Acid-tolerant yeast gene-based synthetic promoter

Abstract

To provide a synthetic promoter capable of controlling the expression of a target gene at various locations in the genome of an acid-resistant strain, and a synthetic promoter including a core promoter derived from an acid-resistant strain and an upstream activating sequence (UAS) element serving as an enhancer, where, in application to a variety of genetic and metabolic engineering techniques for acid-resistant derived yeast, various metabolic networks can be configured as desired while controlling the expression level of the target gene, so that a method enabling production of various metabolites using acid-resistant yeast is provided, and the cost of producing the metabolites can be greatly reduced depending on properties of the acid-resistant yeast.SOLUTION: A synthetic promoter for expression of a target gene comprises: (i) a core promoter represented by a specific sequence; and (ii) an upstream activating sequence (UAS) element represented by another specific sequence.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a synthetic promoter capable of regulating the expression of a target gene at various positions in the genome of an acid-resistant strain, and more particularly, to a synthetic promoter containing a core promoter derived from an acid-resistant strain and a UAS (upstream activating sequence) factor that plays the role of an enhancer.

Background Art

[0002] Using bioprocesses to bioconvert various raw materials into chemicals such as organic acids, alcohols, and amines has attracted attention in terms of environmental friendliness, carbon dioxide reduction, sustainability, and the supply of new platform chemicals. Such bioconversions supply chemicals, polymers, foods, cosmetics, nutraceuticals, and pharmaceutical-related chemicals. However, generally, products produced by bioconversion must undergo a purification process to remove impurities generated during the production process. Moreover, when producing organic acids, in most cases, neutral pH fermentation with a base is carried out to prevent the growth of the strain from being inhibited by the generated organic acid. To separate / purify this, a large amount of neutralization salt is generated as a by-product while further acidifying, and as the process becomes more complex, the production cost increases. Such a high cost burden of the purification process is considered a factor inhibiting the entry of fermentation products into the chemical substance market.

[0003] To solve the above problems, when producing acidic substances such as organic acids, using microorganisms that can grow even at low pH and exhibit high fermentation ability, especially yeasts that thrive in acidic conditions (acid-tolerant yeasts), to produce lactic acid-containing organic acids eliminates the need to maintain the culture medium's pH at 6-7 using neutralizing agents during fermentation, thus simplifying the fermentation process and eliminating the need for a subsequent purification step to remove the neutralizing agent. Furthermore, since yeast produces many of the components necessary for metabolism itself, it can be cultured in culture media with relatively low nutrient levels compared to bacteria, especially Lactobacillus. Therefore, many subsequent purification steps can be omitted, and production costs can be significantly reduced through process simplification and reduction of additives. However, in many cases, microorganisms that survive at low pH have very slow growth rates, making it difficult to obtain the sufficient cell volume required for substance production. This results in low raw material consumption and production rates, making them unsuitable for industrial fermentation processes. Therefore, it is crucial to select microorganisms that exhibit rapid growth at a pH lower than the product's pKa while maintaining a high raw material consumption rate. Such microorganisms can be selected from various strain libraries using various selection pressures. Examples of selection pressures include resistance to the target product concentration, resistance to the raw material concentration, raw material consumption rate, pH conditions, and growth ability in minimal media. Microbial selection can be done manually, but automated screening allows for the rapid selection of strains with superior characteristics from a larger sample size.

[0004] While selected microorganisms possess superior characteristics that allow them to withstand selective pressure, most wild-type microorganisms are unable to produce the target product and often produce other products instead. Therefore, in order to confer the ability to produce the target product to the selected microorganisms, we are conducting research to genetically engineer them by introducing conversion genes to the target product and eliminating their ability to produce the product that was originally being produced. To confer the ability to produce a target product to selected microorganisms, methods are used to either introduce a gene capable of converting the target product or to enhance the microorganism's naturally occurring genes. However, since the activity of the naturally occurring genes and the enzymes they produce is generally low, it is almost always necessary to introduce a strong foreign gene. Furthermore, the introduction of a promoter that can strongly express the foreign DNA is essential in such a process. Generally, when the target microorganism is yeast, the promoter of Saccharomyces cerevisiae (S. cerevisiae), which is well-known in yeast, can be used as a viable promoter, and various genetic engineering techniques developed for S. cerevisiae can also be applied. Furthermore, it is necessary to select a strong promoter from the promoters involved in the main carbon flux of the selected microorganism and to apply the method that most effectively expresses the target gene using various techniques as the top priority. In particular, if relevant genetic engineering studies have not been conducted on the selected acid-resistant yeast, the general approach is to use the S. cerevisiae promoter or the promoter inherent in the selected microorganism. In eukaryotes, promoters generally consist of various regulatory regions, including the core promoter region, and each regulatory gene differs from microorganism to microorganism. Therefore, while it is possible to select a sufficiently long sequence from the top 5' of the ORF and search for the optimal site while confirming the role of the promoter, separate research is needed for long-range regulation (enhancers, silencers, etc.) or complex regulatory mechanisms.

[0005] The inventors used an automated system to select microorganisms with excellent acid resistance and other fermentation characteristics from 700 types of yeast, and established genetic engineering tools for these microorganisms, which include the development of promoters. First, they attempted to express various genes using the above-mentioned S. cerevisiae-derived promoter and endogenous promoter (5'UTR region at the top of the ORF), and while excellent expression levels were confirmed for some, the degree of expression, particularly the discovery of a strong promoter, did not meet their objective. Therefore, the inventors proposed a method using the endogenous promoter itself, and after searching for an appropriate promoter within the genome, they found a novel strong endogenous promoter that met their objective and confirmed its performance (Registered Patent No. 2,140,596 of the Republic of Korea). This promoter strongly expressed various introduced genes, but due to the characteristics of endogenous promoters, it was not possible to apply it to more than one gene due to the expression caused by the substitution of the target gene. Given the general characteristics of genetic engineering, where it is often necessary to express various genes, these researchers aimed to develop promoters that could be used to adjust the expression intensity at various locations within the genome. Expression tests using various promoters in S. cerevisiae confirmed expression for some promoters, but sufficient expression intensity could not be achieved. Therefore, it was concluded that the YBC strain itself likely possesses a unique expression regulatory mechanism, and an attempt was made to discover the YBC strain's own promoter. First, the expression levels of various genes within the YBC strain were examined, and attempts were made to express the target gene by cleaving the promoter (5'UTR) region of the gene showing strong expression by up to 1kb, but this resulted in low expression. In particular, it was confirmed that the g4423 promoter (Registered Patent No. 2,140,596 in the Republic of Korea), a strong promoter that expresses other transgenes besides endogenous genes very strongly in YBC strains, hardly functions when such a 1kb-cut promoter is used. From this, it was concluded that YBC genes are strongly influenced by components that act at a distance (UAS or enhancer) in addition to the core promoter. Therefore, an attempt was made to strengthen the promoter by binding it with a UAS component discovered in existing research (John Blazeck et al., Biotechnology and Bioengineering, 109(11);2884, 2012).

