 promoter as the transformation chassis, and designs and constructs a series of short (<300bp) adjustable synthetic promoter sequences, which can achieve high-intensity activation and avoid the problems caused by the use of natural promoters: potential endogenous regulation, construction and transformation difficulties caused by repeated use, and genomic instability. Even under the condition that the xylose induction system is saturatedly activated, there is no significant inhibitory effect on the growth of Saccharomyces cerevisiae.

[0101] In the present application, hydrolyzate of crop waste (such as corn cobs) rich in xylose can replace pure xylose to activate the xylose induction system, thereby greatly reducing the cost of the system for the production of bulk industrial products.

[0102] In terms of regulation rigor, maximum activation intensity, dynamic range, toxicity to host growth, and response speed, the performance of the dual-regulation xylose induction system developed in this application is superior to the commonly used induction systems of Saccharomyces cerevisiae (such as the natural endogenous system P GAL1 ,P CUP1 ,P MET3 , and the synthetic inducible system LexA-ER-VP16 / B112).

[0103] The XlnR-mediated xylose-induced transcription system of the present application is highly portable and can still respond to xylose at a dose in multiple distantly related unconventional yeasts; moreover, the XlnR-mediated xylose-induced transcription system of the present application is highly practical and can efficiently induce the secretion of the secretory protein msfGFP in Pichia pastoris, and the secretion level is higher than that of the endogenous methanol system.

[0104] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0106] The names and sequences of the elements involved in the following embodiments are shown in Table 1 (see below). In Table 1, "(amino acid sequence)" after the sequence indicates that the sequence is an amino acid sequence, and if there is no "(amino acid sequence)" after the sequence, it means that the sequence is a nucleotide sequence.

[0107] Unless otherwise specified, the methods used in the following embodiments are as follows:

[0108] Table 2: GOLDEN GATE system and conditions used in each example

[0109]

[0110] 1. Substrate induction test culture

[0111] Single clones were picked from the YPD plate and placed in 500 μL SD defective medium in a 2 mL 96-well plate (Axygen, P-2ML-SQ-CS), sealed with a gas permeable membrane (BioTss, SF-200), and cultured at 800 rpm and 30°C for 24 hours. Then 2.5 μL aliquots were inoculated into 497.5 μL fresh SD medium containing the corresponding concentration of inducer (OD 600 =0.005) and cultured for 16 h under the same conditions. Then the culture was diluted 10-fold by adding 20 μL aliquots of 180 μL phosphate buffered saline (PBS, Proteintech, PR20014) containing 10 μg mL -1 Cycloheximide (Maokang Biotechnology, MS0035) was added, and the mixture was then analyzed by flow cytometry.

[0112] 2. Flow cytometry and data processing

[0113] A BD FACSCelestaTM flow cytometer was used in high throughput sampler (HTS) mode (BD Biosciences, Germany) to detect the intensity of yellow fluorescent protein (yEmCitrine) in the treated samples. A 488 nm excitation laser (100 mW) and a 530 / 30 nm emission filter were selected to measure the YFP intensity. Data were recorded using FACSDiva software (BD Biosciences, Germany), and more than 10,000 events were recorded for each sample. Cells of the correct size were selected in the forward scatter area (FSC-A) / side scatter area (SSC-A) plot. Data were obtained using the BD FACSCelestaTM flow cytometer. The data were processed using TreeStar 10.4 (TreeStar, USA), and the median of the fluorescein isothiocyanate (FITC-A) signal was used for yellow fluorescence quantification and statistical analysis. The formula for calculating the induction fold was (YFP 诱导 -YFP 0 ) / (YFP 非诱导 -YFP 0 ), where YFP 诱导 and YFP 非诱导 Represents the fluorescence measured in the presence and absence of 10 mM xylose, respectively. YFP 0 Represents the spontaneous fluorescence of leukocytes. The calculation formula for the inhibition factor is (YFP -抑制因子 -YFP 0 ) / (YFP +抑制因子 -YFP 0 ), where YFP -抑制因子 and YFP +抑制因子 The fluorescence intensity was measured in the absence and presence of transcriptional repressors, respectively. 参考 ) was normalized to RPU, and the calculation formula of RPU was (YFP-YFP 0 ) / (YFP 参考 -YFP 0 ). Therefore, leukocytes, CY671int (P PFY1 -YFP) and CY676int(P TDH3 -YFP) was used as a control strain and was cultured on the same plate as the test strain each time.

[0114] 3. Induction kinetic test culture

[0115] Pick a single clone from the YPD plate and place it in 500 μL SD defective medium in a 2mL 96-well plate (Axygen, P-2ML-SQ-CS), seal it with a breathable membrane (BioTss, SF-200), and culture it at 800 rpm, 30°C for 24 hours. For GAL system-derived strains (GAL80Δ, GAL80Δ & GAL1Δ) without glucose inhibition, use SC medium (SCR medium) supplemented with 2% (w / v) raffinose. Unless otherwise specified, 2% (w / v) glucose is used as the carbon source. Then, 2.5 μL of culture is inoculated into 475.5 μL of fresh SD medium (OD 2000) supplemented with the corresponding inducer in a 2mL 96-well plate. 600=0.005). Subsequently, 25 μL of these cultures were transferred to 475 μL of fresh SD or SCR medium and the corresponding inducers were added in a 2mL 96-well plate and cultured in a shaking incubator (Zhichu, ZQZY-88AH) at 30°C and 800 r.pm. During the initial 3 hours of growth, 50 μL of culture was collected at each specified time point, and then an equal volume of fresh medium with the corresponding inducer added was added. After the initial 3 hours of culture, 20 μL of culture solution was collected from each culture well every 3 hours during the 36-hour culture period. The collected samples were washed with 180 μL containing 10 μg mL -1 Cycloheximide was diluted in PBS and then subjected to flow cytometric analysis.

