Promoter elements and their use in 3-hydroxypropionic acid biosynthesis

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a promoter element and its application in 3-hydroxypropionic acid biosynthesis. Specifically, the present application performs transcriptome analysis on a wild strain of Yarrowia lipolytica under different culture conditions and growth stages, and successfully screens a series of growth-regulated promoters and gradient-intensity promoters. Meanwhile, the screened promoters are applied to modular construction of a 3-hydroxypropionic acid biosynthesis pathway in Yarrowia lipolytica, so as to optimize the metabolic pathway and biological process of Yarrowia lipolytica. In addition, potential new upstream activating sequences (UAS) are mined by fine truncation of the promoters, so as to provide more choices for construction of an artificially synthesized hybrid promoter. In summary, the present application enriches the promoter element library of Yarrowia lipolytica, realizes accurate regulation of gene expression level from multiple angles, and provides a reference for dynamic regulation of metabolic networks and construction of an efficient cell factory.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a promoter element and its application in 3-hydroxypropionic acid biosynthesis. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Yarrowia lipolytica is an unconventional yeast with unique physiological and metabolic advantages, and is a potential eukaryotic microbial cell factory. The development and application of synthetic biology elements greatly improve the flexibility of cell factory construction.

[0004] Among them, the promoter element is the basis of gene expression regulation in synthetic biology and metabolic engineering. However, there is currently a lack of systematic research on natural promoters in Yarrowia lipolytica, and the types of promoters available for selection are very limited, especially there is no report on universal dynamic regulation promoters. Therefore, it is urgent to solve the problem of deep mining of promoter elements in Yarrowia lipolytica and expanding the synthetic biology toolkit. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application aims to provide a promoter element and its application in 3-hydroxypropionic acid biosynthesis. Specifically, the present application performs transcriptome analysis on Yarrowia lipolytica wild strain Po1f under different culture conditions and growth stages, selects different natural promoters as candidates according to the gene expression intensity and trend on the transcription level, and successfully screens a series of growth-regulated promoters and gradient-intensity promoters. At the same time, the screened promoters are applied to the modular construction of 3-hydroxypropionic acid (3-HP) biosynthesis pathway in Yarrowia lipolytica, optimize the metabolic pathway and biological process of Yarrowia lipolytica, and obtain a Yarrowia lipolytica strain for high-efficiency synthesis of 3-HP by using glucose. Based on the above research results, the present application is completed.

[0006] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0007] In a first aspect of the present application, a promoter element is provided, which is as shown in any one of (a)-(b) as follows:

[0008] (a): the promoter shown in the following table:

[0009]

[0010]

[0011]

[0012] (b) based on the promoter shown in (a), the nucleotide sequence of the promoter is subjected to substitution and / or deletion and / or addition of one or more nucleotides;

[0013] The "multiple" can be not more than 400, such as adding (extending) 400bp or 200bp at the 5' end of the promoter, or deleting (shortening) 400bp, 200bp, 100bp, 50bp, 25bp, etc. at the 5' end of the promoter, which is not specifically limited here.

[0014] In a second aspect of the present application, the above-mentioned promoter element is applied in the biosynthesis of 3-hydroxypropionic acid.

[0015] Specifically, the present application takes Yarrowia lipolytica Po1f as the starting strain, introduces and optimizes malonyl-CoA reductase MCR from C.aurantiacus, balances the gene expression amount of MCR-C and MCR-N by constructing different strength promoter combinations, dynamically down-regulates FAS1 gene to weaken the consumption of 3-HP direct precursors by the competitive pathway, and enhances the expression of precursor supply genes. A new strategy of dynamic and balanced regulation successfully constructs an engineering strain for high-efficiency production of 3-HP with glucose as carbon source. Finally, the strain is used for batch feeding fermentation experiment in a 5-L fermenter. In the batch feeding fermentation, the 3-HP yield, conversion rate and production rate reach 100.37g / L, 0.21g / g and 0.48g / L / h, respectively. This is the highest yield reported so far for the fermentation production of 3-HP with glucose as carbon source. Thus, the above-mentioned different types of promoter elements have potential application in metabolic engineering of Yarrowia lipolytica.

[0016] Therefore, in a third aspect of the present application, a high-yield 3-hydroxypropionic acid engineering strain is provided, which takes Yarrowia lipolytica as the chassis strain, expresses malonyl-CoA reductase from C.aurantiacus, balances the gene expression amount of MCR-C and MCR-N by constructing different strength promoter combinations, dynamically down-regulates FAS1 gene by dynamic down-regulation type promoter, and promotes the expression of precursor supply genes based on strong promoter.

[0017] The chassis strain is Yarrowia lipolytica Po1f.

[0018] The expression of the C. aurantiacus-derived malonyl-CoA reductase MCR, and the specific method for balancing the gene expression of MCR-C and MCR-N by combining different strength promoters includes: splitting the MCR into MCR-N (the first-549 amino acid residue part) and MCR-C (the 550-1219 amino acid residue part), using the above-mentioned promoters to regulate the MCR-C and MCR-N genes, constructing an integrated expression vector, and integrating it into the Yarrowia lipolytica genome.

[0019] Further, the malonyl-CoA reductase MCR is mutated to improve the enzyme activity of MCR-C, and the mutation sites are N940V, K1106W and S1114R.

[0020] Further, the MCR-C is regulated by the PC48 promoter; the MCR-N gene is regulated by any one of the PC12 (SEQ ID NO. 9), PC37 (SEQ ID NO. 10), PU01 (SEQ ID NO. 1), PU06 (SEQ ID NO. 3), PU04 (SEQ ID NO. 2) promoters, and the preferred promoter is PC37.

[0021] Further, the FAS1 gene is dynamically down-regulated by a dynamic down-regulation promoter, and the dynamic down-regulation promoter includes PD19 (SEQ ID NO. 6), PD20 (SEQ ID NO. 7) and PD21 (SEQ ID NO. 8), and the preferred one is PD19.

[0022] Further, based on the strong promoter promoting the expression of the precursor supply gene, the strong promoter includes any one of PU12 (SEQ ID NO. 4), PU13 (SEQ ID NO. 5) and PC48 (SEQ ID NO. 11).

[0023] The precursor supply gene includes any one or more of the ACL1, ACL2, ACC1, PDC, ALD and ACS genes;

[0024] More specifically, the use of PU13 to promote the expression of the precursor supply gene ACC1.

