Arabinose responsive transcription factor component and application thereof

Abstract

The invention relates to an arabinose response transcription factor component and application thereof. The arabinose response transcription factor component comprises a transcription activator AraRA, and the transcription activator AraRA is derived from eukaryotes. In the arabinose response transcription factor component, the transcription activator AraRA derived from eukaryotes can be used for an eukaryotic system to perform arabinose induction, so that the blank that the eukaryotic system has no synthetic arabinose induction system is filled up; a novel transcription induction system which is low in price and high in performance is developed in the saccharomyces cerevisiae; the toxic and side effects on the growth of a host are very small, the response speed is high, the specificity is high, the inhibition by other carbon sources such as glucose is avoided, and the decoupling of a growth stage and an induction stage is realized; and by combining with a previously developed xylose induction system, a new simple way is opened up for producing high-value compounds by recycling agricultural wastes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an arabinose-responsive transcription factor assembly and its applications. Background Art

[0002] L-arabinose widely exists in nature, such as in plant pectin, hemicellulose, pectic acid, the heartwood of coniferous trees, bacterial polysaccharides, and certain glycosides. In the food industry, as a low-calorie sweetener, it has been approved as a health food additive. It helps control blood sugar levels and promote the growth of intestinal probiotics. Commercial products of L-arabinose are mainly extracted from raw materials such as corncobs and lignocellulose, with relatively low costs, which is one-third of that of commercial galactose. In addition, neither yeast nor the human digestive tract can directly degrade and absorb it, and this property makes arabinose a specific inducer rather than a nutrient. Therefore, the food-grade safety, stability, and low price of arabinose make it a transcription system inducer with broad prospects.

[0003] Based on the AraC-P BAD system, the arabinose transcriptional expression system is widely used in prokaryotes. This system has the characteristics of low leakage and precise regulation by arabinose, and is a powerful genetic tool in the field of molecular biology and metabolic engineering of bacteria. Due to the food-grade safety of L-arabinose, the AraC-P BAD induction system has been used for the expression of tumor antigens in bacteria to develop oral tumor vaccines. However, no arabinose synthesis induction system has been reported in either yeast or mammalian cells. This situation may stem from two aspects: 1. The transcriptional activation mechanisms of eukaryotes and prokaryotes are very different, and the AraC-P BAD system of prokaryotes cannot be directly transferred to eukaryotes. 2. The research on transcription factors (TFs) that are clearly proven to respond to L-arabinose and related mechanisms in eukaryotes is limited.

[0004] According to the mechanism of action, transcription factors that can respond to L-arabinose can be divided into two types: transcriptional activators and transcriptional repressors; according to the host source, these transcription factors can be further divided into prokaryotic transcriptional activators, prokaryotic transcriptional repressors, eukaryotic transcriptional activators, and eukaryotic transcriptional repressors. A typical example of a prokaryotic transcriptional activator is AraC from Escherichia coli, and examples of prokaryotic transcriptional repressors are AraR from Bacillus subtilis, Corynebacterium glutamicum, and Mycobacterium smegmatis R , and AraR of filamentous fungus Aspergillus belongs to eukaryotic transcriptional activators A . Currently, no eukaryotic transcriptional repressors that respond to L-arabinose have been reported.

[0005] At present, there is no reported arabinose induction system in eukaryotes. The arabinose induction system in prokaryotes is based on the AraC-P BAD system in Escherichia coli, and its principle of action is as Figure 25 shown and is widely used in Gram-negative and Gram-positive bacteria. The current AraC-P BAD cannot be transferred to eukaryotes due to different transcriptional activation mechanisms. Summary of the Invention

[0006] Based on this, in order to solve the problem that the AraC-P BAD system in the field of synthetic biology cannot be transferred to eukaryotes, it is necessary to provide an arabinose-responsive transcription factor component and its application. This arabinose-responsive transcription factor component can be applied to eukaryotic systems for arabinose induction.

[0007] An arabinose-responsive transcription factor component, comprising: a transcriptional activator AraR A , and the transcriptional activator AraR A is derived from eukaryotes.

[0008] In the above-mentioned arabinose-responsive transcription factor component, the transcriptional activator AraR derived from eukaryotes A enables arabinose induction to be used in eukaryotic systems.

[0009] A promoter component, characterized in that it can bind to the above-mentioned arabinose-responsive transcription factor component. The promoter component comprises: a first promoter, and the first promoter can bind to the transcriptional activator AraR A binding.

[0010] A dual-regulated promoter that can bind to the transcriptional activator AraR A and can bind to the transcriptional repressor AraR R binding, the transcriptional activator AraR A is derived from eukaryotes, and the transcriptional repressor AraR R is derived from bacteria.

[0011] A transcription factor expression plasmid, which is a backbone plasmid inserted with the above-mentioned arabinose-responsive transcription factor component.

[0012] A promoter-reporter gene plasmid, which is a reporter gene backbone plasmid inserted with an inducible promoter, and the inducible promoter is selected from any one of the above-mentioned promoter components and the above-mentioned dual-regulated promoter.

[0013] A recombinant bacterium, which is Saccharomyces cerevisiae carrying the above-mentioned transcription factor expression plasmid.

[0014] Application of the above-mentioned recombinant bacterium in the preparation of linalool. Description of the drawings

[0015] Figure 1 It is the design schematic diagram of the existing xylose biosensor. Figure A is the schematic diagram of the action principle of the xylose induction system based on the transcriptional repressor XylR, and Figure B is the schematic diagram of the xylose induction system designed based on the bacterial transcriptional activator.

[0016] Figure 2 It is the schematic diagram of the screening strategy of the biosensor based on TF.

[0017] Figure 3 It is the schematic diagram of the modification strategy based on the natural inducible promoter P ADH2

[0018] Figure 4 It is the schematic diagram of the construction principle of the high-performance xylose induction system based on bacterial XylR.

[0019] Figure 5 It is the schematic diagram of the construction principle of the yeast strain carrying the xylose-responsive transcription factor array in Examples 1-3.

[0020] Figure 6 It is the schematic diagram of the construction principle of the single-regulated xylose induction transcription system in Examples 1-3.

[0021] Figure 7 It is the schematic diagram of the structure of the functional dual-transcription factor regulation topology in Examples 1-4.

[0022] Figure 8 It is the schematic diagram of the structure of the preferred dual-transcription factor regulation topology design in Examples 1-4.

[0023] Figure 9 It is the schematic diagram of the reconstruction of the commonly used induction system in Saccharomyces cerevisiae.

[0024] Figure 10 It is the schematic diagram of the preparation process of corncob hydrolysate in Examples 1-5.

[0025] Figure 11 It is the schematic diagram of the xylose transcription system design and genomic recombination strategy based on XlnR in Pichia pastoris.

[0026] Figure 12 It is the schematic diagram of the xylose transcription system design and genomic recombination strategy based on XlnR in Candida glabrata.

[0027] Figure 13 ​Schematic diagram for constructing a universal plasmid adapted to the GOLDEN-GATE technology in Candida albicans;

[0028] Figure 14 Schematic diagram for the design of an XlnR-based xylose transcriptional system and the genomic recombination strategy in Candida albicans;

[0029] Figure 15 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 1-1;

[0030] Figure 16 Detection result diagram for the optimization of the XlnR xylose transcriptional induction system;

[0031] Figure 17 Schematic diagram of the substrate response curve (i.e., Figure a), induction heterogeneity (i.e., Figure b), kinetic behavior determination and fitting (i.e., Figure c) of the xylose single-regulation induction system in Example 1-3;

[0032] Figure 18 Schematic diagram of the construction design of the xylose dual-regulation induction system in Example 1-4. Figure a is the fitting curve diagram of 6 topologies, and Figure b is the statistical chart of the simulation calculation results of τ1 / 2 (response time at the half-activation level) of 6 topologies;

[0033] Figure 19 Schematic diagram of the topological structure and substrate response curve (i.e., Figure a), kinetic behavior determination and fitting (i.e., Figure b) of the galactose induction system in Saccharomyces cerevisiae and the xylose dual-regulation induction system in Example 1-4;

[0034] Figure 20 Composition of polysaccharides in corncob hydrolysate (i.e., Figure a) and substrate response curve for activating the xylose dual-regulation induction system (i.e., Figure b) in Example 1-5;

[0035] Figure 21 Detection result diagram of the substrate induction curve and fluorescence distribution of the xylose induction transcriptional system and the existing induction systems in Saccharomyces cerevisiae in Example 1-6;

[0036] Figure 22 Multi-dimensional comparison diagram of the xylose induction transcriptional system and the existing induction systems in Saccharomyces cerevisiae. Figure a is the maximum activation level, Figure b is the impact on the growth toxicity of the host, Figure c is the induction speed, and Figure d is a radar chart (visualizing multi-dimensional comparison);

[0037] Figure 23 Substrate induction curve determination and Hill fitting diagram of the XlnR-based xylose induction system in Pichia pastoris (a), Candida glabrata (b), and Candida albicans (c) in Example 1-8;

[0038] Figure 24 Comparison diagram of the ability of the xylose system and the methanol system in Pichia pastoris to induce the secretion of msfGFP in Examples 1-8;

[0039] Figure 25 Schematic diagram of the working principle of the AraC-PBAD induction system;

[0040] Figure 26 Arabinose-responsive eukaryotic transcriptional activator AraR A and prokaryotic transcriptional repressor AraR R Mode of action schematic diagram;

[0041] Figure 27 Schematic diagram of the screening strategy for the arabinose-induced transcriptional system;

[0042] Figure 28 Synthetic transcription factor strategy based on the transcriptional activator AraR A ;

[0043] Figure 29 AraR A Combined synthetic inducible promoter optimization. Figure a shows the architecture based on the ADH2 natural inducible promoter, and Figure b shows the optimization of the promoter by changing the number, position, and spacer sequence of lexo;

[0044] Figure 30 AraR R Combined synthetic inducible promoter optimization. Figure a shows the architecture based on the minimal promoter, and Figure b shows the optimization of the promoter by changing the number, position, and spacer sequence of lexo.

[0045] Figure 31 AraR A -AraR R 2-sensor and XylR-XlnR-AraR A -AraR R Schematic diagram of the specific component configuration of the 4-sensor construction;

[0046] Figure 32 Schematic diagram of the construction of the arabinose single-regulated transcriptional induction system;

[0047] Figure 33 Schematic diagram of the construction of the arabinose single- and double-regulated transcriptional induction system;

[0048] Figure 34 Schematic diagram of the construction principle of the engineered yeast strain for linalool production induced by xylose and arabinose;

[0049] Figure 35Schematic diagram of the principle for recycling crop waste to produce high-value compounds through the xylose and arabinose induction system developed in the example part of "Second Part Arabinose-responsive Transcription Factor Component and Its Application";

[0050] Figure 36 Multi-gene expression cassette assembly strategy - GOLDEN-GATE assembly adopted in the second part of the example;

[0051] Figure 37 Arabinose-responsive transcriptional activator AraR A (i.e., Figure a) and transcriptional repressor AraR R (i.e., Figure b) prototype test results;

[0052] Figure 38 Optimization of the induction system based on the synthetic transcription factor LexA-AraRA. Figure a shows the output test results of the induction system with the natural AraR A (An04g08600) retained or deleted. Figure b shows the functional correspondence between the concentration of LexA-AraR A and the output of the induction system. Figure c shows the output test results of the induction system when different active natural constitutive promoters are used to express LexA-AraR A ;

[0053] Figure 39 Induction test of the single-regulation induction system in Examples 2 - 4. Figure a shows the substrate induction curve determination based on the transcriptional activator AraR A and the transcriptional repressor AraR R . Figure b shows the induction kinetics determination at a specified inducer concentration based on the transcriptional activator AraR A and the transcriptional repressor AraR R ;

[0054] Figure 40 Induction population heterogeneity test of the arabinose single-regulation and dual-regulation induction systems in Examples 2 - 4 and Examples 2 - 5. Figure a shows the fluorescence output (FITC-A histogram) by flow cytometry at different inducer concentrations. Figure b shows the fluorescence output by flow cytometry at different induction times under 100 mM arabinose induction;

[0055] Figure 41 Induction test of the dual-regulation induction system in Example 2 - 5. Figure a shows the schematic diagram of the dual-regulation induction control topology and the action mode of the composite promoter. Figure b shows the substrate induction curve determination, Hill fitting and prediction of the dual-regulation induction system. Figure c shows the kinetic behavior determination and fitting of the dual-regulation system under 100 mM arabinose induction;

[0056] Figure 42For the effects of the arabinose induction system and common induction systems in Saccharomyces cerevisiae on host growth at different inducer concentrations in Examples 2-6;

[0057] Figure 43 For the multi-dimensional comparison of the arabinose induction system and common induction systems in Saccharomyces cerevisiae in Example 2-7. In Figure a, the maximum activation levels of each system under the conditions of basal expression, no growth inhibition, and no induction heterogeneity are shown. In Figure b, the growth toxicity effects on the host are presented. In Figure c, the induction speed is shown. In Figure d, a radar chart visualizes the multi-dimensional comparison. In each figure, the numbers are the results after normalizing the maximum and minimum values in the same dimension;

[0058] Figure 44 For the construction of the linalool-producing yeast engineering strain and the product determination results in Example 2-7. In Figure a, the activity of the composite promoter used to control the pathway metabolic gene shows a xylose dose-dependence. In Figure b, the activity of the synthetic promoter used to control ERG20 shows an arabinose dose-dependence. In Figure c, the growth of the linalool-producing yeast engineering strain is inhibited by the increase in arabinose concentration. In Figure d, the growth and linalool yield of the engineering strain reach their peaks when arabinose is added 12 h after the growth of the engineering strain. In Figure e, the standard curve for determining linalool by the GC-FID method is shown. In Figure f, the production titers of linalool under different inducer and carbon source addition conditions are presented. In Figure g, the GC chromatograms for detecting linalool under different carbon source and induction conditions are shown. In each figure, CCH: corncob hydrolysate, YP: YPD medium without glucose;

[0059] Figure 45 For the determination of the cross-reactivity curve and Hill fitting of the arabinose-induced single-regulation and dual-regulation induction systems to gradient xylose in Example 2-8;

[0060] Figure 46 For the test of the expression sustainability of the arabinose-induced single-regulation and dual-regulation induction systems during long-term fermentation in Example 2-9. Detailed implementation manners

[0061] The following further elaborates on the present application in conjunction with the implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and examples described in the present application. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0062] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by one of ordinary skill in the technical field to which this application pertains. The terms used in the description of this application in this application are only for the purpose of describing embodiments and examples, and are not intended to limit this application. The term

[0063] Unless otherwise stated or there is a contradiction, the terms or phrases used in this application have the following meanings:

[0064] The selection scope of the terms "and / or", "or / and", and "and / or" used in this application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when connecting at least three items with at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0065] In this application, "preferably", "better", "more preferably", and "should be" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0066] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0067] In this application, "optionally", "optional", and "optional" mean optional, that is, it means any one of two parallel solutions of "yes" or "no". If "optional" appears in a technical solution in multiple places, without special instructions, and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0068] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0069] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0070] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this application, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this application should be understood to include any and all sub-ranges subsumed therein.

[0071] For the temperature parameter in this application, unless otherwise specified, it allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0072] 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.

[0073] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the purpose of this application and / or its technical solution, the cited documents involved in this application are incorporated by reference in their entirety and for all purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated into this application as references, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description of this application.

[0074] The first part: Xylose-responsive transcription factor component and its application

[0075] In the first aspect of the first part of the embodiments, a xylose-responsive transcription factor component is provided, including: a xylose-responsive transcription factor XlnR and the binding consensus sequence of the xylose-responsive transcription factor XlnR, wherein the xylose-responsive transcription factor XlnR is a xylose-responsive transcription factor XlnR derived from filamentous fungi.

[0076] In the above xylose-responsive transcription factor component, the xylose-responsive activation transcription factor XlnR derived from the eukaryotic filamentous fungus significantly improves the maximum activation level and has a very rapid induction rate. It has been experimentally verified that the induction of the xylose-responsive activation transcription factor XlnR derived from the eukaryotic filamentous fungus can reach 50% of the maximum activation level in 3 hours.

[0077] In some embodiments, the filamentous fungus includes Aspergillus nidulans. Aspergillus nidulans belongs to the family Moniliaceae and the genus Aspergillus. It should be noted that the xylose-responsive transcription factor XlnR can be a xylose-responsive transcription factor XlnR derived from Aspergillus nidulans, or sequence homologs (orthologs) of the xylose-responsive transcription factor XlnR derived from Aspergillus nidulans and the binding consensus sequence can replace this system and play a similar function.

[0078] Furthermore, the amino acid sequence of the xylose-responsive transcription factor XlnR is as shown in SEQ ID NO.1; and / or, the nucleotide sequence of the binding consensus sequence is as shown in SEQ ID NO.50. The above xylose-responsive transcription factor XlnR and / or its binding consensus sequence can improve 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).

