Recombinant acid-tolerant yeast with improved lactic acid production

Abstract

Disclosed is a recombinant acid-tolerant yeast having improved lactic acid production ability and a method for producing lactic acid using the same. When lactic acid is produced using the recombinant acid-tolerant yeast according to the present invention, not only can a significantly smaller amount of neutralizing agent be used to perform lactic acid fermentation with a similar lactic acid production ability to bacterial fermentation, but also the production of by-products, ethanol and glycerol, can be reduced. Accordingly, fermentation costs can be greatly reduced, and the costs of subsequent purification processes can also be reduced.

Description

Technical Field

[0001] The present invention relates to a recombinant acid-tolerant yeast having an enhanced ability to produce lactic acid and a method for producing lactic acid using the same. More specifically, the present invention relates to a recombinant acid-tolerant yeast in which a specific, bacterially derived lactate dehydrogenase gene has been introduced at a site where a gene encoding pyruvate decarboxylase has been deleted, and a method for producing lactic acid using the recombinant acid-tolerant yeast. Background Art

[0002] Polylactic acid (PLA) is a biodegradable polymer produced by converting lactic acid into lactide and then performing a ring-opening polymerization reaction on it. The raw material for PLA, lactic acid, is produced by fermentation. PLA is widely used in disposable food containers and has the advantage of being able to be used alone or in the form of a combination or copolymer in plastics for various industries, including the automotive and fiber industries. Furthermore, it is a representative example of a polymer used in 3D printing in recent years and is an environmentally friendly polymer that produces low levels of harmful gases and odors when used in 3D printers.

[0003] The traditional lactic acid production process is carried out using lactic acid bacteria and includes fermentation while maintaining a neutral pH of 6 to 8 using various forms of Ca salts / Ma salts or neutralizing agents such as ammonia to prevent bacterial death or slowed growth due to lactic acid produced and accumulated by the lactic acid bacteria. When the fermentation is complete, the microorganism is separated and sulfuric acid is added to convert lactate into lactic acid. At the same time, due to the difficulty in separating salts from water and converting lactic acid into lactide, the Ca salt is removed in the form of CaSO 4. In this method, the amount of the by-product CaSO 4 produced is greater than the amount of lactic acid, thereby reducing the process efficiency.

[0004] Typically, PLA is fermented to produce lactic acid, which is then converted to lactide through a purification process. This conversion requires a process to convert the lactic acid to a hydrogenated form. Since the pH of neutral fermentation is typically between 6 and 7, large amounts of sulfuric acid are used to shift the neutral pH to an acidic one. This process produces a large amount of neutralizing salts, which, due to their low value and the investment costs of removing them, reduces economic viability.

[0005] Lactic acid has both L- and D-type optical isomers. A wide variety of microbial species exist. For example, lactic acid bacteria that primarily produce the L-type optical isomer typically also produce approximately 5-10% of the D-type optical isomer. Strains that primarily produce the D-type optical isomer include strains that produce both the D-type and the L-type optical isomer, and strains that produce both the D-type optical isomer and ethanol (Ellen I. Garvie, Microbiological Reviews, 106-139, 1980).

[0006] Meanwhile, with regard to the lactobacillus (Lactobacillus) that naturally produces lactic acid, in order to produce lactic acid commercially, a large amount of expensive nutrients must be used as culture medium. These excessive nutrients greatly hinder the downstream polymerization process or the lactide conversion process carried out when lactide is used as an intermediate. In order to obtain high yield (yield) and highly purified polymer or its precursor, the cost of the purification process such as adsorption, distillation and ion exchange is produced, thereby further increasing production cost. In order to address these problems, the research using yeast has been proposed. As is well known, even if cheap nutrients are used, yeast can also grow / ferment and also has high acid resistance.