[0116] 4. Growth toxicity test

[0117] Three test strain clones were randomly selected and cultured overnight in 500 μL SD defective medium. The culture was inoculated into SD medium containing gradient concentrations of inducers (no defective nutritional screening) at a ratio of 1:100 and cultured for 24 hours. 20 μL of culture was taken for flow cytometry analysis to detect the correctness of the test strain and the induced heterogeneity. At the same time, 5 μL of culture in each culture well was inoculated one by one into the corresponding culture wells containing 495 μL of culture medium on another plate containing gradient inducer concentrations. After 8 hours of culture, the OD of the culture in each culture well was measured using a Tecan Infinite 200Pro plate reader (Tecan). 600 At the same time, the cultures in the uninduced wells were inoculated into SD medium supplemented with gradient inducer concentrations and grown for another 24 h. Data were collected for three consecutive days and finally the OD of the parental CENPK.2-1C strain cultured in SD medium on the first day was used as the value. 600 To normalize the measurement data of the test strain for three consecutive days. The calculation formula is norm.OD 600 =ave(OD 600 ) 诱导 / OD 600(2-1C) .

[0118] 5. HPLC analysis of corncob hydrolysate

[0119] The concentrations of D-glucose, D-xylose, and L-arabinose in corncob hydrolysates were determined by high performance liquid chromatography (HPLC) on an Agilent 1260 HPLC system (Agilent Technologies) using an external standard-based method. The chromatographic column used was an Aminex HPX 87-H (Bio-Rad, Hercules, USA) connected to a Micro-Guard Cation-H guard column (Bio-Rad, Hercules, USA). 5 mM H 2 SO4 was used as the mobile phase, and the flow rate was set at 0.6 mL min -1 , the temperature was 55° C. The signal was detected by RID (Agilent Technologies, G7162A) at 35° C.

[0120] 6. Sample preparation and Tricine-SDS-PAGE

[0121] Sample preparation: Pick xylose induction system and P AOX1 The system controls the monoclonal strain expressing secreted msfGFP to 2mL 96-well plates (Axygen, P-2ML-SQ-CS) containing SD-His medium, which is then sealed with a breathable film and cultured at 800r.pm, 30°C for 24 hours. Then 5μL of the culture was inoculated into 495μL of fresh YPD and BMGY medium, respectively, and grown for another 24 hours. Subsequently, 5μL of the strain culture carrying the xylose induction system was inoculated into 495μL of fresh YPD medium supplemented with 100mM xylose; at the same time, 5μL of the strain culture carrying the methanol induction system was inoculated into BMMY medium supplemented with 0.5% methanol (v / v). After 24 hours of induction, 0.5% methanol (v / v) was supplemented for the methanol-inducible strain. After induction for a total of 48 hours, 20 μL of supernatant was taken and the extracellular msfGFP fluorescence intensity was measured using a Tecan Infinite 200Pro plate reader (excitation wavelength 488 nm, emission wavelength 510 nm, gain 100). 20 μL of culture medium and 20 μL of undiluted supernatant were taken and mixed with 20 μL of 2× Tris-Tricine-SDS-PAGE loading buffer (BOSTER, AR1143), respectively. These mixtures were then boiled at 100°C for 5 minutes and frozen at -80°C for Tricine-SDS-PAGE analysis.

[0122] Tricine-SDS-PAGE: First, a 1.5 mm thick PAGE gel was prepared, consisting of three gels: stacking gel (4% T, 3% C), spacer gel (10% T, 3% C) and separation gel (16.5% T, 6% C, containing 6 M urea). Tricine gel and gel buffer were provided by a commercial kit (Enogene Biotech, E1WP326). Running buffer, anode buffer (0.1 M Tris, 0.0225 M HCl, pH 8.9) and cathode buffer (0.1 M Tris, 0.1 M Tricine, 0.1% SDS, pH 8.25) were purchased from (Servicebio, G2142-1L). After the electrophoresis, the gel was incubated in a fixative (50% methanol, 10% acetic acid) for 1 hour, and then stained overnight with 0.025% Coomassie Brilliant Blue R-250 in a 10% acetic acid solution. The gel was then eluted twice in 10% acetic acid with each incubation for 45 minutes. The destained gel was imaged on a Bio-Rad ChemiDoc imaging system and the images were analyzed using Bio-Rad ImageLab software (version 6.1).

[0123] Example 1: Prototype substrate response test of xylose-inducible transcription systems from different sources in Saccharomyces cerevisiae

[0124] The basic principle of the xylose transcription induction system prototype response test platform is to efficiently screen the xylose response transcription induction system in Saccharomyces cerevisiae. The candidate transcription factors are designed to introduce restriction endonuclease BpiI sites at both ends of the fragment and remove the BpiI sites in the natural sequence by synonymous codon replacement. The reporter plasmids expressing transcription factors and responding transcription factors are constructed in one step by GOLDEN-GATE technology. TF-based biosensor screening strategy such as Figure 2 As shown, Figure 2 Annotated features of the backbone plasmids used for biosensor screening are shown. The required reporter plasmids and plasmids expressing TFs were generated by one-step assembly by replacing ccdB in the vector with BpiI digestion. The purified BsaI digestion product was directly transformed into yeast. The screening strategy for xylose-responsive transcription factors was performed in Saccharomyces cerevisiae. The transcription factor concentration was controlled by the aTc-TetR inducible system, in which TetR is controlled by the constitutive promoter PFBA1. The output of the transcription factor-responsive promoter depends on the concentration of the TF and the inducer.