[0025] The fourth aspect of the present application provides a method for biosynthesizing 3-hydroxypropionic acid, which comprises: fermenting and culturing the above-mentioned 3-hydroxypropionic acid high-yield engineering bacteria.

[0026] Further, the fermentation culture mode can be fed-batch fermentation, and the specific fermentation conditions are as follows: the culture medium is inorganic salt medium, the initial concentration of glucose is 40 g / L, the pH of the fermentation broth is controlled at 6.0, the stirring speed is 200-700 rpm, the aeration amount is 1 vvm, and when the concentration of glucose in the culture medium is lower than 10 g / L, a proper amount of sterilized 70% glucose mother liquor is timely supplemented.

[0027] The above one or more technical solutions have the following beneficial technical effects:

[0028] (1) The above technical solution obtains a series of natural growth-regulated promoters and gradient-intensity promoters of Yarrowia lipolytica based on transcriptome data analysis combined with fluorescence characterization results. The growth-regulated promoters realize dynamic up-regulation or down-regulation of gene expression with time, which is the first time to obtain a universal growth-regulated promoter in Yarrowia lipolytica. The gradient-intensity promoters expand the range of controllable gene expression, and the expression intensity of three promoters (PU12, PC48, and PU13) is 1.10 times, 1.48 times, and 1.69 times that of the currently reported strong promoter P TEFin The above growth-regulated promoters and new strong promoters provide new universal elements for synthetic biology and metabolic engineering.

[0029] (2) The above technical solution uses the screened new promoter combination to construct a 3-HP biosynthesis pathway, and uses the modular regulation strategy of dynamic down-regulation promoters to regulate the competitive pathway and strong promoters to regulate the precursor supply module to optimize the metabolic pathway and biological process of Yarrowia lipolytica, and obtains a Yarrowia lipolytica strain for efficiently synthesizing 3-HP from glucose, with a yield of 100.37 g / L, which is the highest yield reported so far for the fermentation production of 3-HP from glucose as the carbon source, and lays a good foundation for improving the industrial production of 3-HP by microorganisms.

[0030] (3) By fine truncation of the promoters, potential new upstream activation sequences (UAS) are mined, providing more choices for constructing artificial synthetic hybrid promoters. DETAILED DESCRIPTION

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0032] Figure 1 The schematic diagram for promoter screening in Embodiment 1 of the present application is shown in the following figure:

[0033] Figure 2 Fluorescence characterization results of the growth-regulating promoter in Example 1 of the present application-YPD medium; (a) dynamic up-regulation; (b) dynamic down-regulation;

[0034] Figure 3 Fluorescence characterization results of the gradient strength promoter in Example 1 of the present application-YPD medium-48h;

[0035] Figure 4 Fluorescence characterization results and OD of the strong promoter under shake flask culture conditions in Example 1 of the present application 600 ; (a) fluorescence characterization results of the strong promoter; (b) OD of the strong promoter 600 ;

[0036] Figure 5 Fluorescence characterization results of the potential upstream activation sequence PC48-A1 in the form of 1-5 copies of tandem fusion upstream of the Leu2 minimal core promoter (mLeu2) in Example 1 of the present application;

[0037] Figure 6 Fluorescence characterization results of the potential upstream activation sequence PU13-A1 in the form of 1-5 copies of tandem fusion upstream of the Leu2 minimal core promoter (mLeu2) in Example 1 of the present application;

[0038] Figure 7 Schematic diagram of the strategy for producing 3-HP by metabolic engineering of Yarrowia lipolytica in Example 2 of the present application;

[0039] Figure 8 The promoter strength of MCR-C* is less than that of MCR-N in Example 2 of the present application. (a) Possible catalytic reaction mechanism of MCR. (b) 3-HP shake flask yield of strains containing different strength promoter combinations when the gene expression amount presents MCR-C* < MCR-N;

[0040] Figure 9 The promoter strength of MCR-C* is greater than that of MCR-N in Example 2 of the present application. (a) Possible catalytic reaction mechanism of MCR. (b) 3-HP shake flask yield of strains containing different strength promoter combinations when the gene expression amount presents MCR-C* > MCR-N;

[0041] Figure 10 Fluorescence characterization of the FAS1 natural promoter and three dynamic down-regulation promoters in Example 2 of the present application;

[0042] Figure 11 3-HP yield of the engineering strain for regulating FAS1 gene by dynamic down-regulation promoter in Example 2 of the present application;

[0043] Figure 12To overexpress the precursor supply genes in Example 2 of the present application to improve the production of 3-HP;

[0044] Figure 13 To batch-fed fermentation of 3-HP, biomass and glucose consumption of the engineered strain in Example 2 of the present application;(a) Batch-fed fermentation of 3-HP of the engineered strain H6-4.(b) Batch-fed fermentation of 3-HP of the engineered strain H6-14. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in connection with the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains.

[0046] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0047] The present application is further described in connection with the following examples. The following examples further illustrate the present application but are not meant to limit the application in scope to the examples set forth. Based on the examples in the present application, any variation of the present application by those skilled in the art without creative effort belongs to the protection scope of the present application. Meanwhile, in the examples of the present application, unless otherwise specified, all the raw materials for preparation are commercially available products well known to those skilled in the art.

[0048] Example 1 Screening of endogenous promoters and potential upstream activating sequences in Yarrowia lipolytica based on transcriptomic analysis

[0049] Based on transcriptomic analysis, novel dynamic regulatory promoters and gradient strength promoters with a wide range of transcription levels in Yarrowia lipolytica are mined. These types of promoters will be valuable tools in synthetic biology and metabolic engineering. Among them, dynamic regulatory promoters allow the strain to autonomously adjust metabolic flux with cell growth, thereby realizing the dynamic regulation of Yarrowia lipolytica; gradient strength promoters can provide a wide range of strength for selection and use of different needs, and the development of higher transcription level promoters can greatly promote the efficient expression of biosynthetic genes. In addition, through the study of the length of the promoter sequence, it is expected to simplify the promoter and mine potential upstream activating sequences (UAS), providing a certain reference for the construction of artificial hybrid promoters.

