Xylose-inducible transcription factor component and use thereof

Abstract

Disclosed in the present invention are a xylose-inducible transcription factor component and a use thereof. The xylose-inducible transcription factor component comprises: a xylose-inducible transcription factor XlnR derived from a filamentous fungus and a binding consensus sequence of the xylose-inducible transcription factor XlnR. By combining the transcription factor XlnR with a prokaryotic xylose-inducible transcriptional repressor XylR, a dual-regulated inducible system obtained exhibits an extremely low leakage level, but maintains a maximum activation level comparable to that of an XlnR single-regulated xylose-inducible system. The present invention solves the problems of low induction strength and slow response in transcription factor-dependent xylose transcriptional induction systems in the field of synthetic biology.

Description

Xylose-sensing transcription factor component and its application Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to xylose-sensing transcription factor components and their applications. Background Technology

[0002] Transcription induction systems are transcription factor-dependent induction systems. Due to their high specificity and sensitivity, they have irreplaceable advantages for the dynamic regulation of metabolic engineering and the design of synthetic gene circuits. A transcription induction system generally consists of three parts: an inducer, a transcription factor, and a promoter that can be bound by the corresponding transcription factor. Based on whether the inducer turns gene expression on or off, transcription induction systems can be further divided into transcriptional activation and transcriptional repression systems. For example, the tet system developed by Bujard in 1999, by mutating the transcription factor protein, transformed the originally tet-off system mediated by the transcriptional repressor protein TetR-VP16 into a tet-on system mediated by the transcriptional activator rtTA. Furthermore, even within the same system of turning off or activating, transcription factors can have different modes of action. For example, in the TetR and rtTA-mediated transcriptional activation systems, the former dissociates from the promoter upon binding to an inducer such as tetracycline, allowing transcription machinery such as RNA polymerase to bind to the promoter and initiate gene expression; while the latter binds to the promoter upon binding to the inducer, thereby recruiting transcription machinery to initiate expression. The difference between the two is that the activation level of the former depends only on the promoter, while the latter depends not only on the promoter but also on the strength of the transcriptional activation domain of the transcriptional activator. Therefore, the activation level of the latter is often higher than that of the former, but the leakage level of this system is often not negligible.

[0003] Eukaryotic transcription induction systems are generally classified into three types: 1) Endogenous induction systems. For example, the classic galactose-inducible system in *Saccharomyces cerevisiae*. These endogenous systems have complex signal transduction and regulatory networks, making them difficult to migrate to other hosts. Furthermore, due to their non-orthogonality with their own endogenous systems, they cannot be used for synthetic biology design. 2) Induction systems based on bacterial transcriptional repressors. These systems are generally limited by low transcriptional activation levels, making them unsuitable for applications requiring high output intensity, such as overexpressing key metabolic pathway enzymes in metabolic engineering. 3) Modularly assembled synthetic transcription induction systems. Transcription factors obtained using this strategy typically consist of three parts: a DNA-binding domain (DBD), a ligand-binding domain (LBD), and an activation domain (AD). Each module has a developed library, and these modules are then assembled to obtain a synthetic induction system with the desired performance. This development approach greatly expands the range of induction systems with specific performance characteristics, but the limitations of LBD-based systems and the fact that most successful examples are based on bioactive substances as inducers (such as hormone-based inducers) restrict their widespread application. In summary, universal eukaryotic transcription induction systems are still scarce and cannot meet the growing needs of the synthetic biology field.

[0004] Xylose is the second most abundant monosaccharide in nature, a major pentose, and its low cost, food-grade safety, and stability make it a promising inducer. In particular, *Saccharomyces cerevisiae*, a eukaryotic cell factory, cannot naturally utilize xylose and arabinose, making it a suitable inducer rather than a nutrient. Compared to the commonly used galactose, xylose offers significant advantages as an inducer for large-scale fermentation and cost reduction. Furthermore, the importance of xylose is increasingly evident as a raw material for energy production (H2, ethanol, energy precursors, etc.), food, and the production of high-value compounds.

[0005] To date, several xylose-induced systems have been independently developed in yeast by international and domestic research. For example, in 2014, Matthew Wook Chang et al. from South Korea pioneered the heterologous expression of the xylose-sensing transcriptional repressor XylR, derived from Tetragenococcus halophile, Clostridium difficile, and Lactobacillus pentosus, in Saccharomyces cerevisiae. They expressed XylR from different sources using promoters of varying strengths, and xylose successfully activated the expression of downstream target genes. Subsequently, in 2016, Zhao Huimin et al. expressed the transcriptional repressor XylR from Staphylococcus xylosus, Bacillus subtilis, and Bacillus licheniformis in Saccharomyces cerevisiae. 3Similarly, these transcription factors are specifically regulated by xylose to initiate gene expression. The above studies all utilize a xylose-induced system developed based on the transcriptional repressor protein XylR, and its mechanism of action is shown in Figure 1A. Simply put, in the absence of xylose, XylR... R By binding to the operon sequence xylO on the promoter, gene expression is shut down by preventing the RNA polII transcription machinery from binding to the promoter through steric hindrance; conversely, by adding xylose, XylR... R After dissociation from the promoter, transcription begins, and gene expression commences. The upper limit of the response intensity of transcription factor-based biosensors is also related to the activation domain (AD) of the transcription activator. Therefore, some studies have used XylR... R Chimeric transcription factors are obtained by fusing expression with activation domains; however, such sensors are essentially switches that control gene shutdown, which does not meet the requirements for direct induction of expression. Therefore, in 2020, Ye Bangze et al. developed a sensor based on the E. coli transcription activator XylR. A Turn-on xylose induction system. To increase transcriptional activation levels, XylR... A The system was designed to be fused with a strong transcriptional activation domain (VPR+HSF), and its mechanism of action is shown in Figure 1B. This system is based on a sensor of the TEF core promoter that is strongly responsive to xylose, exhibits high selectivity for xylose (more than 14.82 times more responsive to xylose than glucose) and a wide operability range (0-20 mM, 48-72 h).

[0006] Currently, most xylose-induced transcription systems developed in yeast are based on prokaryotic transcription factors. These systems suffer from low maximum transcriptional activation intensity, narrow dynamic range, and an excessively narrow xylose concentration response range (operable range), hindering their widespread application. Xylose sensors relying on prokaryotic transcription factors require precise regulation of transcription factor expression; otherwise, they can lead to host cell toxicity. Similarly, xylose sensors based on fusion expression strategies involving 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. Furthermore, due to the different transcriptional activation mechanisms in prokaryotes and eukaryotes, there are no established optimization strategies, making further optimization extremely difficult.

[0007] Summary of the Invention

[0008] Therefore, in order to address 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, it is necessary to provide a xylose-sensing transcription factor component with high induction intensity and rapid response, and its application.

[0009] A xylose-sensing transcription factor assembly includes: a xylose-sensing transcription factor XlnR and a binding consistency 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.

[0010] In the aforementioned xylose-sensing transcription factor assembly, the xylose-responsive activation transcription factor XlnR, derived from eukaryotic filamentous fungi, significantly enhances the maximum activation level and exhibits a very rapid induction rate. Experimental verification shows that the xylose-responsive activation transcription factor XlnR from eukaryotic filamentous fungi reaches 50% of its maximum activation level within 3 hours of induction.

[0011] A promoter assembly capable of binding to the xylose-sensing transcription factor assembly described above, the promoter assembly comprising: a first promoter capable of binding to the xylose-sensing transcription factor XlnR, wherein the first promoter is obtained by modifying the ADH2 promoter as a chassis promoter;

[0012] Furthermore, the modification includes modifying at least one of the following: 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 initiation site, the transcription factor binding site motif, and the prokaryotic operon site sequence.

[0013] A dual-regulatory promoter, wherein the dual-regulatory promoter can bind to xylose-sensing transcription factor XlnR and xylose-sensing transcription factor XylR, wherein the xylose-sensing transcription factor XlnR is derived from filamentous fungi and the xylose-sensing transcription factor XylR is derived from bacteria.

[0014] A transcription factor expression plasmid, wherein the transcription factor expression plasmid is an expression backbone plasmid inserted with the xylose-sensing transcription factor component described above.

[0015] A promoter-reporter gene plasmid, wherein the promoter-reporter gene plasmid is a reporter gene backbone plasmid with an inserted inducible promoter, wherein the inducible promoter is selected from any one of the promoter components described above and the dual-regulatory promoters described above.