[0006] Therefore, the inventors made diligent efforts to find a promoter suitable for foreign gene expression in acid-resistant yeast. As a result, they confirmed that when a synthetic promoter is used, which combines a core promoter derived from a yeast gene resistant to organic acids with a UAS factor derived from a foreign gene, the target gene is strongly expressed and the ability to produce the target product is increased even when inserted into a position other than the original position of the core promoter, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a synthetic promoter that can express target genes at various positions in a genetic material in order to efficiently produce various target products in recombinant acid-resistant yeast strains that have lactic acid production ability. Another object of the present invention is to provide a DNA structure for the expression of a target gene, comprising the synthetic promoter and the target gene. A further object of the present invention is to provide a recombinant microorganism in which the synthetic promoter is introduced into the genome or into the DNA structure such that the expression of a target gene is regulated by the synthetic promoter. Another object of the present invention is to provide a method for expressing a target gene, characterized by culturing the recombinant microorganism. Another object of the present invention is to provide a method for producing useful substances using the recombinant microorganisms. [Means for solving the problem]

[0008] To achieve the above objectives, the present invention provides a synthetic promoter for the expression of a target gene, comprising (i) and (ii) below. (i) Core promoters represented by Sequence ID 1 or Sequence ID 2; and (ii) UAS (upstream activating sequence) factors represented by SEQ ID NO: 3 or 4 The present invention also provides a DNA structure for the expression of a target gene, comprising the synthetic promoter and the target gene. The present invention also provides recombinant microorganisms in which the synthetic promoter is introduced into the genome or into the DNA structure such that the expression of a target gene is regulated by the synthetic promoter. The present invention also provides a method for expressing a target gene, characterized by culturing the recombinant microorganism. The present invention also provides a method for producing a useful substance, comprising the following steps. (a) A step of culturing the recombinant microorganism to produce a useful substance by expressing the target gene; and (b) Step of obtaining the useful substance that was produced. The present invention also provides a synthetic promoter for the expression of a target gene in a YBC strain or a recombinant strain derived from a YBC strain, comprising (i) and (ii) below. (i) Core promoter including TATA box; and (ii) UAS (upstream activating sequence) factors represented by SEQ ID NO: 3 or 4 The present invention also provides a recombinant yeast strain with lactate-producing ability in which, in the acid-resistant yeast YBC strain (KCTC13508BP), both the PDC gene, which encodes pirubate decarboxylase, and the promoter of the PDC gene are removed from the genome, and the synthetic promoter and the gene encoding lactate dehydrogenase are introduced into the position where the PDC gene was removed, thereby allowing the expression of lactate dehydrogenase to be regulated by the synthetic promoter. The present invention also provides a method for producing lactic acid, comprising the following steps. (a) A step of culturing the recombinant bacterial strain to produce lactic acid; and (b) Step of obtaining the lactic acid produced. [Effects of the Invention]

[0009] When using the synthetic promoter according to the present invention, various useful substances, especially organic acids, can be produced with high efficiency using acid-resistant microorganisms. Organic acid fermentation can be performed with organic acid production ability similar to bacterial fermentation while significantly reducing the amount of neutralizing agents used in existing bacterial fermentation processes. This significantly reduces not only fermentation costs but also subsequent purification process costs. [Brief explanation of the drawing]

[0010] [Figure 1] In the present invention, an example of a deletion cassette used to delete the target gene, including the g3002-1(PDC1) gene, from the genome of a YBC strain, or to delete the relevant gene, and then insert the target gene containing LDH using the synthetic promoter of the present invention, is shown. [Figure 2] This report describes the results of measuring the expression levels of the mCherry gene after cleaving the promoter from S. cerevisiae and various endogenous promoter regions from YBC strains into 1kb fragments and expressing them within YBC genes, then measuring the expression levels using qRT-PCR. [Figure 3] This study compares the expression levels of the MCRsa1 gene (exponential growth (exp.) and stationary growth (station.) of 1kb of g4423) when the MCRsa1 gene is expressed in a YBC gene body, using two methods: introducing the g4423 gene as a substitute for the g4423 natural promoter (a high-performance promoter for YBC) (g4423 replace), and selecting only 1kb of the g4423 promoter region and expressing it in the ald2 gene region of YBC. [Figure 4A] The present invention involves ligating the core promoter of YBC-derived g4423 with two CITs from UAS, resulting in a gene map for vector expression in plasmid form on YBC. [Figure 4B] The present invention involves conjugating the S. cerevisiae TEF1 promoter with three CITs from UAS to YBC in plasmid form, which provides a gene map for the vector during expression. [Figure 5] This report describes the results of introducing the g3002-1 gene and promoter of the YBC strain into the same position while expressing BtLDH using the 2CIT-pg4423 promoter according to the present invention, after removing the terminator, and then confirming the lactic acid production ability. As a comparison group, the lactic acid production amount was compared while expressing the same BtLDH gene using the endogenous promoter of YBC g3002-1, and the results of introducing it in the reverse direction into the same YBC g3002-1 position are shown. [Modes for carrying out the invention]

[0011] Unless otherwise specified, all technical and scientific terms used in this specification shall have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the relevant technical field. In the present invention, in order to secure a core promoter region in the acid-tolerant yeast YBC strain (KCTC13508BP), the position of the TATA box in the 5'-UTR region was determined from the ORF to secure the core promoter region. For the UAS (upstream activating sequence) factor regions of the CLB2 (mitotic cyclin) gene and the CIT1 (mitochondrial citrate synthase) gene, two or three copies were ligated above the core promoter as an AT-rich region so as to be activated, and a synthetic promoter was prepared to enhance the core promoter. Since the expression intensity of the UAS factor can be regulated by the number of repetitions, the expression level can be adjusted while maintaining the characteristics of the core promoter by changing the number of repetitions of the currently identified UAS. In addition, as the core promoter, in addition to the core promoter extracted from the YBC strain, tests were conducted using the core promoter regions of promoters known in existing S. cerevisiae, and the activity was confirmed. In the future, various combinations can be further expanded by applying promoters derived from S. cerevisiae in addition to those derived from the YBC strain.