[0050] The mining of new promoters will help to exploit more potential of Yarrowia lipolytica, provide powerful tools for the efficient metabolic pathway modification, regulate the overall metabolic balance and reduce the metabolic load in cells, and thus maximize the production of target compounds of interest. The workflow of the characterization of endogenous promoters in this embodiment is shown in Figure 1

[0051] Among them, the specific method of promoter characterization and library construction is: using Yarrowia lipolytica episomal vector to characterize the promoter, and using the mined different promoters to express the hrGFP gene. The circular plasmid is transformed into Yarrowia lipolytica Po1f using the kit transformation method, and the transformants are screened on the plate containing 600 μg / mL of hygromycin B. The correct monoclonal strain is inoculated into the YPD liquid medium containing the corresponding resistance, and cultured at 30°C, 220 rpm for 24h. Inoculate into a 48-well plate at a ratio of 1% (vol / vol), with 600 μL of YPD liquid medium containing the corresponding resistance in each well. After inoculation, set the enzyme marker program, 30°C continuous shaking culture for 72h, and detect OD 600 and green fluorescence (excitation light 485nm, emission light 528nm) every 20min. Normalize the fluorescence intensity to OD 600 , and calculate the relative fluorescence intensity (Relative fluorescence intensity, RFI) of the promoter.

[0052] According to GO and KEGG analysis, a lot of useful information on gene expression at the transcriptional level can be obtained in the comparison group of different transcriptome data, but for the research purpose of this subject, we need to select some differentially expressed genes by certain means and develop corresponding standards for classification analysis to obtain candidate promoters.

[0053] Starting with the "YPD-60h" transcriptome data, the promoter screening and characterization work is carried out. According to the gene expression level of Po1f strain cultured in YPD medium for 60h, the promoters of genes with high to low FPKM value are randomly selected as candidate promoters. Considering that the length of the commonly used promoter of Yarrowia lipolytica is about 1000bp, the length of the candidate promoter is also set to about 1000bp, and the corresponding promoter sequence is cloned from the Po1f genome.

[0054] Since the combination expression of endogenous or exogenous genes to form a new metabolic pathway requires the development of functional promoters, at the same time, strong promoters are needed to obtain high level of protein expression, both of which together optimize the metabolic pathway, therefore, the following targets are constructed, screened and characterized: 1) screening growth-regulated promoters; 2) screening gradient strength promoters.

[0055] ​According to the transcription level gene expression quantity, the FPKM value of YPD-60h is sorted from high to low, and 100 genes with strong and weak transcription levels are selected as candidates. The corresponding promoter sequences are found in the NCBI and KEGG databases, and are annotated on the genome. According to the results of transcriptome sequencing, the candidate promoters are named P01-P100.

[0056] The candidate promoter sequence is amplified from the Yarrowia lipolytica Po1f genomic DNA as a template, assembled with the YLEP-Hyg plasmid backbone, and constructed into a free expression vector for characterizing the promoter strength. The vector takes hrGFP as the reporter gene of the candidate promoter, and takes hygromycin resistance as the resistance for screening the Yarrowia lipolytica transformants.

[0057] After the construction of the candidate promoter characterization plasmid, the Frozen-EZ Yeast Transformation II yeast transformation kit is used to transform them into Yarrowia lipolytica Po1f, respectively. The correct transformants are selected and tested by the microplate reader in a 48-well plate. It should be noted that not all candidate promoter characterization vectors can be successfully transformed and grown into single colonies during the transformation of Yarrowia lipolytica, and some single colonies fail to detect fluorescence. Finally, 82 promoters have fluorescence characterization results. The microplate reader measurement data are processed as follows: the fluorescence measurement value of the test group is subtracted from the background measurement value of the control Po1f, and then the OD 600 standardization.

[0058] The characterization results show that the expression of the fluorescence reporter protein of 21 promoters is first increased and then decreased with growth, the expression of the fluorescence reporter protein of 13 promoters is always increased, and the expression of the fluorescence reporter protein of 48 promoters is not much changed with growth. Thus, we find some unusual promoters in Yarrowia lipolytica, and classify them according to the fluorescence characterization results, as shown in Table 1. Among them, the growth-regulated promoters include dynamic up-regulation (PU series) and dynamic down-regulation (PD series); and the dynamic up-regulation type promoters and the steady expression promoters with different strengths over time (PC series) are classified into the gradient strength type promoter category.

[0059] Table 1 Characterization of promoter classification and naming

[0060]

[0061] The gene sources and gene functions of the above promoters are arranged in the summary section.

[0062] Thus, we obtained two categories of natural promoters according to the fluorescence characterization: growth-regulated promoters and gradient-intensity promoters. Among them, 61 gradient-intensity natural promoters were divided into strong (>2100, 8), medium (700-2100, 28), and weak (<700, 25) according to the Flu / OD value, including two categories of time-up (13) and time-stable (48); the typical growth-regulated natural promoters include dynamic up-regulation (13) and dynamic down-regulation (21).

[0063] According to the fluorescence characterization at 5 time points (7h, 32h, 48h, 60h, 72h) in YPD medium, there are 13 up-regulation promoters (PU01-PU13) and 21 down-regulation promoters (PD01-PD21), and the fluorescence intensity of these promoters shows a significant upward trend or a significant downward trend over time, with time-series regulation.

[0064] Among the dynamic up-regulation promoters, the fluorescence intensity of the PU13 promoter, which up-regulates significantly with growth, at 72h is 7.67 times that at 7h, the fluorescence intensity of the PU12 promoter at 72h is 33.67 times that at 7h, and the fluorescence intensity of the PU11 promoter at 72h is 4.18 times that at 7h. Among the dynamic down-regulation promoters, the fluorescence intensity of the PD01 promoter at 72h is 63.34% lower than that at 32h, the fluorescence intensity of the PD03 promoter at 72h is 68.27% lower than that at 32h, and the fluorescence intensity of the PD04 promoter at 72h is 52.48% lower than that at 32h.

[0065] In addition to the dynamic promoters obtained above, there are some promoters that express stably over time. These constitutive promoters are placed together with the dynamic up-regulation promoters to form a gradient-intensity natural promoter toolkit, which expands the strength and breadth of Yarrowia lipolytica promoters and provides more options for gene expression and fine regulation of metabolic engineering strains. Then, the RFI values are divided into strong, medium, and weak categories according to the interval range (2100, 28), and weak (<700, 25) according to the Flu / OD value, including two categories of time-up (13) and time-stable (48); the typical growth-regulated natural promoters include dynamic up-regulation (13) and dynamic down-regulation (21). Figure 3

[0066] 8 strong expression promoters - RFI value >2100: PC43, PC44, PC45, PC46, PC47, PU12, PC48, PU13.