[0016] A recombinant bacterium, wherein the recombinant bacterium is a host bacterium carrying the above-mentioned transcription factor expression plasmid, and the host bacterium is a Saccharomyces cerevisiae carrying the TetR expression cassette.

[0017] A recombinant bacterium, wherein the recombinant bacterium is a host bacterium with a recombinant plasmid integrated on its chromosome, wherein the recombinant plasmid is a backbone plasmid containing a xylose-sensing transcription factor XlnR expression cassette and a promoter-driven reporter gene expression cassette, and wherein the xylose-sensing transcription factor XlnR is a xylose-sensing transcription factor XlnR derived from filamentous fungi. Attached Figure Description

[0018] Figure 1 shows the design principle of an existing xylose biosensor, where: A is the working principle of the xylose induction system based on the transcriptional repressor factor XylR, and B is the working principle of the xylose induction system designed based on the transcriptional activator factor;

[0019] Figure 2 is a schematic diagram of a TF-based biosensor screening strategy;

[0020] Figure 3 shows the results based on the natural inductive promoter P. ADH2 A schematic diagram of the transformation strategy;

[0021] Figure 4 is a schematic diagram of the construction principle of the high-performance xylose induction system based on bacteria XylR;

[0022] Figure 5 is a schematic diagram illustrating the construction principle of the yeast strain carrying the xylose-responsive transcription factor array in Example 3;

[0023] Figure 6 is a schematic diagram of the construction principle of the single-regulatory xylose-induced transcription system in Example 3;

[0024] Figure 7 is a schematic diagram of the functional dual transcription factor regulatory topology in Example 4;

[0025] Figure 8 is a schematic diagram of the preferred dual transcription factor regulatory topology design in Example 4;

[0026] Figure 9 is a schematic diagram of the structure of commonly used induction systems for reconstructing Saccharomyces cerevisiae;

[0027] Figure 10 is a schematic diagram of the preparation process of corn cob hydrolysate in Example 5;

[0028] Figure 11 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Pichia pastoris;

[0029] Figure 12 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Candida glabrata;

[0030] Figure 13 is a schematic diagram of the construction of universal plasmids adapted to GOLDEN-GATE technology in Candida albicans;

[0031] Figure 14 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Candida albicans;

[0032] Figure 15 is a statistical chart of the activation / inhibition fold detection results under xylose induction and non-induction conditions of different xylose-responsive transcription factor screening systems in Example 1;

[0033] Figure 16 shows the detection results of the optimization based on the XlnR xylose transcription induction system, where: a) is the detection result of response promoter optimization; b) is the detection result of transcription factor concentration optimization.

[0034] Figure 17a 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 induction heterogeneity, and c is a schematic diagram of the determination and fitting of kinetic behavior.

[0035] Figure 18 is a schematic diagram of the construction design of the xylose dual-regulation induction system in Example 4, where: a is the fitting curve of 6 topologies, and b is the statistical diagram of the simulated calculation structure of τ1 / 2 (response time at half-activation level) of 6 topologies;

[0036] Figure 19a shows the topology and substrate response curves of the Saccharomyces cerevisiae galactose-induced system and xylose dual-regulation-induced system in Example 4, and b shows the kinetic behavior measurement and fitting.

[0037] Figure 20a is a schematic diagram of the polysaccharide composition of corn cob hydrolysate in Example 5, and b is a schematic diagram of the substrate response curve of the xylose dual-regulation induction system.

[0038] Figure 21 shows the detection results of substrate induction curves and fluorescence distribution of the xylose-induced transcription system and the existing induction system in Saccharomyces cerevisiae in Example 6;

[0039] Figure 22 is a multidimensional comparison of the xylose-induced transcription system and existing induction systems in Saccharomyces cerevisiae, where: a is a schematic diagram of the maximum activation level, b is a schematic diagram of the effect on host growth toxicity, c is a schematic diagram of the induction rate, and d is a schematic diagram of the radar chart (visualizing the multidimensional comparison).

[0040] Figure 23 shows the substrate induction curves and Hill fitting diagrams of the XlnR-based xylose induction system in Pichia pastoris (Figure 23a), Candida glabrata (Figure 23b), and Candida albicans (Figure 23c) in Example 8.

[0041] Figure 24 is a comparison of the ability of the xylose system and methanol system of Pichia pastoris in Example 8 to induce the secretion of msfGFP. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application's description is for descriptive purposes only and is not intended to be limiting of the application. Terminology

[0044] Unless otherwise stated or in case of conflict, the terms or phrases used in this application shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These 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 using 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 solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0047] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0048] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0049] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0050] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0051] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous, and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this application, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed in this application should be understood to include any and all subranges included therein.

[0052] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0053] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0054] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0055] A first aspect of this application provides a xylose-sensing transcription factor assembly, comprising: a xylose-sensing transcription factor XlnR and a binding consistency 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.

[0056] In the aforementioned xylose-sensing transcription factor assembly, the xylose-responsive activation transcription factor XlnR, derived from eukaryotic filamentous fungi, significantly enhances the maximum activation level and exhibits a very rapid induction rate. Experimental verification shows that the xylose-responsive activation transcription factor XlnR from eukaryotic filamentous fungi reaches 50% of its maximum activation level within 3 hours of induction.

[0057] In some embodiments, the filamentous fungus includes *Aspergillus nidulans*. *Aspergillus nidulans* is a fungus belonging to the family Aspergillusceae and the genus *Aspergillus*. It should be noted that the xylose-sensing transcription factor XlnR can be derived from *Aspergillus nidulans*, or its sequence homologs and binding-consistent sequences can replace this system to perform a similar function.

[0058] Further, the amino acid sequence of the xylose-sensing transcription factor XlnR is shown in SEQ ID NO.1; and / or, the nucleotide sequence of the binding concordance sequence is shown in SEQ ID NO.50. The above-mentioned xylose-sensing transcription factor XlnR and / or its binding concordance sequence can enhance the maximum activation level and induction rate. Specifically, the sequence shown in SEQ ID NO.50 is: 5'-GGCTAAW-3', where W represents a mixture of adenine (A) and thymine (T).

[0059] In some embodiments, the xylose-sensing transcription factor assembly further includes a xylose-sensing transcription factor XylR, which is a bacterial xylose-sensing transcription factor XylR. Combining the bacterial xylose-sensing transcription factor XylR can reduce the leakage level of the transcription induction system.

[0060] The bacteria in question is *Bacillus licheniformis*. Furthermore, the amino acid sequence of the xylose-sensing transcription factor XylR is shown in SEQ ID NO.2. The dual-regulatory induction system combining *Bacillus licheniformis* transcription factor XylR significantly reduces leakage levels, ultimately achieving an induction dynamic range of up to 4000-fold, while the maximum activation intensity and response rate remain unaffected compared to the action of a single XlnR. It should be noted that the aforementioned xylose-sensing transcription factor XylR is not limited to those derived from Bacillus licheniformis. It can also be sequence homologs of the xylose-sensing transcription factor XylR derived from Bacillus licheniformis and binding homologous sequences that can replace this system and perform similar functions. It can also be derived from other bacteria, such as Lactobacillus pentosus, Clostridium difficile, Tetragenococcus halophile, Bacillus subtilis, Caulobacter crescentus, etc.

[0061] In the aforementioned xylose-sensing transcription factor component, the xylose-responsive activation transcription factor XlnR, derived from eukaryotic filamentous fungi, is used, which significantly increases the maximum activation level and induces the response very rapidly (reaching 50% of the maximum activation level within 3 hours of induction). Based on the XlnR system, the dual-regulation induction system of Bacillus licheniformis transcription factor XylR is combined to greatly reduce the leakage level, resulting in an induction dynamic range of up to 4000 times. Furthermore, the maximum activation intensity and response rate are not affected by the action of XlnR alone.

[0062] A second aspect of this application provides a promoter component capable of binding to the aforementioned xylose-sensing transcription factor component to monoregulate xylose induction in a host bacterium. The promoter component includes a first promoter capable of binding to the xylose-sensing transcription factor XlnR.

[0063] Due to natural promoters such as P xylPExcessively long, unstructured promoters increase the difficulty of further modification and may also be subject to unknown endogenous regulation. Therefore, it is necessary to rationally design structured synthetic promoters. Thus, in some embodiments, a structurally well-defined inducible natural ADH2 promoter is first selected. This first promoter is obtained by modifying a Saccharomyces cerevisiae-derived ADH2 promoter as the chassis promoter. The Saccharomyces cerevisiae-derived ADH2 promoter is specifically an ADH2 promoter derived from Saccharomyces cerevisiae.