[0007] When lactic acid is produced using yeast that thrives in acid (hereinafter referred to as "acid-tolerant yeast"), there is no need to use neutralizers to maintain the pH of the culture medium at 6 to 7 during fermentation. This simplifies the fermentation process and eliminates the need for downstream purification steps to remove neutralizers. Furthermore, yeast naturally produces many of the components required for metabolism, allowing it to be cultured in media with relatively low nutrient levels compared to bacteria (particularly lactobacilli), thus avoiding downstream purification steps and significantly reducing production costs.

[0008] However, using yeast to produce lactic acid has certain technical requirements. The requirement is that the yield, productivity, and concentration of lactic acid, which are indicators of the fermentation performance of the strain, must be maintained at a high level similar to that of lactic acid bacteria in order for the technology to be commercially applicable.

[0009] Although technology for producing lactic acid using acid-tolerant yeast has been developed, in practice, in many cases, high fermentation performance can only be exhibited when the pH value is maintained at at least 3.7 (not lower than the pKa value of lactic acid) by performing a neutralization reaction during fermentation. Therefore, it is unreasonable to identify this technology as a practical method for achieving acid resistance, and it is difficult to expect the effect of reducing production costs in the process (Michael Sauer et al., Biotechnology and Genetic Engineering Reviews, 27: 229-256, 2010).

[0010] Therefore, acid-tolerant yeasts that can reduce process costs can only be commercially applied if they can complete fermentation without using neutralizers or using neutralizers in minimal amounts when the pH of the fermentation solution does not exceed the pKa value, and the three main fermentation indicators reach levels similar to those of lactic acid bacteria.

[0011] Typically, when fermenting glucose, yeast produces ethanol as the main product, glycerol as a byproduct, and virtually no lactic acid. Furthermore, since it is very difficult to select a lactic acid-producing strain from microorganisms with high acid tolerance, the present inventors selected a yeast strain with excellent acid tolerance and attempted to construct a strain through genetic engineering that is endowed with the ability to produce lactic acid while suppressing the production of ethanol and glycerol.

[0012] Therefore, as a result of efforts to produce yeast strains that have strong acid resistance while showing lactic acid production ability (lactic acid production rate and concentration) similar to that of bacterial strains and a reduced ability to produce by-products ethanol and glycerol, the present inventors found that lactic acid production is improved due to increased lactate dehydrogenase activity by introducing a lactate dehydrogenase gene derived from Staphylococcus epidermidis at the position of the gene encoding pyruvate conversion enzyme in acid-tolerant yeast, and based on this finding, the present invention was completed. Summary of the Invention

[0013] Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide a recombinant strain having an increased lactic acid production rate and an increased lactic acid concentration, which is constructed by imparting enhanced tolerance to high concentrations of lactic acid to a recombinant acid-tolerant yeast strain having lactic acid production ability.

[0014] Another object of the present invention is to provide a method for producing a recombinant yeast strain having lactic acid production ability and enhanced lactic-acid resistance using an adaptive evolution method.

[0015] Another object of the present invention is to provide a recombinant yeast strain produced by this method and having an improved lactic acid production ability in a high-concentration lactic acid culture medium.

[0016] According to one aspect of the present invention, the above and other objects can be achieved by providing a recombinant strain having the ability to produce lactic acid, which is constructed by deleting the gene encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain (KCTC13508BP) and introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis in the position of the gene encoding pyruvate decarboxylase.

[0017] According to another aspect of the present invention, a recombinant strain having the ability to produce lactic acid is provided, the recombinant strain being constructed by deleting the GPD1 gene encoding an enzyme that converts dihydroxyacetone phosphate to glycerol-3-phosphate, the CYB2 gene encoding an enzyme that converts lactate to pyruvate, the ADH gene encoding alcohol dehydrogenase, and the PDC gene encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain (KCTC13508BP), and introducing a gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain.

[0018] wherein the gene encoding lactate dehydrogenase is introduced into the position of the deleted ADH gene, the position of the deleted PDC gene and the position of the deleted GPD1 gene, and

[0019] The gene encoding lactate dehydrogenase introduced in place of the PDC gene is a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis.