[0012] When producing useful substances containing lactic acid using the recombinant acid-resistant yeast with the synthetic promoter, the desired target gene can be strongly expressed to epochally enhance the production performance of useful substances. In particular, even when the gene related to the production of the useful substance has a pathway composed of multiple genes, the promoter of the present invention can express the gene while adjusting the expression intensity by various promoter combinations, and can diversify the products of acid-resistant microorganisms compared with the case of using the promoter existing in the existing endogenous gene. According to the present invention, when producing various useful substances, particularly organic acids, using acid-resistant microorganisms with high efficiency, while significantly reducing the amount of neutralizing agent used in existing bacterial fermentation, organic acid fermentation can be carried out with an organic acid production ability similar to bacterial fermentation, and the cost of the subsequent purification process can also be significantly reduced in addition to the fermentation cost.

[0013] In addition, the present invention provides a solution that can be used independently by enhancing the expression of various promoters derived from acid-resistant strains, enabling this promoter to be used in other yeasts to strongly express various target genes, and providing various promoter libraries within the yeast. Therefore, the present invention relates to a synthetic promoter for the expression of a target gene including the following (i) and (ii) from one perspective. (i) The core promoter represented by SEQ ID NO: 1 or SEQ ID NO: 2; and (ii) The UAS (upstream activating sequence) factor represented by SEQ ID NO: 3 or SEQ ID NO: 4.

[0014]

Table 1

[0015] More specifically, in this invention, core promoter sequences were extracted from g4423 (ADH gene) and g2947 (CYB2 gene) in the acid-resistant yeast YBC strain (KCTC13508BP). By ligating these core promoter sequences with a sequence in which the UAS factor of the CLB gene or the UAS factor of the CIT gene is repeated 1 to 3 times in an active state, a promoter that expresses the target gene was created, and a plasmid was produced that makes this promoter activatable in the YBC strain. In the present invention, the UAS factor may be characterized by being located 1 to 4 times repeatedly in the upper part (upsteram) of the core promoter. In the present invention, the synthetic promoter is preferably for the expression of a target gene in a yeast strain, and more preferably for the expression of a target gene in acid-resistant yeast. In the present invention, the acid-resistant yeast may be characterized by being an acid-resistant yeast selected from the group consisting of the genera Saccharomyces, Casaxtania saccharomyces, and Candida. In this invention, a core promoter sequence was extracted from a known promoter of S. cerevisiae, and this sequence was linked to one, two, or three sequences of UAS factors, including CLB and CIT, in an active state to create a promoter that functions to express the target gene in the YBC strain. Therefore, in other respects, the present invention relates to a synthetic promoter for the expression of a target gene in a YBC strain or a recombinant strain derived from a YBC strain, comprising (i) and (ii) below. (i) Core promoter including TATA box; and (ii) The UAS (upstream activating sequence) factor represented by SEQ ID NO: 3 or SEQ ID NO: 4. In the present invention, the core promoter is characterized by being a core promoter for a gene located on the genome of acid-resistant yeast. In the present invention, the acid-resistant yeast is characterized by being an acid-resistant yeast selected from the group consisting of the genera Saccharomyces, Casaxtania saccharomyces, and Candida, and the core promoter may be characterized by being the core promoter of S. cerevisiae TEF1 (SEQ ID NO: 29) or the core promoter of the CYB2 gene (g2947 gene) of the YBC strain (SEQ ID NO: 33 or SEQ ID NO: 34). In one aspect of the present invention, a core promoter extracted from the acid-resistant yeast strain YBC (KCTC13508BP) and S. cerevisiae is provided, which activates in YBC to express a target gene by linking a sequence in which the UAS factor of the CLB gene or the UAS factor of the CIT gene is repeated 1 to 3 times in an active state. Furthermore, this promoter can be designed to be introduced into a desired target site in the YBC gene by general homologous recombination. In addition, this promoter enables the expression of the target gene at this target site with a high expression rate, and the expression rate can be adjusted by adjusting the number of UAS repeats. In other words, the present invention relates to a DNA structure for the expression of a target gene, comprising the synthetic promoter and the target gene. In another respect, the present invention relates to recombinant microorganisms in which the synthetic promoter is introduced into the genome or into the DNA structure such that the expression of a target gene is regulated by the synthetic promoter. In the present invention, the recombinant microorganism may be characterized in that a gene encoding an enzyme capable of producing useful substances has been introduced as the target gene. In another respect, the present invention relates to a method for expressing a target gene, characterized by culturing the recombinant microorganism. In another respect, the present invention relates to a method for producing a useful substance, comprising the following steps. (a) A step of culturing the recombinant microorganism and producing a useful substance by expressing the target gene; and (b) The step of obtaining the useful substance that was produced. In another aspect, the present invention relates to a recombinant strain of acid-resistant yeast YBC strain (KCTC13508BP) in which both the PDC gene, which encodes pirubate decarboxylase, and the promoter of the PDC gene are removed from the genome, and the synthetic promoter and the gene encoding lactate dehydrogenase are introduced into the position where the PDC gene was removed, and the expression of lactate dehydrogenase can be regulated by the synthetic promoter, thereby having lactate-producing ability. In another respect, the present invention relates to a method for producing lactic acid, comprising the following steps. (a) A step of culturing the recombinant bacterial strain to produce lactic acid; and (b) The step of obtaining the lactic acid produced. Acid-tolerant yeast exhibits a high growth rate by rapidly consuming sugar even at acidic pH levels, and under fermentation conditions, it converts the consumed sugar into products. In our previous research, we selected the acid-tolerant yeast YBC strain (KCTC13508BP) using multiple yeast libraries, and the acid-tolerant yeast YBC strain (KCTC13508BP) is a strain that shows high growth and sugar consumption rate even under conditions where the lactic acid concentration is 40 g / L to 80 g / L (Registered Patent No. 2,140,597 of the Republic of Korea).