[0064] Due to the structured sequence characteristics, the naturally inducible ADH2 promoter of *Saccharomyces cerevisiae* was selected as the chassis for the design of a fully synthetic promoter. The principle was to ensure that the characteristic sequences remained unchanged: the nucleosome depletion region (NDR)-poly(A) sequence, the TATA box, the transcription start site (TSS), and to modify the spacer sequences between these characteristic sequences, thereby obtaining a fully synthetic promoter that met the desired performance. Furthermore, depending on the induction system, the upstream activation sequence (UAS) could be replaced. Therefore, the modification described in this application includes modifying at least one of the following: 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 operon site sequence.

[0065] In some embodiments, the first promoter is obtained primarily through 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 through 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 this semi-synthetic promoter is not negligible.

[0066] Since the leakage level of the aforementioned semi-synthetic promoters is not negligible, in some embodiments, the first promoter is mainly obtained through 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 altering the spacer sequence between the TATA box and the nucleosome deletion region and / or the spacer sequence between the TATA box and the transcription initiation site. These modifications aim to improve the promoter leakage level.

[0067] Furthermore, the first promoter is selected from any one of the promoters shown in SEQ ID NO.5-SEQ ID NO.11 and SEQ ID NO.18-SEQ ID NO.20.

[0068] Specifically, the sequence between NDR and TATA-box was subjected to high-throughput screening using synthesized random primers (hereinafter referred to as random primer 1, with the base sequence: 5'GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAGCA-3'), ultimately yielding low-background, strongly activated P. xln.1b (Base sequences are shown in SEQ ID NO. 5). Previous literature reported that the core promoter determines the maximum activation intensity; therefore, this application replaced the core promoter region of ADH2 with other highly active promoter core regions, resulting in six synthetic promoters (their base sequences are shown in SEQ ID NO. 6-SEQ ID NO. 11). Although all of these support xylose-induced activation, the maximum activation level is lower than that of the original ADH2 core promoter. These results also suggest that the core promoter region has programmability. To obtain fully synthetic promoters, the TATA-BOX and TSS region sequences were further synthesized using multiple random primer stacking for high-throughput screening. Finally, a series of fully synthetic inducible promoters were obtained, especially the three fully synthetic promoters with base sequences shown in SEQ ID NO. 18-SEQ ID NO. 20.

[0069] Studies have found that appropriately increasing the binding sites of transcription factors is a reliable strategy to enhance transcriptional activation levels. Therefore, in some embodiments, the first promoter is obtained primarily through the following steps: inserting the xylose-sensing transcription factor XlnR binding site xlno into the ADH2 promoter. Further, the number of inserted binding sites xlno is 1-4. Specifically, the first promoter is selected from at least one promoter with the base sequences shown in SEQ ID NO.4-SEQ ID NO.5 and SEQ ID NO.12-SEQ ID NO.15. Among these, promoters containing 2 xlno have significantly increased activation levels, especially those containing 2 xlno. xln.2b (Base sequence shown in SEQ ID NO.13), 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, therefore, P xln.4 It has the largest dynamic range.

[0070] The ADH2 promoter chassis architecture and promoter optimization strategy adopted in this application refer specifically to the characteristic sequences (kozak, TSS, TATA-Box, NDR, UAS, see Table 1 notes) and the spacing lengths between the characteristic sequences (see Figure 3). High-throughput screening was performed on the spacer sequences (Spacer.a and Spacer.b in Figure 3) between the characteristic sequences (TSS, TATA-Box, NDR) using randomly designed primers with fixed spacer sequences.

[0071] The first promoter described above is a series of short (<300bp) tunable promoters designed and constructed using the natural ADH2 promoter from Saccharomyces cerevisiae as the modified chassis. It can achieve high-intensity activation and avoids the problems caused by using natural promoters: potential endogenous regulation, difficulties in construction and transformation due to repeated use, and genomic instability.

[0072] It should be noted that the ADH2 promoter architecture derived from Saccharomyces cerevisiae used in this application has a certain degree of compatibility in terms of the length and sequence of its spacer sequences (spacer.a and spacer.b in Figure 3), and its UAS sequence can be replaced with other transcription factor binding sites for use in other transcription induction systems.

[0073] In some embodiments, the promoter component further includes a second promoter capable of binding to the xylose-sensing transcription factor XylR.

[0074] Further, the second promoter contains a binding site xylo for the xylose-sensing transcription factor XylR, located upstream of the transcription promoter site and near the TATA box. Even further, the XylR binding sequence (operator) is positioned downstream of the TATA box and upstream of the TSS. In some specific examples, the base sequence of the binding site xylo is as shown in SEQ ID NO. 49; and / or, the base sequence of the second promoter is as shown in SEQ ID NO. 3.

[0075] A third aspect of this application provides a dual-regulatory promoter capable of binding to both xylose-sensing transcription factor XlnR and xylose-sensing transcription factor XylR. XlnR is derived from filamentous fungi, while XylR is derived from bacteria. This dual-regulatory promoter can dual-regulate xylose induction in the host bacterium.

[0076] In some embodiments, the dual-regulatory promoter is selected from at least one of the promoters whose base sequences are shown in SEQ ID NO.16-SEQ ID NO.17.

[0077] A fourth aspect of this application provides a transcription factor expression plasmid, wherein the transcription factor expression plasmid is an expression backbone plasmid in which the xylose-sensing transcription factor component described above is inserted. The xylose-sensing transcription factor component can be expressed through this transcription factor expression plasmid.

[0078] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid in which both xylose-sensing transcription factor XlnR and xylose-sensing transcription factor XylR are inserted. That is, xylose-sensing transcription factor XlnR and xylose-sensing transcription factor XylR are inserted into the same backbone plasmid, which can be used to construct a yeast strain with a transcription factor array.

[0079] In other embodiments, the transcription factor expression plasmid includes a first expression plasmid and a second expression plasmid. The first expression plasmid is an expression backbone plasmid in which the xylose-sensing transcription factor XlnR is inserted, and the second expression plasmid is an expression backbone plasmid in 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 inserted into different backbone plasmids, which can be used to construct a prototype response testing platform for a xylose transcription-induced system. Further, the expression backbone plasmid corresponding to the xylose-sensing transcription factor XlnR is the pGD137 plasmid with the base sequence shown in SEQ ID NO.23, and the expression backbone plasmid corresponding to the xylose-sensing transcription factor XylR is the pGS001 plasmid with the base sequence shown in SEQ ID NO.24.

[0080] A fifth aspect of this application provides a promoter-reporter gene plasmid, which is a reporter gene backbone plasmid with an inserted inducible promoter, wherein the inducible promoter is selected from any one of the promoter components described above and the dual-regulatory promoters described above. This promoter-reporter gene plasmid can be used together with transcription factor expression plasmids to construct a xylose transcription-inducible system.

[0081] A sixth aspect of this application provides a recombinant bacterium, which is a host bacterium carrying the aforementioned transcription factor expression plasmid, wherein the host bacterium is a Saccharomyces cerevisiae carrying a TetR expression cassette. The transcription factor expression plasmid is transferred into the host bacterium to express the aforementioned xylose-sensing transcription factors XlnR and XylR.

[0082] In some embodiments, the recombinant bacteria also carry a promoter-reporter gene plasmid, which is a reporter gene backbone plasmid with the aforementioned inducible promoter inserted. The inducible promoter is selected from any one of the aforementioned promoter components and the aforementioned dual-regulatory promoters. This recombinant bacteria can be used to construct yeast strains for transcription factor arrays or to construct a prototype response testing platform for a xylose transcription induction system.

[0083] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid containing both the xylose-sensing transcription factor XlnR and the xylose-sensing transcription factor XylR, with the expression cassettes of XlnR and XylR integrated into the NRT1 gene spacer site of *Saccharomyces cerevisiae*. This recombinant strain can express both xylose-sensing transcription factor XlnR and xylose-sensing transcription factor XylR.

[0084] Furthermore, in some examples, based on the recombinant bacteria of the above embodiments, the recombinant bacteria are further transformed 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. Detailed descriptions of the first and second promoters are provided above and will not be repeated here. Recombinant bacteria transformed with the aforementioned first and second promoters can single-regulate xylose-induced expression.