[0125] The specific operation process of constructing the prototype response test platform of xylose transcription induction system is as follows:

[0126] The sequence of the xylose-responsive transcription factor XlnR (AN7610) (amino acid sequence as shown in SEQ ID NO. 1) was obtained by segmented PCR amplification from the Aspergillus nidulans genome by designing primers, removing introns and BpiI restriction sites; different bacteria (such as Fig.15 The xylose-responsive transcription factor XylR derived from Bacillus licheniformis was obtained by gene synthesis after codon optimization (see https: / / sg.idtdna.com / pages / tools / codon-optimization-tool for the codon optimization method), wherein the amino acid sequence of the xylose-sensitive transcription factor XylR derived from Bacillus licheniformis is shown in SEQ ID NO.2.

[0127] The obtained transcription factor fragment was reacted with the backbone plasmid pGD137 (the base sequence is shown in SEQ ID NO.23) and pGS001 (the base sequence is shown in SEQ ID NO.24) through the GOLDEN-GATE system, and the product was transformed into E. coli trans10 to obtain the target plasmid, which was then digested with BsaI and transformed into Saccharomyces cerevisiae CYE90 (from Chen, Y. et al. Genetic circuit design automation for yeast. Nat Microbiol 5, 1349-1360, doi: 10.1038 / s41564-020-0757-2 (2020)) to obtain a yeast strain carrying the xylose transcription factor, wherein the xylose-responsive transcription factor XlnR was connected to the backbone plasmid pGD137, and the xylose-responsive transcription factor XylR was connected to the backbone plasmid pGS001.

[0128] From the native promoter P of Penicillin chrysogenum xylP (711 bp, the base sequence of which is SEQ ID NO.55) was also designed to remove the BpiI restriction site in the promoter region and introduce a BpiI restriction site at the end, and the reporter plasmid was obtained by reacting with the existing backbone plasmid pXJH1 in the laboratory through the GOLDEN-GATE system; for XylR of different bacterial species, its promoter (the amino acid sequence of the xylose sensing transcription factor XylR shown in SEQ ID NO.2 is P xyl, the base sequence is shown in SEQ ID NO.3) was designed according to a previously published paper (reference: Chen, Y. et al. Genetic circuit design automation for yeast. Nat Microbiol 5, 1349-1360, doi: 10.1038 / s41564-020-0757-2 (2020).), and the reporter plasmid was obtained in the same way. The obtained reporter plasmid was transferred into the yeast strain with xylose transcription factor mentioned above, and the activation / inhibition multiples under xylose induction and non-induction were tested as described in the method section. The test results are shown in Fig.15 As shown in the figure. Among them, the schematic diagram of the construction of a high-performance xylose induction system based on bacterial XylR is shown in Figure 4 ( Figure 4 In the example, T20: 20 consecutive thymine bases. TATA: TATA-box (5'-TATAAAA-3', sequence number is SEQ ID NO.51), TSS: transcription start site (5'-AGAATATCAAGCTACAAAAA-3', sequence number is SEQ ID NO.52), the length between characteristic sequences is shown in the above numbers), for the XylR transcription factor from bacteria, reference: Chen, Y. et al. Genetic circuit design automation for yeast. Nat Microbiol 5, 1349-1360, doi: 10.1038 / s41564-020-0757-2 (2020), the binding sequence (operator) of XylR is placed downstream of TATA-box and upstream of TSS respectively.

[0129] from Fig.15 It can be seen that XlnR from Aspergillus nidulans showed an activation factor of more than 100 times, while XylR from Bacillus licheniformis had the largest inhibition factor, so both were used for subsequent further optimization.

[0130] Example 2: Design and optimization of synthetic promoters responsive to XlnR

[0131] Since the natural promoter P xylP Too long, unstructured frameworks increase the difficulty of further modification, and may also be subject to unknown endogenous regulation, so it is necessary to rationally design structured synthetic promoters. ADH2 For details on the transformation strategy, see Figure 3 The results of the optimized XlnR xylose transcription induction system are as follows: Fig.16As shown, where: a is a graph showing the detection results of response to synthetic promoter optimization; b is a graph showing the detection results of transcription factor concentration optimization.

[0132] The specific optimization process of startup is as follows:

[0133] First, we selected the inducible ADH2 natural promoter with a clear structure and replaced the original ADR1-binding UAS region sequence with the XlnR binding sequence (e.g. Figure 3 As shown), the semisynthetic promoter P xln.1a (The base sequence is shown in SEQ ID NO.4). Figure 3 In the present invention, the ADR1 binding site of the natural promoter ADH2 was replaced with the XlnR binding site to obtain the semisynthetic promoter P. xln.1a By performing high-throughput screening and replacement of the TFBS array, spacer.a and spacer.b in the figure, fully synthetic promoters with different induction systems can be obtained.

[0134] However, the semisynthetic promoter P xln.1a The leakage level cannot be ignored, so the sequence between NDR and TATA-box was screened by high-throughput screening with a synthetic random primer (5'GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAG CA-3', sequence numbered as SEQ ID NO.48, referred to as random primer sequence 1), and finally a P with low background and strong activation was obtained. xln.1b (The base sequence is shown in SEQ ID NO.5).

[0135] It has been previously reported that the core promoter determines the maximum activation strength, so in this example, the core promoter region of ADH2 was replaced with other highly active promoter core regions to obtain 6 synthetic promoters (whose base sequences are shown in SEQ ID NO.6-SEQ ID NO.11). Although all of them can support xylose-induced activation, the maximum activation level is not as good as the original ADH2 core promoter. These results also suggest that the core promoter part has programmability.