[0016] In the present inventors' previous invention, metabolic pathways were modified in the acid-resistant yeast YBC strain to increase lactic acid production ability and decrease ethanol production ability. By deleting the genes encoding alcohol dehydrogenase and pirubate decarboxylase from the YBC strain, and deleting the gene encoding the cytochrome b2 enzyme that converts lactate to pirubate from a strain into which the lactate dehydrogenase gene had been introduced, recombinant strains with lactic acid production ability were created (Korean Patent Application 2018-0119721). Furthermore, the present inventors have previously created recombinant bacterial strains in which the gene encoding the glycerol 3-phosphate dehydrogenase enzyme, which converts dihydroxyacetone phosphate to glycerol 3-phosphate, is deleted in order to suppress glycerol production in the recombinant bacterial strains created as described above (Korean Patent Application No. 2020-0046779). In a previous application by the present inventors, in order to restore the lactic acid tolerance of the recombinant strain, strains with high lactic acid tolerance were selected while subculturing the recombinant strain in culture media containing various lactic acid concentrations up to ~80 g / L, and a new recombinant strain was created by replacing the foreign lactate dehydrogenase gene that had been substituted in the PDC locus of the selected strain with a lactate dehydrogenase gene derived from S. epidermidis. It was confirmed that the recombinant strain simultaneously possessed high lactic acid tolerance and high lactic acid production ability, while suppressing ethanol production ability and glycerol production ability (Korean Patent Application No. 2020-0077331). In this invention, in addition to the method of using the endogenous promoter of the acid-resistant strain, we have further developed an artificial promoter that can adjust the expression intensity in the gene and express the target gene at various intensities and variations, while utilizing the acid-resistant characteristics of the recombinant bacterial strain that has been prepared in the past to enhance its lactic acid production ability and to produce various useful substances, namely various biocompounds including organic acids, alcohols and aldehydes, by introducing production pathways for each useful substance.

[0017] In one embodiment of the present invention, the location of the TATA box is identified in the upper 5'-UTR region of the g4423(ADH) gene locus of the acid-resistant yeast strain YBC (KCTC13508BP), a core promoter sequence containing the transcription start locus is searched for, and this is linked to one, two, or three repeats of the UAS factor CIT or CLB, thereby providing a method for maintaining the characteristics of the core promoter while adjusting its performance by the number of UAS repeats. In another aspect of the present invention, the promoter is introduced into a uracil-dependent plasmid to express the LDH gene, thereby introducing a recombinant strain with lactic acid-producing ability introduced into YBC, along with related genes capable of producing lactic acid-producing genes and other useful products, including LDH, into the genetic material at a target position in YBC or a mutant strain of YBC using the promoter. In yet another aspect of the present invention, a strain was created in which the gene encoding the gene encoding pirubate decalpoxylase and its associated promoter were deleted from YBC, and other target genes, including Bos taurus-derived LDH, were introduced with high activity into the locus of the target gene, including the pirubate decalpoxylase locus, of the YBC strain using the aforementioned artificial promoter or various modified promoters thereof. In the present invention, the core promoter region of the 5'-UTR of the g4423(ADH) gene is represented by SEQ ID NO: 1 and SEQ ID NO: 2, and the CIT and CLB regions that bind to this core promoter are represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. These can actively bind to constitute the promoter of the present invention. As an example, 2CIT-pg4423, an example of a synthetic promoter bound to two CITs, is shown in SEQ ID NO: 5.

[0018] The promoter of the present invention constitutes a DNA construct that is introduced into yeast together with the gene encoding the target protein. Such DNA constructs include constructs suitable for various known yeast transformation methods, and as an example, an example of a DNA construct for homologous recombination is shown in Figure 1. The DNA construct may consist of a synthetic promoter in the promoter region, particularly a synthetic promoter comprising two CIT and g4423 core promoters. Various target genes, such as the LDH gene, can be used, with Bos taurus-derived LDH (BtLDH) as the target gene and S. cerevisiae-derived CYC1t as the terminator. A cassette can be constructed for introducing the gene while deleting it at various genetic locations of YBC strains, such as the g3002-1 gene. For this purpose, an example in which BtLDH is used as the target gene and 2CIT-pg4423 and the CYC1t terminator are bound is shown in Sequence ID No. 6. Furthermore, when modifying and inserting the target DNA into the aforementioned cassette, or when a target gene location-specific site is inserted into the homologous recombination site, or when a synthetic promoter of a different combination is inserted into the synthetic promoter site, a cassette for inserting the target gene can be created in each case, and this is a well-known fact among researchers with knowledge in this field. Among the configurations for such different combinations, as an example of a synthetic promoter, the 3CIT-pTEF1 promoter, consisting of CIT, the TEF1 gene derived from S. cerevisiae, and a core promoter in the UAS site, is shown in SEQ ID NO: 7, and an example in which BtLDH is bound to the CYC1 terminator as the target gene is shown in SEQ ID NO: 8.

[0019] In the present invention, the cassette is characterized by containing a promoter represented by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7, and the cassette can contain various target genes and can be introduced into the genes of various yeasts, including YBC, by changing the HR site in Figure 1. The promoter of the present invention can constitute a DNA construct to be introduced into yeast together with a gene encoding a target protein. Such DNA constructs include constructs suitable for various known yeast transformation methods, and examples of DNA constructs for introduction in plasmid form are shown in SEQ ID NOs: 9 and 10. The DNA construct is mainly composed of the synthetic promoter, in particular a synthetic promoter consisting of two CITs and a g4423 core promoter, and can constitute a plasmid-form vector that expresses uracil as an auxotroph marker for the expression of various target genes, for example LpLDH, in YBC strains, as shown in SEQ ID NO: 9. Another example of the synthetic promoter is a synthetic promoter mainly composed of three CITs, a TEF1 gene derived from S. cerevisiae, and a core promoter, and can constitute a plasmid-form vector that expresses uracil as an auxotroph marker for the expression of various target genes, for example LpLDH, in YBC strains, as shown in SEQ ID NO: 10. Furthermore, when modifying and inserting target DNA into this cassette, or when auxotrophic markers for introduction into YBC are altered, or when synthetic promoters of other combinations are inserted into the synthetic promoter site, a cassette for the desired gene insertion can be created, and this is a self-evident fact in this field.