[0085] In some examples, based on the recombinant bacteria described in the above embodiments, the recombinant bacteria also carry a promoter-reporter gene plasmid. This promoter-reporter gene plasmid is a reporter gene backbone plasmid with an inserted inducible promoter, and the inducible promoter is selected from any of the dual-regulatory promoters described above (details are described above and will not be repeated here). In this recombinant bacteria, the signals of transcription activators and transcription repressors are integrated into the same synthetic promoter. That is, the binding site of the transcription repressor is located in the core promoter region, while the binding site of the transcription activator is located in the UAS region. Furthermore, the selected transcription activator and transcription repressor do not compete for binding; they interact independently with the same synthetic promoter. The above recombinant bacteria can dual-regulate xylose-induced expression.

[0086] A dual transcription factor regulatory system can be adopted, integrating the advantages of both to further optimize the xylose transcription regulation system. However, there are multiple design schemes for the dual regulation mode (Figure 18a), among which six functional topologies exist (Figure 7). The dual regulation topology of the recombinant bacteria is selected from any one of topologies 1 to 6. Topology 1 is the most effective dual regulation topology. The fitting equations for topologies 1 to 6 are shown in Table 5 below. In the fitting equations, m... a It is the mRNA concentration of transcription activator a; m[yfp] β1 is the mRNA concentration of fluorescent reporter protein YFP; β2 is the mRNA synthesis rate driven by transcription activator a; β3 is the mRNA synthesis rate driven by transcription repressor; β4 is the mRNA synthesis rate driven by transcription repressor. p This refers to the protein synthesis rates of transcription factor protein and fluorescent reporter protein YFP; it is assumed here that their protein synthesis rates are the same; γ m It refers to the rate of mRNA degradation and dilution; γ p is the rate of protein degradation and dilution; k1 is the concentration of transcription activators when the transcription output reaches half the activation intensity; n1 is the Hill coefficient in the output function and the concentration of transcription activators.

[0087] Based on the dual transcription factor regulatory system, the kinetic model formula 1 for the substrate response curve of the recombinant bacteria is as follows:

[0088] The meanings of each letter are: y min y represents the output of the induction system without the addition of an inducing agent. max This represents the output of the induction system when it reaches steady state after the addition of the inducer. x is the concentration of the inducer. k1, k2, n1, and n2 are the Hill parameters of the xylose-based single-regulation system (XlnR-based and XylR-based induction systems), respectively.

[0089] Agricultural wastes such as corn, wheat, and cotton stalks are rich in xylose. Recycling the xylose in their hydrolysates could yield significant economic returns. In some embodiments, the inducing substrate for the recombinant bacteria includes corn cob hydrolysate, which is prepared primarily by the following steps: treating 3g of 20-mesh corn cob powder with a 2% (w / w) H₂SO₄ solution at 120°C for 45 minutes, then adding calcium carbonate, followed by hydrolysis with 1,800U of cellulase and 1,500U of hemicellulase, then performing solid-liquid separation, collecting the supernatant to obtain the corn cob hydrolysate. Using xylose-rich agricultural waste (such as corn cob) hydrolysate can replace pure xylose in activating the xylose-inducing system, thereby significantly reducing the cost of using this system for large-scale industrial production.

[0090] To verify the transferability of the XlnR-based xylose-inducible system in other yeast species, an integrated single-regulatory XlnR-based xylose-inducible system was incorporated into the genomes of *Pichia pastoris*, *Candida glabrata*, and *Candida albicans* to detect the xylose response of the system. Therefore, in a seventh aspect of this application, a recombinant bacterium is provided, wherein the recombinant bacterium is a host bacterium with a recombinant plasmid integrated onto its chromosome. The recombinant plasmid is a backbone plasmid containing a xylose-sensing transcription factor XlnR expression cassette and a promoter-driven reporter gene expression cassette. The xylose-sensing transcription factor XlnR is derived from filamentous fungi. A detailed description of the xylose-sensing transcription factor XlnR is provided above and will not be repeated here.

[0091] In some embodiments, the host bacterium 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 shown in SEQ ID NO.15.

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

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

[0095] The recombinant strains described above demonstrate that the XlnR-mediated xylose-induced transcription system has high transferability and can still respond to xylose in doses in several distantly related unconventional yeasts. The XlnR-mediated xylose-induced transcription system has strong practicality and can efficiently induce the secretion of the secretory protein msfGFP in Pichia pastoris, with a higher secretion level than the endogenous methanol system.

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

[0097] This application develops a highly efficient and inexpensive transcription induction system: it uses xylose-based transcription factor XlnR, derived from eukaryotic filamentous fungi, to activate the transcription factor, which significantly increases the maximum activation level and induces the transcription very rapidly (50% of the maximum activation level can be achieved in 3 hours of induction).

[0098] Based on the XlnR system, this application combines the dual-regulation induction system of Bacillus licheniformis transcription factor XylR, which greatly reduces the leakage level and ultimately achieves an induction dynamic range of up to 4000 times. Moreover, the maximum activation intensity and response speed are not affected by the action of single XlnR.

[0099] The dual-mode induction system designed in this application allows the substrate response function to be predicted using parameters of the single-mode substrate response.

[0100] This application utilizes the natural ADH2 promoter as a modified platform to design and construct a series of short (<300bp) tunable synthetic promoter sequences that achieve high-intensity activation while avoiding the problems associated with using natural promoters: potential endogenous regulation, difficulties in construction and transformation due to repeated use, and genomic instability. Even under conditions of saturated activation of the xylose-induced system, the growth of *Saccharomyces cerevisiae* was not significantly inhibited.

[0101] In this application, xylose-rich crop waste (such as corn cob) hydrolysate can replace pure xylose to activate the xylose induction system, thereby greatly reducing the cost of using the system for the production of bulk industrial products.

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

[0103] The XlnR-mediated xylose-induced transcription system of this application has high transferability and can still respond to xylose in doses in several unconventional yeasts with distant phylogenetic relationships. Furthermore, the XlnR-mediated xylose-induced transcription system of this application has strong practicality and can efficiently induce the secretion of the secretory protein msfGFP in Pichia pastoris, with the secretion level being higher than that of the endogenous methanol system.

[0104] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[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 the absence of “(amino acid sequence)” after the sequence indicates 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 embodiment

[0109] 1. Substrate induction test culture

[0110] Single colonies were picked from YPD plates and transferred to 500 μL of SD deficiency medium in 2 mL 96-well plates (Axygen, P-2ML-SQ-CS). The plates were sealed with a breathable membrane (BioTss, SF-200) and incubated at 800 rpm and 30°C for 24 hours. Then, 2.5 μL aliquots were inoculated into 497.5 μL of fresh SD medium (OD) containing the appropriate concentration of inducer. 600 =0.005), and cultured for 16 h under the same conditions, then diluted 10-fold, i.e., 20 μL of 180 μL phosphate-buffered saline (PBS, Proteintech, PR20014) was added to a 96-well U-shaped plate (corning, 3799), containing 10 μg mL of PBS. -1 Cycloheximine (Mocom Biotech, MS0035) was used, and the mixture was then analyzed by flow cytometry.

[0111] 2. Flow cytometry and data processing

[0112] The intensity of yellow fluorescent protein (yEmCitrine) in treated samples was detected using a BD FACSCelesta™ flow cytometer in High Throughput Sampler (HTS) mode (BD Biosciences, Germany). A 488 nm excitation laser (100 mW) and 530 / 30 nm emission filters were selected to measure YFP intensity. Data were recorded using FACSDiva software (BD Biosciences, Germany), with over 10,000 events recorded for each sample. Cells of the correct size were selected in the forward scattering area (FSC-A) / side scattering area (SSC-A) plot. Data were used... Software 10.4 (TreeStar, USA) was used for processing. The median signal in the FITC-A region (fluorescein isothiocyanate-based region) was used for quantitative and statistical analysis of yellow fluorescence. The formula for calculating the fold increase is (YFP... 诱导 -YFP0) / (YFP 非诱导 -YFP0), where YFP 诱导 and YFP 非诱导 These represent fluorescence measured in the presence and absence of 10 mM xylose, respectively. YFP0 represents the autofluorescence of leukocytes. The inhibition factor is calculated using the formula (YFP...). -抑制因子 -YFP0) / (YFP +抑制因子 -YFP0), where YFP -抑制因子 and YFP +抑制因子 The values ​​represent fluorescence measured in the absence and presence of transcription repressors, respectively. Fluorescence intensity is expressed using the endogenous constitutive PFY1 promoter (YFP). 参考 The fluorescence intensity of the sample was normalized and expressed in RPU. The formula for calculating RPU is (YFP - YFP0) / (YFP0). 参考 -YFP0). Therefore, white blood cells, CY671int(P PFY1 -YFP) and CY676int(P TDH3 -YFP) was used as a control strain and was cultured on the same plates as the test strain each time.