[0136] In order to obtain a fully synthetic promoter, the TATA-BOX and TSS region sequences were screened by multiple random primer stacking synthesis, and finally a series of fully synthetic inducible promoters were obtained, especially three fully synthetic promoters with base sequences as shown in SEQ ID NO.18-SEQ ID NO.20.

[0137] In addition, appropriately increasing the binding sites of transcription factors is a reliable strategy to increase the level of transcriptional activation. By testing promoters containing 1, 2, 3, and 4 XlnR binding sites (xlno) (base sequences are shown in SEQ ID NO.4-SEQ ID NO.5 and SEQ ID NO.12-SEQ ID NO.15), the results (such as Fig.16 a) shows that the promoter containing two xlno has significantly increased the activation level, especially the P containing two xlno xln.2b (The base sequence is shown in SEQ ID NO.13), and its activation level even exceeds that of the promoter P containing 4 xlno xln.4 (base sequence as shown in SEQ ID NO.15), but its background leakage level is too high, so P xln.4 Has the largest dynamic range.

[0138] In addition, the corresponding functional relationship between the concentration of XlnR and the system output was tested (such as Fig.16 b), and with this as a reference, select a suitable constitutive promoter to ensure that XlnR has stable expression throughout the yeast stage. Three constitutive promoters with different activities were initially selected: P ACT1 (the base sequence is shown in SEQ ID NO.26), P PFY1 (the base sequence is shown in SEQ ID NO.53) and P PXR1 (Base sequence is shown in SEQ ID NO.54), and finally it is shown that when P ACT1 When used to express XlnR, the system has the largest dynamic range (e.g. Fig.16 c).

[0139] Example 3: Substrate response curve, induction heterogeneity, kinetic behavior determination and fitting of xylose single regulation induction system

[0140] 1. Construction of transcription factor array yeast strain, schematic diagram of its construction principle (see Figure 5 , Figure 5 The genetic elements constituting the XylR and XlnR expression cassettes are shown, and the sequences of the individual elements are shown in Table 1 ): Based on the optimal transcription factor expression level, the transcription factor XlnR from eukaryotes and the bacterial XylR expression cassette were assembled on a plasmid using GOLDEN-GATE technology, and the fragments were transformed into Saccharomyces cerevisiae (i.e., CENPK.2-1C strain) after being digested with restriction endonuclease BsaI, and integrated into the NRT1 gene intergenic site to obtain a transcription factor array yeast strain (i.e., a xylose-sensing yeast).

[0141] 2. Construction of a single-regulatory xylose-induced transcription system, schematic diagram of its construction principle (see Figure 6): In the transcription factor array yeast strain, the yellow fluorescent reporter gene YFP expression frame that responds to XlnR and XylR was respectively transferred and integrated into the original ura3 genomic site to obtain a single regulatory transformation strain.

[0142] 3. For the optimized P xln.2b -XlnR system and P xyl -XylR (i.e. single regulation xylose inducible transcription system) performance characterization:

[0143] The obtained single-regulatory transformed strain was induced with 16 different concentrations of xylose, and the output signal was analyzed by flow cytometry (methods), and the substrate concentration induction curve was drawn and fitted with the Hill function (e.g. Fig.17 The Hill parameters and EC50 values ​​were obtained (see Table 3). The results showed that compared with P xyl , P xln.2b The leakage level is two orders of magnitude higher than that of P xln.2b， P xyl The maximum activation level of is 25% of the former. For a given transcriptional activator, the relationship between its protein amount (A) and the output amount (y) of the response promoter can be described by the Hill function:

[0144]

[0145] For the promoter XlnR-P xln.2b The system has a functional relationship such as Fig.16 b. The fitted k 1 and n 1 They are 3.42 and 1.82 respectively (see Table 3).

[0146] In addition, the heterogeneity of yeast populations under different xylose concentrations was observed and analyzed. Fig.17 As shown in b, under the induction of xylose concentrations of 5 different orders of magnitude, a single peak appeared constantly, and no population heterogeneity appeared; finally, the kinetic behavior under the induction of 10mM xylose concentration that can make the induction system reach saturation activation was measured and fitted. Fig.17 As shown in Figure c, the fitting parameters are shown in Table 3. The results show that the observed kinetic data can be fitted by the standard transcription-translation model, namely, Formula 2 and Formula 3.

[0147] Wherein, formula 2 is as follows:

[0148] Formula 3 is as follows:

[0149] The meanings of the letters in Formula 2 and Formula 3 are as follows: m, p are the concentrations of mRNA and protein, respectively; β m,β p are constants describing the rates of mRNA and protein synthesis, respectively; γ m ,γ p are constants describing the degradation and dilution rates of mRNA and protein, respectively.

[0150] Table 3: Substrate response curve fitting parameters

[0151]

[0152] Example 4: Construction and design of xylose dual-regulation induction system and substrate response curve, kinetic behavior determination and fitting

[0153] 1. In view of P xln.2b -XlnR system high leakage and P xyl -XylR system has low activation ability, and the dual transcription factor regulation system is adopted to integrate the advantages of both to further optimize the xylose transcription regulation system. However, there are many design schemes for the dual regulation mode (such as Fig.18 There are 6 functional topological structures (see Figure 7 ), according to the standard transcription-translation model (see Table 5) and the kinetic fitting parameters of single regulation (see Table 4), the induced kinetic simulation was performed on the dual regulation systems of these six topological structures. The final simulation results are shown in Fig.18 As shown in b, topology 1 has the largest induction rate (see Fig.18 b, c) and maximum activation level (see Fig.18 b). Therefore, the simple topology 1 is selected for further construction and performance characterization.