[0020] According to another aspect of the present invention, a method for producing a recombinant strain with improved lactic acid tolerance and improved lactic acid production ability is provided, the method comprising: (a) inducing adaptive evolution of the recombinant yeast strain toward high lactic acid concentration by sequentially culturing the recombinant yeast strain with lactic acid production ability from a low-concentration lactic acid medium to a high-concentration lactic acid medium; (b) selecting a recombinant yeast strain with improved lactic acid production ability in the high-concentration lactic acid medium; and (c) introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis at the position of the PDC gene in the genome of the selected strain.

[0021] According to another aspect of the present invention, a recombinant strain #26-5 (accession number: KCTC 14215BP) is provided, which is constructed by adaptive evolution of a recombinant strain with lactic acid production ability under high lactic acid concentration. The recombinant strain with lactic acid production ability is constructed by deleting the GPD1 gene (which encodes an enzyme that converts dihydroxyacetone phosphate into glycerol-3-phosphate), the CYB2 gene (which encodes an enzyme that converts lactate into pyruvate), the ADH gene (which encodes alcohol dehydrogenase), and the PDC gene (which encodes pyruvate decarboxylase) from the acid-tolerant yeast YBC strain (KCTC13508BP), and introducing the gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain.

[0022] According to another aspect of the present invention, a recombinant yeast YBC6 strain is provided, which is constructed by introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis at the position of the PDC gene in the genome of the recombinant strain #26-5 (accession number: KCTC 14215BP), wherein the recombinant yeast YBC6 strain has an improved lactic acid production ability and a reduced ethanol and glycerol production ability under high lactic acid concentration compared with the YBC strain (KCTC13508BP) or the YBC5 strain.

[0023] According to another aspect of the present invention, there is provided a method for producing lactic acid, comprising: (a) culturing the strain to produce lactic acid; and (b) collecting the produced lactic acid.

[0024] Effects of the present invention

[0025] When lactic acid is produced using the recombinant acid-tolerant yeast according to the present invention, significantly less neutralizing agent can be used than in conventional bacterial fermentation to produce lactic acid at a similar lactic acid production capacity, thereby significantly reducing fermentation costs. Furthermore, the production of byproducts such as ethanol and glycerol can be reduced, thereby reducing the cost of subsequent purification processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0027] Figure 1 A schematic diagram illustrating the process of forced adaptation of the YBC5 strain to increase tolerance to lactic acid at various lactic acid concentrations according to the present invention, specifically, Figure 1(a) shows the growth of cells during subculture in the second round of adaptive evolution to ensure that strain #26-5 was selected during the lactate concentration adaptation process, Figure 1 (b) shows the growth of cells at lactate concentrations of 70 g / L to 80 g / L during subculture in the third round of adaptive evolution to further improve tolerance to lactate.

[0028] Figure 2 Shown are the results of comparison of lactic acid production curves between strain #26-1 and strain #26-5, which are two strains selected in the second round of adaptive evolution according to the present invention, and strain #26 was selected in the first round of adaptive evolution.

[0029] Figure 3 shows the fermentation curve of strain #26-5, which was selected from a colony conferred with improved lactic acid tolerance by adaptive evolution of the YBC5 strain according to the present invention;

[0030] Figure 4 Shown are the results of a comparison of fermentation curves between strain #26-5 and strain YBC5 according to the present invention;

[0031] Figure 5 Shown are examples of cassettes for deleting the PDC1 (g3002-1) gene from the genome of strain #26-5, which is an improved variant of the YBC strain, the YBC1 strain, and the YBC5 strain, for inserting an LDH gene at the position where the corresponding gene is deleted, or for exchanging an already inserted LDH gene with another LDH gene;

[0032] Figure 6 shows the fermentation curve of YBC6, which is a strain constructed by introducing LDH derived from Staphylococcus epidermidis into the #26-5 strain, which was selected from a strain flora endowed with improved lactic acid tolerance by adaptive evolution of the YBC5 strain according to the present invention;

[0033] Figure 7 The results of comparison of the lactic acid production capabilities of the YBC5 strain, the #26-5 strain, and the YBC6 strain during fermentation according to the present invention are shown. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Generally, the terms used in this specification are well known in the art and are commonly used in this art.