[0020] In the present invention, the recombinant strain may be a strain genetically modified to produce lactic acid, as described above, or a strain modified to produce other useful products. Recombinant microorganisms into which the DNA structure of the present invention has been introduced can be cultured to produce organic acids.

[0021] As used herein, “homology” refers to the percentage of identity between two comparison amino acids or polynucleotide molecules. “Similarity” refers to the degree to which sequences are functionally or structurally identical based on amino acid or polynucleotide sequences using a comparison window. Sequence homology or similarity can be verified by comparing sequences using standard software, for example, a program called BLASTN or BLASTX, developed based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993). In the present invention, the g4423 core promoter may include a TATA box and a transcription initiation sequence, and may have a sequence that preferably exhibits 90% or more, 92% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence homology with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A promoter can be considered substantially equivalent if it exhibits 90% or more homology to the g4423 promoter of the present invention while showing equivalent levels of expression efficiency in YBC gene bodies or other yeasts. Depending on the circumstances, the g4423 promoter according to the present invention may be mutated by applying known techniques familiar to the art in order to increase the expression efficiency of the target gene. The UAS sites CIT and CLB used in the same instance may have sequences that exhibit sequence homology of preferably 90% or more, 92% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% with the sequences of sequence numbers 2 and 3. A promoter can be considered substantially homogeneous if it has 90% or more homology to the UAS site of the present invention and, when bound to the explicitly stated core promoter, a known promoter, or a promoter having 90% or more homology thereto, exhibits equivalent levels of expression efficiency in YBC gene bodies or other yeasts. Depending on the circumstances, the UAS region according to the present invention can be mutated by applying known techniques to the promoter in order to increase the expression efficiency of the target gene.

[0022] In the present invention, recombinant yeast is characterized by having acid resistance, and in order to produce acid-resistant recombinant yeast that conforms to the present invention, it is preferable to use a host yeast that has acid resistance to organic acids. The acid-resistant yeast may be an acid-resistant yeast selected from the group consisting of the genera Saccharomyces, Kazaxstania saccharomyces, and Candida, and may, for example, be selected from the group consisting of Saccharomyces cerevisiae, Kazaxstania exigua, Kazaxstania bulderi, and Candida humilis, but is not limited thereto. In the present invention, 'acid-tolerant yeast' is defined as yeast that can maintain a biomass depletion rate (such as sugar depletion rate) or a non-growth rate of at least 10% when the culture medium contains 1M or more of organic acid (especially lactic acid) at a pH below the pKa value of an organic acid, compared to when the culture medium does not contain any organic acid. More specifically, in the present invention, 'acid-tolerant yeast' is defined as yeast that can maintain a biomass depletion rate (such as sugar depletion rate) or a non-growth rate of at least 10% at a pH of 2 to 4, compared to when the pH is 5 or higher. Recombinant yeast according to the present invention can be produced by conventional methods, either by inserting the gene onto the chromosome of a host yeast or by introducing a vector containing the gene into the host yeast. The host yeast typically used is a host cell with high DNA introduction efficiency and high expression efficiency of the introduced DNA. In one embodiment of the present invention, acid-resistant yeast was used, but the invention is not limited to this; any type of yeast is acceptable as long as the target DNA can be sufficiently expressed. The recombinant yeast can be produced by any transformation method. "Transformation" refers to the process of introducing DNA into a host organism so that the DNA can be replicated as a chromosomal component or through the completion of chromosome integration. It is a phenomenon in which genetic changes are artificially induced by introducing external DNA into cells. Common transformation methods include electroporation and lithium acetate-PEG. Furthermore, in the present invention, any commonly known genetic engineering method can be used to insert a gene onto the chromosome of a host microorganism. Examples include methods using retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, poxvirus vectors, lentiviral vectors, and nonviral vectors. A “vector” means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence that can express DNA in a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, be integrated into the genome itself. Currently, plasmids are the most commonly used form of vector, and linearized DNA is also a form commonly used for yeast genome integration. A typical plasmid vector has a structure that includes (a) a replication origin to ensure efficient replication so that each host cell contains the plasmid vector, (b) an antibiotic resistance gene or auxotrophic marker gene to allow selection of host cells transformed with the plasmid vector, and (c) restriction enzyme cleavage sites to allow insertion of foreign DNA sections. Even if suitable restriction enzyme cleavage sites are not present, the vector and foreign DNA can be easily ligated (Gibson assembly) using conventionally synthesized oligonucleotide adapters or linkers, and if necessary, methods for synthesizing and using the desired complete sequence are also commonly employed.

[0023] Furthermore, the aforementioned gene is "operably linked" when it is positioned in a functional relationship with other nucleic acid sequences. This may be a gene and regulatory sequence linked in a manner that enables gene expression when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence. For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-protein participating in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of secretory readers, are in contact and located within the reading frame. However, enhancers do not need to be in contact. Linking of these sequences is performed by ligation at convenient restriction enzyme sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used by conventional methods. Of course, not all vectors function equally well in expressing the DNA sequence of the present invention, and similarly, not all hosts function identically to the same expression system. However, those skilled in the art can appropriately select and apply various other vectors, regulatory sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, the host must be considered because the vector needs to be replicated within that host, and the number of copies of the vector, the ability to regulate the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