[0113] 3. Induction kinetics test culture

[0114] Single colonies were picked from YPD plates and transferred to 500 μL of SD-deficient medium in 2 mL 96-well plates (Axygen, P-2ML-SQ-CS). The plates were sealed with a breathable membrane (BioTss, SF-200) and incubated at 800 rpm and 30°C for 24 hours. For GAL system derivatized strains (GAL80Δ, GAL80Δ & GAL1Δ) without glucose inhibition, SC medium (SCR medium) supplemented with 2% (w / v) raffinose was used. Unless otherwise specified, 2% (w / v) glucose was used as the carbon source. Then, 2.5 μL of culture was aliquoted into 475.5 μL of fresh SD medium (OD medium) supplemented with the appropriate inducer in 2 mL 96-well plates. 600=0.005). Then, 25 μL of these cultures were transferred to 475 μL of fresh SD or SCR medium, and the corresponding inducer was added to 2 mL of 96-well plates. The plates were then incubated at 30°C and 800 rpm in a shaking incubator (Zhichu, ZQZY-88AH). During the first 3 hours of growth, 50 μL of culture was collected at each designated time point and then an equal volume of fresh medium containing the corresponding inducer was added. After the first 3 hours of incubation, 20 μL of culture was collected from each well every 3 hours during the 36-hour incubation period. The collected samples were then mixed with 180 μL of medium containing 10 μg / mL of SD or SCR medium. -1 Cycloheximine was diluted with PBS and then analyzed by flow cytometry.

[0115] 4. Growth toxicity test

[0116] Three experimental bacterial clones were randomly selected and cultured overnight in 500 μL of SD nutrient-deficient medium. The cultures were then inoculated at a 1:100 ratio into SD medium (for nutrient-deficient screening) containing gradient concentrations of inducers and cultured for 24 hours. 20 μL of culture was analyzed by flow cytometry to detect the correctness of the experimental strains and induction heterogeneity. Simultaneously, 5 μL of culture from each well was inoculated one-to-one into the corresponding wells of 495 μL medium containing gradient concentrations of inducers on another plate. After 8 hours of culture, the OD of the culture in each well was measured using a Tecan Infinite 200Pro plate reader (Tecan). 600 Simultaneously, the cultures from the uninduced wells were inoculated into SD medium supplemented with gradient concentrations of inducers, and allowed to regrow for 24 hours. 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 OD value. 600 The normalized test strains were measured over three consecutive days. The calculation formula is norm.OD. 600 =ave(OD) 600 ) 诱导 / OD 600(2-1C) .

[0117] 5. HPLC analysis of corn cob hydrolysates

[0118] The concentrations of D-glucose, D-xylose, and L-arabinose in corn cob hydrolysate 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), coupled with a Micro-Guard Cation-H guard column (Bio-Rad, Hercules, USA). The separation process used 5 mM H₂SO₄ as the mobile phase, with a flow rate set at 0.6 mL / min. -1 The temperature was 55°C. The signal was detected by RID (Agilent Technologies, G7162A) at 35°C.

[0119] 6. Sample preparation and Tricine-SDS-PAGE

[0120] Sample preparation: Picking xylose-inducing system and P AOX1 A single clone of the controlled-expressing secretory msfGFP strain was cultured in 2 mL 96-well plates (Axygen, P-2ML-SQ-CS) containing SD-His medium, sealed with a breathable membrane, and incubated at 800 rpm and 30°C for 24 hours. Then, 5 μL of the culture was inoculated into 495 μL of fresh YPD and BMGY media, respectively, and allowed to grow for another 24 hours. Subsequently, 5 μL of the strain culture carrying the xylose-inducible system was inoculated into 495 μL of fresh YPD medium supplemented with 100 mM xylose; simultaneously, 5 μL of the strain culture carrying the methanol-inducible system was inoculated into BMMY medium supplemented with 0.5% methanol (v / v). After 24 hours of induction, 0.5% methanol (v / v) was added to the methanol-inducible strain. After 48 hours of induction, 20 μL of supernatant was collected and the extracellular msfGFP fluorescence intensity was measured using a Tecan Infinite 200 Pro plate reader (excitation wavelength 488 nm, emission wavelength 510 nm, gain 100). 20 μL of culture medium and 20 μL of undiluted supernatant were then mixed with 20 μL of 2×Tris-Tricine-SDS-PAGE loading buffer (BOSTER, AR1143). These mixtures were then boiled at 100 °C for 5 minutes and frozen at -80 °C for Tris-SDS-PAGE analysis.

[0121] Tricine-SDS-PAGE: First, a 1.5 mm thick PAGE gel was prepared, consisting of three gels: a stacked gel (4% T, 3% C), a spacer gel (10% T, 3% C), and a separating gel (16.5% T, 6% C, containing 6M urea). Tricine gels and gel buffers were provided by a commercial kit (Enogene Biotech, E1WP326). Running buffer, anolyte buffer (0.1M Tris, 0.0225M HCl, pH 8.9), and catholyte buffer (0.1M Tris, 0.1M Tricine, 0.1% SDS, pH 8.25) were purchased from Servicebio (G2142-1L). After electrophoresis, the gel was incubated in fixative (50% methanol, 10% acetic acid) for 1 hour, and then stained overnight with 0.025% Coomassie Brilliant Blue R-250 in 10% acetic acid solution. The gel was then eluted twice in 10% acetic acid, incubating for 45 minutes each time. The destaining gel was imaged on the Bio-Rad ChemiDoc imaging system, and the images were analyzed using Bio-Rad ImageLab software (version 6.1).

[0122] Example 1: Prototype substrate response test of xylose-induced transcription systems from different sources in Saccharomyces cerevisiae

[0123] The basic principle of constructing the xylose-responsive transcriptional induction system prototype response testing platform is to efficiently screen xylose-responsive transcriptional induction systems in *Saccharomyces cerevisiae*. Candidate transcription factors are introduced with restriction endonuclease BpiI sites at both ends of fragments through primer design and removal of BpiI sites from the natural sequence through codon synonymy substitution. Reporter plasmids expressing the transcription factors and responding transcription factors are constructed in one step using the GOLDEN-GATE technique. The TF-based biosensor screening strategy is shown in Figure 2, which illustrates the annotation features of the backbone plasmids used for biosensor screening. The required reporter plasmid and the plasmid expressing TF are generated in one step by replacing ccdB in the vector with BpiI digestion. The purified BsaI digestion product is directly transformed into yeast. The xylose-responsive transcription factor screening strategy is performed in *Saccharomyces cerevisiae*. The transcription factor concentration is controlled by the aTc-TetR induction system, where TetR expression is controlled by the constitutive promoter PFBA1. The output of the transcription factor-responsive promoter depends on the concentrations of TF and the inducer.

[0124] The specific steps for constructing the xylose transcription induction system prototype response testing platform are as follows:

[0125] The xylose-responsive transcription factor XlnR (AN7610) sequence (amino acid sequence as shown in SEQ ID NO.1) was obtained from the extracted Aspergillus nidulans genome by designing primers, removing introns and BpiI restriction sites, and then amplifying by segmental PCR. Xylose-responsive transcription factors XylR from different bacteria (as shown in Figure 15) were synthesized after codon optimization (codon optimization method can be found at https: / / sg.idtdna.com / pages / tools / codon-optimization-tool). Among them, the amino acid sequence of the xylose-sensing transcription factor XylR from Bacillus licheniformis is shown in SEQ ID NO.2.

[0126] The obtained transcription factor fragments were reacted with the backbone plasmids pGD137 (base sequence shown in SEQ ID NO. 23) and pGS001 (base sequence shown in SEQ ID NO. 24) using a GOLDEN-GATE system. 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 xylose transcription factors. Among them, the xylose-responsive transcription factor XlnR was ligated into the backbone plasmid pGD137, and the xylose-responsive transcription factor XylR was ligated into the backbone plasmid pGS001.