[0035] Acid-tolerant yeast is characterized by consuming sugar at a high rate even at acidic pH, showing a high growth rate, and converting the consumed glucose into the desired product under fermentation conditions. In the inventors' previous research, an acid-tolerant yeast strain (KCTC13508BP) was selected from yeasts with these characteristics in several yeast libraries. The acid-tolerant yeast strain (KCTC13508BP) had a high growth rate and a high sugar consumption rate even at lactic acid concentrations of 40 g / L to 80 g / L (Korean Patent Application No. 10-2018-0044509).

[0036] In the inventors' previous patent application, the lactic acid production ability of the acid-tolerant yeast YBC strain was improved and its ethanol production ability was suppressed by controlling the metabolic circuit of the strain. The inventors obtained a recombinant strain by deleting the gene encoding the cytochrome b2 enzyme that converts lactate into pyruvate from the strain obtained as follows: deleting the gene encoding alcohol dehydrogenase and the gene encoding pyruvate decarboxylase from the YBC strain, and introducing the lactate dehydrogenase gene into the YBC strain.

[0037] Furthermore, in order to suppress the production of glycerol in the constructed strain, the present inventors constructed a recombinant strain by deleting a gene encoding glycerol-3-phosphate dehydrogenase (which converts phosphate hydroxyacetone into glycerol 3-phosphate) from the strain.

[0038] In the present invention, in order to restore the lactic acid tolerance of the recombinant strain, the recombinant strain was subcultured in a culture medium containing various concentrations of lactic acid up to 80 g / L, and a strain with excellent lactic acid tolerance was selected. The exogenous lactate dehydrogenase gene replaced at the PDC genomic position of the selected strain was replaced with a lactate dehydrogenase gene derived from Staphylococcus epidermidis to construct a new recombinant strain, and it was found that the recombinant strain had high lactic acid tolerance, high lactic acid production ability and inhibited ethanol and glycerol production ability.

[0039] Therefore, on the one hand, the present invention relates to a recombinant strain having the ability to produce lactic acid, which is constructed by deleting the gene encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain (KCTC13508BP) and introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis in the position of the gene encoding pyruvate decarboxylase.

[0040] In the present invention, the gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis can be represented by SEQ ID NO: 1.

[0041] In the present invention, the recombinant strain is characterized by further deletion or inactivation of a gene encoding alcohol dehydrogenase, and further deletion or inactivation of a gene encoding an enzyme converting dihydroxyacetone phosphate into glycerol-3-phosphate.

[0042] In the present invention, the recombinant strain is characterized by further deletion or inactivation of a gene encoding an enzyme that converts lactate into pyruvate.

[0043] In another aspect, the present invention relates to a recombinant strain capable of producing lactic acid, which is constructed by deleting the GPD1 gene (which encodes an enzyme that converts dihydroxyacetone phosphate into glycerol-3-phosphate), the CYB2 gene (which encodes an enzyme that converts lactate into pyruvate), the ADH gene (which encodes alcohol dehydrogenase), and the PDC gene (which encodes pyruvate decarboxylase) from the acid-tolerant yeast YBC strain (KCTC13508BP), and introducing a gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain.

[0044] wherein the gene encoding lactate dehydrogenase is introduced into the position of the deleted ADH gene, the position of the deleted PDC gene and the position of the deleted GPD1 gene, and

[0045] The gene encoding lactate dehydrogenase introduced at the position of the deleted PDC gene is a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis.

[0046] In the present invention, the gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis is represented by SEQ ID NO: 1, and its protein sequence is represented by SEQ ID NO: 2, wherein codon usage is adjusted for gene expression in the acid-resistant YBC strain.

[0047] In the present invention, the gene encoding lactate dehydrogenase introduced at the position of the deleted ADH gene and the position of the deleted GPD1 gene may be derived from Staphylococcus epidermidis or Lactobacillus plantarum.