[0024] In the present invention, the carbon source may be one or more selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, cellulose, galactose, glucose oligomers, and glycerol, but is not limited thereto. In the present invention, the culture may be carried out under conditions that prevent microorganisms, such as Escherichia coli, from acting further (e.g., making metabolite production impossible). For example, the culture may be characterized by a pH of 1.0 to 6.5, preferably 1.0 to 6.0, and more preferably 2.6 to 4.0, but is not limited thereto. Furthermore, in this invention, the target product and related genes contain organic acids such as succinic acid, adipic acid, acrylic acid, methyl methacrylic acid, 3-hydroxypropionic acid, acetic acid, butyric acid, isobutyric acid, valeric acid, hexanoic acid, isovaleric acid, malic acid, fumaric acid, and itaconic acid, in addition to lactic acid. Moreover, since the wild type of the acid-resistant strain is a yeast capable of fermenting ethanol at high concentrations, it is also advantageous for the production of other alcohols. Therefore, it is possible to genetically modify the strain to efficiently produce alcohols such as butanediol, propanediol, propanol, butanol, isobutanol, and hexanol, and develop high-productivity strains. [Examples]

[0025] The present invention will be described in more detail below with reference to examples. It will be obvious to those of ordinary skill in the art that these examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0026] Example 1: Confirmation of cleavage YBC promoter performance in acid-resistant yeast strains We attempted to discover promoters capable of regulating the expression intensity and expression pattern of target genes using the acid-resistant yeast strain YBC (KCTC13508BP) and genetically engineered and forcibly evolved strains derived from the YBC strain (e.g., KCTC14215BP). Various promoters derived from Saccharomyces cerevisiae (S. cerevisiae S288C) were used, and a 1kb segment from the 5'-UTR region of genes identified as major genes based on annotation in YBC genesets was cleaved and bound to the gene to be expressed. The terminator selected the mCherry gene (SEQ ID NO: 31) as the gene to be expressed by the promoter. The mCherry gene is well expressed in S. cerevisiae, and since the intensity of the color development due to the protein changes depending on the promoter strength, it was thought to have the advantage of being able to easily measure the promoter strength by the degree of color development. However, in the YBC strain, fluorescence was difficult to measure in all transformed colonies. This could be due to poor mCherry expression itself, or it could be that the tested promoter expressed the bacteria very weakly. Therefore, qRT-PCR was performed to compare the promoter strengths. The experimental method was as follows: Each promoter to be tested was introduced into the promoter region of a vector expressing mCherry, which has homologous recombination sites at the 5' and 3' UTR of Ura3, using restriction enzymes (AscI & SbfI). The promoters used were ENO1 / 2, ILV5, EFT2, FBA1, ADH1 (g4423), PDC1, PGI1, TDH3, TEF1, PYK1, and GPM1 from YBC, with the 5' UTR portion of each gene cut into 1kb segments and cloned by PCR. The promoters used from S. cerevisiae were GPM1, ADH1, PYK1, TPI1, ENO2, TDH3, and TEF1. After inserting the respective segments into the YBC gene, each insertion was confirmed by PCR. Cells containing the target promoter were cultured, cDNA was synthesized after RNA prep, and real-time PCR was performed. The concentration of each protein was measured individually, and the expression levels at different concentrations were compared. Figure 2 shows the expression levels of the promoter in question, relative to the expression level of mCherry expressed by TPI1p in S. cerevisiae. Although there are differences in expression levels for each promoter, it was found that the maximum never exceeded 13. Considering that strong promoters typically show expression levels several hundred times higher than housekeeping genes when the original target gene of the promoter is expressed (referencing the expression level of the g4423 gene in Figure 6 of Registered Patent No. 2,140,596 of the Republic of Korea), it was confirmed that the tested promoters could not ensure sufficient expression levels.

[0027] Example 2: Performance comparison of a highly acid-resistant g4423 promoter and a 1kb-cleaved g4423 promoter in an acid-resistant yeast strain. Since the expression of the mCherry gene itself may be weak in YBC strains, in this example, we focused on MCR and the g4423 promoter, which were strongly expressed within YBC. The g4423 gene was strongly expressed in the YBC gene, and the MCR gene was expressed using this promoter. For comparison, the g4423 promoter region was cleaved to 1kb, and the same MCR gene was expressed in the YBC gene. In this case, the promoter and MCR gene were introduced at the ALD2 location in the target gene. The method for preparing the aforementioned strain is as follows: First, when introducing MCR at the g4423 position, the g4423 gene (sequences 11 and 12), which is the main ADH gene of the YBC strain, was removed, and the MCR gene, sequence number 13 from Sulfolobales archaeon Acd 1, was introduced at the g4423 position. Based on the information of the g4423 strain and its UTRs, the ORF of each gene was removed to create a gene cassette containing the 5' and 3' UTRs, which was used as donor DNA. For each allele of g4423, the corresponding 5' UTR is shown in sequence numbers 14 and 15, and the 3' UTR is shown in sequence numbers 16 and 17. As mentioned above, restriction enzyme cloning, Gibson assembly, and gene synthesis methods were used to prepare the donor DNA. As a comparison group, strains were created in which the g4423 promoter was cleaved into 1kb segments (SEQ ID NOs. 18 and 19), bound to the MCR gene (SEQ ID NO. 13), and then introduced into the ald2 position of the YBC gene. For each allele at the ald2 position, the corresponding 5'UTR is shown in SEQ ID NOs. 20 and 21, and the 3'UTR is shown in SEQ ID NOs. 22 and 23. As previously mentioned, restriction enzyme cloning, Gibson assembly, and gene synthesis methods were used to prepare the donor DNA. The prepared bacterial strains were cultured for 120 hours at 30°C and 150 rpm in YP (10 g / L yeast extract, 20 g / L peptone) medium with 15 μM cerulenin and 40 g / L glucose added. For both bacterial strains, the expression level of the MCR gene during culture was confirmed by qPCR, as in Example 1, and the results are shown in Figure 3. As shown in Figure 3, the MCR gene, utilizing the g4423 promoter as is, was strongly expressed and experimentally confirmed to bind to other genes in the 3-hydroxypropionic acid (3-HP) related production pathway, enabling fermentation of 3-HP at a high concentration of 710 mg / L in a flask using glucose as a substrate. In contrast, strains expressing the MCR gene using a 1 kb cleavage promoter of g4423 showed relatively very low MCR expression levels. In 3-HP production flask experiments where it was bound to other 3-HP pathway genes, it was confirmed to produce very low 3-HP concentrations of less than 10 mg / L at the same glucose concentration, confirming that the difference in MCR expression levels directly affects the product concentration of the pathway.