[0079] In some embodiments, the xylose-sensing transcription factor component further includes the xylose-sensing transcription factor XylR, which is a xylose-sensing transcription factor XylR derived from bacteria. The principle of its action is shown in Figure 1 . Combining with the xylose-sensing transcription factor XylR derived from bacteria can reduce the leakage level of the transcriptional induction system.

[0080] Among them, the bacteria are Bacillus licheniformis. Further, the amino acid sequence of the xylose-sensing transcription factor XylR is shown in SEQ ID NO.2. The dual-regulation induction system combined with the Bacillus licheniformis transcription factor XylR greatly reduces the leakage level, and finally the induction dynamic range is as high as 4000 times, and the maximum activation intensity and response speed are not affected compared with the action of a single XlnR. It should be noted that the above-mentioned xylose-sensing transcription factor XylR is not limited to being derived from Bacillus licheniformis, and can also be sequence orthologs of the xylose-sensing transcription factor XylR derived from Bacillus licheniformis and binding consensus sequences that can replace this system to play a similar function, and can also be derived from other bacteria, such as Lactobacillus pentosus, Clostridium difficile, Tetragenococcus halophile, Bacillus subtilis, Caulobacter crescentus and other bacteria.

[0081] In the above xylose-sensing transcription factor component, the xylose-responsive activating transcription factor XlnR derived from the eukaryotic filamentous fungus is used, which greatly improves the maximum activation level and has a very rapid induction speed (it can reach 50% of the maximum activation level in 3 hours of induction); on the basis of the XlnR system, the dual-regulation induction system combined with the Bacillus licheniformis transcription factor XylR greatly reduces the leakage level, and finally the induction dynamic range is as high as 4000 times, and the maximum activation intensity and response speed are not affected compared with the action of a single XlnR.

[0082] In the second aspect of the first part of the embodiments, a promoter component is provided, which can bind to the above-mentioned xylose-sensing transcription factor component to be able to singly regulate the xylose induction of the host bacterium. The promoter component includes: a first promoter, which can bind to the xylose-sensing transcription factor XlnR.

[0083] Since natural promoters such as P xylPThe overly long and unstructured framework increases the difficulty of further modification and may also be subject to unknown endogenous regulation. Therefore, it is necessary to rationally design a structured synthetic promoter. Thus, in some embodiments, an inducible ADH2 natural promoter with a clear structure was first selected. The first promoter was obtained by modifying the ADH2 promoter from Saccharomyces cerevisiae as a chassis promoter. Among them, the ADH2 promoter from Saccharomyces cerevisiae is the ADH2 promoter derived from Saccharomyces cerevisiae.

[0084] Due to the structured sequence characteristics, the natural inducible ADH2 promoter of Saccharomyces cerevisiae was selected as the chassis for modification to design a fully synthetic promoter. The principle is to ensure that the characteristic sequences remain unchanged: the nucleosome depletion region (NDR)-poly(A) sequence, the TATA-box, and the transcription start site (TSS), and to change the spacer sequence between the characteristic sequences, so as to obtain a fully synthetic promoter that meets the required performance. In addition, according to the different induction systems, the upstream activation sequence (UAS) can be replaced. Therefore, the modification described in this application includes modifying at least one of the core promoter sequence of the chassis promoter, the spacer sequence between the TATA-box and the nucleosome depletion region, the spacer sequence between the TATA-box and the transcription start site, the transcription factor binding site motif, and the prokaryotic operon site sequence.

[0085] In some embodiments, the first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the ADH2 promoter from Saccharomyces cerevisiae with the binding sequence of the xylose-responsive transcription factor XlnR. The promoter obtained by this step is a semi-synthetic promoter. Further, the nucleotide sequence of the first promoter is shown in SEQ ID NO.4. However, the leakage level of this semi-synthetic promoter cannot be ignored.

[0086] Since the leakage level of the above semi-synthetic promoter cannot be ignored, in some embodiments, the first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the ADH2 promoter from Saccharomyces cerevisiae with the binding sequence of the xylose-responsive transcription factor XlnR, and then changing the spacer sequence between the TATA-box and the nucleosome depletion region and / or the spacer sequence between the TATA-box and the transcription start site. The above modification is used to improve the leakage level of the promoter.

[0087] Further, the first promoter is selected from any one of the promoters shown in the base sequences such as SEQ ID NO.5 - SEQ ID NO.11 and SEQ ID NO.18 - SEQ ID NO.20.

[0088] Specifically, by performing high-throughput screening on the sequence between NDR and TATA-box using a synthetic random primer (hereinafter referred to as random primer 1, with the base sequence: 5’GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAGC A-3’), a P with low background and strong activation was finally obtained. xln.1b (with the base sequence shown in SEQ ID NO.5). Previous literature reported that the core promoter determines the maximum activation intensity. Therefore, in this application, the core promoter region of ADH2 was replaced with the core regions of other highly active promoters, resulting in 6 synthetic promoters (with their base sequences shown in SEQ ID NO.6 - SEQ ID NO.11). Although all of these can 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 part has programmability. To obtain a fully synthetic promoter, high-throughput screening was performed on the sequences of the TATA-box and TSS regions by superimposing multiple segments of random primers. Finally, a series of fully synthetic inducible promoters were obtained, especially three fully synthetic promoters with the base sequences shown in SEQ ID NO.18 - SEQ ID NO.20.

[0089] The study found that appropriately increasing the binding sites of transcription factors is a reliable strategy to improve the transcriptional activation level. Therefore, in some embodiments, the first promoter is mainly obtained through the following steps: inserting the binding site xlno of the xylose-sensing transcription factor XlnR 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 of the promoters shown in the base sequences such as SEQ ID NO.4 - SEQ ID NO.5 and SEQ ID NO.12 - SEQ ID NO.15. Among them, the promoter containing 2 xlno has significantly increased the activation level, especially P xln.2b (with the base sequence shown in SEQ ID NO.13), whose activation level even exceeds that of the promoter P xln.4 (with the base sequence shown in SEQ ID NO.15) containing 4 xlno, but its background leakage level is too high. Therefore, P xln.4 has the largest dynamic range.

[0090] The ADH2 promoter chassis architecture of Saccharomyces cerevisiae and the optimization strategy for synthetic promoters used in this application specifically refer to the characteristic sequences (kozak, TSS, TATA-box, NDR, UAS, see Table 1 Notes) contained in the architecture and the length of the intervals between the characteristic sequences (see Figure 3 ). For the spacer sequences between characteristic sequences (TSS, TATA-box, NDR) Figure 3 Spacer.a and Spacer.b) in the assay were subjected to high-throughput screening by designing random primers with fixed spacer sequences.

[0091] The first promoter is a series of short (<300bp) adjustable synthesized promoters designed and constructed with the natural ADH2 promoter from Saccharomyces cerevisiae as the modified chassis, which can achieve high-intensity activation and avoid the problems caused by the use of natural promoters: potential endogenous regulation, construction and transformation difficulties caused by repeated use, and genomic instability.

[0092] It should be noted that the Saccharomyces cerevisiae ADH2 promoter architecture used in this application has an intervening sequence ( Figure 3 The length and sequence of spacer.a and spacer.b) in the expression vector have certain compatibility within a certain range, and the UAS sequence can be replaced with other transcription factor binding sites, thereby being used in other transcription induction systems.

[0093] In some embodiments, the promoter component further includes a second promoter capable of binding to the xylose sensing transcription factor XylR.

[0094] Furthermore, the second promoter contains the binding site xylo of the xylose sensing transcription factor XylR, and the binding site xylo is located upstream of the adjacent transcription initiation site and near the TATA-box. Furthermore, the binding sequence (operator) of XylR is placed downstream of the TATA-box and upstream of the TSS. In some specific examples, the base sequence of the binding site xylo is shown in SEQ ID NO.49; and / or, the base sequence of the second promoter is shown in SEQ ID NO.3.

[0095] The third aspect of the first part of the embodiment provides a dual-regulated promoter, which can bind to the xylose sensing transcription factor XlnR and can bind to the xylose sensing transcription factor XylR, wherein the xylose sensing transcription factor XlnR is a xylose sensing transcription factor XlnR derived from filamentous fungi, and the xylose sensing transcription factor XylR is a xylose sensing transcription factor XylR derived from bacteria. The dual-regulated promoter can dual-regulate the xylose induction of the host bacteria.

[0096] In some embodiments, the dual-regulatory promoter is selected from at least one of the promoters having the base sequences shown in SEQ ID NOs. 16-17.

[0097] In the fourth aspect of the first part of the embodiments, a transcription factor expression plasmid is provided. The transcription factor expression plasmid is an expression backbone plasmid inserted with the above-mentioned xylose-responsive transcription factor assembly. The above-mentioned xylose-responsive transcription factor assembly can be expressed by this transcription factor expression plasmid.

[0098] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid inserted with both the xylose-responsive transcription factor XlnR and the xylose-responsive transcription factor XylR. That is, the xylose-responsive transcription factor XlnR and the xylose-responsive transcription factor XylR are inserted into the same backbone plasmid, which can be used to construct a transcription factor array yeast strain.

[0099] 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 inserted with the xylose-responsive transcription factor XlnR, and the second expression plasmid is an expression backbone plasmid inserted with the xylose-responsive transcription factor XylR. That is, the xylose-responsive transcription factor XlnR and the xylose-responsive transcription factor XylR are inserted into different backbone plasmids respectively, which can be used to construct a prototype response test platform for the xylose transcriptional induction system. Further, the expression backbone plasmid corresponding to the xylose-responsive transcription factor XlnR is the pGD137 plasmid having the base sequence shown in SEQ ID NO. 23, and the expression backbone plasmid corresponding to the xylose-responsive transcription factor XylR is the pGS001 plasmid having the base sequence shown in SEQ ID NO. 24.

[0100] In the fifth aspect of the first part of the embodiments, a promoter-reporter gene plasmid is provided. The promoter-reporter gene plasmid is a reporter gene backbone plasmid inserted with an inducible promoter, and the inducible promoter is selected from any one of the above-mentioned promoter assemblies and the above-mentioned dual-regulatory promoters. This promoter-reporter gene plasmid can be used to construct a xylose transcriptional induction system together with the transcription factor expression plasmid.

[0101] In the sixth aspect of the first part of the embodiments, a recombinant bacterium is provided. The recombinant bacterium is a host bacterium carrying the above-mentioned transcription factor expression plasmid, and the host bacterium is Saccharomyces cerevisiae carrying a TetR expression cassette. By transferring the transcription factor expression plasmid into the host bacterium, the above-mentioned xylose-responsive transcription factor XlnR and the xylose-responsive transcription factor XylR are expressed.

[0102] In some embodiments, the recombinant bacterium also carries a promoter - reporter gene plasmid, and the promoter - reporter gene plasmid is a reporter gene backbone plasmid inserted with the above - mentioned inducible promoter. The inducible promoter is selected from any one of the above - mentioned promoter components and the above - mentioned dual - regulation promoter. This recombinant bacterium can be used to construct a yeast strain with a transcription factor array or to construct a prototype response test platform for a xylose transcriptional induction system.

[0103] In some embodiments, the transcription factor expression plasmid is an expression backbone plasmid inserted with both the xylose - sensing transcription factor XlnR and the xylose - sensing transcription factor XylR, and the expression cassettes of the xylose - sensing transcription factor XlnR and the xylose - sensing transcription factor XylR are integrated at the NRT1 gene spacer locus of Saccharomyces cerevisiae. This recombinant bacterium can express both the xylose - sensing transcription factor XlnR and the xylose - sensing transcription factor XylR.

[0104] Further, in some examples, based on the recombinant bacterium of the above - mentioned embodiments, a first promoter and a second promoter are further introduced into the recombinant bacterium. 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. For the specific descriptions of the first promoter and the second promoter, see the above text and will not be elaborated here. The recombinant bacterium introduced with the above - mentioned first promoter and second promoter can regulate xylose - induced expression singly.

[0105] In some examples, based on the recombinant bacterium of the above - mentioned embodiments, the recombinant bacterium also carries a promoter - reporter gene plasmid, and the promoter - reporter gene plasmid is a reporter gene backbone plasmid inserted with an inducible promoter. The inducible promoter is selected from any one of the above - mentioned dual - regulation promoters (for the specific description, see the above text and will not be elaborated here). In this recombinant bacterium, the signals of the transcriptional activator and the transcriptional repressor are integrated onto the same synthetic promoter, that is, the binding site of the transcriptional repressor is located in the core promoter region, while the binding site of the transcriptional activator is located in the UAS region, and the selected transcriptional activator and transcriptional repressor do not have competitive binding, that is, they interact with the same synthetic promoter independently. The above - mentioned recombinant bacterium can regulate xylose - induced expression doubly.

[0106] Using a dual - transcription - factor regulation system and integrating the advantages of both can further optimize the xylose transcriptional regulation system. However, there are multiple design schemes for the dual - regulation mode (such as Figure 18 a), and there are 6 functional topological structures (see Figure 7 ), that is, the dual - regulation topological structure of the recombinant bacterium is selected from any one of Topology 1 to Topology 6. Among them, Topology 1 is the most effective dual - regulation topological structure. The fitting equations of Topology 1 to Topology 6 are shown in Table 5 below. In the fitting equations, m ais the mRNA concentration of transcriptional activator a; m [yfp] is the mRNA concentration of fluorescent reporter protein YFP; a is the protein concentration of transcriptional activator a; [YFP] is the protein concentration of fluorescent reporter protein YFP; β 1 is the mRNA synthesis rate driven by the dual regulatory promoter; β 2 is the mRNA synthesis rate driven by the response transcriptional activator; β 3 is the mRNA synthesis rate driven by the response transcriptional repressor; β p is the protein synthesis rate of the transcription factor protein and the fluorescent reporter protein YFP, assuming that the protein synthesis rates of the two are the same; γ m is the degradation and dilution rate of mRNA; γ p is the degradation and dilution rate of protein; k 1 is the concentration of the transcriptional activator when the transcriptional output reaches half of the activation intensity; n 1 is the concentration of the transcriptional activator and the Hill coefficient in the output function.

[0107] Based on the dual transcription factor regulation system, the kinetic model formula 1 of the substrate response curve of the recombinant bacterium is as follows:

[0108]

[0109] Among them, the meanings of each letter are: y min is the output when the inducer is not added to the induction system, y max is the output when the induction system reaches a steady state after adding the inducer. x is the inducer concentration. k 1 , k 2 , n 1 , and n 2 are the Hill parameters of the xylose single regulation system (the induction systems based on XlnR and XylR), respectively.

[0110] Agricultural wastes such as corn, wheat, cotton straw, etc. are rich in xylose. If the xylose in their hydrolysates can be recycled, it will bring huge economic returns. In some embodiments, the induction substrate of the recombinant bacterium includes corn cob hydrolysate, and the corn cob hydrolysate is mainly prepared by the following steps: Treat 3 g of 20-mesh corn cob powder with an H 2 SO 4 solution with a mass percentage of 2% at 120 °C for 45 min, then add calcium carbonate for treatment, then add 1,800 U of cellulase and 1,500 U of hemicellulase for hydrolysis, and then perform solid-liquid separation to collect the supernatant to obtain the corn cob hydrolysate. Using the hydrolysate of crop wastes rich in xylose (such as corn cobs) can replace pure xylose to activate the xylose induction system, thereby greatly reducing the cost of this system for bulk industrial product production.

[0111] To verify the transferability of the XlnR-based xylose induction system in other yeast species, the single-regulated XlnR-based xylose induction system was integrated into the genomes of Pichia pastoris, Candida glabrata, and Candida albicans to detect the xylose response of the system. Therefore, in the seventh aspect of the first part of the embodiments, a recombinant bacterium is provided. The recombinant bacterium is a host bacterium with a recombinant plasmid integrated into its chromosome. The recombinant plasmid is a backbone plasmid containing a xylose-responsive transcription factor XlnR expression cassette and a reporter gene expression cassette driven by a responsive promoter. The xylose-responsive transcription factor XlnR is a xylose-responsive transcription factor XlnR derived from filamentous fungi. For the specific description of the xylose-responsive transcription factor XlnR, please refer to the above text and will not be elaborated here.

[0112] In some embodiments, the host bacterium is Pichia pastoris or Candida glabrata, and the xylose-responsive transcription factor XlnR expression cassette is located upstream of the reporter gene expression cassette;

[0113] Furthermore, the reporter gene is yEmCitrine; and / or, the base sequence of the promoter is as shown in SEQ ID NO. 15.

[0114] In some embodiments, the host bacterium is Candida albicans, and the xylose-responsive transcription factor XlnR expression cassette is located downstream of the reporter gene expression cassette;

[0115] Furthermore, 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.

[0116] The above recombinant bacterium can illustrate that the XlnR-mediated xylose induction transcription system has a high degree of transferability and can still respond to xylose in a dose-dependent manner in multiple distantly related non-conventional yeasts; the XlnR-mediated xylose induction transcription system has strong practicability and can efficiently induce the secretion of the secreted protein msfGFP in Pichia pastoris, and the secretion level is higher than that of the endogenous methanol system.