[0154] For a given induction system, γ p is a fixed value of 0.29, according to the equation It is deduced that τ is the cell doubling time, which is 90 min here (reference McDonald, PNTwo-hybrid systems. Methods and protocols. Introduction. Methods Mol Biol177, v-viii (2001).). The other parameters (β m ,γ m ,β p ) is obtained by fitting the formula (3) and (4) and the measured kinetic data (see Table 4). Therefore, through the following parameters and values, β 1 =2.22; β 2 =2.19; β 3 =0.49; β p =1.62;γ m =0.28;γ p=0.29; k 1 =3.42; n 1 =1.82.

[0155] According to Table 3, when t = 0h, P(a)[t = 0] = 0.28, P(YFP)[t = 0] = 0.28 (P xln.2b ) or 0.001(P xln.2b-xyl ). Therefore, when P(YFP)[t=0]=0.28, β m [t=0]=0.28*0.283*0.29 / 1.618=0.014, and m(a)[t=0]=m(YFP)[t=0]=β m [t=0] / γ m = 0.050; when P(YFP)[t=0]=0.001, m(YFP)[t=0]=0.00018. Dynamic data fitting is performed using the above differential equations and fitting values. The fitting results are shown in Fig.18 a. Since it is impossible to obtain an analytical solution to the above dynamic simulation function, the dichotomy method is used to calculate τ 1 / 2 The results are as follows Fig.18 As shown in b.

[0156] 2. Construction of the topological structure of dual transcription factors 1 (the schematic diagram of its framework design is shown in Figure 8 ), just follow Fig.19 The structure shown in a is the sequence P xln.2b-xyl (The base sequence is shown in SEQ ID NO.17) to construct a promoter sequence, and the sequence P xln.2b-xyl The fragment and the backbone plasmid pXJH1 (constructed in-house in our laboratory, the schematic diagram of its plasmid structure is as follows Figure 2 ) was subjected to a GOLDEN-GATE one-step reaction to obtain the target reporter plasmid, and then the existing transcription factor array strain (i.e., the transcription factor array yeast strain obtained by step 1 of Example 3, the construction diagram of which is shown in FIG. Figure 5 Then, the strain containing the dual regulatory system was induced by 16 different concentrations of xylose and analyzed by flow cytometry to draw a substrate response curve ( Fig.19 In a), the measured data can also be well fitted by the Hill function ( Fig.19 a), and the EC50 is 0.93 mM. Assuming that XlnR and XylR interact with the promoter P xln.2b-xyl The binding of is an independent event, then the steady-state induction of the promoter can be predicted using the partition function, i.e., Formula 1. The actual prediction results are as follows: Fig.19 As shown in (a), the substrate-induced behavior of the dual-regulatory system can be well predicted by the fitting parameters of the single-regulatory system.

[0157] 3. Due to the P in the classic GAL system of Saccharomyces cerevisiae GAL1 It is also at least a dual-regulatory system, that is, glucose repression mediated by Mig1p and galactose activation mediated by Gal4p. In terms of steady-state substrate-induced response and kinetic induction, it was introduced as a control to better evaluate P xln.2b-xyl -XlnR-XylR performance. GAL1 The output of P is determined by the ratio of galactose to glucose. Therefore, when the total sugar is controlled at 2%, the P of P under different ratios of galactose and glucose is measured. GAL1 Output. The result is as follows Fig.19 As shown in a, the results show that only when galactose: glucose>31, P GAL1 reached the saturation activation level, but was still significantly lower than P xln.2b-xyl activation level.

[0158] 4. In addition, the P GAL1 The kinetics of the dual-regulation xylose system under the induction of 100 mM xylose were determined. Fig.19 As shown in b. GAL1 The memory of glucose inhibition has a great influence on the induction kinetic behavior. Specifically, strains without a history of glucose inhibition are induced very quickly, and the induction level reaches 50% of the maximum activation after 5 hours of induction. However, for strains with a history of glucose inhibition, there is at least 1 hour of induction hysteresis. The induction behavior of the dual-regulated xylose induction system is not affected by glucose, and the increase in fluorescence output can be detected within 15 minutes after the addition of xylose, and the induction level reaches 50% of the maximum activation after only 3 hours of induction. Similarly, the kinetic test data of the dual-regulated xylose induction system can be fitted by the standard transcription-translation model, namely, Exp. 2 and 3 (see Fig.19 (b)

[0159] Table 4: Xylose-induced kinetic fitting parameters

[0160]

[0161] Table 5: Fitting equations corresponding to the six topological structures

[0162]

[0163]

[0164]

[0165] Example 5: Substrate response curve of corncob hydrolysate activating xylose-induced transcription system

[0166] Agricultural waste such as corn, wheat, and cotton straw are rich in xylose. If the xylose in their hydrolyzates can be recycled, it will bring huge economic returns. In view of the high sensitivity of the xylose induction regulation system of the present application (EC50 is about 1mM), the hydrolyzates of these straws may replace pure xylose to activate the induction system. Therefore, 3g of 20-mesh corn cob powder was selected as the raw material, and the xylose in the hydrolyzate was 100% ethanol, and ... Fig.10 The production process was followed to obtain 50 mL of hydrolyzate, and the concentrations of glucose, xylose, and arabinose in the hydrolyzate were 178.9 mM, 53.3 mM, and 8.3 mM, respectively, as determined by HPLC (see Fig. 20 (a) Take different volumes of CCH as inducer and obtain the response substrate concentration curve as shown in Fig. 20 As shown in b. 50 μL of non-concentrated corncob hydrolysate can reach the maximum activation level (see Fig. 20 (b)

[0167] in, Fig.10 Preparation steps: 3g 20-mesh corn cob powder was mixed with 2% H 2 SO 4 The solution was treated at 120°C for 45 min, then treated with calcium carbonate, and then 1,800U of cellulase (from Beijing Solebow Technology Co., Ltd., item number C8270) and 1,500U of hemicellulase (from Beijing Solebow Technology Co., Ltd., item number H8110) were added for hydrolysis. Then, the solid-liquid separation was performed, and the supernatant was collected to obtain corn cob hydrolysate.