[0067] ​28 moderately expressed promoters with RFI values ​​between 700 and 2100: PU04, PC23, PC24, PC25, PC26, PC27, PU05, PC28, PC29, PC30, PC31, PC32, PC33, PU06, PC34, PC35, PU07, PU08, PC36, PU09, PC37, PC38, PU10, PU11, PC39, PC40, PC41, PC42.

[0068] 25 weak expression promoters with RFI values ​​< 700: PC01, PC02, PC03, PC04, PC05, PC06, PC07, PC08, PC09, PC10, PC11, PC12, PC13, PC14, PC15, PC16, PU01, PC17, PC18, PU02, PC19, PC20, PC21, PU03, PC22.

[0069] In YPD medium, among 61 gradient intensity promoters, the fluorescence fold change between the strongest and weakest promoters reached 93.40-fold. Among them, the expression intensities of promoters PU12, PC48, and PU13 were higher than those of commonly reported strong promoters. TEFin P TEFin 1.10 times, 1.48 times and 1.69 times.

[0070] Among the strong promoters, when characterized in YPG medium, only the fluorescence intensity of promoters PU12, PC48, and PU13 was higher than that of promoter P. TEFin In addition, there are PC43, PC44, PC45, and PC46; when characterized in SD medium, the fluorescence intensity of the PC45, PC46, and PU12 promoters was higher than that of the control P. TEFin .

[0071] Three strains with greater activity than *Yarrowia lipolyticis* P, selected during YPD culture, were used in the well plates. TEFin The strong promoters PU12, PU13, and PC48 were used, and shake-flask characterization was performed to further verify their expression effects. Figure 4 As shown, the results indicate that even after amplification using the culture system, the relative fluorescence intensity of the three strong promoters remains stronger than that of the commonly used strong promoter P. TEFin and P Ut8 This further demonstrates the consistency between the characterization results of well plates and shake flasks, indicating that these promoters have the potential to be applied to regulate strong gene expression in large systems.

[0072] The 3 strong promoters PU12, PU13 and PC48 screened were respectively extended at 5' end, L1 represents 5' end extension of 200 bp; L2 represents 5' end extension of 400 bp. It was found that when the 5' end of the 3 strong promoters was extended, the fluorescence was not enhanced, which was speculated to be that the upstream sequence affected the expression, or the increase in length brought the expression burden, or the extended sequence contained some repressor sequences of transcription factors, etc.

[0073] In order to explore potential upstream activating sequences, 6 promoters PU11, PU12, PU13, PD03, PC11 and PC48 were selected for truncation at 5' end, each time by 200 bp, s1 represents 5' end truncation of 200 bp; s2 represents 5' end truncation of 400 bp. The results of fluorescence intensity of truncated promoters of different types showed that after the first truncation and the second truncation, the fluorescence intensity of the promoters corresponding to 48 h showed different trends, including increase, decrease and basically unchanged. For the fluorescence change, it can be explored whether it is a potential activating or repressing sequence; for the fluorescence basically unchanged, the sequence of the promoter can be considered to be simplified.

[0074] Then, PC48 and PU13 were selected for fine truncation, according to the rule of truncation at 5' end every 25 bp. Except PC48-675 bp, all transformed spread plates grew single colonies.

[0075] The PC48 promoter is derived from P TEFin The promoter sequences overlap, so the length of P TEFin When PC48 was truncated, it stopped at 525 bp. With the truncation moving towards the core promoter, the fluorescence intensity increased or decreased in different intervals. Referring to the length of the reported yeast UAS, the length of the potential activating sequence was artificially defined. Between PC48(600bp) and PC48(525bp), PC48(775bp) and PC48(625bp), and PC48(996bp) and PC48(850bp), the fluorescence intensity decreased by 38.50%, 85.24% and 68.16% respectively, indicating that there are potential upstream activating sequences in these intervals, which are called PC48-A1, PC48-A2 and PC48-A3 respectively.

[0076] The PU13 promoter was truncated from the 5' end by 25 bp each time, and the truncation stopped at 100 bp. As the truncation moved towards the core promoter, the fluorescence intensity generally decreased, and the truncation of different sequences also showed fluorescence enhancement and weakening. Between 350 bp and 100 bp, the fluorescence intensity was basically unchanged, suggesting that this sequence may be the core promoter sequence of PU13. Between PU13 (650 bp) and PU13 (600 bp), between PU13 (750 bp) and PU13 (675 bp), and between PU13 (998 bp) and PU13 (850 bp), the fluorescence intensity decreased by 80.88%, 62.05%, and 52.98%, respectively, indicating that there are potential upstream activating sequences in these intervals, which are referred to as PU13-A1, PU13-A2, and PU13-A3, respectively.

[0077] When the promoters in different regions were truncated, the fluorescence levels changed differently, indicating that the truncation was closely related to the strength of the promoter. According to the fluorescence characterization results of the fine truncation, the sequence interval with decreased transcription activity during truncation was defined as a potential activating sequence, named A1, A2, and A3 (Activating sequences), as shown in Table 2.

[0078] Table 2 Naming and corresponding regions of potential activating sequences

[0079]

[0080] Then, 6 potential activating sequences were fused upstream of the Leu2 minimal core promoter (mLeu2) in the form of 1 copy, and the expression of the reporter gene hrGFP was performed. The results showed that the relative fluorescence intensity of PC48-A1-mLeu2 promoter at different time points of 32h, 48h, 60h, and 72h was 1.30 times, 1.39 times, 1.53 times, and 1.74 times that of the control mLeu2 promoter, respectively. The relative fluorescence intensity of PU13-A1-mLeu2 promoter at different time points of 32h, 48h, 60h, and 72h was 1.73 times, 2.07 times, 2.31 times, and 2.51 times that of the control mLeu2 promoter, respectively. This result shows that compared with the control mLeu2 promoter, the hybrid promoters constructed by fusing the potential upstream activating sequences PC48-A1 and PU13-A1 have obvious fluorescence enhancement effect at different time points, further proving the potential activating effect of the two sequences. The fluorescence activation effect of the positive control hp1d promoter (fusing one copy of UAS1B sequence upstream of the mLeu2 promoter) is not obvious, which may be related to the copy number of the upstream activating sequence.