[0117] The technical solutions of the first part have the following advantages:

[0118] Developed an efficient and inexpensive transcription induction system: By using the xylose-responsive activating transcription factor XlnR derived from eukaryotic filamentous fungi, the maximum activation level is greatly increased, and the induction speed is very rapid (it can reach 50% of the maximum activation level in 3 hours of induction).

[0119] Based on the XlnR system, the dual-regulation induction system combined with the Bacillus licheniformis transcription factor XylR greatly reduces the leakage level. Eventually, the induction dynamic range is as high as 4000-fold, and the maximum activation intensity and response speed are not affected compared with the action of a single XlnR.

[0120] For the designed dual-regulation mode induction system, its substrate response function can be predicted by the parameters of the single-regulation substrate response.

[0121] Using the natural ADH2 promoter as the transformation chassis, a series of short (<300bp) adjustable synthetic promoter sequences are designed and constructed, which can achieve high-intensity activation and avoid the problems brought by using natural promoters: potential endogenous regulation, construction and transformation difficulties caused by repeated use, and genomic instability. Even under the condition that the xylose induction system is saturated and activated, the growth of Saccharomyces cerevisiae is not significantly inhibited.

[0122] Using the hydrolysate of xylose-rich crop wastes (such as corncobs) can replace pure xylose to activate the xylose induction system, thus greatly reducing the cost of using this system for bulk industrial product production.

[0123] In terms of regulation rigor, maximum activation intensity, dynamic range, toxicity to host growth, and response speed, the performance of the dual-regulation xylose induction system developed in this application has comprehensively exceeded the commonly used induction systems in Saccharomyces cerevisiae (such as the natural endogenous systems P GAL1 ,P CUP1 ,P MET3 , and the synthetic induction system LexA-ER-VP16 / B112).

[0124] The xylose-induced transcription system mediated by XlnR has high migrability and can still respond to xylose in a dose-dependent manner in multiple distantly related unconventional yeasts; moreover, the XlnR-mediated xylose-induced transcription system of this application has strong practicability and can efficiently induce the secretion of the secreted protein msfGFP in Pichia pastoris, and the secretion level is higher than that of the endogenous methanol system.

[0125] The second part: Arabinose-responsive transcription factor components and their applications

[0126] In the first aspect of the second part of the examples, an arabinose-responsive transcription factor component is provided, including: the transcriptional activator AraR A , and the transcriptional activator AraR A is derived from eukaryotes.

[0127] In the above arabinose-responsive transcription factor component, the transcriptional activator AraR derived from eukaryotes AEnable its use in eukaryotic systems for arabinose induction.

[0128] In some embodiments, the eukaryote is selected from one of Aspegillus nidulans, Aspegillus niger, and Meyerozyma guilliermondii. In a specific example, the eukaryote is Aspegillus niger. It has been experimentally verified that AraR in Aspergillus niger A exhibits an activation fold of more than 8.4 - fold.

[0129] Furthermore, the transcriptional activator AraR A is the transcriptional activator AraR with the amino acid sequence as shown in SEQ ID NO.56 A (An04g08600), or the transcriptional activator AraR with the nucleotide sequence as shown in SEQ ID NO.69 A (AN0388).

[0130] Since the consensus sequence bound by AraR A is not clear, the possible AraR binding motifs proposed in the existing literature (K. Ishikawa, E. Kunitake, T. Kawase, M. Atsumi, Y. Noguchi, S. Ishikawa, M. Ogawa, Y. Koyama, M. Kimura, K. Kanamaru, M. Kato, T. Kobayashi, Curr Genet 2018, 64(6), 1245, https: / / doi.org / 10.1007 / s00294 - 018 - 0837 - 5) were introduced into the promoter with ADH2 as the chassis for testing, but all showed constitutive output and could not achieve inducible output. Therefore, to rule out the inability to respond to arabinose caused by incorrect reference of the DBD binding motif, the full - length AraR A was used. AIt is fused and expressed with the well-characterized LexA DBD (LexA1-87) from bacteria through the linker 3x(ggggs). The output promoter encodes the binding sequence lexo of LexA DBD. The test results of such a designed induction system show that arabinose induction achieves an 8.4-fold output. Due to the presence of two DBDs, the natural DBD may interfere with the function of LexA DBD. Therefore, combined with the structure prediction of Alphafold2, it is designed to delete the natural AraR DBD (1-67), and the components in other systems remain unchanged. The test results show that the arabinose induction level is improved, from 1.03 RPU before deletion to 3.07 RPU. However, because the background level also increases after deleting the natural DBD, the dynamic range itself changes little.

[0131] Therefore, based on this, in some embodiments, the transcriptional activator AraR A is linked with an exogenous DNA binding domain, and the exogenous DNA binding domain is derived from the bacterial TF LexA or the CI repressor of phage 434.

[0132] Furthermore, the exogenous DNA binding domain and the transcriptional activator AraR A are linked through a linking sequence, and the linking sequence is 3x(ggggs).

[0133] Furthermore, the transcriptional activator AraR A does not contain a DNA binding domain. Specifically, the transcriptional activator AraR A has the natural AraR DBD (1-67) deleted.

[0134] In some specific examples, the transcriptional activator AraR linked with the exogenous DNA binding domain A is the synthetic transcriptional activator LexA-AraR with the amino acid sequence as shown in SEQ ID NO.57 A or the synthetic transcriptional activator CI434-AraR with the nucleotide sequence as shown in SEQ ID NO.68 A .

[0135] It should be noted that the exogenous DNA binding domain is not limited to being derived from the bacterial TF LexA or the CI repressor of phage 434, and can also be other well-characterized DBDs to perform similar functions.

[0136] In some embodiments, it includes the transcriptional repressor AraR R , and the transcriptional repressor AraR R is derived from bacteria;

[0137] Further, the bacterium is selected from Thermotoga sp. RQ2, Bacillus subtilis, Bacillus licheniformis, Clostridium acetobutylicum, Shewanella sp. Sh95, and Bacillus amyloliquefaciens;

[0138] Further, the transcriptional repressor AraR R is selected from AraR having a nucleotide sequence as set forth in SEQ ID NOs. 70 to 76 R -NLS.

[0139] It should be noted that the bacterium is not limited to the above-mentioned bacteria, and other bacteria may also be used. The transcriptional repressor AraR of these bacteria R can replace this system and perform a similar function.

[0140] In some embodiments, it further includes a binding site sequence linked to the transcriptional repressor AraR R ;

[0141] Further, the amino acid sequence of the transcriptional repression component composed of the transcriptional repressor AraR R and its binding site sequence is as shown in SEQ ID NO. 58.

[0142] In the second aspect of the second part of the examples, a promoter component is provided, which can bind to the arabinose-responsive transcription factor component described in the first aspect of the second part of the above examples. The promoter component includes: a first promoter, and the first promoter can bind to the transcriptional activator AraR A ;

[0143] In some embodiments, the first promoter is the constitutive promoter -P TDH3 , the constitutive promoter P PGK1 , the constitutive promoter P ACT1 , the promoter P ANladR , or the promoter P ANlxrA ;

[0144] Further, the nucleotide sequence of the constitutive promoter P ACT1 is as shown in SEQ ID NO. 26.

[0145] In some embodiments, the first promoter is obtained by modifying the Saccharomyces cerevisiae-derived ADH2 promoter as a chassis promoter;

[0146] Further, the modification includes modifying at least one of the UAS region sequence of the chassis promoter, the spacer sequence between the TATA-box and the nucleosome-depleted region, the spacer sequence between the TATA-box and the transcription start site, and the transcription factor binding site motif.

[0147] In some embodiments, the first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the Saccharomyces cerevisiae-derived ADH2 promoter with the binding site lexo of the transcriptional activator AraR A of;

[0148] Further, the number of inserted binding sites xlno is 1-4;

[0149] Further, the first promoter is selected from at least one of the promoters shown in the base sequences such as SEQ ID NO.60-SEQ ID NO.63.

[0150] In some embodiments, a second promoter is further included, and the second promoter can bind to the transcriptional repressor AraR R bind;

[0151] Further, the second promoter is obtained by modifying the minimal promoter as the chassis;

[0152] Further, the second promoter is mainly obtained by the following steps: inserting the binding sequence of the transcriptional repressor AraR into the minimal promoter, and the binding sequence of the transcriptional repressor AraR R is located downstream of the TATA-box of the minimal promoter and upstream of the transcription start site; R of;

[0153] Further, the base sequence of the second promoter is as shown in SEQ ID NO.59.

[0154] In the third aspect of the second part of the embodiments, a dual-regulated promoter is provided, and the dual-regulated promoter can bind to the transcriptional activator AraR A bind, and can bind to the transcriptional repressor AraR R bind, the transcriptional activator AraR A is derived from eukaryotes, and the transcriptional repressor AraR R is derived from bacteria.

[0155] In some embodiments, in the dual-regulated promoter, the sequence upstream of the TATA-box can bind to the transcriptional activator AraR AIn combination, the sequence downstream of the TATA-box can bind to the transcriptional repressor AraR R bind;

[0156] Furthermore, the base sequence of the dual regulatory promoter is as shown in SEQ ID NO.64.

[0157] In the fourth aspect of the second part of the embodiments, a transcription factor expression plasmid is provided, which is a backbone plasmid inserted with the arabinose-responsive transcription factor assembly described in the first aspect of the second part of the embodiments.

[0158] In some embodiments, the transcriptional activator AraR A and the transcriptional repressor AraR R are simultaneously inserted into the transcription factor expression plasmid; furthermore, the backbone plasmid is the pGS077 plasmid with the nucleotide sequence as shown in SEQ ID NO.65;

[0159] Alternatively, the transcription factor expression plasmid includes a first expression plasmid and a second expression plasmid. The first expression plasmid is a backbone plasmid inserted with the transcriptional activator AraR A , and the second expression plasmid is a backbone plasmid inserted with the transcriptional repressor AraR R ; furthermore, the backbone plasmid is the pGS001 plasmid.

[0160] In some embodiments, the transcriptional activator AraR A , the transcriptional repressor AraR R , the expression backbone plasmid, the xylose transcriptional activator XlnR and the xylose transcriptional repressor XylR are simultaneously inserted into the transcription factor expression plasmid;

[0161] Furthermore, the xylose transcriptional activator XlnR is derived from filamentous fungi, and the xylose transcriptional repressor XylR is derived from bacteria;

[0162] Furthermore, the backbone plasmid is the pGS078 plasmid with the nucleotide sequence as shown in SEQ ID NO.66;

[0163] In the fifth aspect of the second part of the embodiments, a promoter-reporter gene plasmid is provided, which is characterized in that the promoter-reporter gene plasmid is a reporter gene backbone plasmid inserted with an inducible promoter, and the inducible promoter is selected from any one of the promoter assemblies described in the second aspect of the second part of the embodiments and the dual regulatory promoters described in the third aspect of the second part of the embodiments.

[0164] In the sixth aspect of the second part of the embodiments, a recombinant bacterium is provided, and the recombinant bacterium is Saccharomyces cerevisiae carrying the transcription factor expression plasmid described in the fourth aspect of the second part of the embodiments above.

[0165] In some embodiments, the transcription factor expression plasmid is integrated at the NRT1 gene spacer locus of the Saccharomyces cerevisiae; the transcription activator AraR is inserted into the transcription factor expression plasmid A and the transcription repressor AraR R .

[0166] In some embodiments, the recombinant bacterium also carries a promoter-reporter gene plasmid, and the promoter-reporter gene plasmid is a reporter gene backbone plasmid inserted with an inducible promoter, and the inducible promoter is selected from a first promoter capable of binding to the transcription activator AraR A and a second promoter capable of binding to the transcription repressor AraR R . At least one of the first promoter and / or the second promoter is integrated at the ura3 genomic locus of the Saccharomyces cerevisiae. Among them, the specific descriptions of the first promoter and the second promoter are as detailed in the description of the second aspect of the second part of the embodiments above, and will not be repeated here.

[0167] In some embodiments, the recombinant bacterium also carries a promoter-reporter gene plasmid, and the promoter-reporter gene plasmid is a reporter gene backbone plasmid inserted with a dual-regulated promoter, and the dual-regulated promoter can bind to the transcription activator AraR A and can bind to the transcription repressor AraR R . The transcription activator AraR A is derived from eukaryotes, and the transcription repressor AraR R is derived from bacteria. Among them, the promoter-reporter gene plasmid is as detailed in the description of the fifth aspect of the second part of the embodiments above, and will not be repeated here.

[0168] In some embodiments, the kinetic model formula 1 of the substrate response curve of the recombinant bacterium is as follows:

[0169]

[0170] where y min is the output when the inducer is not added to the induction system, y max is the output when the induction system reaches a steady state after adding the inducer, x is the inducer concentration, k 1 , k 2 , n 1 , and n 2 are respectively based on the transcription activator AraR A, the transcriptional repressor AraR R The Hill parameter of the arabinose single-regulation induction system.

[0171] In some embodiments, the transcriptional factor expression plasmid is integrated into the NRT1 gene spacer locus of the Saccharomyces cerevisiae; the transcriptional factor expression plasmid is inserted with the transcriptional activator AraR A , the transcriptional repressor AraR R expression backbone plasmid, the xylose transcriptional activator XlnR and the xylose transcriptional repressor XylR. The xylose transcriptional activator XlnR is derived from filamentous fungi, and the xylose transcriptional repressor XylR is derived from bacteria.

[0172] In some embodiments, the recombinant bacterium also carries a first promoter-reporter gene plasmid and a second promoter-reporter gene plasmid;

[0173] The first promoter-reporter gene plasmid is inserted with a first dual-regulation promoter, and the first dual-regulation promoter can bind to the transcriptional activator AraR A , and can bind to the transcriptional repressor AraR R ;

[0174] The second promoter-reporter gene plasmid is inserted with a second dual-regulation promoter, and the second dual-regulation promoter can bind to the xylose transcriptional activator XlnR and can bind to the xylose transcriptional repressor XylR. Among them, the description of the first dual-regulation promoter can be found in the third aspect of the first part of the examples; the second dual-regulation promoter can be found in the third aspect of the second part of the examples, which will not be elaborated here.

[0175] In some embodiments, the induction substrate of the recombinant bacterium includes corncob hydrolysate, and the corncob hydrolysate is mainly prepared by the following steps: treating 3 g of 20-mesh corncob powder with a 2% by mass H 2 SO 4 solution at 120 °C for 45 min, then adding calcium carbonate to adjust the pH value to 5.0, then adding 1,800 U of cellulase and 1,500 U of hemicellulase for hydrolysis, and then performing solid-liquid separation to collect the supernatant to obtain the corncob hydrolysate.

[0176] The above-mentioned recombinant bacterium can be used to prepare linalool.

[0177] AraC-P BAD system shows transcriptional heterogeneity under low-concentration arabinose induction. AraC-P BAD system is inhibited by glucose. AraC-P BADThe induction level is not high enough, limiting its use in application scenarios requiring high yields.

[0178] This section solves the above problems through the following solutions:

[0179] (1) Literature research on arabinose-responsive transcription factors: Through literature and related databases (such as RegPrecise and FungiDB), we found the core elements that constitute the arabinose transcription induction system: arabinose-responsive transcription factors (derived from the transcription activator AraR in eukaryotes) A and homologous variants, a series of transcriptional repressors AraR in bacteria R ) and the consensus sequence (motif or operator) of the transcription factor binding or the corresponding sequence that has been verified to be able to pass AraR A The natural promoter that responds to arabinose. A with AraR R The working principle of Figure 26 .

[0180] (2) Eukaryotic transcription activator AraR from different bacterial species A and prokaryotic transcriptional repressor AraR R The yeast arabinose induction system was constructed and the prototype response test was conducted: First, two backbone plasmids were constructed to express the transcription factor (TF) and the fluorescent reporter gene YFP controlled by the promoter that binds to the transcription factor; then the transcription factor AraR A (AN0388, An04g08600, pGUG03166 were amplified from the genomes of Aspegillus nidulans, Aspegillus niger, and Meyerozyma guilliermondii, respectively) and AraR from seven different bacterial sources R (Gene synthesis, codon optimization) inserted into the TF expression backbone plasmid; at the same time, responding to AraR A The natural promoter (P ANladR ,P ANlxrA ) and respond to different AraR R The synthetic promoter was inserted into the reporter gene backbone plasmid; finally, the two paired plasmids were successively transformed and integrated into the Saccharomyces cerevisiae genome, and the expression under arabinose-induced and non-induced conditions was quantitatively tested by flow cytometry.

[0181] (3) Construction of synthetic arabinose-responsive transcription factors: AraR A(AN0388, An04g08600, pGUG03166) The binding consensus sequence has not been clearly defined and lacks support from EMSA or ChIP-seq data. The full-length AraR or AraR without the DNA-binding domain (DBD) A was fused and expressed with well-characterized DBDs from different sources. In this study, the DBD of the bacterial TF LexA (the first 87 amino acids at the N-terminus, LexA 1-87 ), and the DBD of the CI repressor from phage 434 (CI434 1-70 ) were selected. The resulting synthetic TFs and the corresponding promoters containing the respective binding sites were constructed and tested according to the content in (2).