[0127] From the natural promoter P of Penicillin chrysogenum xylP (711bp, its base sequence is SEQ ID NO.55) Similarly, the BpiI restriction site in the promoter region was removed by primer design and a BpiI restriction site was introduced at the end. The reporter plasmid was obtained by reacting with the existing backbone plasmid pXJH1 in the laboratory using the GOLDEN-GATE system; for XylR of different bacterial species, its promoter (where the amino acid sequence is as shown in SEQ ID NO.2, the promoter corresponding to the xylose-sensing transcription factor XylR is P) xylThe base sequence is shown in SEQ ID NO.3. The reporter plasmid 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 using the same method. The obtained reporter plasmid was transformed into the yeast strain already carrying the xylose transcription factor, and the activation / inhibition fold under xylose-induced and non-induced conditions was tested according to the method section. The detection results are shown in Figure 15. The schematic diagram of the construction of the high-performance xylose induction system based on bacterial XylR is shown in Figure 4 (in Figure 4, T20: 20 consecutive thymine bases; TATA: TATA-box (5'-TATAAAA-3', sequence number SEQ ID NO.51); TSS: transcription start site (5'-AGAATATCAAGCTACAAAAA-3', sequence number SEQ ID NO.52); the lengths between characteristic sequences are shown in the numbers above). For the bacterial XylR transcription factor (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 XylR binding sequence (operator) was placed downstream of the TATA-box and upstream of the TSS.

[0128] As shown in Figure 15, XlnR in Aspergillus nidulans showed an activation factor of over 100-fold, while XylR in Bacillus licheniformis had the largest inhibition factor. Therefore, both were used for further optimization.

[0129] Example 2: Design and optimization of synthetic promoters responding to XlnR

[0130] Due to the natural promoter P xylP Excessive length and unstructured architecture increase the difficulty of further modification and may also be subject to unknown endogenous regulation. Therefore, it is necessary to rationally design structured synthetic promoters. Based on the naturally inducible promoter P... ADH2 The modification strategy is detailed in Figure 3. The detection results of the optimization based on the XlnR xylose transcription induction system are shown in Figure 16, where: a is the detection result of the response to the synthetic promoter optimization; b is the detection result of the transcription factor concentration optimization.

[0131] The specific optimization process for startup is as follows:

[0132] First, a well-defined inducible natural promoter for ADH2 was selected. The original UAS region sequence for ADR1 binding was replaced with the XlnR binding sequence (as shown in Figure 3), resulting in the semi-synthetic promoter P. xln.1a (Base sequence as shown in SEQ ID NO.4). In Figure 3, the semi-synthetic promoter P was obtained by replacing the ADR1 binding site of the natural promoter ADH2 with the XlnR binding site. xln.1a By performing high-throughput filtering and replacement on the TFBS array, spacer.a, and spacer.b in the figure, total synthetic promoters for different induction systems can be obtained.

[0133] However, this semi-synthetic promoter P xln.1a The leakage level is not negligible. Therefore, high-throughput screening was performed on the sequence between NDR and TATA-box using synthetic random primers (5'GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAGCA-3', sequence number SEQ ID NO.48, abbreviated as random primer sequence 1). This ultimately yielded P with low background and strong activation. xln.1b (The base sequence is shown in SEQ ID NO.5).

[0134] Previous literature has reported that the core promoter determines the maximum activation intensity. Therefore, in this embodiment, the core promoter region of ADH2 was replaced with other highly active promoter core regions, resulting in six synthetic promoters (their base sequences are shown in SEQ ID NO. 6-SEQ ID NO. 11). Although all of these support xylose-induced activation, the maximum activation level is lower than that of the original ADH2 core promoter. These results also suggest that the core promoter region has programmability.

[0135] To obtain fully synthetic promoters, high-throughput screening was performed on the TATA-BOX and TSS region sequences using multiple random primer stacking. Ultimately, a series of fully synthetic inducible promoters were obtained, especially the three fully synthetic promoters with base sequences shown in SEQ ID NO.18-SEQ ID NO.20.

[0136] Furthermore, appropriately increasing the number of transcription factor binding sites is a reliable strategy to enhance transcriptional activation levels. By testing promoters containing 1, 2, 3, and 4 XlnR binding sites (xlno) (base sequences shown in SEQ ID NO.4-SEQ ID NO.5 and SEQ ID NO.12-SEQ ID NO.15), the results (Figure 16a) indicate that promoters containing 2 xlno sites significantly increased activation levels, especially the P promoter containing 2 xlno sites. xln.2b (Base sequence shown in SEQ ID NO.13), 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, therefore, P xln.4 It has the largest dynamic range.

[0137] Furthermore, the functional relationship between XlnR concentration and system output was tested (as shown in Figure 16b), and based on this, suitable constitutive promoters were selected to ensure stable XlnR expression throughout the yeast process. Three constitutive promoters with different activities were initially selected: P... ACT1 (Base sequence as shown in SEQ ID NO.26), P PFY1 (Base sequence as shown in SEQ ID NO.53) and P PXR1 (Base sequence as shown in SEQ ID NO.54), finally indicating that when P ACT1 When used to express XlnR, the system has the largest dynamic range (as shown in Figure 16c).

[0138] Example 3: Substrate response curves, induction heterogeneity, kinetic behavior determination and fitting of the xylose-controlled induction system

[0139] 1. Construction of the transcription factor array yeast strain, and a schematic diagram of its construction principle (see Figure 5, which shows the gene elements constituting the XylR and XlnR expression cassettes, and the sequences of each element are shown in Table 1): Based on the optimal transcription factor expression level, the eukaryotic transcription factor XlnR and the bacterial XylR expression cassette were assembled into a plasmid using the GOLDEN-GATE technology. The fragment was then digested with the restriction endonuclease BsaI and transformed into Saccharomyces cerevisiae (i.e., CENPK.2-1C strain), and integrated into the NRT1 gene spacer site to obtain the transcription factor array yeast strain (i.e., xylose-sensitive yeast).

[0140] 2. Construction of a single-regulated xylose-induced transcription system, the principle of which is illustrated in Figure 6: In a yeast strain with a transcription factor array, the yellow fluorescent reporter gene YFP, which responds to XlnR and XylR, was transformed into the original ura3 genomic locus, respectively, to obtain a single-regulated transformed strain.

[0141] 3. For the optimized P xln.2b -XlnR system and P xyl The performance of -XylR (i.e., the single-regulatory xylose-induced transcription system) was characterized:

[0142] The obtained single-regulatory transformed strains were induced with 16 different concentrations of xylose, and the output signals were analyzed by flow cytometry (method). Substrate concentration induction curves were plotted, and the Hill function was used for fitting (as shown in Figure 17a) to obtain the Hill parameter and EC50 values ​​(see Table 3). The results showed that compared to P... xyl P xln.2b The leakage level is two orders of magnitude higher; while compared to P xln.2b P xyl The maximum activation level is 25% of the former. Specifically, for a given transcription activator, the relationship between its protein mass (A) and the output (y) in response to the promoter can be described by the Hill function:

[0143] For the promoter XlnR-P xln.2b The system's functional relationship is shown in Figure 16b. The fitted k1 and n1 are 3.42 and 1.82, respectively (see Table 3).

[0144] Furthermore, the heterogeneity of yeast populations under different xylose concentrations was observed and analyzed. The results are shown in Figure 17b. Under induction at five different orders of magnitude of xylose concentration, a single peak appeared consistently, and no population heterogeneity was observed. Finally, the kinetic behavior induced by a 10 mM xylose concentration that could saturate the induction system was measured and fitted. The results are shown in Figure 17c, and the fitting parameters are shown in Table 3. The results show that the observed kinetic data can be fitted by the standard transcription-translation models, namely Equations 2 and 3.

[0145] Formula 2 is as follows:

[0146] Formula 3 is as follows:

[0147] The letters in Formulas 2 and 3 represent the following: m and p are the concentrations of mRNA and protein, respectively; β... m ,β p These are constants describing the rates of mRNA and protein synthesis, respectively; γ m ,γp These are constants describing the degradation and dilution rates of mRNA and protein, respectively.

[0148] Table 3: Parameters for Fitting Substrate Response Curves

[0149] Example 4: Construction and design of a xylose dual-regulation induction system, substrate response curves, kinetic behavior determination and fitting.