[0168] Example 6: Multi-dimensional comparison of the xylose-induced transcription system with existing induction systems in Saccharomyces cerevisiae

[0169] In order to better evaluate the single-regulation and dual-regulation xylose induction system, this application reconstructed the endogenous induction system of Saccharomyces cerevisiae - P GAL1 ,P MET3 ,P CUP1 and a synthetic inducible system based on the ER system (LexA-ER-VP16 / B112). The configuration of the gene elements and the integration sites are shown in Fig. 9 (The sequences of the relevant elements are shown in Table 1.) This application makes a comprehensive comparison with the developed xylose induction system in terms of five aspects: regulation stringency, induction activation level, dynamic range, growth toxicity, and induction speed.

[0170] In order to determine the maximum activation level, this application sets a test standard: when the maximum activation level is reached, the population should not show fluorescence heterogeneity, and the growth of the host should not be severely inhibited. To this end, the output and fluorescence distribution of each system at different inducer concentrations were tested and examined (see Fig.21), thus determining the inducer concentration that achieved the maximum activation level, at which the induced output level was measured, and the dynamic range of each system was calculated based on the output level when no inducer was added, and the strain growth OD was tested for three days by continuous transfer. 600 , the original transformed strain CENPK.2-1C was used as a reference to evaluate the growth of the strain under induction. Finally, the induction kinetics of each strain within 36 hours were tested under this concentration of induction. The results of the test are as follows Fig. 22 As shown, the results indicate that the dual-regulation xylose system has the best comprehensive performance in the above five aspects.

[0171] Example 7: Determination and fitting of substrate response functions of xylose-induced transcription system in Pichia pastoris, Candida glabrata and Candida albicans

[0172] XlnR protein has no homologs in Pichia pastoris, Candida glabrata and Candida albicans, and the former two cannot degrade and metabolize xylose. All three yeasts are non-conventional yeasts, with distant evolutionary relationships and relatively scarce genetic tools such as induction systems. Therefore, these three yeasts were selected to test the mobility of the XlnR-based xylose induction system. In order to facilitate prototype testing, the XlnR expression cassette and the reporter gene expression cassette driven by the response promoter were designed to be included on a plasmid. The gene elements, subelements and backbone plasmids used were as follows: Figure 11-14 In addition, in order to obtain a stable inducible system, all plasmids or restriction fragments containing the inducible system expression cassette were designed to be integrated into the genome. The integration strategy and site are shown in Fig.11 , as shown in 12 and 14. Fig.11 Schematic diagram of the design of the xylose transcription system based on XlnR and the genome recombination strategy in Pichia pastoris. The configuration of the gene elements in the dashed box on the right is as shown on the left; Fig.12 Schematic diagram of the design of the XlnR-based xylose transcription system and genome recombination strategy in Candida glabrata. The configuration of the gene elements in the dashed box on the right is as shown on the left; Fig.13 Schematic diagram of the construction of a universal plasmid adapted to the GOLDEN-GATE technology in Candida albicans. The specific plasmid sequences are shown in Table 1; Fig.14 This is a schematic diagram of the design of the XlnR-based xylose transcription system and genome recombination strategy in Candida albicans. The configuration of the gene elements within the dotted box in the right figure is shown in the left figure.

[0173] Finally, substrate induction curves were tested for three strains with an integrated xylose induction system, e.g. Fig. 22The induction times in Pichia pastoris, Candida glabrata, and Candida albicans were 189, 368, and 327 times, respectively. Similarly, in the three strains, the xylose induction curves can be well fitted by the hill function, and the fitting parameters are shown in Table 3. The results show that the EC50 of the xylose induction system in the first two strains is around 1mM, while the EC50 in Candida albicans is 13.7mM.

[0174] Example 8: Comparison of the ability of the xylose system and the methanol system to induce secretion of msfGFP in Pichia pastoris

[0175] Pichia pastoris is a powerful platform for the production of exogenous proteins. The methanol system is the main and powerful induction system of Pichia pastoris, but the system has inherent problems. First, the methanol system is an endogenous system and is inhibited by glucose and glycerol. In industrial production, the production stage that relies on glucose and glycerol must be separated from the methanol induction stage, thus lengthening the production cycle. Secondly, methanol itself is both a nutrient and an inducer, and its flammability and toxicity increase the safety risk of storage. Therefore, the practicality of the xylose induction system in inducing and driving the secretion of exogenous proteins in Pichia pastoris has far-reaching industrial value.