[0182] (4) Design, construction, and optimization of high-performance adjustable synthetic promoters: For eukaryotic transcriptional activators, the strongly inducible endogenous ADH2 promoter of Saccharomyces cerevisiae was used as the chassis promoter for modification; for prokaryotic transcriptional repressors, a fully synthetic minimal promoter was used for chassis modification. Through library construction, high-throughput screening was performed on the core promoter sequence of the chassis promoter for modification, the spacer sequence between the TATA-box and the nucleosome depletion region (NDR), the spacer sequence between the TATA-box and the transcription start site (TSS), the number and sites of transcription factor binding site motifs (motifs), and the prokaryotic operator sequence to optimize the performance of the induction system.

[0183] (5) Performance characterization of the single-regulated arabinose sensor: The arabinose response curve and induction kinetic behavior of the optimized single transcription factor regulation system were measured and fitted. Among them, the induction response curve was fitted by the Hill equation, and the induction kinetic curve was fitted by a basic transcription and translation model.

[0184] (6) Design, construction, and performance characterization of the dual-regulated arabinose induction expression system: After analyzing the single-regulated induction system, it was found that for the induction system based on AraR A , the leaky expression level was relatively high; while for the induction system based on AraR R , the maximum induction intensity was relatively low. Therefore, to obtain a low-leakage and high-induction expression, that is, an induction system with a large dynamic range, a dual-regulation system was constructed. First, yeast strains containing constitutive expression of AraR A and AraR R were constructed. Secondly, a composite promoter containing the binding sites of AraR A and AraR R was constructed; finally, the composite promoter was transformed into AraR A-AraR R Yeast strains. To ensure the stability of the system, AraR A -AraR R and the composite promoter were integrated into the yeast genome. The resulting final strains were induced with arabinose at gradient concentrations and fitted by the Hill equation; and their induction kinetic behaviors were measured under 100 mM arabinose induction and fitted according to the basic transcription and translation model.

[0185] (7) Multidimensional comprehensive comparison of the single-regulated and dual-regulated arabinose-inducible expression systems with the existing endogenous and synthetic inducible systems in Saccharomyces cerevisiae: By reconstructing the endogenous systems (P GAL1 , P CUP1 , P MET3 ) and synthetic inducible systems (LexA-ER-VP16, LexA-ER-B112) uniformly, comparisons were made with the single-regulated and dual-regulated arabinose-inducible systems in terms of five dimensions: basal expression, maximum activation level, dynamic range, induction response speed, and toxicity to host growth.

[0186] (8) The arabinose-inducible system is combined with the xylose-inducible system developed within the group to control the production of linalool to demonstrate its practicality for metabolic engineering: The arabinose-inducible system is used to control the expression of regulatory proteins to redirect the metabolic flux due to its strictly regulated characteristics; while the xylose-inducible system is used to overexpress metabolic genes due to its extremely strong inducibility. The optimal arabinose concentration and addition time were tested, and finally the titer of linalool produced by the genetically modified Saccharomyces cerevisiae strains was measured. In addition, since agricultural wastes such as corncobs have hydrolysates mainly composed of glucose, xylose, and arabinose, as a proof of concept, corncob hydrolysates were used as the sole carbon source and inducer source for the production of linalool, and the titer of production was measured.

[0187] (9) Cross-reactivity test of the arabinose-inducible system to xylose and sustainable expression test of the arabinose-inducible system.

[0188] The second part of the technical solution has the following advantages:

[0189] 1. It fills the gap in the eukaryotic system without a synthetic arabinose-inducible system.

[0190] 2. A new low-cost and high-performance transcriptional induction system has been developed in Saccharomyces cerevisiae: As a non-metabolic inducer and one of the components of hemicellulose hydrolysates in crop wastes, the price of arabinose is 1 / 3 of that of galactose, the commonly used inducer in Saccharomyces cerevisiae at present, and there is no need for continuous addition during the long-term fermentation process. Using the arabinose-responsive transcriptional activator AraR derived from the eukaryote Aspergillus niger A, such that the maximum activation level is comparable to the transcriptional level of the strongest TDH3 promoter in Saccharomyces cerevisiae. Compared with the slow de-repression process of the endogenous GAL system in Saccharomyces cerevisiae, its induction speed is rapid (it can reach 50% of the maximum activation level after 5.5 h of induction); based on the AraR A system, by combining the transcription factor AraR R from Thermotoga sp. RQ2, the dual-regulation induction system significantly reduces the leakage level, and finally the induction dynamic range exceeds 300-fold.

[0191] 3. The developed inducible regulatory synthetic promoter is short (<400 bp) and can achieve high-intensity activation, avoiding the problems brought by using natural promoters.

[0192] 4. It has little growth toxicity to the host: integrating the induction system based on AraR A has no toxic or side effects on the growth of yeast strains under both induced and non-induced conditions; although integrating the induction system with AraR R has an inhibitory effect on the growth of yeast strains under non-induced conditions, adding 1 mM arabinose can restore the growth rate of yeast strains to the wild-type growth level.

[0193] 5. In terms of regulatory rigor, dynamic range, toxicity to host growth, and response speed, the dual-regulation arabinose induction system developed in the present invention has comprehensively surpassed the commonly used induction systems in Saccharomyces cerevisiae (such as the natural endogenous systems P CUP1 , P MET3 , and the synthetic induction system LexA-ER-VP16 / B112), and is comparable to P GAL1 of the GAL system.

[0194] 6. The developed arabinose induction system is not inhibited by other carbon sources such as glucose, realizing the decoupling of the growth stage and the induction stage. The developed arabinose induction system does not show the phenomenon of population heterogeneity under inducer concentrations spanning 5 orders of magnitude and during long induction periods. The developed arabinose induction system has been proven to have strong practicability and can be effectively used in metabolic engineering (such as for the production of linalool). It should be noted that the developed system is not limited to the production of linalool, and other high-value compounds can also be produced using other agricultural wastes rich in xylose.

[0195] 7. Combining with the previously developed xylose induction system, the present invention opens up a new and simple way for the recycling of agricultural wastes to produce high-value compounds.

[0196] 8. The arabinose induction system developed in the present invention has high specificity and no cross-reactivity with xylose, which is very similar in structure, even at a high concentration (100 mM). The arabinose induction system developed in the present invention can achieve continuous and stable expression in Saccharomyces cerevisiae, that is, no additional inducer needs to be supplemented after the initial addition of arabinose to maintain stable expression.

[0197] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following examples, the guidance given in the present application is preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0198] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0199] The names and sequences of the elements involved in the following examples are shown in Table 1 (see later). In Table 1, the “(amino acid sequence)” after the sequence indicates that the sequence is an amino acid sequence, and if there is no “(amino acid sequence)” after the sequence, it means that the sequence is a nucleotide sequence.

[0200] Unless otherwise specified, the methods used in the following examples are as follows:

[0201] Table 2: The GOLDEN GATE systems and conditions used in each example

[0202]

[0203]

[0204] The first part: Xylose-sensing transcription factor components and their applications

[0205] Unless otherwise specified, the methods used in the following examples are as follows:

[0206] 1. Substrate induction test culture

[0207] Pick monoclonal colonies on the YPD plate into 500 μL of SD defective medium in a 2 mL 96-well plate (Axygen, P-2ML-SQ-C-S), seal with a breathable membrane (BioTss, SF-200), and culture at 800 r.p.m and 30 °C for 24 hours. Then, inoculate 2.5 μL aliquots into 497.5 μL of fresh SD medium containing the corresponding concentration of inducer (OD 600= 0.005), cultured for 16 h under the same conditions, and then the culture was diluted 10-fold, i.e., 20 μL aliquots of 180 μL phosphate-buffered saline (PBS, Proteintech, PR20014) containing 10 μg mL -1 cycloheximide (Maokang Biology, MS0035) were added to a 96-well U-bottom plate (Corning, 3799), and then the mixture was analyzed by flow cytometry.

[0208] 2. Flow cytometry and data processing

[0209] The intensity of yellow fluorescent protein (yEmCitrine) in the treated samples was detected using a BD FACSCelestaTM flow cytometer in high-throughput sampler (HTS) mode (BD Biosciences, Germany). A 488 nm excitation laser (100 mW) and a 530 / 30 nm emission filter were selected to measure the YFP intensity. Data were recorded using FACSDiva software (BD Biosciences, Germany), and more than 10,000 events were recorded for each sample. Cells of the correct size were selected in the forward scatter area (FSC-A) / side scatter area (SSC-A) plot. The data were processed using software 10.4 (TreeStar, USA), and the median of the fluorescein isothiocyanate region (FITC-A) signal was used for yellow fluorescence quantification and statistical analysis. The formula for the induction fold is (YFP 诱导 -YFP 0 ) / (YFP 非诱导 -YFP 0 ), where YFP 诱导 and YFP 非诱导 represent the fluorescence measured in the presence and absence of 10 mM xylose, respectively. YFP 0 represents the autofluorescence of white blood cells. The formula for the inhibition fold is (YFP -抑制因子 -YFP 0 ) / (YFP +抑制因子 -YFP 0 ), where YFP -抑制因子 and YFP +抑制因子 represent the fluorescence measured in the absence and presence of a transcriptional repressor, respectively. The fluorescence intensity was normalized to the fluorescence intensity of the endogenous constitutive PFY1 promoter (YFP 参考 ), in units of RPU, and the formula for RPU is (YFP - YFP 0 ) / (YFP 参考 -YFP 0 ). 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 plate as the test strain each time.

[0210] 3. Induction kinetics test culture

[0211] Single colonies were picked on YPD plates and transferred to 500 μL of SD-deficient medium in a 2 mL 96-well plate (Axygen, P-2ML-SQ-C-S). The plate was sealed with a breathable membrane (BioTss, SF-200) and cultured at 800 r.p.m. and 30 °C for 24 hours. For GAL system derivative strains (GAL80Δ, GAL80Δ&GAL1Δ) under non-glucose repression conditions, 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 aliquots of the culture were inoculated into 475.5 μL of fresh SD medium supplemented with the corresponding inducer in a 2 mL 96-well plate (OD 600 = 0.005). Subsequently, 25 μL of these cultures were transferred to 475 μL of fresh SD or SCR medium, and the corresponding inducer was added in a 2 mL 96-well plate. The cultures were then cultured in an oscillating incubator (Zhichu, ZQZY-88AH) at 30 °C and 800 r.p.m. During the initial 3-hour growth period, 50 μL of the culture was collected at each specified time point and then an equal volume of fresh medium supplemented with the corresponding inducer was added. After the initial 3-hour culture, 20 μL of the culture solution was collected from each culture well every 3 hours during the 36-hour culture period. The collected samples were diluted with 180 μL of PBS containing 10 μg mL -1 cycloheximide and then subjected to flow cytometry analysis.

[0212] 4. Growth toxicity test

[0213] Three test strain clones were randomly selected and cultured overnight in 500 μL of SD-deficient medium. The culture was inoculated into SD medium containing a gradient concentration of inducer (without defective nutrient screening) at a ratio of 1:100 and cultured for 24 hours. 20 μL of the culture was taken for flow cytometry analysis to detect the correctness and induction heterogeneity of the test strains. At the same time, 5 μL of the culture in each culture well was inoculated one-to-one into the corresponding culture well of another plate containing 495 μL of medium with a gradient concentration of inducer. After culturing for 8 hours, the OD of the culture in each culture well was measured using a Tecan Infinite 200Pro plate reader (Tecan). 600Meanwhile, the cultures in the non-induced pores were inoculated into SD medium supplemented with gradient concentrations of inducer, and grown for another 24 hours. Data were collected continuously for three days, and finally, the OD of the parental CENPK.2-1C strain cultured in SD medium on the first day was used 600 to normalize the measured data of the test strains for three consecutive days. The calculation formula is norm.OD 600 = ave(OD 600 ) 诱导 / OD 600(2-1C) .

[0214] 5. HPLC analysis of corncob hydrolysate

[0215] The concentrations of D-glucose, D-xylose, and L-arabinose in corncob 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 Aminex HPX 87-H (Bio-Rad, Hercules, USA), which was connected to a Micro-Guard Cation-H guard column (Bio-Rad, Hercules, USA). The separation process used 5 mM H 2 SO4 as the mobile phase, and the flow rate was set at 0.6 mL min -1 , and the temperature was 55 °C. The signal was detected by RID (Agilent Technologies, G7162A) at 35 °C.

[0216] 6. Sample preparation and Tricine-SDS-PAGE

[0217] Sample preparation: Pick the xylose induction system and P AOX1The monoclonal strain that systemically controls the expression and secretion of msfGFP was transferred into 2 mL of SD-His medium in a 96-well plate (Axygen, P-2ML-SQ-C-S), sealed with a breathable film, and cultured at 800 r.p.m. 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 grown for another 24 hours. Subsequently, 5 μL of the strain culture carrying the xylose induction 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 induction system was inoculated into BMMY medium supplemented with 0.5% methanol (v / v). After 24 hours of induction, 0.5% methanol (v / v) was supplemented to the methanol-induced strain. After a total of 48 hours of induction, 20 μL of the supernatant was taken to measure the extracellular msfGFP fluorescence intensity using a Tecan Infinite 200Pro plate reader (excitation wavelength 488 nm, emission wavelength 510 nm, gain 100). 20 μL of the culture solution and 20 μL of the undiluted supernatant were respectively mixed with 20 μL of 2×Tris-Tricine-SDS-PAGE loading buffer (BOSTER, AR1143). Then these mixtures were boiled at 100 °C for 5 minutes and frozen at -80 °C for Tricine-SDS-PAGE analysis.

[0218] Tricine-SDS-PAGE: First, a 1.5 mm thick PAGE gel was prepared, consisting of three gels: stacking gel (4% T, 3% C), spacer gel (10% T, 3% C), and separating gel (16.5% T, 6% C, containing 6 M urea). The Tricine gel and gel buffer were provided by a commercial kit (Enogene Biotech, E1WP326). The running buffer, anode buffer (0.1 M Tris, 0.0225 M HCl, pH 8.9), and cathode buffer (0.1 M Tris, 0.1 M Tricine, 0.1% SDS, pH 8.25) were purchased from (Servicebio, G2142-1L). After electrophoresis, the gel was incubated in the fixing solution (50% methanol, 10% acetic acid) for 1 hour, and then stained overnight with 0.025% Coomassie Brilliant Blue R-250 added to a 10% acetic acid solution. Then the gel was washed twice in 10% acetic acid, with each incubation for 45 minutes. The decolorized gel was imaged on a Bio-Rad ChemiDoc imaging system, and the image was analyzed using Bio-Rad ImageLab software (version 6.1).

[0219] Example 1-1: Prototype Substrate Response Test of Xylose-Inducible Transcription Systems from Different Sources in Saccharomyces cerevisiae

[0220] The basic principle for constructing the prototype response test platform for the xylose transcriptional induction system is for the high-throughput screening of the xylose-responsive transcriptional induction system in Saccharomyces cerevisiae. Candidate transcription factors are introduced with restriction endonuclease BpiI sites at both ends of the fragment through primer design and the BpiI sites in the natural sequence are removed by synonymous codon substitution. The expression plasmid of the transcription factor and the reporter plasmid responsive to the transcription factor are constructed in one step by GOLDEN-GATE technology. The TF-based biosensor screening strategy is as Figure 2 shown Figure 2 which shows the annotated features of the backbone plasmid for biosensor screening. The required reporter plasmid and the plasmid expressing TF are generated by one-step assembly through BpiI digestion to replace ccdB in the vector. The purified BsaI digestion product is directly transformed into yeast. The screening strategy for xylose-responsive transcription factors is carried out in Saccharomyces cerevisiae. The concentration of the transcription factor is controlled by the aTc-TetR induction system, in which TetR is expressed under the control of the constitutive promoter PFBA1. The output of the transcription factor-responsive promoter depends on the concentrations of TF and the inducer.

[0221] The specific operation process for constructing the prototype response test platform for the xylose transcriptional induction system is as follows:

[0222] The sequence of the xylose-responsive transcription factor XlnR (AN7610) (amino acid sequence as shown in SEQ ID NO.1) was obtained by designing primers from the extracted genome of Aspergillus nidulans, removing introns and BpiI restriction sites, and amplifying by segmental PCR; the xylose-responsive transcription factor XylR from different bacteria (such as Figure 15 shown

[0223] The obtained transcription factor fragment, the backbone plasmids pGD137 (base sequence shown in SEQ ID NO.23) and pGS001 (base sequence shown in SEQ ID NO.24) are reacted through the GOLDEN-GATE system. The product is transformed into E. coli trans10 to obtain the target plasmid, which is then digested with BsaI and transformed into the Saccharomyces cerevisiae CYE90 (from Chen, Y. et al. Genetic circuit design automation for yeast. Nat Microbiol 5, 1349-1360, doi:10.1038 / s41564-020-0757-2 (2020)) to obtain a yeast strain carrying the xylose transcription factor. Among them, the xylose-responsive transcription factor XlnR is ligated into the backbone plasmid pGD137, and the xylose-responsive transcription factor XylR is ligated into the backbone plasmid pGS001.