[0081] Further, the potential upstream activation sequence PC48-A1 was fused in tandem upstream of the Leu2 minimal core promoter (mLeu2) in the form of 1-5 copies, and the promoters were named PC48-1A-mLeu2, PC48-2A-mLeu2, PC48-3A-mLeu2, PC48-4A-mLeu2, and PC48-5A-mLeu2, respectively, followed by expression of the reporter gene hrGFP, and fluorescence characterization was performed in YPD medium. It was found that, upstream of the Leu2 minimal core promoter (mLeu2), with the increase in the number of copies of PC48-A1, the relative fluorescence intensity had a trend of increasing at different time points, and the effect of 4 or 5 copies was the most obvious. When the potential upstream activation sequence PC48-A1 was fused in tandem upstream of the minimal core promoter mLeu2 in the form of 4 copies, the relative fluorescence intensity at 60 h in the stationary phase was 1.15 times that of the positive control P TEFin , and the fluorescence intensity of PC48-4A-mLeu2 at 72 h was 1.26 times that of P TEFin . When the potential upstream activation sequence PC48-A1 was fused in tandem upstream of the minimal core promoter mLeu2 in the form of 5 copies, the relative fluorescence intensity at 60 h in the stationary phase was 1.15 times that of the positive control P TEFin , and the fluorescence intensity of PC48-5A-mLeu2 at 72 h was 1.27 times that of P TEFin .

[0082] Meanwhile, the potential upstream activation sequence PU13-A1 was fused in tandem upstream of the Leu2 minimal core promoter (mLeu2) in the form of 1-5 copies, and the promoters were named PU13-1A-mLeu2, PU13-2A-mLeu2, PU13-3A-mLeu2, PU13-4A-mLeu2, and PU13-5A-mLeu2, respectively, followed by expression of the reporter gene hrGFP, and fluorescence characterization was performed in YPD medium. It was found that, upstream of the Leu2 minimal core promoter (mLeu2), with the increase in the number of copies of PU13-A1, the relative fluorescence intensity had a trend of increasing at different time points, and the effect of 3 or 5 copies was obvious. When there were 3 copies, PU13-3A-mLeu2 had the best fluorescence intensity effect at different time points.

[0083] Example 2 Construction and optimization of 3-hydroxypropionic acid synthesis pathway in Yarrowia lipolytica

[0084] 3-hydroxypropionic acid (3-HP) is a three-carbon, optically inactive organic compound, containing carboxyl and hydroxyl groups in the molecule, which is an isomer of lactic acid. As a precursor of numerous chemicals, 3-HP is a high-value-added compound, which can produce chemicals such as acrylic acid, 1,3-propanediol, malonic acid, and propiolactone. Although 3-HP has wide application value, commercial production is limited due to high production cost. With the continuous development of metabolic engineering and synthetic biology, the use of microbial hosts to produce organic acid compounds heterologously has become a promising method.

[0085] In this embodiment, Yarrowia lipolytica was used as a chassis for metabolic engineering to construct a "microbial cell factory" for efficient biosynthesis of 3-HP. In this part, Yarrowia lipolytica Po1f was used as the starting strain, and malonyl-CoA reductase MCR from C. aurantiacus was introduced and optimized to balance the gene expression of MCR-C and MCR-N by combining different strength promoters, dynamically down-regulate FAS1 gene to weaken the competition pathway for the consumption of 3-HP direct precursors, and enhance the expression of precursor supply genes. A new strategy for dynamic and balanced regulation was successfully used to construct an engineered strain for efficient production of 3-HP using glucose as a carbon source. Finally, fed-batch fermentation experiments were carried out in a 5-L fermenter using the strain. The 3-HP biosynthesis pathway modification strategy is shown in Figure 7

[0086] I. Strains, plasmids

[0087] Table 3. Main Yarrowia lipolytica strains used in this embodiment

[0088]

[0089]

[0090] Table 4. Main plasmids used in this embodiment

[0091]

[0092]

[0093] II. Construction of 3-HP biosynthesis pathway in Yarrowia lipolytica

[0094] ​Malonyl-CoA is the common precursor of fatty acid and 3-HP biosynthesis pathway, Yarrowia lipolytica has all the enzymes from glucose to malonyl-CoA. As a key enzyme of 3-HP synthesis pathway, malonyl-CoA reductase (MCR) catalyzes malonyl-CoA to generate 3-HP directly. Since Y. lipolytica does not contain MCR, we selected MCR from Chloroflexus aurantiacus in this study, which was synthesized after codon optimization and expressed in Y. lipolytica.

[0095] 2.1 Full-length MCR Ca Expression of the gene

[0096] Y. lipolytica itself can convert acetyl-CoA to malonyl-CoA, so theoretically, by heterologous expression of MCR gene (SEQ ID NO. 12), the malonyl-CoA pathway can be opened to synthesize 3-HP. In this experiment, we first constructed an integrated plasmid containing the 3-HP biosynthesis pathway gene MCR Ca , selected two strong promoters PC48 and PU13, two control promoters P TEFin and P Ut8 , respectively, to regulate the expression of MCR Ca , constructed four integrated expression vectors, and integrated them into the Y. lipolytica Po1f genome. We screened the correct transformants and named them H0-1, H0-2, H0-3, and H0-4, respectively. Using fermentation medium, we performed 72h shake flask fermentation at 30℃ and 220rpm. The OD 600 , sugar consumption and 3-HP production of the control strain and the strain containing the expression gene were detected. The results showed that almost no 3-HP accumulation was detected. We speculated that the reason for the lack of 3-HP accumulation in the engineered strain may be caused by problems in MCR expression or low activity.

[0097] 2.2 Split expression of MCR-C and MCR-N

[0098] MCR protein can be divided into two independent functional units, N-terminal and C-terminal, which play the role of alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Therefore, we split MCR into MCR-N (part of the first-549 amino acid residues) and MCR-C (part of the 550-1219 amino acid residues). MCR-C catalyzes malonyl-CoA to generate malonate semialdehyde, which is synthesized to 3-HP under the action of MCR-N. We used the same strong promoter for MCR-C and MCR-N genes, constructed integrated expression vectors, and integrated them into the Y. lipolytica Po1f genome. We screened the correct transformants and named this part of the strain as H1 series (from H1-1 to H1-3).