[0150] 1. Given P xln.2b -XlnR system has high leakage and P xyl The low activation capacity of the -XylR system necessitates the use of a dual transcription factor regulatory system. Integrating the advantages of both systems can further optimize the xylose transcriptional regulation system. However, there are multiple design schemes for the dual regulation mode (as shown in Figure 18a), among which six functional topologies exist (see Figure 7). Based on the standard transcription-translation model (see Table 5) and the kinetic fitting parameters of single regulation (see Table 4), induction kinetics simulations were performed on these six dual regulation systems. The final simulation results are shown in Figure 18b, where topology 1 exhibits the highest induction rate (see Figure 18b, c) and the highest activation level (see Figure 18b). Therefore, the simple topology 1 was chosen for further construction and performance characterization.

[0151] For a given induction system, γ p It is a fixed value of 0.29, according to the equation. The formula τ represents the cell doubling time, which is 90 min here (reference: McDonald, PN Two-hybrid systems. Methods and protocols. Introduction. Methods Mol Biol 177, v-viii (2001)). Other parameters (β...) m ,γ m ,β p The result is obtained by fitting the measured dynamic data using formulas (3) and (4) (see Table 4). Therefore, with the following parameters and assignments, β1 = 2.22; β2 = 2.19; β3 = 0.49; β p =1.62; γ m =0.28; γ p =0.29; k1=3.42; n1=1.82.

[0152] According to Table 3, when t = 0, 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, βm [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 fitted values, and the fitting results are shown in Figure 18a. Since the analytical solution of the above dynamic simulation function cannot be obtained, the bisection method is used to calculate τ. 1 / 2 The results are shown in Figure 18b.

[0153] 2. Construct the topological structure 1 of the dual transcription factor (its framework design is shown in Figure 8). This only requires following the architecture shown in Figure 19a, i.e., sequence P. xln.2b-xyl (The base sequence is shown in SEQ ID NO.17) Construct the promoter sequence, and then construct the sequence P. xln.2b-xyl The fragment and the backbone plasmid pXJH1 (constructed internally in our laboratory, its plasmid structure is shown in Figure 2) were subjected to a one-step GOLDEN-GATE reaction to obtain the target reporter plasmid, which was then transformed into an existing transcription factor array strain (i.e., the transcription factor array yeast strain constructed in step 1 of Example 3, its construction diagram is shown in Figure 5). Next, the strain containing this dual regulatory system was induced with 16 different concentrations of xylose and analyzed by flow cytometry to plot the substrate response curve (Figure 19a). The measurement data were also well fitted by the Hill function (Figure 19a), with an EC50 of 0.93 mM. It is assumed that XlnR and XylR are related to the promoter P... xln.2b-xyl If the combination of the two is an independent event, then the steady-state induction of the promoter can be predicted using the partition function, i.e., Equation 1. The actual prediction result is shown in Figure 19a. The substrate-induced behavior of this dual-regulated promoter can be well predicted by the fitting parameters of the single-regulated promoter.

[0154] 3. Due to the presence of P in the classic GAL system of brewing yeast... GAL1 It also belongs to at least a dual-regulatory system, namely glucose inhibition mediated by Mig1p and galactose activation mediated by Gal4p. It was introduced as a control in both steady-state substrate-induced response and kinetic induction to better evaluate P. xln.2b-xyl The performance of -XlnR-XylR. Due to P GAL1 The output is determined by the ratio of galactose to glucose. Therefore, with the total sugar content controlled at 2%, the P values ​​induced by different ratios of galactose and glucose were measured. GAL1 Output. The results are shown in Figure 19a. The results show that P only occurs when the galactose:glucose ratio > 31. GAL1 It reached the saturation activation level, but was still significantly lower than P.xln.2b-xyl The activation level.

[0155] 4. In addition, for P induced by 2% galactose GAL1 Kinetic measurements were performed on a dual-regulated xylose system induced by 100 mM xylose, and the results are shown in Figure 19b. For P... GAL1 The presence or absence of glucose inhibition memory significantly impacts induction kinetics. Specifically, strains without a history of glucose inhibition exhibit very rapid induction, reaching 50% of maximum activation within 5 hours. However, strains with a history of glucose inhibition show at least a 1-hour induction lag. In contrast, the induction behavior of the dual-regulated xylose induction system is unaffected by glucose, with a detectable increase in fluorescence output within 15 minutes of xylose addition, and reaching 50% of maximum activation within just 3 hours. Furthermore, the kinetic data of the dual-regulated xylose induction system can be fitted by standard transcription-translation models, namely Formulas 2 and 3 (see Figure 19b).

[0156] Table 4: Fitting parameters of xylose-induced kinetics

[0157] Table 5: Fitting equations for the 6 topologies

[0158] Example 5: Substrate response curve of corn cob hydrolysate to xylose-induced transcription system

[0159] Agricultural wastes such as corn, wheat, and cotton stalks are rich in xylose, and recycling the xylose in their hydrolysates could bring significant economic benefits. Given the high sensitivity (EC50 of approximately 1 mM) of the xylose-induced regulation system described in this application, these stalk hydrolysates could potentially replace pure xylose in activating the system. Therefore, 3 g of 20-mesh corn cob powder was selected as the raw material, and 50 mL of hydrolysate was obtained according to the process described in Figure 10. HPLC analysis revealed that the concentrations of glucose, xylose, and arabinose in the hydrolysate were 178.9 mM, 53.3 mM, and 8.3 mM, respectively (see Figure 20a). Different volumes of CCH were used as inducers, and the response substrate concentration curves are shown in Figure 20b. 50 μL of non-concentrated corn cob hydrolysate was sufficient to achieve the maximum activation level (see Figure 20b).

[0160] The preparation steps in Figure 10 are as follows: 3g of 20-mesh corn cob powder is treated with 2% H2SO4 solution at 120℃ for 45min, then calcium carbonate is added for further treatment, followed by hydrolysis with 1,800U cellulase (from Beijing Solarbio Technology Co., Ltd., product number C8270) and 1,500U hemicellulase (from Beijing Solarbio Technology Co., Ltd., product number H8110), and then solid-liquid separation is performed. The supernatant is collected to obtain corn cob hydrolysate.

[0161] Example 6: A multidimensional comparison between the xylose-induced transcription system and existing induction systems in Saccharomyces cerevisiae.

[0162] To better evaluate single- and dual-regulated xylose induction systems, this application reconstructs the endogenous induction system of Saccharomyces cerevisiae - P GAL1 ,P MET3 ,P CUP1 The ER-based synthetic induction system (LexA-ER-VP16 / B112) is also shown in Figure 9, with gene element configuration and integration sites (related element sequences are shown in Table 1). This application comprehensively compares the system with the developed xylose induction system in five aspects: regulatory rigor, induction activation level, dynamic range, growth toxicity, and induction rate.

[0163] To determine the maximum activation level, this application established a test criterion: the population should not exhibit fluorescence heterogeneity while reaching the maximum activation level, and host growth 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 Figure 21), thereby determining the inducer concentration required to achieve the maximum activation level. At this concentration, the induced output level was measured. Based on the output level without any inducer, the dynamic range of each system was calculated, and the OD of strain growth was tested over three consecutive days through continuous transfer. 600 The original transformed strain CENPK.2-1C was used as a reference to evaluate the growth of the strain under induction conditions. Finally, the induction kinetics of each strain were tested over 36 hours at this concentration. The results are shown in Figure 22. The results indicate that the dual-regulatory xylose system has the best overall performance in the above five aspects.

[0164] Example 7: Determination and Fitting of Substrate Response Function of Xylose-Induced Transcription System in Pichia pastoris, Candida glabrata, and Candida albicans

[0165] The XlnR protein has no ortholog in *Pichia pastoris*, *Candida glabrata*, or *Candida albicans*, and the former two cannot degrade xylose. These three yeasts are all non-conventional yeasts with distant evolutionary relationships, and genetic tools such as induction systems are relatively scarce. Therefore, these three yeasts were chosen to test the migration of the XlnR-based xylose induction system. For ease of prototype testing, the XlnR expression cassette and the promoter-driven reporter gene expression cassette were designed and contained on a single plasmid. The gene elements, subelements, and backbone plasmid used are shown in Figures 11-14. Furthermore, to obtain a stable induction system, all plasmids or restriction fragments containing the induction system expression cassette were designed to be integrated into the genome. The integration strategy and sites are shown in Figures 11, 12, and 14. Figure 11 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Pichia pastoris. The gene element configuration within the dashed box in the right figure is shown in the left figure. Figure 12 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Candida glabrata. The gene element configuration within the dashed box in the right figure is shown in the left figure. Figure 13 is a schematic diagram of the construction of a universal plasmid adapted to GOLDEN-GATE technology in Candida albicans. The specific plasmid sequences are shown in Table 1. Figure 14 is a schematic diagram of the design of the XlnR-based xylose transcription system and the genome recombination strategy in Candida albicans. The gene element configuration within the dashed box in the right figure is shown in the left figure.