[0224] The natural promoter P from Penicillin chrysogenum xylP (711bp, whose base sequence is SEQ ID NO.55) also removes the BpiI restriction site within the promoter region through primer design and introduces a BpiI restriction site at the end, and reacts with the existing backbone plasmid pXJH1 in the laboratory through the GOLDEN-GATE system to obtain the reporter plasmid; for XylR of different bacterial species, its promoter (where the promoter corresponding to the xylose-sensing transcription factor XylR with the amino acid sequence shown in SEQ ID NO.2 is P xyl , with the base sequence shown in SEQ ID NO.3) is designed according to the 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 is obtained in the same way. The obtained reporter plasmid is transferred into the above-mentioned yeast strain carrying the xylose transcription factor, and the activation / inhibition multiples under xylose induction and non-induction are tested as described in the method section. The detection results are as Figure 15 shown. Among them, the schematic diagram of the construction of the high-performance xylose induction system based on bacterial XylR is shown in Figure 4 ( Figure 4Among them, T20: 20 consecutive thymine bases. TATA: TATA-box (5’-TATAAAA-3’, sequence number is SEQ ID NO.51), TSS: transcription start site (5’-AGAATATCAAGCTACAAAAA-3’, sequence number is SEQ ID NO.52), the length between the characteristic sequences is shown in the above numbers). For the XylR transcription factor from bacteria, reference: Chen, Y. et al. Genetic circuit design automation for yeast. Nat Microbiol 5, 1349-1360, doi:10.1038 / s41564-020-0757-2 (2020). The binding sequence (operator) of XylR was placed downstream of the TATA-box and upstream of the TSS respectively.

[0225] From Figure 15 It can be seen that XlnR in Aspergillus nidulans shows an activation multiple of more than 100 times, while XylR in Bacillus licheniformis has the largest inhibition multiple. Therefore, both are used for subsequent further optimization.

[0226] Example 1-2: Design and optimization of the synthetic promoter responsive to XlnR

[0227] Since the natural promoter P xylP is too long, the unstructured framework increases the difficulty of further modification. In addition, it may also be subject to unknown endogenous regulation. Therefore, it is necessary to rationally design a structured synthetic promoter. Based on the natural inducible promoter P ADH2 The modification strategy is detailed in Figure 3 . Based on the optimized detection results of the XlnR xylose transcriptional induction system as shown in Figure 16 shown, Figure 16 In, Figure a is the detection result diagram of the optimization of the responsive synthetic promoter; Figure b is the detection result diagram of the optimization of the transcription factor concentration.

[0228] The specific optimization process is as follows:

[0229] First, the inducible ADH2 natural promoter with a clear structure was selected, and the UAS region sequence originally bound by ADR1 was replaced with the binding sequence of XlnR (as shown in Figure 3 ), to obtain the semi-synthetic promoter P xln.1a (the base sequence is as shown in SEQ ID NO.4). Figure 3 In, after replacing the ADR1 binding site of the natural promoter ADH2 with the XlnR binding site, the semi-synthetic promoter P xln.1aBy performing high-throughput screening and replacement on the TFBS array, spacer.a, and spacer.b in the figure, fully synthetic promoters for different induction systems can be obtained.

[0230] However, the leakage level of this semi-synthetic promoter P xln.1a cannot be ignored. Therefore, by performing high-throughput screening on the sequence between NDR and TATA-box using a synthetic random primer (5’GTCACTGAAGACAAGGCAANNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNTCACAATGTCTTCCGAGGCAGAG CA-3’, with the sequence number being SEQ ID NO.48, abbreviated as random primer sequence 1), P xln.1b (with the base sequence shown in SEQ ID NO.5) with low background and strong activation was finally obtained.

[0231] Previously, it was reported in the literature that the core promoter determines the maximum activation intensity. Therefore, in this example, the core promoter region of ADH2 was replaced with the core regions of other highly active promoters, resulting in 6 synthetic promoters (with their base sequences shown in SEQ ID NO.6 - SEQ ID NO.11). Although all of these can 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 part is programmable.

[0232] To obtain a fully synthetic promoter, high-throughput screening was carried out on the sequences of the TATA-box and TSS regions through multiplex random primer stacking synthesis. A series of fully synthetic inducible promoters were finally obtained, especially three fully synthetic promoters with base sequences shown in SEQ ID NO.18 - SEQ ID NO.20.

[0233] In addition, appropriately increasing the binding sites of transcription factors is a reliable strategy to improve the transcriptional activation level. By testing promoters containing 1, 2, 3, and 4 XlnR binding sites (xlno) (with base sequences shown in SEQ ID NO.4 - SEQ ID NO.5 and SEQ ID NO.12 - SEQ ID NO.15), the results (as Figure 16 a) showed that the promoter containing 2 xlno has significantly increased the activation level. Especially, P xln.2b (with the base sequence shown in SEQ ID NO.13), whose activation level even exceeds that of the promoter P xln.4 (with the base sequence shown in SEQ ID NO.15) containing 4 xlno. However, its background leakage level is too high. Therefore, P xln.4has the largest dynamic range.

[0234] In addition, the corresponding functional relationship between the concentration of XlnR and the system output was tested (as shown in Figure 16 b), and based on this, a suitable constitutive promoter was selected to ensure stable expression of XlnR throughout the yeast stage. 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 IDNO.53), and P PXR1 (base sequence as shown in SEQ ID NO.54). Finally, it was shown that when P ACT1 was used to express XlnR, the system had the largest dynamic range.

[0235] Examples 1 - 3: Determination and fitting of substrate response curve, induction heterogeneity, and kinetic behavior of the xylose single-regulation induction system

[0236] 1. Construction of the transcription factor array yeast strain, the schematic diagram of the construction principle (see Figure 5 , 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 frames were assembled on a plasmid through GOLDEN-GATE technology, and the fragments after digestion with the restriction enzyme BsaI were transformed into Saccharomyces cerevisiae (i.e., the CENPK.2-1C strain), and integrated at the NRT1 gene spacer site to obtain the transcription factor array yeast strain (i.e., the xylose-sensing yeast).

[0237] 2. Construction of the single-regulation xylose-induced transcription system, the schematic diagram of the construction principle (see Figure 6 ): In the transcription factor array yeast strain, the yellow fluorescent reporter gene YFP expression frames responsive to XlnR and XylR were respectively transferred and integrated into the original ura3 genomic locus to obtain the single-regulation transformed strain.

[0238] 3. Characterization of the performance of the optimized P xln.2b -XlnR system and P xyl -XylR (i.e., the single-regulation xylose-induced transcription system):

[0239] The obtained single-regulation transformed strains were induced with 16 different concentrations of xylose, and the output signals were analyzed by flow cytometry (method), the substrate concentration induction curve was plotted, and it was fitted with the hill function (as shown in Figure 17 a) to obtain the Hill parameter and EC50 value (see Table 3). The results showed that compared with P xyl , Pxln.2b has a leakage level two orders of magnitude higher; while compared to P xln.2b , P xyl 's maximum activation level is 25% of the former. Among them, for a given transcriptional activator, the relationship between the protein mass (A) and the output (y) of the responsive promoter can be described by the Hill function:

[0240]

[0241] For the promoter XlnR-P xln.2b system, this functional relationship is as shown in Figure 16 b. The fitted k 1 and n 1 are 3.42 and 1.82 respectively (see Table 3).

[0242] In addition, the heterogeneity of yeast populations at different xylose concentrations was observed and analyzed. The results are as shown in Figure 17 b. Under the induction of xylose concentrations at 5 different orders of magnitude, a single peak constantly appears and there is no population heterogeneity. Finally, the kinetic behavior under the induction of 10 mM xylose concentration that can saturate the induction system was measured and fitted. The results are as shown in Figure 17 c. The fitting parameters are shown in Table 3. The results show that the observed kinetic data can be fitted by the standard transcription-translation model, namely Equation 2 and Equation 3.

[0243] Among them, Equation 2 is as follows:

[0244] Equation 3 is as follows:

[0245] The meanings of the letters in Equation 2 and Equation 3 are: m and p are the concentrations of mRNA and protein respectively; β m , β p are the constants describing the synthesis rates of mRNA and protein respectively; γ m , γ p are the constants describing the degradation and dilution rates of mRNA and protein respectively.

[0246] Table 3: Fitting parameters of substrate response curves

[0247]

[0248] Examples 1-4: Construction design of xylose dual-regulation induction system and determination and fitting of substrate response curves and kinetic behaviors

[0249] 1. Given the high leakage of the P xln.2b -XlnR system and the P xyl- The low activation ability of the XylR system. By adopting a dual transcription factor regulation system and integrating the advantages of both, the xylose transcriptional regulation system can be further optimized. However, there are multiple design schemes for the dual regulation mode (as shown in Figure 18 a), and there are 6 functional topologies (see Figure 7 ). According to the standard transcription and translation model (see Table 5) and the kinetic fitting parameters of single regulation (see Table 4), the induction kinetics of these 6 topologies of the dual regulation system were simulated. The final simulation results are as shown in Figure 18 b, where topology 1 has the maximum induction rate and the maximum activation level (see Figure 18 b). Therefore, the simple topology 1 was selected for further construction and performance characterization.

[0250] For a given induction system, γ p is a fixed value of 0.29, derived from the equation , where τ is the bacterial doubling time, which is 90 min here (reference: McDonald, P. N. Two - hybrid systems. Methods and protocols. Introduction. Methods Mol Biol 177, v - viii (2001)). The other parameters (β m , γ m , β p ) were obtained by fitting with equations (3) and (4) and the measured kinetic data (see Table 4). Therefore, through the following parameters and assignments, β 1 = 2.22; β 2 = 2.19; β 3 = 0..49; β p = 1.62; γ m = 0.28; β p = 0.29; k 1 = 3.42; n 1 = 1.82.

[0251] Among them, according to Table 3, when t = 0 h, P(a)[t = 0] = 0.28, P(YFP)[t = 0] = 0.28 (P xln.2b ) or 0.001 (P xln.2b-xyl ). Therefore, when P(YFP)[t = 0] = 0.28, β m [t = 0] = 0.28 * 0.283 * 0.29 / 1.618 = 0.014, and m(a)[t = 0] = m(YFP)[t = 0] = β m [t = 0] / γ m= 0.050; when P(YFP)[t = 0] = 0.001, m(YFP)[t = 0] = 0.00018. Dynamic data fitting was performed using the above differential equations and fitting values, and the fitting results are shown in Figure 18 a. Since the analytical solution of the above dynamic simulation function cannot be obtained, the bisection method was used to calculate τ 1 / 2 , and the obtained results are as shown in Figure 18 b.

[0252] 2. Construct the topological structure 1 of the double transcription factor (the schematic diagram of its framework design is shown in Figure 8 ). Only need to follow the architecture in Figure 19 a, that is, the promoter sequence was constructed according to sequence P xln.2b-xyl (the base sequence is shown in SEQ ID NO.17). The sequence P xln.2b-xyl fragment was subjected to a GOLDEN-GATE one-step reaction with the backbone plasmid pXJH1 (constructed in our laboratory, and the schematic diagram of its plasmid structure is Figure 2 shown) to obtain the target reporter plasmid, and then it was transformed into the existing transcription factor array strain (that is, the transcription factor array yeast strain constructed in step 1 of Example 3, and its construction schematic diagram is shown in Figure 5 ). Then, the strain containing this double regulatory system was induced with 16 different concentrations of xylose and analyzed by flow cytometry to measure and draw the substrate response curve (such as Figure 19 a). Similarly, the measured data can be well fitted by the Hill function ( Figure 19 a), and the EC50 is 0.93 mM. Assuming that the binding of XlnR and XylR to the promoter P xln.2b-xyl is an independent event, the steady-state induction of this promoter can be predicted by the partition function, that is, formula 1, and the actual prediction results are shown in Figure 19 a. The substrate induction behavior of this double regulation can be well predicted by the fitting parameters of single regulation.

[0253] 3. Since P GAL1 in the classical GAL system in Saccharomyces cerevisiae also belongs to at least a double regulatory system, that is, glucose repression mediated by Mig1p and galactose activation mediated by Gal4p on the other hand. In terms of both steady-state substrate induction response and kinetic induction, it was introduced as a control to better evaluate the performance of P xln.2b-xyl -XlnR-XylR. Since the output of P GAL1 is determined by the ratio of galactose and glucose, therefore, under the condition of controlling the total sugar to be 2%, the output of P GAL1 induced by different ratios of galactose and glucose was measured. The results are shown in Figure 19 a. The results show that only when galactose:glucose > 31, P GAL1 reaches the saturated activation level, but it is still significantly lower than Pxln.2b-xyl Activation level.

[0254] 4. In addition, kinetic measurements were performed on the P induced by 2% galactose GAL1 and the dual-regulated xylose system induced by 100 mM xylose, and the results are as Figure 19 shown in b. For P GAL1 , the memory of the presence or absence of glucose inhibition has a great impact on the induced kinetic behavior. Specifically, for strains without a history of glucose inhibition, the induction is very rapid. After 5 hours of induction, the induction level reaches 50% of the maximum activation. However, for strains with a history of glucose inhibition, there is at least a 1-hour induction lag. The induction behavior of the dual-regulated xylose induction system is not affected by glucose, and an increase in fluorescence output can be detected within 15 minutes after the addition of xylose. Moreover, after only 3 hours of induction, the induction level reaches 50% of the maximum activation. Similarly, the kinetic test data of the dual-regulated xylose induction system can be fitted by the standard transcription-translation models, namely Formulas 2 and 3 (see Figure 19 b).

[0255] Table 4: Fitting parameters for xylose induction kinetics

[0256]

[0257] Table 5: Fitting equations corresponding to 6 topologies

[0258]

[0259]

[0260] Examples 1-5: Substrate response curve of corn cob hydrolysate activating the xylose-induced transcription system

[0261] Agricultural wastes such as corn, wheat, and cotton straw are rich in xylose. If the xylose in their hydrolysates can be recycled, it will bring huge economic returns. Given the high sensitivity of the xylose-induced regulatory system in this application (with an EC50 of about 1 mM), the hydrolysates of these straws may replace pure xylose to activate this induction 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 Figure 10 production process. The concentrations of glucose, xylose, and arabinose in the hydrolysate were detected by HPLC analysis to be 178.9 mM, 53.3 mM, and 8.3 mM, respectively (see Figure 20 a). Different volumes of CCH were taken as the inducer, and the response substrate concentration curve was obtained as shown in Figure 20 b. A 50 μL non-concentrated corn cob hydrolysate can reach the maximum activation level (see Figure 20 b).

[0262] Among them, Figure 10Preparation steps: Treat 3 g of 20-mesh corncob powder with an H 2 SO 4 solution with a mass percentage content of 2% at 120 °C for 45 min, then add calcium carbonate for treatment, then add 1,800 U of cellulase (from Beijing Solarbio Science & Technology Co., Ltd., product number C8270) and 1,500 U of hemicellulase (from Beijing Solarbio Science & Technology Co., Ltd., product number H8110) for hydrolysis, then perform solid-liquid separation, collect the supernatant to obtain corncob hydrolyzate.

[0263] Examples 1 - 6: Multidimensional comparison of the xylose-inducible transcription system with existing inducible systems in Saccharomyces cerevisiae

[0264] To better evaluate the single-regulation and dual-regulation xylose-inducible systems, this application reconstructed the endogenous inducible system of Saccharomyces cerevisiae - P GAL1 ,P MET3 ,P CUP1 and the synthetic inducible system (LexA-ER-VP16 / B112) based on the ER system. The gene element configuration and integration sites are shown in Figure 9 (the relevant element sequences are shown in Table 1). This application comprehensively compared the developed xylose-inducible system in terms of regulation rigor, induction activation level, dynamic range, growth toxicity, and induction speed.

[0265] To determine the maximum activation level, this application set a test criterion: while reaching the maximum activation level, there should be no fluorescence heterogeneity in the population, and the growth of the host should not be severely inhibited. For this purpose, the outputs and fluorescence distributions of each system at different inducer concentrations were tested and examined (see Figure 21 ), thereby determining the inducer concentration at which the maximum activation level is reached. At this concentration, its induction output level was measured. According to the output level without adding any inducer, the dynamic range of each system was calculated, and the growth OD 600 of the strains was tested by continuous subculture for three days. The original transformed strain CENPK.2-1C was used as a reference to evaluate the growth status of the strains under induction. Finally, the induction kinetic behavior of each strain within 36 hours was tested under the induction of this concentration. The test results are as shown in Figure 22 , and the results show that the dual-regulation xylose system has the best comprehensive performance in the above five aspects.