[0176] This application compares the induction activation levels of the xylose induction system and the methanol system. The results show that the maximum activation level of xylose is twice that of the methanol system ( Fig. 22 Middle a, Fig.23 In addition, the present application compared the ability of the two inducible systems to secrete msfGFP, and the results are as follows Fig.23 As shown in b. The results showed that under the respective optimal induction conditions, the ability of xylose to induce the secretion of msfGFP was significantly higher than that of the methanol system (such as Fig.23 (b, c). Fig.23 The xylose induction system based on XlnR in Example 8 was expressed in Pichia pastoris ( Fig.23 a), Candida glabrata ( Fig.23 b) and Candida albicans ( Fig.23 Substrate induction curve determination and Hill fitting diagram in c). Fig.24 This is a comparison of the ability of the xylose system and the methanol system in Pichia pastoris to induce secretion of msfGFP in Example 8; Fig.24 a is P AOX1 Substrate induction curve of Fig.24 b in the middle is a rycine SDS-PAGE image, debri: Pichia pastoris cell body, sup: culture supernatant, LS01-C3, C5, C6: xylose-induced system positive clones, LS03-C5, C7: methanol-induced system positive clones; Fig.24Middle c is the statistical analysis of the expression of msfGFP in the culture supernatant of the xylose-induced system and the methanol-induced system. Unpaired t-test. ***: p value = 0.0002.

[0177] In summary, in the present application, the xylose induction system based on the xylose-sensing transcription factor XlnR derived from filamentous fungi can reach 50% of the maximum activation level after 3 hours of xylose induction in the presence of 2% glucose, and can reach the maximum activation level after 6 hours of induction, so that the maximum activation level is greatly improved and the induction speed is rapid; and, by combining the xylose-sensing transcription factor XlnR derived from filamentous fungi and the prokaryotic xylose-sensing transcription repressor XylR, the leakage level of the dual-regulation induction system is extremely low, but the maximum activation level is equivalent to that of the XlnR single-regulation xylose induction system, which greatly expands the dynamic range by up to 4,000 times; and solves the problems of low induction intensity and slow response of the xylose transcription induction system dependent on transcription factors in the field of synthetic biology.

[0178] Table 1 Names and sequences of components involved in each embodiment

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0197] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A xylose-sensing transcription factor assembly, characterized in that include: A xylose-sensing transcription factor XlnR and a binding consensus sequence of the xylose-sensing transcription factor XlnR, wherein the xylose-sensing transcription factor XlnR is a xylose-sensing transcription factor XlnR derived from filamentous fungi.

2. The xylose-sensing transcription factor assembly according to claim 1, characterized in that The filamentous fungi include Aspergillus nidulans; Furthermore, the amino acid sequence of the xylose-sensing transcription factor XlnR is shown as SEQ ID NO.1; and / or the nucleotide sequence of the binding consensus sequence is shown as SEQ ID NO.

50.

3. The xylose sensing transcription factor assembly according to any one of claims 1 to 2, characterized in that The invention comprises a xylose sensing transcription factor XylR, wherein the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria.

4. The xylose-sensing transcription factor assembly according to claim 3, characterized in that The bacteria is Bacillus licheniformis; Furthermore, the amino acid sequence of the xylose sensing transcription factor XylR is shown in SEQ ID NO.

2.

5. A promoter component, characterized in that A promoter component capable of binding to the xylose sensing transcription factor component according to any one of claims 1 to 4, wherein the promoter component comprises: a first promoter capable of binding to the xylose sensing transcription factor XlnR, wherein the first promoter is obtained by modifying the ADH2 promoter from Saccharomyces cerevisiae as a chassis promoter; Furthermore, the modification includes modifying at least one of the core promoter sequence of the chassis promoter, the spacer sequence between the TATA box and the nucleosome deletion region, the spacer sequence between the TATA box and the transcription start site, the transcription factor binding site motif, and the prokaryotic operator site sequence.

6. The promoter assembly according to claim 5, characterized in that The first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the ADH2 promoter from Saccharomyces cerevisiae with the binding sequence of the xylose sensing transcription factor XlnR; Furthermore, the base sequence of the first promoter is shown in SEQ ID NO.

4.

7. The promoter assembly according to claim 5, characterized in that The first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the Saccharomyces cerevisiae-derived ADH2 promoter with the binding sequence of the xylose-sensing transcription factor XlnR, and then changing the spacer sequence between the TATA box and the nucleosome-deficient region and / or the spacer sequence between the TATA box and the transcription initiation site; Furthermore, the first promoter is selected from any one of the promoters with base sequences as shown in SEQ ID NO.5-SEQ ID NO.11 and SEQ ID NO.18-SEQ ID NO.

20.

8. The promoter assembly according to claim 5, characterized in that The first promoter is mainly obtained by the following steps: inserting the binding site xlno of the xylose sensing transcription factor XlnR into the ADH2 promoter; Furthermore, the number of inserted binding sites xlno is 1-4; Furthermore, the first promoter is selected from at least one of the promoters with base sequences such as SEQ ID NO.4-SEQ ID NO.5 and SEQ ID NO.12-SEQ ID NO.

15.

9. The promoter assembly according to any one of claims 5 to 8, characterized in that Also includes a second promoter, which is capable of binding to the xylose sensing transcription factor XylR; Furthermore, the second promoter contains a binding site xylo of the xylose sensing transcription factor XylR, and the binding site xylo is located upstream of the adjacent transcription initiation site and near the TATA box; Furthermore, the base sequence of the binding site xylo is shown as SEQ ID NO.49; and / or the base sequence of the second promoter is shown as SEQ ID NO.

3.

10. A dual-regulatory promoter, characterized in that: The dual-regulatory promoter can bind to a xylose sensing transcription factor XlnR and can bind to a xylose sensing transcription factor XylR. The xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi, and the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria.

11. The dual-regulatory promoter according to claim 10, characterized in that The dual-regulated promoter is selected from at least one of the promoters whose base sequences are shown in SEQ ID NO.16-SEQ ID NO.

17.

12. A transcription factor expression plasmid, characterized in that: The transcription factor expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor component according to any one of claims 1 to 4 is inserted.