[0028] [Table 2]

[0029] Therefore, we confirmed that the current method of cleaving the target promoter region by 1kb alone is insufficient to utilize the strong promoter within YBC in other gene bodies.

[0030] Example 3: Preparation and activity confirmation of a g4423-based synthetic promoter. To enable the use of the YBC strain-derived promoter in other gene variants of the same strain, and to enhance the performance of other promoters already used in yeast when used in YBC, we attempted to apply a strategy to strengthen the core promoter using UAS in a synthetic promoter form. To achieve this, the core promoter region was further extracted from the 1kb portion of g4423. After identifying common TATA box regions and initiators (Transcription start sites) using various TATA box prediction tools (Eukaryotic core promoter predictor (YAPP), ElementNT-Elements Navigation Tool), the core promoter (SEQ ID NO: 1 and SEQ ID NO: 2) was selected as the region containing these elements. For use as synthetic promoters, we selected UAS genes for CLB2 (mitotic cyclin) and CIT1 (mitochondrial cytrate synthase). The UAS region of CIT (SEQ ID NO: 3) and the UAS region of CLB (SEQ ID NO: 4) were repeated twice and placed on top of the core promoter to create promoters 2CIT-pg4423 (SEQ ID NO: 5) and 2CLB-pg4423, respectively. Furthermore, new synthetic promoters were created by placing CIT and CLB three times at the top of the promoters of TEF1 and TDH3 derived from S. cerevisiae, and these were named 3CIT-pTEF1 (SEQ ID NO: 7) and 3CLB-pTDH3, respectively. Furthermore, pCCW14v5 (Arun S. Rajkumar et al., Nucleic Acids Research, 44(17); e136, 2016), a literature-reported acid-resistant promoter, was used as a comparison group. To express the aforementioned promoters in plasmid form in YBC strains, they were introduced into a vector containing a uracil auxotroph marker (pSK699 (pRS426 variant) from VTT), and the Lactobacillus plantarum-derived LDH gene (SEQ ID NO: 32) was expressed via the promoter. Of the forms introduced into the vector, the plasmid with the g4423 core promoter in the CIT2 repeat is shown in Figure 4A, and the plasmid sequence is shown in SEQ ID NO: 9. Furthermore, the plasmid with the S. cerevisiae-derived TEF1 promoter (SEQ ID NO: 29) in the CIT3 repeat is shown in Figure 4B, and the plasmid sequence is shown in SEQ ID NO: 10. Similar forms were prepared for other promoters and introduced into YBC strains. The vector possesses a Ura marker, and the YBC strain was prepared using uracil auxotrophy, ensuring that only the strain into which the vector was introduced grew on the selective medium. Flask culture evaluation was performed on colonies selected for each plasmid. The culture conditions were as follows: 50 g / L glucose was added to yeast nitrogen base medium (without Uracil) in a 250 ml flask to a total volume of 50 ml. Microorganisms were then inoculated at an initial OD of 0.5 and cultured at 30°C and 250 rpm for 24 hours. The results of the lactic acid production analysis for flask cultures are shown in Table 3.

[0031] [Table 3]

[0032] Table 3 shows a comparison of LDH expression levels using synthetic promoters. 2CIT-pg4423: A synthetic promoter formed by linking two UAS factors (CIT) with a core promoter derived from YBC-derived g4423; 2CLB-pg4423: A synthetic promoter formed by linking two UAS factors (CLB) with a core promoter derived from YBC-derived g4423; 3CIT-pTEF1: A synthetic promoter formed by linking three UAS factors (CIT) with a core promoter from S. cerevisiae-derived TEF1; 3CLB-pTDH3: A synthetic promoter formed by linking three UAS factors (CLB) with a core promoter from S. cerevisiae-derived TDH3; pCCW14v5: A previously reported acid-resistant promoter, used as a reference group. From the flask results mentioned above, it was found that the two active promoters strongly expressed LpLDH, and we attempted to compare their performance by directly inserting the selected 2CIT-pg4423 and 3CIT-pTEF1 into the genomes of acid-resistant bacterial strains.