[0266] Examples 1 - 7: Determination and fitting of the substrate response function of the xylose-inducible transcription system in Pichia pastoris, Candida glabrata, and Candida albicans

[0267] The XlnR protein has no orthologs in Pichia pastoris, Candida glabrata, and Candida albicans, and the first two cannot degrade and metabolize xylose. All three yeasts belong to non-conventional yeasts, with relatively distant evolutionary relationships and a relative lack of genetic tools such as induction systems. Therefore, these three yeasts were selected to test the transferability of the xylose induction system based on XlnR. For the convenience of prototype testing, the expression cassette of XlnR and the expression cassette of the reporter gene driven by the responsive promoter were designed to be included on a plasmid, and the gene elements, sub-elements, and backbone plasmids used are as Figure 11 - 14 shown. In addition, 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, and the integration strategy and sites are as Figure 11 , 12, 14 shown. Figure 11 Schematic diagram of the design of the xylose transcription system based on XlnR and the genome recombination strategy in Pichia pastoris; the configuration of the gene elements within the dashed box on the right is as shown on the left; Figure 12 Schematic diagram of the design of the xylose transcription system based on XlnR and the genome recombination strategy in Candida glabrata; the configuration of the gene elements within the dashed box on the right is as shown on the left; Figure 13 Schematic diagram of the construction of a universal plasmid adapting the GOLDEN-GATE technology in Candida albicans; the specific plasmid sequence is shown in Table 1; Figure 14 Schematic diagram of the design of the xylose transcription system based on XlnR and the genome recombination strategy in Candida albicans; the configuration of the gene elements within the dashed box on the right is as shown on the left.

[0268] Finally, the substrate induction curves of the three strains integrated with the xylose induction system were tested, as Figure 22 shown. The induction multiples in Pichia pastoris, Candida glabrata, and Candida albicans reached 189, 368, and 327 times, respectively. Similarly, in all three strains, the xylose induction curve could be well fitted by the hill function, and the fitting parameters are shown in Table 3. The results show that the EC50 of the xylose induction system in the first two strains is about 1 mM, while in Candida albicans, the EC50 is 13.7 mM.

[0269] Examples 1-8: Comparison of the ability of the xylose system and the methanol system to induce the secretion of msfGFP in Pichia pastoris

[0270] Pichia pastoris is a powerful platform for the production of heterologous proteins. The methanol system is the main and powerful induction system in Pichia pastoris, but this system has inherent problems. First, the methanol system is an endogenous system and is inhibited by glucose and glycerol. In industrial production, the production stage relying on glucose and glycerol and the methanol induction stage must be separated, thus lengthening the production cycle. Second, methanol itself serves as both a nutrient and an inducer, and its flammability and toxicity increase the safety risk of storage. Therefore, the xylose induction system has profound industrial value in inducing and driving the secretion of heterologous proteins in Pichia pastoris.

[0271] This application compared the induction activation levels of the xylose induction system and the methanol system. The results showed that the maximum activation level of xylose was twice that of the methanol system ( Figure 22 a, Figure 23 a). In addition, this application compared the abilities of the two induction systems to secrete msfGFP. The results are as Figure 23 shown in Figure 23 b. The results showed that under their respective optimal induction conditions, the ability of xylose to induce the secretion of msfGFP was significantly higher than that of the methanol system (as Figure 23 shown in Figure 24 b, c). Among them, AOX1 is the substrate induction curve determination and Hill fitting diagram of the XlnR-based xylose induction system in Pichia pastoris (a), Candida glabrata (b), and Candida albicans (c) in Example 8;

[0272] To sum up, for the xylose induction system based on the filamentous fungus-derived xylose-sensing transcription factor XlnR, in the presence of 2% glucose, the maximum activation level can reach 50% after 3 hours of xylose induction, and the maximum activation level can be reached after 6 hours of induction, resulting in a significant increase in the maximum activation level and a rapid induction speed. Moreover, by combining the filamentous fungus-derived xylose-sensing transcription factor XlnR and the prokaryotic xylose-sensing transcriptional repressor XylR, a dual-regulation induction system with an extremely low leakage level is obtained, but the maximum activation level is equivalent to that of the XlnR single-regulation xylose induction system, greatly expanding the dynamic range to 4,000-fold, solving the problems of low induction intensity and slow response of the xylose transcriptional induction system dependent on transcription factors in the field of synthetic biology.

[0273] The Second Part: Arabinose-responsive Transcription Factor Components and Their Applications

[0274] The main technical route of this part is as follows:

[0275] Arabinose Transcription Induction System Prototype Response Test Strategy (see Figure 27 , Figure 27 , which involves the GOLDEN-GATE one-step assembly of transcription factor and response promoter plasmids, and the concentration of the transcription factor is regulated by the aTc-tetR system): for the high-throughput screening of the arabinose-responsive transcription induction system in Saccharomyces cerevisiae. Candidate transcription factors introduce restriction endonuclease BpiI sites at both ends of the fragment through primer design and remove the BpiI sites in the natural sequence through codon synonymous substitution, and a reporter plasmid expressing the transcription factor and the response transcription factor is constructed in one step by GOLDEN-GATE technology.

[0276] Design and Optimization of the Induction System Based on Synthetic Transcription Factors (see Figure 28 ): The synthetic transcription factor is expressed by fusing AraR A without the natural DBD with DBDs from different sources, and the output promoter is a synthetic promoter based on the ADH2 promoter encoding the corresponding DBD binding sequence.

[0277] Design, Construction and Optimization of Eukaryotic High-performance Adjustable Synthetic Promoters (see Figure 29 a): Select the natural inducible ADH2 promoter of Saccharomyces cerevisiae as the chassis for the transformation of the inducible synthetic promoter, and its original characteristic sequences (nucleosome-depleted region, TATA-box, transcription start site) remain unchanged. The spacer sequences between these characteristic sequences are screened by high throughput to obtain a synthetic promoter that meets the requirements. In addition, the upstream activation sequence (UAS) of the ADH2 promoter is replaced with cis-regulatory elements (motifs) that bind AraR A . And the number and different positions of these motifs are tested to obtain an induction system with the best performance.

[0278] Design, Construction and Optimization of a High-performance Arabinose Induction System Based on the Bacterial Transcription Repressor AraR R (see Figure 30 a): For the transcription factor AraR R from bacteria, the binding sequences (operators) of AraR R are placed downstream of the TATA-box and upstream of the TSS of the minimal promoter, respectively.

[0279] Construction of Transcription Factor Array Yeast Strains (see Figure 31):Based on the optimal transcription factor expression levels, yeast strains with AraR A -AraR R 2-sensor and XylR-XlnR-AraR A -AraR R 4-sensor were constructed. The eukaryotic transcription factor AraR A and the bacterial AraR R expression cassettes were assembled onto a de novo constructed backbone plasmid (the backbone plasmid is pGS077 plasmid, and the nucleotide sequence is shown in SEQ ID NO.65) by GOLDEN-GATE technology; meanwhile, based on AraR A -AraR R , the transcription factors XylR and XlnR (specifically see the examples in "Part I Xylose-Sensing Transcription Factor Components and Their Applications") on which the xylose induction system developed in our laboratory depends were introduced. Similarly, the 4 transcription factors were assembled onto a de novo constructed backbone plasmid (the backbone plasmid is pGS078 plasmid, and the nucleotide sequence is shown in SEQ ID NO.66) by GOLDEN-GATE technology. The two obtained plasmids were digested with the restriction endonuclease BsaI and then transformed into Saccharomyces cerevisiae, and integrated into the NRT1 gene spacer locus to obtain two yeast strains that can sense arabinose and can sense xylose and arabinose simultaneously.

[0280] Construction and performance characterization of the single-regulated arabinose-induced transcription system (see Figure 32 ):In the obtained arabinose-sensing array yeast strains, the yellow fluorescent reporter plasmids responsive to AraR A and AraR R were respectively introduced, that is, the reporter plasmids containing P lex.3 -YFP and P ara -YFP. The sequences of the synthetic promoters P lex.3 (nucleotide sequence shown in SEQ ID NO.62) and P ara (nucleotide sequence shown in SEQ ID NO.59) are shown in Table 1 and were integrated into the original ura3 genomic locus.

[0281] Construction and performance characterization of the dual-regulated arabinose-induced expression system (see Figure 33, which involves the component configuration of the synthetic promoter fluorescence reporter expression cassette being the same as that of the single-regulation induction system): Reference (K. Forster, V. Helbl, T. Lederer, S. Urlinger, N. Wittenburg, W. Hillen, Nucleic acids research 1999, 27(2), 708). The composite promoter was constructed as follows: The sequence upstream of the TATA-box was from the synthetic promoter P A corresponding to the part in the single-regulation system that responds to AraR lex.3 , while the sequence downstream of the TATA-box was taken from the synthetic promoter P R corresponding to the part in the single-regulation system that responds to AraR ara . The correctly sequenced composite promoter construct was transferred into the induction strain containing AraR A -AraR R to obtain an arabinose-induced dual-regulation induction system, and its substrate induction curve and reaction kinetic behavior were measured. Among them, the substrate induction behavior was predicted by Equation 1, and its induction kinetics could also be fitted by a basic transcription-translation model.

[0282] Multi-dimensional comparison of the single-regulation and dual-regulation arabinose-induced expression systems with the existing endogenous and synthetic induction systems in Saccharomyces cerevisiae (see the method section for details): To better evaluate the performance of the constructed single-regulation and dual-regulation arabinose-induced systems, in Saccharomyces cerevisiae, fluorescence reporter plasmids for measuring the activities of natural induction promoters (P GAL1 , P MET3 , P CUP1 ) and the commonly used synthetic ER induction systems (LexA-ER-VP16, LexA-ER-B112) were reconstructed. The arabinose-induced system and the above-mentioned commonly used induction systems in yeast were compared in terms of leakage, induction ability, dynamic range, induction speed, and toxicity to host growth in five dimensions.

[0283] Verification of the practicality of the arabinose system in the field of metabolic engineering: As a proof of concept, monoterpene linalool was selected as the target product (see Figure 34 ). In addition, in the yeast strain integrated with the XylR-XlnR-AraR A -AraR R sensor element array, glucose in corn cob hydrolysate can be used for cell growth, xylose can induce the overexpression of metabolic pathway enzymes, and arabinose can be used for metabolic flux redirection by connecting a NOT logic gate to implement function inversion (see Figure 35 ). Similarly, as a proof of concept, monoterpene linalool was selected as the target product. The production titer of linalool in Saccharomyces cerevisiae is currently not more than 150 mg L -1, The main reasons may be as follows: 1. The expression level of linalool synthase is insufficient; 2. The supply of geranyl diphosphate (GPP) as a precursor is insufficient. Therefore, the xylose induction system developed by our research group before was used to control the key enzyme genes in the linalool metabolic pathway: tHMG1, IDI1, ERG20ww and linalool synthase t67McLIS; Conditional control of the closure of the GPP competitive pathway (the ergosterol synthesis pathway essential for growth) was confirmed to be helpful for the synthesis of monoterpenes. Since the arabinose induction system realizes the "turn-on" function after adding the inducer and cannot directly realize the gene closed expression, we introduced a repressor protein PhlF to achieve functional inversion, and this requires the induction control system to have extremely low leakage. Therefore, the dual-regulated arabinose system meets this requirement and is used for metabolic flux redirection.

[0284] Specific embodiments set according to the above technical route are as follows:

[0285] Unless otherwise specified, the methods used in the following examples are as follows:

[0286] 1. Plasmid construction

[0287] All synthetic DNA fragments, primers and Sanger sequencing services used in the following examples were provided by Tsingke Biotechnology Co., Ltd.

[0288] The plasmids used in this study were created by three different strategies.

[0289] 1) Overlap polymerase chain reaction (Overlap PCR): The backbone universal vector plasmid (the initial plasmid carrying the ccdB gene) and the plasmids that cannot be assembled from the gene element library created in the laboratory were constructed by this strategy. Q5 DNA polymerase (New England Biolabs, M0491L) was used, and the amplification program strictly followed the protocol provided by the manufacturer. The amplification products were digested with DpnI (New England Biolabs, R0176) to remove the template plasmid, and the digestion reaction program strictly followed the protocol provided by the manufacturer. The digestion products were directly used for cloning transformation. It should be noted that for the backbone plasmid carrying ccdB, the digestion products need to be transformed into Escherichia coli carrying the ccdA antitoxin gene such as Trans DB3.1 (TransGenBiotech, CD531). The obtained plasmids were subjected to whole plasmid sequencing.

[0290] 2) GOLDEN-GATE assembly (GGA): Most of the plasmids in this study were constructed based on the hierarchical assembly strategy of type IIS restriction enzymes (BsaI, New England Biolabs, R3733L and BpiI, Fermentas, ER1012) (see Figure 36)。To adapt to this strategy, the BpiI and BsaI enzyme digestion sites in the backbone vector and the insert were removed by synonymous codon replacement (https: / / sg.idtdna.com / pages / tools / codon-optimization-tool). To avoid false positive colonies, different bacterial selection markers were designed for plasmids at different levels (the 0-level plasmid carries AmpR; the 1-level plasmid carries KanR; the 2-level plasmid carries sdCmR). Gene elements from different sources (promoters, gene open reading frames, terminators) were assembled with the backbone vector through different 4-bp sticky ends and thus stably retained in the 0-level plasmid ( Figure 36 )。The GGA mixture was transformed into Escherichia coli Trans10 and the correct plasmids were identified by Sanger sequencing. Among them, the GOLDEN GATE system and conditions are shown in Table 2.

[0291] 3) Restriction enzyme digestion and ligation: This strategy was used to construct large molecular weight plasmids or plasmids that are incompatible with the GGA system. The general expression vector usually has a multiple cloning site (MCS), and appropriate restriction enzymes can be selected for linearization. Shorter insert fragments were amplified by PCR and the corresponding digestion sites were introduced. After product purification (Omega, D6293), the insert and the linearized general vector were ligated with T4 DNA ligase (New England Biolabs, M0202L) strictly according to the procedures provided by the manufacturer. The final ligation mixture was used for Escherichia coli transformation and the correctness of the clones was verified by sanger sequencing.

[0292] 2. Substrate-induced test culture

[0293] Single colonies were picked from the YPD plate into 500 μL of SD-deficient medium in a 2 mL-96 well plate (Axygen, P-2ML-SQ-C-S), sealed with a breathable membrane (BioTss, SF-200), and cultured at 800 r.p.m and 30 °C for 24 h. Then, 2.5 μL of the culture solution was inoculated into 497.5 μL of fresh SD medium containing gradient concentrations of arabinose (OD 600 = 0.005), cultured under the same conditions for 16 h, and then the culture was diluted 10-fold, that is, 20 μL of the culture was added to 180 μL of phosphate buffered saline (PBS, Proteintech, PR20014) in a 96-well U-bottom plate (corning, 3799), which contained 10 μg mL -1 cycloheximide (Maokangbio, MS0035), and then the mixture was analyzed by flow cytometry.

[0294] 3. Flow cytometry and data processing

[0295] The BD FACSCelestaTM flow cytometer was used, and the intensity of yellow fluorescent protein (yEmCitrine) in the treated samples was detected in the high-throughput sampler (HTS) mode (BD Biosciences, Germany). A 488 nm excitation laser (100 mW) and a 530 / 30 nm emission filter were selected to measure the YFP intensity. Data were recorded using FACSDiva software (BD Biosciences, Germany), and more than 10,000 events were recorded for each sample. Cells of the correct size were selected in the forward scatter area (FSC-A) / side scatter area (SSC-A) plot. The data were processed using software 10.4 (TreeStar, USA), and the median fluorescein isothiocyanate signal (FITC-A) was used for yellow fluorescence quantification and statistical analysis. The calculation formula for the induction fold is (YFP 诱导 -YFP 0 ) / (YFP 非诱导 -YFP 0 ), where YFP 诱导 and YFP 非诱导 represent the fluorescence measured in the presence and absence of 100 mM arabinose, respectively. YFP 0 represents the autofluorescence of white blood cells. The calculation formula for the inhibition fold is (YFP -抑制因子 -YFP 0 ) / (YFP +抑制因子 -YFP 0 ), where YFP -抑制因子 and YFP +抑制因子 represent the fluorescence measured in the absence and presence of the transcriptional repressor, respectively. The fluorescence intensity was normalized using the fluorescence intensity of the endogenous constitutive PFY1 promoter (YFP 参考 ), in units of RPU, and the calculation formula for RPU is (YFP - YFP 0 ) / (YFP 参考 -YFP 0 ). In the experiment, white blood cells, CY671int (P PFY1 -YFP) and CY676int (P TDH3 -YFP) were used as control strains and were cultured on the same plate as the test strains each time.

[0296] 4. Induction kinetics test culture

[0297] Pick a single colony on a YPD plate and transfer it to 500 μL of SD dropout medium in a 2 mL 96-well plate (Axygen, P-2ML-SQ-C-S). Seal it with a breathable membrane (BioTss, SF-200) and culture at 800 r.p.m. and 30 °C for 24 h. For GAL system test strains (GAL80Δ, GAL80Δ&GAL1Δ) under conditions without glucose repression, use SC medium (SCR medium) supplemented with 2% raffinose. Unless otherwise specified, 2% glucose is used as the carbon source. Then inoculate 2.5 μL of the culture into 475.5 μL of fresh SD medium supplemented with 100 mM arabinose in a 2 mL-96 well plate (OD 600 = 0.005). Subsequently, transfer 25 μL of these cultures to 475 μL of fresh SD or SCR medium, add the corresponding inducer in a 2 mL 96-well plate, and culture in an oscillating incubator (Zhichu, ZQZY-88AH) at 30 °C and 800 r.p.m. During the initial 3 h of growth, collect 50 μL of the culture at each specified time point, and then supplement with an equal volume of fresh medium supplemented with the corresponding inducer. After the initial 3 h of culture, during the 36 h culture period, collect 20 μL of the culture solution from each culture well every 3 h. The collected samples are diluted with 180 μL of PBS containing 10 μg mL -1 cycloheximide and then subjected to flow cytometry analysis.