[0099] To compare the ability of the engineered strains to produce 3-HP under the same conditions, shake flask fermentation experiments were performed. MCR was split into two parts, N-terminal and C-terminal, MCR-N (SEQ ID NO. 13) and MCR-C (SEQ ID NO. 14) genes were regulated by the same strength of promoters. H1-1 strain, H1-2 and H1-3 strains used PC48, PC13 and P TEFin The promoters regulated the expression of two genes, and the 3-HP yield after 72h fermentation was 0.571g / L, 0.262g / L and 0.753g / L, respectively. The results showed that splitting MCR into MCR-N and MCR-C helped to improve the yield of 3-HP synthesis in Y. lipolytica. Although the 3-HP yield was still low, proper functional domain splitting of multifunctional protein molecules was beneficial to increase enzyme catalytic activity and improve fermentation yield.

[0100] 2.3 Mutation of MCR-C

[0101] The mutation of the three amino acid residue sites N940V, K1106W and S1114R helped to improve the enzyme activity of MCR-C, and all three sites were located in the C-terminal region of MCR-C protein, far from the active site and NADPH binding site. We mutated the three sites of MCR-C to obtain MCR-C* (SEQ ID NO. 15). MCR-C* and MCR-N genes were regulated by the same strong promoter, and integrated expression vectors were constructed and integrated into the Y. lipolytica Po1f genome, and the correct transformants were screened, and this part of the strain was named as H2 series (from H2-1 to H2-3).

[0102] To compare the ability of the engineered strains to produce 3-HP under the same conditions, shake flask fermentation experiments were performed. MCR was split into two parts, N-terminal and C-terminal, MCR-N (SEQ ID NO. 13) and MCR-C (SEQ ID NO. 14) genes were regulated by the same strength of promoters. H1-1 strain, H1-2 and H1-3 strains used PC48, PC13 and P TEFin The promoters regulated the expression of two genes, and the 3-HP yield after 72h fermentation was 0.571g / L, 0.262g / L and 0.753g / L, respectively. The results showed that splitting MCR into MCR-N and MCR-C helped to improve the yield of 3-HP synthesis in Y. lipolytica. Although the 3-HP yield was still low, proper functional domain splitting of multifunctional protein molecules was beneficial to increase enzyme catalytic activity and improve fermentation yield.

[0103] Three, modular regulation of metabolic pathways to promote the production of 3-HP

[0104] 3.1 Promoter combination balances the gene expression of MCR-C* and MCR-N

[0105] Malonate semialdehyde (MSA) is a byproduct of the MCR-catalyzed reaction of malonyl-CoA to 3-HP Figure 8 a、 Figure 9 a) Promoters can be used as an effective tool to regulate the expression strength of multi-gene biosynthetic pathways in microorganisms. An effective metabolic engineering strategy relies on the balanced expression of endogenous and exogenous genes to achieve biomass accumulation while guiding metabolic flux to the target product of interest. Therefore, it is important to control the expression of MCR-C* and MCR-N genes at appropriate levels, and the expression levels of MCR-C* and MCR-N proteins directly affect the 3-HP productivity of the strain. Here, we used different strength promoters to express MCR-C* and MCR-N genes to achieve a balance in their catalytic activities. By optimizing protein expression levels through promoter strength regulation, we effectively adjusted the catalytic balance of the two genes, thereby improving the ability of the engineered strain to produce 3-HP.

[0106] First, we used a medium / weak promoter to regulate the expression of MCR-C gene and a strong promoter to regulate the expression of MCR-N gene. This part of the strain is named H3 series (from H3-1 to H3-21), and the strain name and specific promoter combination are shown in Table 3-1. The shake flask fermentation results are shown in Figure 8 As shown in the table, the H3-14 strain regulated by the PD09 promoter for MCR-C* and the PU13 promoter for MCR-N had the highest 3-HP yield in the same batch fermentation, reaching 0.631 g / L, but it was not as high as the yield of the previous strain without different promoter combinations. It can be seen that this combination did not improve the 3-HP yield, but instead led to an imbalance in the catalytic activities of the two enzymes, resulting in a decrease in 3-HP yield.

[0107] Next, we used the opposite promoter strength combination to regulate the expression of the two genes, i.e., using a strong promoter to regulate the expression of MCR-C* gene and a medium / weak promoter to regulate the expression of MCR-N gene. This part of the strain is named H4 series (from H4-1 to H4-15). The shake flask fermentation results are shown in Figure 9As shown, the 3-HP production of the five strains H4-1 to H4-5 is better than that of other strains in the same batch, and the five strains are all regulated by the PC48 promoter to control MCR-C* and the MCR-N is regulated by the PC12, PC37, PU01, PU06 and PU04 promoters respectively, and the 3-HP production reaches 4.301 g / L, 5.236 g / L, 1.473 g / L, 2.226 g / L and 2.379 g / L respectively. It can be seen that after reducing the expression amount of MCR-N protein, the 3-HP production is significantly increased. Among them, when the strong promoter PC48 is used to express the MCR-C* gene and the medium promoter PC37 is used to express the MCR-N gene, the H4-2 strain has the highest production, reaching 5.236 g / L, which is 1.12 times higher than that of the strain H2-1 before modification. However, it is worth noting that when the other two strong promoters PC45 and PU13 are used to regulate MCR-C* and different medium / weak promoters are used to regulate MCR-N, the effect is not obvious, and the reason is not yet known, which is probably caused by the complexity of the metabolic network and the adaptability of the promoter combination.