[0166] Finally, the substrate induction curves were tested for three strains with a xylose induction system, as shown in Figure 22. The induction folds in *Pichia pastoris*, *Candida glabrata*, and *Candida albicans* reached 189, 368, and 327 times, respectively. Similarly, the xylose induction curves for all three strains were well fitted by the Hill function, with the fitting parameters shown in Table 3. The results indicate that the EC50 of this xylose induction system was around 1 mM in the first two strains, while it was 13.7 mM in *Candida albicans*.

[0167] Example 8: Comparison of the ability of the xylose system and the methanol system to induce the secretion of msfGFP in Pichia pastoris.

[0168] Pichia pastoris is a powerful platform for exogenous protein production. The methanol system is a primary and powerful induction system for Pichia pastoris, but it has inherent problems. First, the methanol system is an endogenous system, inhibited by glucose and glycerol. In industrial production, the glucose- and glycerol-dependent production stages must be separated from the methanol induction stage, thus lengthening the production cycle. Second, methanol itself acts as both a nutrient and an inducer; its flammability and toxicity increase storage safety risks. Therefore, the xylose-induced system has profound industrial value in inducing and driving exogenous protein secretion in Pichia pastoris.

[0169] This application compared the induction activation levels of the xylose-induced system and the methanol system. The results showed that the maximum activation level of xylose was twice that of the methanol system (Figure 22a, Figure 23a). Furthermore, this application compared the ability of the two induction systems to secrete msfGFP, as shown in Figure 23b. The results showed that, under their respective optimal induction conditions, the ability of xylose to induce msfGFP secretion was significantly higher than that of the methanol system (Figure 23b, c). Figure 23 shows the substrate induction curve determination and Hill fitting plot of the XlnR-based xylose-induced system in Pichia pastoris (Figure 23a), Candida glabrata (Figure 23b), and Candida albicans (Figure 23c) in Example 8. Figure 24 is a comparison of the ability of the xylose system and the methanol system to induce msfGFP secretion in Pichia pastoris in Example 8; Figure 24a shows the ability of Pylose system to induce msfGFP secretion. AOX1 The substrate induction curves are shown in Figure 24b, where debri represents Pichia pastoris cells, sup represents culture supernatant, LS01-C3, C5, C6 represent positive clones in the xylose-induced system, and LS03-C5, C7 represent positive clones in the methanol-induced system. Figure 24c shows the statistical analysis of msfGFP expression levels in the culture supernatants of the xylose-induced and methanol-induced systems. Unpaired t-test was used. ***: p = 0.0002.

[0170] In summary, the xylose-inducible system based on the xylose-sensing transcription factor XlnR from filamentous fungi in this application achieves 50% of the maximum activation level after 3 hours of xylose induction and reaches the maximum activation level after 6 hours of induction in the presence of 2% glucose, resulting in a significant increase in the maximum activation level and rapid induction speed. Furthermore, by combining the xylose-sensing transcription factor XlnR from filamentous fungi with the xylose-sensing transcriptional repressor XylR from prokaryotes, the leakage level of the dual-regulated induction system is extremely low, while the maximum activation level is comparable to that of the XlnR-monoregulated xylose induction system, greatly expanding the dynamic range to 4,000 times. This solves the problems of low induction intensity and slow response of transcription factor-dependent xylose transcription induction systems in the field of synthetic biology.

[0171] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0172] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A xylose-sensing transcription factor assembly, characterized in that, include: Xylose-sensing transcription factor XlnR and its binding sequence, 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 nidus; Further, the amino acid sequence of the xylose-sensing transcription factor XlnR is shown in SEQ ID NO.1; and / or, the nucleotide sequence of the binding concordance sequence is shown in SEQ ID NO.

50.

3. The xylose-sensing transcription factor assembly according to any one of claims 1-2, characterized in that, This includes the xylose-sensing transcription factor XylR, which is a bacterial xylose-sensing transcription factor XylR.

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 starter component, characterized in that, The promoter assembly is capable of binding to the xylose-sensing transcription factor assembly according to any one of claims 1-4, wherein the promoter assembly comprises: a first promoter, the first promoter being capable of binding to the xylose-sensing transcription factor XlnR, wherein the first promoter is obtained by modifying the Saccharomyces cerevisiae ADH2 promoter as the chassis promoter; Furthermore, the modification includes modifying at least one of the following: 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 initiation site, the transcription factor binding site motif, and the prokaryotic operon site sequence.

6. The promoter component according to claim 5, characterized in that, The first promoter is obtained mainly through 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; Furthermore, the base sequence of the first promoter is shown in SEQ ID NO.

4.

7. The promoter component according to claim 5, characterized in that, The first promoter is obtained mainly through 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 deletion 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 shown in SEQ ID NO.5-SEQ ID NO.11 and SEQ ID NO.18-SEQ ID NO.

20.

8. The promoter component according to claim 5, characterized in that, The first promoter is obtained mainly through the following steps: inserting the xylose-sensing transcription factor XlnR binding site xlno 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 shown in SEQ ID NO.4-SEQ ID NO.5 and SEQ ID NO.12-SEQ ID NO.

15.

9. The promoter component according to any one of claims 5-8, characterized in that, It also includes a second promoter that can bind to the xylose-sensing transcription factor XylR; Furthermore, the second promoter contains a binding site xylo for the xylose-sensing transcription factor XylR, and the binding site xylo is located upstream of the transcription promoter site and near the TATA box. Furthermore, 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.

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

11. The dual-regulation promoter according to claim 10, characterized in that, The dual-regulatory 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 containing the xylose-sensing transcription factor component as described in any one of claims 1-4.

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

24.

14. A promoter-reporter gene plasmid, characterized in that, The promoter-reporter gene plasmid is a reporter gene backbone plasmid with an inserted inducible promoter, wherein 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.

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

16. The recombinant bacteria according to claim 15, characterized in that, The recombinant bacteria also carries a promoter-reporter gene plasmid, which is a reporter gene backbone plasmid with an inserted inducible promoter. 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 bacteria according to claim 15, characterized in that, The transcription factor expression plasmid is an expression backbone plasmid containing both the xylose-sensing transcription factor XlnR and the xylose-sensing transcription factor XylR. The expression cassettes of the xylose-sensing transcription factor XlnR and the xylose-sensing transcription factor XylR are integrated into the NRT1 gene spacer site of the yeast.

18. The recombinant bacteria according to claim 17, characterized in that, The recombinant bacteria also contain 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.

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

20. The recombinant bacteria 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 topologies 1 to 6, m a It is the mRNA concentration of transcription activator a; m [yfp] β1 is the mRNA concentration of fluorescent reporter protein YFP; β2 is the mRNA synthesis rate driven by transcription activator a; β3 is the mRNA synthesis rate driven by transcription repressor; β4 is the mRNA synthesis rate driven by transcription repressor. p This refers to the protein synthesis rates of transcription factor protein and fluorescent reporter protein YFP; it is assumed here that their protein synthesis rates are the same; γ m It refers to the rate of mRNA degradation and dilution; γ p is the rate of protein degradation and dilution; k1 is the concentration of transcription activators when the transcription output reaches half the activation intensity; n1 is the Hill coefficient in the output function and the concentration of transcription activators.

21. The recombinant bacteria according to claim 19, characterized in that, The kinetic model formula 1 for the substrate response curve of the recombinant bacteria is as follows: Among them, y min y represents the output of the induction system without the addition of an inducing agent. max Induction system after adding inducing agent The output at steady state is given by x, where x is the inducer concentration, and k1, k2, n1, and n2 are the Hill parameters of the xylose-based single-regulation induction system based on XlnR and XylR, respectively.

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

23. A recombinant bacterium, characterized in that, The recombinant bacteria are host bacteria with recombinant plasmids integrated on their chromosomes. The recombinant plasmid is a backbone plasmid containing a xylose-sensing transcription factor XlnR expression cassette and a promoter-driven reporter gene expression cassette. The xylose-sensing transcription factor XlnR is a xylose-sensing transcription factor XlnR derived from filamentous fungi.

24. The recombinant bacteria according to claim 23, characterized in that, The host bacterium 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 shown in SEQ ID NO.

15.

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

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