[0298] 5. Growth toxicity test

[0299] Randomly select monoclonal colonies of three test strains and culture them overnight in 500 μL of SD dropout medium. Inoculate the culture at a ratio of 1:100 into SD medium containing a gradient concentration of inducer (without defective nutrient screening) and culture for 24 h. Take 20 μL of the culture for flow cytometry analysis to detect the correctness of the test strains and induction heterogeneity. At the same time, inoculate 5 μL of the culture in each culture well into the corresponding culture well of another plate with 495 μL of medium containing a gradient concentration of inducer, and measure the OD 600 of the culture in each culture well after 8 h of culture using a Tecan Infinite 200Pro plate reader (Tecan). At the same time, inoculate the culture in the uninduced well into SD medium supplemented with a gradient concentration of inducer and grow for another 24 h. Collect data continuously for three days, and finally use the OD 600 of the CENPK.2-1C strain cultured in SD medium on the first day to correct the measured data of the treated test strains for three consecutive days. The calculation formula is norm.OD 600 = ave(OD 600 ) 诱导 / OD600(2-1C) .

[0300] 6. Conditional knockout of ERG20

[0301] Since ERG20 is an essential gene, it cannot be directly deleted. We designed a strategy to turn off the expression of ERG20 using the arabinose induction system during the linalool production stage by inhibiting the protein PhlF. First, native ERG20 was amplified from CENPK.2-1C genomic DNA and expressed under the control of the synthetic promoter P phlo . This expression cassette (pGS121) was targeted to the leu2 locus of a strain containing a 4-sensor array of XylR-XlnR-AraR A -AraR R (see Figure 31 ). Second, the full length of ERG20 at the original locus was knocked out by the CRISPR / Cas9 system. After transforming the Cas9 plasmid (Addgene, 83946), a gRNA expression plasmid (Addgene, 43803) carrying a 20-bp sequence (i.e., ERG20ΔgRNA, shown in nucleotide sequence SEQ ID NO. 67) targeting the ERG20 promoter and repair DNA of 500-bp on each side of the ERG20 gene were co-transformed. Selection was carried out on an SMSG-Ura / G418 agar plate, and the correct clones were verified by clone PCR (2x Phanta Flash Master Mix (Vazyme, #P510), 35x (98℃ 10s, 60℃ 5s; 72℃ 20s)). The gRNA and Cas9 expression plasmids were lost by counter-selection with 5-FOA (1mg mL -1 ) and continuous YPD culture, respectively. Finally, an expression cassette of the inhibitory protein PhlF controlled by the combined promoter P lex.3-ara was integrated at the ura3 locus. Growth tests in media containing gradient concentrations of arabinose were performed to verify the successful shutdown of ERG20 gene expression by arabinose induction.

[0302] 7. Preparation of corncob hydrolysate

[0303] Corncob hydrolysate was obtained by a method combining high-temperature cooking, sulfuric acid treatment, and enzymatic hydrolysis. Specifically, 3 g of 20-mesh corncob powder (Lianyungang, Jiangsu) was added to 50 mL of 2% H 2 SO 4Then, the mixture was autoclaved at 121 °C for 45 min (Zealway, GR110DP). The autoclaved product was centrifuged at 4,000 rpm for 5 min, and the supernatant was taken out. The pH value was adjusted to 5.0 with calcium carbonate (CaCO 3 , Sigma-Aldrich, 398101) because the optimal activities of cellulase (Solarbio, C8270) and hemicellulase (Solarbio, H8110) were achieved at pH 5.0. Corncob residue was hydrolyzed with cellulase (1,800 U) and hemicellulase (1,500 U) in a 50 mL Falcon tube and then incubated overnight at 37 °C and 220 r.p.m. in an oscillator. After centrifugation at 4,000 rpm for 5 min, the supernatant was collected. Milli-Q water was added to 50 mL and then filtered through a 0.22 μm syringe filter (Millipore, SLGP033RB). Subsequently, the hydrolysate was stored at 4 °C for further analysis. For linalool fermentation, the hydrolysate was further concentrated to 10 mL by incubating the Falcon tube at 65 °C overnight.

[0304] 8. Construction of linalool yeast engineering strains and GC-FID determination

[0305] The construction process of the linalool synthesis pathway is as follows: In Saccharomyces cerevisiae integrated with the XylR-XlnR-AraR A -AraR R array, the xylose dual-regulation induction system was used to overexpress the four pathway genes IDI1, HMG1, ERG20, and McLIS to produce linalool (see Figure 34 ). The isopentenyl diphosphate δ-isomerase IDI1 gene (YPL117C) was amplified from the genomic DNA of CENPK.2-1C and tHMGR obtained by truncating the HMG-CoA reductase gene (YML075C) at the 554th amino acid. The mutant gene ERG20 F96W-N127W encoding farnesyl diphosphate synthase and truncated citronellal linalool synthase (t67McLIS) were synthesized by the company (Tsingke Biotechnology Co., Ltd.) after codon optimization. The construction carrying the linalool metabolic pathway genes was assembled by the hierarchical GGA strategy and targeted to the ho locus. The obtained plasmid was linearized by digestion with BsaI, and after column purification (Omega, D6293) of the digestion mixture, the product was transformed into the ERG20 conditional control strain obtained above. The transformants were screened on SD-Ura / -Leu / -His plates. The sequences of the pathway genes are shown in Table 1.

[0306] The production of linalool adopted a two-phase shake-flask fermentation strategy. A single colony of recombinant yeast was inoculated into 5 mL of YPD medium and cultured overnight at 30 °C and 220 r.p.m. Then the pre-culture was inoculated into 10 mL of YPD medium containing 100 mM xylose, with an initial OD 600 of 0.05. To reduce product volatilization, 2 mL of isopropyl myristate (IPM, Macklin, I811858) was added to the medium. Subsequently, the culture was grown in a shaker at 30 °C and 220 r.p.m. After 12 h of culture, 40 mM arabinose was added to turn off the expression of ERG20. In addition, in the control group, neither xylose nor arabinose ("- / -") or one of them ("- / +" or "+ / -") was added. The linalool in the IPM phase was collected and filtered through a 0.22 μm syringe filter (Haire, HR-YJZTLQ13). 100 μL of the filtered product was transferred to a gas chromatography vial (Agilent, HBDY3100), the vial cap (Agilent, 5182-0717) was put on, and it was immediately stored in a freezer at -80 °C.

[0307] The gas chromatography-flame ionization detection (GC-FID) method was as follows: The gas chromatograph used for detecting linalool was equipped with an HP-5 chromatographic column (5%-phenyl-methyl polysiloxane non-polar chromatographic column; 30 m × 0.32 mm × 0.25 μm, Agilent, 19091S-433) and a hydrogen flame ionization detector. The temperatures of the injector and the detector were both set at 280 °C, and 1 μL of sample was injected in splitless mode. The program used was as follows: The oven temperature was maintained at 80 °C for 2 min, then increased to 110 °C at a rate of 10 °C / min, then increased to 250 °C at a rate of 40 °C / min, and finally maintained at 250 °C for 3 min. The carrier gas was helium, and the flow rate was 1 mL / min. The solvent delay time was set at 5 min. The peak area was calculated using MSD ChemStation (Agilent). A linear calibration curve was plotted using linalool standard (Shanghai Yuanye Bio-Technology Co., Ltd., R3101S) diluted with ethyl acetate. Standard samples with different linalool concentrations (0, 25, 50, 100, 250, 400, and 500 mg l -1 ) were prepared and measured according to the above method.

[0308] Example 2-1: Prototype Substrate Response Test of Arabinose-Inducible Transcription Systems from Different Sources in Saccharomyces cerevisiae

[0309] Arabinose-responsive transcriptional activator AraR A (nucleotide sequence as shown in SEQ ID NO.81) from different eukaryotic species and its synthetic transcription factor LexA-AraR A(The amino acid sequence is as shown in SEQ ID NO. 57), CI434-AraR A and seven response transcriptional repressors AraR from different prokaryotic species R Through the GOLDEN-GATE system reaction, it was assembled with the backbone plasmid pGD137 (see Table 1) to obtain the target plasmid, that is, a transcription factor expression cassette controlled by the aTc-TetR system; while the natural or synthetic promoter fragments responding to these transcription factors were obtained through the GOLDEN-GATE system reaction with the backbone plasmid pGS001 (see Table 1) to obtain the fluorescent gene reporter plasmid, that is, a yellow fluorescent reporter protein gene expression cassette controlled by the output promoter. After the obtained reporter plasmid was digested with enzymes, it was transferred into a yeast strain already carrying the arabinose transcription factor, and the activation / inhibition multiples were tested under the conditions of arabinose induction and non-induction as described in the method section. The results are as Figure 37 shown. AraR in Aspergillus niger A showed an activation multiple of more than 8.4 times, while AraR of Thermotoga sp. RQ2 R had the largest inhibition multiple of 21.1, so both were used for further optimization in the follow-up.

[0310] Example 2-2: Construction based on AraR A Synthetic transcription factor construction

[0311] Since the AraR A binding consensus sequence is not clear, the possible AraR binding motifs proposed in the reference (K. Ishikawa, E. Kunitake, T. Kawase, M. Atsumi, Y. Noguchi, S. Ishikawa, M. Ogawa, Y. Koyama, M. Kimura, K. Kanamaru, M. Kato, T. Kobayashi, Curr Genet 2018, 64(6), 1245, https: / / doi.org / 10.1007 / s00294-018-0837-5;) were introduced into the promoter with ADH2 as the chassis for testing, but all showed constitutive output and could not achieve inducible output. Therefore, in order to exclude the inability to respond to arabinose caused by incorrect reference of the DBD binding motif, the present invention first fused and expressed the full-length AraR A with the well-characterized LexA DBD (LexA1-87) from bacteria through linker3x (ggggs). The output promoter encodes the binding sequence lexo of LexA DBD (see Table 1). The test results of the inducible system designed in this way are shown in ( A Figure 37 ​), and the results showed that arabinose induction achieved an 8.4-fold output. Since there are two DBDs, the natural DBD may interfere with the function of the LexA DBD. Therefore, combined with the structural prediction of Alphafold2, it was designed to delete the natural AraR DBD (1-67), and the components in other systems remained unchanged. The test results showed that the arabinose induction level was improved, from 1.03 RPU before deletion to 3.07 RPU. However, because the background level also increased after deleting the natural DBD, the dynamic range itself changed little ( Figure 38 a).

[0312] In addition, the concentration of the transcriptional activator also determines the maximum output ability. To further improve the induction ability, the concentration of AraR A corresponding to the system output was tested (as shown in Figure 38 b), and based on this, a suitable constitutive promoter was selected to ensure that AraR A had stable expression throughout the yeast stage. Three constitutive promoters with different activities - P TDH3 , P PGK1 and P ACT1 were initially selected. The results showed that the activity of the selected promoter was positively correlated with the final output. When the strong promoter TDH3 promoter was used to express the synthetic transcription factor, the output promoter P lex.3 had the maximum output ( Figure 38 c), and the dynamic range also reached 64.4-fold.

[0313] Example 2-3: Design and optimization of the synthetic promoter responsive to AraR A Similar to the xylose induction system, we introduced the well-defined inducible ADH2 natural promoter as the chassis for modification, and replaced the UAS region sequence originally bound by ADR1 with lexo (as shown in

[0314] ), and obtained the semi-synthetic promoter P Figure 29 series (see Table 1). Appropriately increasing the binding sites of transcription factors is a reliable strategy to improve the transcriptional activation level. By testing the promoters containing 1, 2, 3, and 4 lexo (nucleotide sequences are shown in SEQ ID NO.60 to SEQ ID NO.63), the results (as shown in lex b) showed that the promoter containing 2 lexo had significantly increased the activation level. When the promoter encoded 1-3 lexo, the induction activation level was further enhanced with the increase in the number of motifs, and its leakage level decreased with the increase in the number of lexo. When encoding 4 lexo, due to the elevation of the background leakage level, the induction kinetics of the system was lower than that when the output promoter encoded 3 lexo. Figure 29 b) indicated that the promoter containing 2 lexo had significantly increased the activation level. When the promoter encoded 1-3 lexo, the induction activation level was further enhanced with the increase in the number of motifs, and its leakage level decreased with the increase in the number of lexo. When encoding 4 lexo, due to the elevation of the background leakage level, the induction kinetics of the system was lower than that when the output promoter encoded 3 lexo.

[0315] Examples 2-4: Substrate response curves, induction heterogeneity, kinetic behavior determination and fitting of the arabinose single-regulation induction system

[0316] For the optimized P lex.3 -AraR A system and P ara -AraR R performance was characterized. The obtained transformed strains were induced with 16 different concentrations of arabinose, and the output signals (methods) were analyzed by flow cytometry to plot the substrate concentration induction curves, which were then fitted with the Hill function (as Figure 39 a) The Hill parameters and EC50 values were obtained (see Table 6). The results showed that compared with P ara , P lex3 had a leakage level x orders of magnitude higher. In addition, according to the specific description in the method section, the kinetic behavior under the induction of 100 mM arabinose concentration that could saturate the activation of the induction system was determined and fitted, and the results were as Figure 39 shown in b, and the fitting parameters are shown in Table 7. The results showed that the observed kinetic data could be fitted by the standard transcription-translation models, namely Equations 2 and 3. Finally, the heterogeneity of the yeast population at different arabinose concentrations was observed and analyzed, and the results were as Figure 40 shown in a. Under the induction of arabinose concentrations in 5 different orders of magnitude, a single peak constantly appeared, and there was no population heterogeneity. In addition, a single induction peak was also observed during the induction period of up to 33 h, and no induced transcriptional population heterogeneity occurred( Figure 40 b).

[0317] Table 6: Fitting parameters of the arabinose substrate response curve

[0318]

[0319] Table 7: Fitting parameters of arabinose induction kinetics

[0320]

[0321] Examples 2-5: Design and construction of the arabinose dual-regulation induction system, substrate response curve, kinetic behavior determination and fitting

[0322] In view of the high leakage of the P lex.3 -AraR A system and the low activation ability of the P ara -AraR R system, a dual-transcription factor regulation system was adopted to integrate the advantages of both to further optimize the arabinose transcriptional regulation system. Referring to the first part: the design scheme of the xylose dual-regulation system, topological structure 1 was adopted( Figure 41a) Perform further construction and performance characterization.

[0323] To construct the topology 1 of the dual transcription factor, only follow the Figure 41 architecture in a), i.e., sequence P lex.3-ara (the nucleotide sequence is shown as SEQ ID NO.63) to construct the promoter sequence, and react the sequence P lex.3-ara fragment with the backbone plasmid pXJH1GOLDEN-GATE in one step to obtain the target reporter plasmid, and then transform the existing transcription factor array (AraR A -AraR R ) strain (see Figure 31 a). Then, the strain containing this dual regulatory system is induced with 16 different concentrations of xylose and measured by flow cytometry to plot the substrate response curve. Similarly, the measurement data can be well fitted by the Hill function ( Figure 41 b), and the EC50 is 52.1 mM. Assuming that the binding of AraR A to AraR R and the promoter P lex.3-ara is an independent event, the steady-state induction of this promoter can be predicted by the partition function, i.e., formula 1. The actual prediction results are as shown in Figure 41 b. The substrate induction behavior of this dual regulation can be well predicted by the fitting parameters of the single regulation.

[0324] Among them, formula 1 is as follows:

[0325]

[0326] Among them, y min is the output of the induction system without the inducer, y max is the output of the induction system when it reaches the steady state after adding the inducer, x is the inducer concentration, k 1 , k 2 , n 1 , and n 2 are the Hill parameters of the arabinose single-regulation induction system based on the transcription activator AraR A and the transcription repressor AraR R , respectively.

[0327] The induction behavior of the dual-regulation arabinose induction system is not affected by glucose, and the induction level reaches 50% of the maximum activation 5.5 h after adding arabinose induction. Similarly, the kinetic test data of the dual-regulation arabinose induction system can be fitted by the standard transcription and translation models, i.e., formulas 2 and 3 (see Figure 41 c).

[0328] Among them, formula 2 is as follows:

[0329] Formula 3 is as follows:

[0330] The meanings of the letters in Formula 2 and Formula 3 are as follows: m and p are the concentrations of mRNA and protein respectively; β m , β p are the constants describing the synthesis rates of mRNA and protein respectively; γ m , γ p are the constants describing the degradation and dilution rates of mRNA and protein respectively.