13. The transcription factor expression plasmid according to claim 12, characterized in that: The transcription factor expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are inserted simultaneously; Alternatively, the transcription factor expression plasmid includes a first expression plasmid and a second expression plasmid, the first expression plasmid is an expression backbone plasmid into which the xylose-sensing transcription factor XlnR is inserted, and the second expression plasmid is an expression backbone plasmid into which the xylose-sensing transcription factor XylR is inserted; further, the expression backbone plasmid corresponding to the xylose-sensing transcription factor XlnR is a pGD137 plasmid having a base sequence as shown in SEQ ID NO.23, and the expression backbone plasmid corresponding to the xylose-sensing transcription factor XylR is a pGS001 plasmid having a base sequence as shown in SEQ ID NO.

24.

14. A promoter-reporter gene plasmid, characterized in that: The promoter-reporter gene plasmid is a reporter gene skeleton plasmid with an inducible promoter inserted therein, and the inducible promoter is selected from any one of the promoter components described in claims 5-9 and the dual-regulated promoters described in claims 10-11.

15. A recombinant bacterium, characterized in that: The recombinant bacteria is a host bacteria carrying the transcription factor expression plasmid according to any one of claims 12 to 14, and the host bacteria is Saccharomyces cerevisiae carrying the TetR expression box.

16. The recombinant bacterium according to claim 15, characterized in that The recombinant bacteria also carry a promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with an inducible promoter inserted therein, and the inducible promoter is selected from any one of the promoter components described in claims 5-9 and the dual-regulatory promoters described in claims 10-11.

17. The recombinant bacterium according to claim 15, characterized in that The transcription factor expression plasmid is an expression backbone plasmid into which the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are inserted simultaneously. The expression frames of the xylose sensing transcription factor XlnR and the xylose sensing transcription factor XylR are integrated into the NRT1 gene interval site of the Saccharomyces cerevisiae.

18. The recombinant bacterium according to claim 17, characterized in that The recombinant bacteria also have a first promoter and a second promoter introduced therein. The first promoter can bind to the xylose sensing transcription factor XlnR, and the second promoter can bind to the xylose sensing transcription factor XylR.

19. The recombinant bacterium according to claim 17, characterized in that The recombinant bacteria also carry a promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with an inducible promoter inserted therein, and the inducible promoter is selected from any one of the dual-regulatory promoters described in claims 10-11.

20. The recombinant bacterium according to claim 19, characterized in that The dual regulatory topology of the recombinant bacteria is selected from any one of topology 1 to topology 6; The fitting equation for topology 1 is as follows: The fitting equation for topology 2 is as follows: The fitting equation for topology 3 is as follows: The fitting equation for topology 4 is as follows: The fitting equation for topology 5 is as follows: The fitting equation for topology 6 is as follows: In the fitting equations of topology 1 to topology 6, m a is the mRNA concentration of transcription activator a; m [yfp] is the mRNA concentration of the fluorescent reporter protein YFP; a is the protein concentration of the transcription activator a; [YFP] is the protein concentration of the fluorescent reporter protein YFP; β1 is the mRNA synthesis rate driven by the dual-regulated promoter; β2 is the mRNA synthesis rate driven by the response transcription activator; β3 is the mRNA synthesis rate driven by the response transcription repressor; β p is the protein synthesis rate of transcription factor protein and fluorescent reporter protein YFP. Here, it is assumed that the protein synthesis rates of the two are the same; γ m is the degradation and dilution rate of mRNA; γ p is the degradation and dilution rate of the protein; k1 is the concentration of the transcription activator when the transcription output reaches half the activation intensity; n1 is the Hill coefficient in the function of transcription activator concentration and output.

21. The recombinant bacterium according to claim 19, characterized in that The kinetic model formula 1 of the substrate response curve of the recombinant bacteria is as follows: Among them, y min is the output of the induction system when no inducer is added, y max is the output when the induction system reaches steady state after the addition of inducer, x is the inducer concentration, k1, k2, n1, and n2 are the Hill parameters of the xylose-only regulated induction system based on XlnR and XylR, respectively.

22. The recombinant bacterium according to any one of claims 15 to 21, characterized in that: The induction substrate of the recombinant bacteria includes corn cob hydrolyzate, and the corn cob hydrolyzate is mainly prepared by the following steps: 3g of 20-mesh corn cob powder is treated with a 2% H2SO4 solution at 120°C for 45 minutes, then calcium carbonate is added for treatment, and then 1,800U of cellulase and 1,500U of hemicellulase are added for hydrolysis, and then solid-liquid separation is performed, and the supernatant is collected to obtain the corn cob hydrolyzate.

23. A recombinant bacterium, characterized in that: The recombinant bacteria is a host bacteria with a recombinant plasmid integrated into the chromosome, the recombinant plasmid is a backbone plasmid containing a xylose sensing transcription factor XlnR expression box and a reporter gene expression box driven by a response promoter, and the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi.

24. The recombinant bacterium according to claim 23, characterized in that The host bacteria is Pichia pastoris or Candida glabrata, and the xylose sensing transcription factor XlnR expression cassette is located upstream of the reporter gene expression cassette; Furthermore, the reporter gene is yEmCitrine; and / or the base sequence of the promoter is as shown in SEQ ID NO.

15.

25. The recombinant bacterium according to claim 23, characterized in that The host bacteria is Candida albicans, and the xylose sensing transcription factor XlnR expression cassette is located downstream of the reporter gene expression cassette; Furthermore, the reporter gene is mNeonGreen; and / or the base sequence of the promoter is shown in SEQ ID NO.21; and / or the backbone plasmid is pGS173 plasmid, and the base sequence of the backbone plasmid is shown in SEQ ID NO.22.

Images