[0108] 3.2 Dynamic down-regulation of FAS1 gene expression to weaken the competitive pathway

[0109] Malonyl-CoA is a direct precursor of 3-HP synthesis, which is generated from acetyl-CoA by acetyl-CoA carboxylase. At the same time, malonyl-CoA is also an essential precursor for fatty acid synthesis, and the fatty acid synthesis pathway, as a necessary pathway for cell growth, gradually synthesizes long-chain fatty acids from acetyl-CoA in cells to provide the main components of cell membranes, and also participates in important biological processes such as cell signal transduction and energy storage. Therefore, fatty acid synthesis is essential for cell growth. As a growth essential gene of Yarrowia lipolytica, FAS1 encodes a subunit of fatty acid synthase complex, which cannot be knocked out or mutated, otherwise it may affect the normal growth and function of cells. In order to reduce the metabolic flux of the fatty acid cycle, the expression of the FAS1 gene is controlled by the screened dynamic down-regulation promoter, which presents a trend from strong to weak regulation of the gene over time. In the cell growth stage, the metabolic flux meets the growth demand, and in the later stage, more malonyl-CoA and NADPH are used to produce 3-HP to ensure the rational use of cell resources.

[0110] The fatty acid synthesis pathway is the main competitive pathway for malonyl-CoA to synthesize 3-HP, and it is also an indispensable pathway for bacterial growth. We first compared the fluorescence characterization of the natural promoter of the FAS1 gene with the 21 dynamic down-regulation promoters screened, excluded the promoters higher than the expression level of the natural promoter of the FAS1 gene in the later stage, and finally selected the PD19, PD20 and PD21 promoters to control the expression of the FAS1 gene, as shown in Table 3. Figure 10The promoters were shown in Figure 1. The transcriptional strength of the promoters varied significantly at different cell growth stages, and the strength decreased from the early stage to the late stage. The strength of the early stage was comparable to or slightly higher than that of the native promoter, and the strength of the late stage was lower than that of the native promoter. FAS1 FAS1

[0111] The native promoter of the FAS1 gene (YALI0B15059g) was replaced by the dynamic down-regulation promoter through homologous recombination. The successful replacement of the dynamic down-regulation promoter was verified by colony PCR and sequencing, so that the strain could dynamically down-regulate the expression of the FAS1 gene with the cell growth to decouple the cell growth and 3-HP synthesis. This part of the strain was named as the H5 series (from H5-1 to H5-3). The fermentation results are shown in Figure 2. The 3-HP production of the H5-1, H5-2 and H5-3 strains regulated by the PD19, PD20 and PD21 promoters, respectively, was increased by 41.36%, 31.30% and 30.69% compared with the control H4-2 strain (PC48 expressing MCR-C* and PC37 expressing MCR-N), and the production reached 7.401 g / L, 6.875 g / L and 6.843 g / L, respectively. Figure 11

[0112] The results showed that the dynamic down-regulation promoter had a relatively high transcriptional strength in the early stage of cell growth (exponential growth phase), and the transcriptional strength decreased significantly in the stationary phase. Replacing the native promoter of the FAS1 gene with the PD19, PD20 and PD21 promoters helped to optimize the carbon efficiency of the 3-HP production strain, so that the cell had sufficient carbon flux in the growth stage and did not have a negative impact on the cell growth, and more carbon flux flowed to the product synthesis in the production stage. The use of the dynamic regulation promoter could improve the selectivity and control of gene expression, and did not need to add any exogenous substance, but could achieve regulation only with the cell growth period, which provided a useful and general dynamic regulation tool for the research of metabolic engineering and synthetic biology.

[0113] 3.3 Strong promoter expression of upstream genes to enhance precursor supply

[0114] The synthesis of metabolic pathway precursors was very important for improving the yield of target products. In this part, the related genes were combined to overexpress to enhance the upstream acetyl-CoA supply, and the engineering strain was named as the H6 series (from H6-1 to H6-15). The H5-1, H5-2 and H5-3 strains were used as the starting strains, and different strong promoters were used to overexpress the ACL1 (YALI0D24431g), ACL2 (YALI0E34793g), ACC1 (YALI0C11407g), PDC (YALI0D06930g), ALD (YALI0D07942g) and ACS (YALI0F05962g) genes. The shake flask fermentation results are shown in Figure 3.​​​Figure 12 As shown.

[0115] In the H5-1 strain, the strains H6-1 to H6-5 obtained by overexpressing ACL1, ACL2, ACC1, PDC, ALD and ACS related genes, the 3-HP yield in the shake flask was improved as a whole, among which the 3-HP yield of the H6-4 strain overexpressing ACC1 using the PU13 promoter was most obviously improved, reaching 10.551 g / L, which was 1.43 times that of the control strain; the 3-HP yield of the H6-1 strain overexpressing ACL1 using the PU12 promoter reached 9.403 g / L, which was 1.27 times that of the control strain; the 3-HP yield of the H6-2 strain overexpressing ACL2 using the PU13 promoter reached 8.413 g / L, which was 1.14 times that of the control strain; the 3-HP yield of the H6-2 strain overexpressing ACL1 and ACL2 simultaneously was slightly decreased; the 3-HP yield of the H6-5 strain overexpressing PDC gene regulated by PC48 promoter, ALD gene regulated by PU13 promoter and ACS gene regulated by PU12 promoter reached 9.107 g / L, which was 1.23 times that of the control strain.

[0116] In the H5-2 strain, the strains H6-6 to H6-10 obtained by overexpressing ACL1, ACL2, ACC1, PDC, ALD and ACS related genes, the 3-HP yield in the shake flask was 1.631 g / L, 5.519 g / L, 4.106 g / L, 6.055 g / L, 5.244 g / L, respectively, which was not obviously improved compared with the control strain, but decreased instead.

[0117] In the H5-3 strain, the strains H6-11 to H6-15 obtained by overexpressing ACL1, ACL2, ACC1, PDC, ALD and ACS related genes, the 3-HP yield in the shake flask was 7.421 g / L, 7.266 g / L, 6.251 g / L, 8.705 g / L, 7.320 g / L, respectively, which was improved compared with the control strain except for the H6-13 strain, among which the yield of the H6-14 strain overexpressing ACC1 using the PU13 promoter was most obviously improved, reaching 8.705 g / L, which was 1.27 times that of the control strain.

[0118] In summary, overexpression of ACC1 gene has the most positive effect on the improvement of 3-HP production. In any of H5-1, H5-2, H5-3, the effect of overexpression of ACC1 gene is better than that of ACL1, ACL2, PDC, ALD and ACS genes. In H5-1 strain, the production is improved when ACL1, ACL2, ACC1, PDC, ALD and ACS genes are overexpressed respectively. In H5-2 strain, overexpression of genes encoding 3-HP precursors does not improve the production, and even causes the decrease of production. In H5-3 strain, overexpression of genes regulating the supply of precursors has certain effect on the improvement of production, and overexpression of ACC1 gene has the most prominent effect. In H5-1 and H5-3 strains, 3-HP production is improved when ACL1 and ACL2 are overexpressed respectively, but when ACL1 and ACL2 are overexpressed simultaneously, the production is not as good as the control and the strains overexpressing ACL1 or ACL2 alone.