[0033] Example 4: Confirmation of in vivo expression of 2CIT-pg4423 and 3CIT-pTEF1 in YBC genes. Using two promoters selected from Example 3, we attempted to confirm the performance of the YBC strain-derived promoter by expressing it in the gene of the strain. The YBC gene locus was selected as g3002-1, and the target gene was selected as BtLDH. The reason for changing the target gene from LpLDH, which was tested in the plasmid, to BtLDH is that the researchers discovered that LpLDH expression is suppressed by g3002-1, and this is described in detail in the researchers' prior patent application (Republic of Korea Patent Application No. 2020-0077331). It was determined that it would be difficult to compare the activity of the introduced promoter using the suppressed target gene, so comparative experiments were conducted using BtLDH, whose activity has been confirmed. The BtLDH gene (SEQ ID NO: 30) was placed as the target gene in the above-mentioned selective promoter, and CYC1t derived from S. cerevisiae was used as the terminator. To introduce this gene, the 5'-UTR and 3'-UTR regions of the g3002-1 gene were utilized. To suppress the activity of the original g3002-1 promoter, the gene was introduced either by removing a portion of the 5'-UTR region of the g3002-1 locus, or by introducing the promoter and gene in the opposite direction to the endogenous promoter of the g3002-1 locus. The method for preparing the aforementioned strain is as follows: First, when introducing BtLDH as the target gene in the reverse direction at the g3002-1 position, using 2CIT-pg4423p and 3CIT-pTEF1 as promoters, the g3002-1 gene (SEQ ID NO: 24), which is the main PDC gene of the YBC strain, was removed. The BtLDH target gene, which has the aforementioned promoter and CYC1t as a terminator, was then introduced at the g3002-1 position. For reverse introduction, the complementary sequence of the 3'-UTR region of g3002-1 (Reverse complement, SEQ ID NO: 25) was introduced into the upper part (5'-UTR) of the introduced gene, and the complementary sequence of the 5'-UTR region of g3002-1 (Reverse complement, SEQ ID NO: 26) was introduced into the lower part (3'-UTR) of the introduced gene. Based on this UTR information, a gene cassette was created in which the target genes (SEQ ID NO: 6 and SEQ ID NO: 8) were introduced in the reverse direction while the ORFs of each gene were removed, and this was used as donor DNA. As mentioned above, the donor DNA was prepared using restriction enzyme cloning, Gibson assembly, and gene synthesis methods. First, when introducing BtLDH as the target gene with 2CIT-pg4423p as the promoter in the forward direction at the g3002-1 position, the g3002-1 gene, which is the main PDC gene of the YBC strain, was removed, and the BtLDH target gene, which has the aforementioned promoter and CYC1t as the terminator, was introduced at the g3002-1 position. For forward introduction, the intrinsic promoter was inactivated by introducing a sequence obtained by cutting a portion of the 5'-UTR region, which acts as the intrinsic promoter at the top of the existing g3002-1 gene, as the target site for HR. Similarly, a sequence obtained by cutting a portion of the 3'-UTR region, which acts as the intrinsic terminator at the bottom of the existing g3002-1 gene, was introduced as the target site for HR, inactivating the intrinsic terminator during introduction. The sequences of the UTR region of g3002-1 for this purpose are shown as Sequence ID No. 27 and Sequence ID No. 28 for 5'-UTR and 3'-UTR, respectively. Based on the UTR information, gene cassettes were constructed in which the ORFs of each gene were removed while the target gene was introduced in the forward direction, and these were used as donor DNA. As mentioned above, restriction enzyme cloning, Gibson assembly, and gene synthesis methods were used to prepare the donor DNA. First, we compared the case where 3CIT-pTEF1 was introduced as a promoter with the case where it was introduced while directly replacing the intrinsic g3002-1 promoter. Flask culture evaluation was performed on the selected colonies, under the following conditions: Glucose was added to YPDU medium (20 g / L peptone, 10 g / L yeast extract, 0.15 g / L uracil) to a final concentration of 50 g / L, and the mixture was prepared in a 500 ml baffled flask to a total volume of 50 ml. Microorganisms were then inoculated, and the mixture was cultured for 24 hours at 30°C and 150 rpm.

[0034] [Table 4]

[0035] As shown in Table 4, when 3CIT-pTEF1 was introduced as the promoter, lactic acid expression within the same gene was found to be weaker compared to the inherent promoter. However, considering that it was not easy to measure expression levels within YBC genes when introducing other existing promoters, this result is considered significant as it demonstrates that various promoters from existing yeast can be used within YBC. Next, we compared the case where 2CIT-pg4423 was introduced as a promoter with the case where it was introduced in both the forward and reverse directions, and with the case where it was introduced while directly replacing the intrinsic g3002-1 promoter. Selected colonies were evaluated in flask culture, under the following conditions: Glucose was added to YPDU medium (20 g / L peptone, 10 g / L yeast extract, 0.15 g / L uracil) to a final concentration of 50 g / L, and the mixture was prepared in a 500 ml baffled flask to a total volume of 50 ml. Microorganisms were then inoculated, and the mixture was cultured for 24 hours at 30°C and 150 rpm. As a result, as shown in Figure 5, the expression rate of BtLDH expressed by 2CIT-pg4423 was clearly confirmed by the difference in lactic acid production yield, compared to the expression rate obtained by g3002-1, an endogenous promoter that strongly expresses the major PDC1 gene of the YBC strain. In particular, when introduced in the forward direction while removing the endogenous promoter and terminator region, it was confirmed that expression was achieved at three times the intensity compared to existing promoters, demonstrating that it can be fully used as a strong promoter that can be independently used within YBC, a problem that has been sought to solve in various studies to date. In particular, by presenting a method for creating various synthetic promoters by utilizing other promoters within YBC when using such a synthetic promoter method, it will be possible to utilize it in various ways in the future, and there is also the possibility that the expression intensity can be adjusted by changing the number of UAS repeats. Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Synthetic promoters for the expression of the target gene, including the following: (i) A core promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; and (ii) A UAS (upstream activating sequence) factor consisting of the nucleotide sequence shown in Sequence ID No.

3.

2. The synthetic promoter according to claim 1, characterized in that the UAS factor is located 1 to 4 times repeatedly in the upper part (upsteram) of the core promoter.

3. The synthetic promoter according to claim 1, characterized in that it is a promoter for expressing a target gene in a yeast strain.

4. The synthetic promoter according to claim 3, characterized in that the yeast strain is an acid-resistant yeast.

5. The synthetic promoter according to claim 4, characterized in that the acid-resistant yeast is an acid-resistant yeast selected from the group consisting of the genera Saccharomyces, Casaxtania saccharomyces, and Candida.

6. A DNA structure for expressing a target gene, comprising a synthetic promoter and a target gene according to any one of claims 1 to 4.

7. A recombinant microorganism in which the synthetic promoter described in claim 1 is introduced into the genome, or the DNA structure described in claim 6 is introduced.

8. The recombinant microorganism according to claim 7, characterized in that it is an acid-resistant yeast selected from the group consisting of the genera Saccharomyces, Casaxtania saccharomyces, and Candida.

9. The recombinant microorganism according to claim 7, characterized in that a gene encoding an enzyme capable of producing useful substances has been introduced as the target gene.

10. A method for expressing a target gene, characterized by culturing the recombinant microorganism described in claim 7.

11. A method for producing a useful substance, including the following steps: (a) A step of culturing the recombinant microorganism described in claim 9 and producing a useful substance by expressing a target gene; and (b) The step of obtaining the useful substance that was generated.

12. A recombinant yeast strain having lactic acid production ability, wherein in acid-resistant yeast YBC strain (KCTC13508BP), both the PDC gene, which encodes pirubate decarboxylase, and the promoter of the PDC gene are removed from the genome, and the synthetic promoter described in claim 1 and the gene encoding lactate dehydrogenase are introduced at the position where the PDC gene was removed, and the expression of lactate dehydrogenase can be regulated by the synthetic promoter.

13. A method for producing lactic acid, including the following steps: (a) the step of culturing the recombinant strain described in claim 12 to produce lactic acid; and (b) The step of obtaining the lactic acid produced.

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