[0331] Examples 2-6: Multidimensional Comparison between Arabinose Induction System and Existing Induction Systems in Saccharomyces cerevisiae

[0332] To better evaluate the single-regulation and dual-regulation arabinose induction systems, the present invention reconstructed the endogenous induction system-P GAL1 , P MET3 , P CUP1 and the synthetic induction system (LexA-ER-VP16 / B112) based on the ER system in Saccharomyces cerevisiae. The present invention made a comprehensive comparison with the developed xylose induction system in terms of five aspects: regulation rigor, induction activation level, dynamic range, growth toxicity, and induction speed.

[0333] To determine the maximum activation level, the present invention set a test criterion: while reaching the maximum activation level, there should be no fluorescence heterogeneity in the population, and the growth of the host should not be severely inhibited (see Figure 42 ). For this purpose, the outputs and fluorescence distributions of each system at different inducer concentrations were tested and examined, and thus the inducer concentration reaching the maximum activation level was determined. At this concentration, its induction output level( Figure 43 a) was measured. According to the output level without adding any inducer, the dynamic range of each system was calculated, and the growth OD 600 of the strain was tested by continuous subculture for three days, taking the original transformed strain CENPK.2-1C as a reference to evaluate the growth status of the strain under induction( Figure 43 b). Finally, under the induction of this concentration, the induction kinetic behaviors of each strain within 36 h were tested. The test results are as shown in Figure 43 c, and the results show that the dual-regulation arabinose system is superior to P MET3 , P CUP1 , LexA-ER-VP16 / B112 system in four aspects: leakage level, dynamic range, impact on host growth toxicity, and induction speed, and is comparable to P GAL1 of the GAL system(see Figure 43 d).

[0334] Example 2-7: Verification of the Practicality of the Arabinose Induction System in Metabolic Engineering - High-Titer Linalool Production

[0335] To test the practicality of the xylose and arabinose induction systems in metabolic engineering, we applied them to the dynamic control of linalool production. In Saccharomyces cerevisiae, the linalool titer is low mainly because of the insufficient supply of monoterpene synthase (LIS) and geranyl diphosphate (GPP). Conditional knockout of the essential gene ERG20 by inhibiting the competitive ergosterol biosynthesis pathway is beneficial for linalool biosynthesis. Providing sufficient pathway enzymes and adjusting metabolic flux are crucial for increasing the linalool titer. In Saccharomyces cerevisiae integrated with the XyIR-XInR-AraR A -AraR R array, the xylose dual-mode induction promoter P xln.2b-xyl (base sequence as shown in SEQ ID NO.17) was used to control the linalool synthesis pathway genes (see Figure 34 ), including tHMG1, IDI1, the mutant GPP synthase gene ERG20 F96W / N127W (ERG20ww) and the linalool synthase gene (t67McLIS), while the arabinose induction system used the repressor PhlF to control native ERG20 because the synthetic P phlo showed an expression level comparable to that of native ERG20.

[0336] We monitored the expression of the pathway genes and native ERG20 in the reporter strains controlling yEmCitrine by P xln.2b-xyl and P phlo respectively. The outputs of the two systems were dose-related, and the genes controlled by the xylose composite promoter were induced by xylose ( Figure 44 a), while the genes controlled by the synthetic promoter P phlo were repressed by arabinose ( Figure 44 b). When the concentration of arabinose exceeded 20 mM, the output of P phlo was completely repressed, which was consistent with the inhibition of the growth of the linalool-producing strain with the increase in arabinose concentration ( Figure 44 c). This indicates that the arabinose dual-mode induction system can achieve strict control of ERG20 expression through a NOT gate.

[0337] We adopted several strategies to increase the linalool yield, including expressing the pathway genes in the early growth stage, delaying the inhibition of the sterol biosynthesis pathway by 12 h before inducing the linalool pathway ( Figure 44 d), and maintaining the expression ratio of ERG20WW to t67McLIS at 2:1 according to the design regulations of RIDD-RIAD. These methods increased the linalool titer to more than 100 mg L-1 ( Figure 44 d). If xylose and arabinose are not introduced simultaneously, linalool cannot be detected or the detected linalool titer is very low. Figure 44 f&g).

[0338] Example 2 - 8: Cross - reactivity test of arabinose induction system to D - xylose

[0339] Given the highly similar chemical structures of xylose and arabinose, it is speculated that the arabinose induction system may have cross - reactivity to xylose. We quantified this cross - reactivity by measuring the response function of the arabinose system at 16 different xylose gradients. The results showed that even in the presence of 100 mM xylose,

[0340] Based on AraR R - P ara the single - regulated arabinose induction system had an output of only 0.08 RPU. Figure 45 a), while based on AraR A - P lex.3 the single - regulated arabinose induction system had an output of only 0.34 RPU. Figure 45 b), and the double - regulated arabinose induction system showed little response to high - concentration xylose, with an output of only 0.04 RPU. Figure 45 c). These data indicate that the arabinose induction system developed in this invention has strong inducer specificity.

[0341] Example 2 - 9: Sustainable expression test of arabinose induction system in long - term fermentation

[0342] In Saccharomyces cerevisiae, as a non - metabolizable inducer, arabinose may support the sustainable expression of the induction system. To verify this hypothesis, the reaction kinetics of the single - regulated and double - regulated arabinose induction systems for 4 days were measured. A fluorescence reporter gene expression cassette integrated with the strong promoter TDH3 promoter was introduced, and the designated strains were inoculated into YPD medium in a 10 mL Erlenmeyer flask. Specified amounts of xylose and arabinose were introduced at the start of growth. At 6 specified time points, 20 μL of the culture was diluted in 180 μL of PBS buffer for flow cytometry analysis. The results showed that all systems maintained stable expression throughout the fermentation test period. Even at the end of fermentation (96 h), the induced expression level did not show a significant decrease. Among them, the output level of the single - regulated arabinose induction system based on AraR A - P lex3 was 2 times that of P TDH3 . Figure 46 This indicates that the arabinose induction system is an economical and efficient tool for gene overexpression.

[0343] In summary, the arabinose induction system in the second part of this application fills the gap in the eukaryotic system without a synthetic arabinose induction system; a new, low-cost and high-performance transcriptional induction system has been developed in Saccharomyces cerevisiae: as a non-metabolic inducer and as one of the components of hemicellulose hydrolysates in crop waste, the price of arabinose is 1 / 3 of that of galactose, the commonly used inducer in Saccharomyces cerevisiae at present, and it does not need to be continuously added during the long-term fermentation process. The arabinose-responsive transcriptional activator AraR derived from the eukaryote Aspergillus niger is used A , such that the maximum activation level is comparable to the transcriptional level of the strongest TDH3 promoter in Saccharomyces cerevisiae. Compared with the slow de-repression process of the endogenous GAL system in Saccharomyces cerevisiae, its induction speed is rapid (it can reach 50% of the maximum activation level in 5.5 h of induction); on the basis of the AraR A system, the dual-regulation induction system combining the transcriptional activator AraR R from Thermotoga sp. RQ2 greatly reduces the leakage level, and finally the induction dynamic range exceeds 300-fold; it has very little growth toxicity to the host, a fast response speed, high specificity, is not inhibited by other carbon sources such as glucose, and realizes the decoupling of the growth stage and the induction stage; combined with the previously developed xylose induction system, it opens up a new and simple way for the recycling of agricultural waste to produce high-value compounds.

[0344] Table 1 Names and sequences of elements involved in each example

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as the scope described in this specification.

[0369] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An arabinose-responsive transcription factor assembly, characterized in that include: Transcription activator AraR A , the transcriptional activator AraR A Derived from eukaryotic organisms.

2. The arabinose-responsive transcription factor assembly according to claim 1, characterized in that The eukaryotic organism is selected from one of Aspegillus nidulans, Aspegillus niger and Meyerozyma guilliermondii; Furthermore, the transcription activator AraR A The amino acid sequence of the transcription activator AraR is shown in SEQ ID NO.56 A (An04g08600), or, the transcription activator AraR whose nucleotide sequence is shown in SEQ ID NO.69 A (AN0388).

3. The arabinose-responsive transcription factor assembly according to claim 1, characterized in that The transcriptional activator AraR A An exogenous DNA binding domain is connected, and the exogenous DNA binding domain is derived from bacterial TF LexA or the CI inhibitory factor of bacteriophage 434; Furthermore, the exogenous DNA binding domain binds to the transcription activator AraR A The connection is carried out through a connection sequence, and the connection sequence is 3x (ggggs); Furthermore, the transcription activator AraR A Does not contain a DNA binding domain.

4. The arabinose-responsive transcription factor assembly according to claim 3, characterized in that The transcription activator AraR connected with the exogenous DNA binding domain A The amino acid sequence of the synthetic transcription activator LexA-AraR is shown in SEQ ID NO.57 A Or the synthetic transcription activator CI434-AraR whose nucleotide sequence is shown in SEQ ID NO.68 A .

5. The arabinose-responsive transcription factor assembly according to any one of claims 1 to 4, characterized in that AraR R , the transcriptional repressor AraR R Originated from bacteria; Further, the bacteria is selected from one of Thermotoga sp.RQ2, Bacillus subtilis, Bacilluslicheniformis, Clostridium acetobutylicum, Shewanella sp.Sh95 and Bacillusamyloliquefaciens; Furthermore, the transcriptional repressor AraR R AraR selected from the group consisting of nucleotide sequences as SEQ ID NO.70 to SEQ ID NO.76 R -One of the NLS.

6. The arabinose-responsive transcription factor assembly according to claim 5, characterized in that Also included is the transcriptional repressor AraR R The binding site sequence of the ligation; Furthermore, the transcriptional repressor AraR R The amino acid sequence of the transcription inhibition component composed of its binding site sequence is shown in SEQ ID NO.

58.

7. A promoter component, characterized in that The promoter component can be combined with the arabinose response transcription factor component according to any one of claims 1 to 4, wherein the promoter component comprises: a first promoter, wherein the first promoter can be combined with the transcription activator AraR A Combine.

8. The promoter component according to claim 7, characterized in that The first promoter is a constitutive promoter-P TDH3 , constitutive promoter P PGK1 , constitutive promoter P ACT1 , promoter P ANladR , or promoter P ANlxrA ; Furthermore, the constitutive promoter P ACT1 The nucleotide sequence is shown in SEQ ID NO.

26.

9. The promoter assembly according to claim 7, characterized in that The first promoter is obtained by modifying the ADH2 promoter from Saccharomyces cerevisiae as a chassis promoter; Furthermore, the modification includes modifying at least one of the UAS region sequence of the chassis promoter, the spacer sequence between the TATA-box and the nucleosome missing region, the spacer sequence between the TATA-box and the transcription start site, and the transcription factor binding site motif.

10. The promoter assembly according to claim 9, characterized in that The first promoter is mainly obtained by the following steps: replacing the UAS region sequence of the ADH2 promoter from Saccharomyces cerevisiae with the transcription activator AraR A The binding site of lexo; Furthermore, the number of inserted binding sites xlno is 1-4; Furthermore, the first promoter is selected from at least one of the promoters whose base sequences are shown in SEQ ID NO.60 to SEQ ID NO.

63.

11. The promoter assembly according to any one of claims 7 to 10, characterized in that Also includes a second promoter, which can bind to the transcriptional repressor AraR R Combine; Furthermore, the second promoter is obtained by modification using the minimal promoter as the chassis; Furthermore, the second promoter is mainly obtained by the following steps: inserting the transcription inhibitor AraR into the minimal promoter R The binding sequence of the transcriptional repressor AraR R The binding sequence is located downstream of the minimal promoter TATA-box and upstream of the transcription initiation site; Furthermore, the base sequence of the second promoter is shown in SEQ ID NO.

59.

12. A dual-regulatory promoter, characterized in that: The dual-regulated promoter can interact with the transcriptional activator AraR A Binds to the transcriptional repressor AraR R Binds to the transcriptional activator AraR A The transcriptional repressor AraR is derived from eukaryotic organisms. R Derived from bacteria.

13. The dual-regulatory promoter according to claim 12, characterized in that: In the dual-regulatory promoter, the sequence upstream of the TATA-box can interact with the transcriptional activator AraR A Binding, the sequence downstream of the TATA-box can bind to the transcriptional repressor AraR R Combine; Furthermore, the base sequence of the dual-regulated promoter is shown in SEQ ID NO.

64.

14. A transcription factor expression plasmid, characterized in that: The transcription factor expression plasmid is a backbone plasmid into which the arabinose-responsive transcription factor component according to any one of claims 1 to 6 is inserted.

15. The transcription factor expression plasmid according to claim 14, characterized in that: The transcription factor expression plasmid is also inserted with the transcription activator AraR A and the transcriptional repressor AraR R ; Further, the backbone plasmid is a pGS077 plasmid having a nucleotide sequence as shown in SEQ ID NO.65; Alternatively, the transcription factor expression plasmid comprises a first expression plasmid and a second expression plasmid, wherein the first expression plasmid is a plasmid into which the transcription activator AraR A The second expression plasmid is a backbone plasmid into which the transcription inhibitor AraR R backbone plasmid; further, the backbone plasmid is pGS001 plasmid.

16. The transcription factor expression plasmid according to claim 14, characterized in that: The transcription factor expression plasmid is also inserted with the transcription activator AraR A , the transcriptional repressor AraR R The expression backbone plasmid, xylose transcription activator XlnR and xylose transcription repressor XylR; Furthermore, the xylose transcription activator XlnR is derived from filamentous fungi, and the xylose transcription repressor XylR is derived from bacteria; Furthermore, the backbone plasmid is a pGS078 plasmid having a nucleotide sequence as shown in SEQ ID NO.

66.

17. A promoter-reporter gene plasmid, characterized in that: The promoter-reporter gene plasmid is a reporter gene backbone plasmid with an inducible promoter inserted therein, and the inducible promoter is selected from any one of the promoter components described in claims 7-11 and the dual-regulated promoters described in claims 12-13.

18. A recombinant bacterium, characterized in that: The recombinant bacteria is Saccharomyces cerevisiae carrying the transcription factor expression plasmid according to any one of claims 14 to 16.

19. The recombinant bacterium according to claim 18, characterized in that The recombinant bacteria also carry a promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with an inducible promoter inserted therein, and the inducible promoter is selected from a promoter that can bind to the transcription activator AraR A The first promoter binds to the transcriptional repressor AraR R At least one of the combined second promoters, the first promoter and / or the second promoter are integrated into the ura3 genomic site of the Saccharomyces cerevisiae.

20. The recombinant bacterium according to claim 18, characterized in that The transcription factor expression plasmid is integrated into the NRT1 gene spacer of the Saccharomyces cerevisiae; the transcription activator AraR is also inserted into the transcription factor expression plasmid A and the transcriptional repressor AraR R .

21. The recombinant bacterium according to claim 20, characterized in that The recombinant bacteria also carry a promoter-reporter gene plasmid, which is a reporter gene skeleton plasmid with a double-regulated promoter inserted therein. The double-regulated promoter can interact with the transcription activator AraR A Binds to the transcriptional repressor AraR R Binds to the transcriptional activator AraR A The transcriptional repressor AraR is derived from eukaryotic organisms. R Derived from bacteria.

22. The recombinant bacterium according to claim 21, characterized in that The kinetic model formula 1 of the substrate response curve of the recombinant bacteria is as follows: Among them, y min is the output of the induction system when no inducer is added, y max is the output when the induction system reaches steady state after the addition of inducer, x is the inducer concentration, k1, k2, n1, and n2 are based on the transcription activator AraR A , the transcriptional repressor AraR R Hill parameters of the arabinose-only induction system.

23. The recombinant bacterium according to claim 18, characterized in that The transcription factor expression plasmid is integrated into the NRT1 gene spacer of the Saccharomyces cerevisiae; the transcription activator AraR is also inserted into the transcription factor expression plasmid A , the transcriptional repressor AraR R The invention discloses an expression backbone plasmid, a xylose transcription activator XlnR and a xylose transcription repressor XylR, wherein the xylose transcription activator XlnR is derived from filamentous fungi, and the xylose transcription repressor XylR is derived from bacteria.

24. The recombinant bacterium according to claim 23, characterized in that The recombinant bacteria also carry a first promoter-reporter gene plasmid and a second promoter-reporter gene plasmid; The first promoter-reporter gene plasmid is inserted with a first dual-regulated promoter, which can interact with the transcription activator AraR A Binds to and is able to bind to the transcriptional repressor AraR R Combine; The second promoter-reporter gene plasmid is inserted with a second dual-regulated promoter, and the second dual-regulated promoter can bind to the xylose transcription activator XlnR and can bind to the xylose transcription repressor XylR.

25. The recombinant bacterium according to any one of claims 18 to 24, characterized in that: The induction substrate of the recombinant bacteria includes corn cob hydrolyzate, and the corn cob hydrolyzate is mainly prepared by the following steps: 3g of 20-mesh corn cob powder is treated with a 2% H2SO4 solution at 120°C for 45 minutes, and then calcium carbonate is added to adjust the pH value to 5.0, and then 1,800U of cellulase and 1,500U of hemicellulase are added for hydrolysis, and then solid-liquid separation is performed, and the supernatant is collected to obtain the corn cob hydrolyzate.

26. Use of the recombinant bacterium according to any one of claims 18 to 25 in the preparation of linalool.

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