[0119] IV. Fed-batch fermentation for synthesis of 3-HP

[0120] Finally, we selected H6-4 and H6-14 strains which have the most prominent effect on 3-HP synthesis in shake flask culture to carry out fed-batch fermentation in 5-L fermenter. Figure 13 The specific fermentation conditions are as follows: the initial volume of the fermenter is 2 L, the culture medium is inorganic salt medium, the initial concentration of glucose is 40 g / L, the pH of the fermentation broth is controlled at 6.0, the stirring speed is 200-700 rpm, the aeration rate is 1 vvm, and when the concentration of glucose in the culture medium is lower than 10 g / L, the appropriate amount of sterilized 70% glucose mother liquor is timely supplemented. In the fermentation process of H6-4 strain, the accumulation of 3-HP increases with the extension of fermentation time, and the yield reaches 100.371 g / L at 207 h, the conversion rate and production rate are 0.21 g / g and 0.48 g / L / h respectively, and the maximum productivity is about 1.76 g / L / h. From 54 h, the biomass OD 600 maintains at about 230. In addition, 22.002 g / L of erythritol, 6.134 g / L of mannitol and 6.873 g / L of arabitol are detected in the final fermentation broth. In the fermentation of H6-14 strain, the yield of 3-HP is only 54.134 g / L at 165 h, OD 600 is 244.65, and 10.301 g / L of erythritol, 7.185 g / L of mannitol and 7.952 g / L of arabitol are detected in the fermentation broth containing fermentation byproducts. The obvious difference between the two strains in the fermentation process indicates that the optimal fermentation conditions of each engineered strain are different, and need to be properly controlled.

[0121] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is explained in detail with reference to the examples, the technical solutions of the present application can be modified or equivalently replaced by those skilled in the art according to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A promoter element, characterized in that The promoter element is a promoter as shown in the following table:

2. An engineered bacterium producing 3-hydroxypropionic acid, characterized in that, The high-yield 3-hydroxypropionic acid engineered strain uses *Yarrowia lipolytica* Po1f as the substrate strain and heterologously expresses the MCR gene (SEQ ID NO.12). C. aurantiacus Malonyl-CoA reductase of different origins was constructed, and a balance was achieved by combining promoters of varying strengths. MCR-C and MCR-N Gene expression levels are dynamically downregulated by dynamically downregulated promoters. FAS1 Genes; and based on dynamically downregulated promoters, FAS1 gene is dynamically downregulated, and strong promoters promote the expression of precursor supply genes; The promoter element is a promoter as shown in the following table: The malonyl-CoA reductase MCR is mutated, and the mutation sites are N940V, K1106W and S1114R; The expression C. aurantiacus MCR from different sources, and the balance of different strength promoter combinations MCR-C With MCR-N The specific method of gene expression amount includes: MCR is divided into two parts of N terminal and C terminal, MCR-N (SEQ ID NO. 13) and MCR-C (SEQ ID NO. 14), MCR-C And MCR-N The genes are regulated by the same strong promoter, and the integrated expression vector is integrated into the genome of Yarrowia lipolytica Po1f. The MCR-C Regulated by PC48 promoter; The MCR-N The gene is regulated by any one of the promoters PC12, PC37, PU01, PU06, PU04. the dynamic down-regulation by the dynamic down-regulation promoter FAS1 in the gene, the dynamic down-regulation promoter comprises PD19, PD20 and PD21; The FAS1 gene is dynamically down-regulated based on a dynamic down-regulation type promoter, and a strong promoter promotes the expression of a precursor supply gene, and the promoter comprises: (1) the expression of the FAS1 gene is controlled by the PD19 promoter, and the ACL1 gene overexpression is regulated by the PU12 promoter; (2) the expression of the FAS1 gene is controlled by the PD19 promoter, and the ACL2 gene overexpression is regulated by the PU13 promoter; (3) the expression of the FAS1 gene is controlled by the PD19 promoter, and the ACC1 gene overexpression is regulated by the PU13 promoter; (4) the expression of the FAS1 gene is controlled by the PD19 promoter, and the PDC gene regulated by the PC48 promoter, the ALD gene regulated by the PU13 promoter and the ACS gene regulated by the PU12 promoter are co-overexpressed; (5) the expression of the FAS1 gene is controlled by the PD21 promoter, and the ACL1 gene overexpression is regulated by the PU12 promoter; (6) the expression of the FAS1 gene is controlled by the PD21 promoter, and the ACL2 gene overexpression is regulated by the PU13 promoter; (7) the expression of the FAS1 gene is controlled by the PD19 promoter, and the ACC1 gene overexpression is regulated by the PU13 promoter; (8) the expression of the FAS1 gene is controlled by the PD21 promoter, and the PDC gene regulated by the PC48 promoter, the ALD gene regulated by the PU13 promoter and the ACS gene regulated by the PU12 promoter are co-overexpressed.

3. The engineered bacterium of claim 2, wherein the bacterium is capable of producing 3-hydroxypropionic acid at a yield of at least 0.5 g / g. The MCR-N The gene is regulated by the PC37 promoter.

4. The engineered bacterium of claim 2, wherein the bacterium is selected from the group consisting of Escherichia coli, Klebsiella, Pantoea, and Pseudomonas. by the dynamic down-regulation type promoter FAS1 In the gene, the dynamic down-regulation type promoter is PD19.

5. The application of the 3-hydroxypropionic acid engineering bacteria with high yield of 3-hydroxypropionic acid in 3-hydroxypropionic acid biosynthesis according to any one of claims 2-4.

6. A method of biosynthesizing 3-hydroxypropionic acid, characterized by, The method comprises: fermenting and culturing the 3-hydroxypropionic acid engineering bacteria with high yield of 3-hydroxypropionic acid according to any one of claims 2-4.

7. The method of biosynthesizing 3-hydroxypropionic acid according to claim 6, wherein, The fermentation and culture mode is fed-batch fermentation.

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