[0048] Recombinant strains with lactic acid production ability show high lactic acid production. However, the huge changes in lactic acid produced in large quantities in the cell and the carbon flux in the cell affect the redox balance and cell growth and regulatory mechanisms, resulting in changes in cell growth rate and sugar consumption rate (and ultimately, lactic acid production rate) reduction. The reasons why genetic engineering causes a decrease in lactic acid tolerance are as follows. Traditional wild-type microorganisms are strains that can grow well even in an environment where the extracellular lactic acid concentration is high (40-80 g / L) and the pH is 2 to 3 (lower than pKa). This strain actively produces lactic acid in the cell after genetic engineering, so the production of lactic acid is inhibited by the lactic acid produced in the cell and the lactic acid that penetrates the cell membrane (by mass transfer) from outside the cell. The increased lactic acid concentration in the cell reduces the pH of the cell, thereby showing the effect of inhibiting various intracellular activities, including gene replication and protein production, resulting in reduced lactic acid tolerance.

[0049] In addition, when extracellular lactic acid concentration increases or external pH acidity is higher, therefore when most of total lactic acid exists in hydrated form, this effect becomes stronger.In order to solve the problem brought by recombinant strain, bacterial strain is continuously cultured in target environment, such as adaptive evolution / forced evolution, constantly selects cells modified while adapting to the environment to improve performance (Zhengming Zhu et al., Applied Microbiology and Biotechnology, 102:4615-4627,2018;Eugene Fletcher et al., Metabolic Engineering 39(2017)19-28,2017;Christopher P Long, Current Opinions in Chemical Engineering, 22:209-215,2018). Compounds causing mutations or physical factors causing mutations (such as ultraviolet rays) can be used for this forced evolution (Zhengming Zhu et al., Applied Microbiology and Biotechnology(2018) 102:4615-4627). Initially, attempts were made to apply other methods rather than the adaptive evolution described above in the present invention. However, random mutations that simultaneously increase desired performance aspects (e.g., acid tolerance) and other performance aspects (e.g., productivity) are very rare. Although approximately 100 strains were monitored individually, it was difficult to select useful strains from them. Considering the need to develop automated high-throughput systems capable of selecting superior colonies and systems capable of culturing and mutating from 10 strains to 10 strains, it is not feasible to develop a system that can select superior colonies. 8Genetic systems that detect superior colonies in a cell population with colonies / mL or higher (e.g., fluorescent reporters proportional to LDH expression levels) make it difficult to expand the panel for selection.

[0050] Therefore, an adaptive evolution method is used, rather than a method using mutants, to continuously culture cells of the recombinant yeast strain under high sugar and high lactic acid concentrations, and when the cells grow well, the lactic acid concentration increases. In addition, the process including inoculating the corresponding cells on a solid medium containing lactic acid in the middle stage of the culture, selecting colonies with a high growth rate (with a large size) from the solid medium, and testing the lactic acid production ability of the colonies using flasks is repeated. The selected strains are directly compared with the parent strain of the culture. When this operation is repeated, a strain with improved lactic acid production ability and the desired lactic acid tolerance can be selected. The improvement in fermentation performance was detected by fermentor-based culture (see Figure 7 ).

[0051] In another aspect, the present invention relates to a method for producing a recombinant yeast strain with improved lactic acid tolerance and improved lactic acid production ability. The method comprises: (a) inducing adaptive evolution of the recombinant yeast strain toward high lactic acid concentrations by sequentially culturing the recombinant yeast strain with lactic acid production ability in a low-concentration lactic acid medium to a high-concentration lactic acid medium; (b) selecting a recombinant yeast strain with improved lactic acid production ability in the high-concentration lactic acid medium; and (c) introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis into the genome of the selected strain at the position of the PDC gene.

[0052] In the present invention, the recombinant yeast strain having the ability to produce lactic acid in step (a) is a YBC5 strain constructed by deleting the GPD1 gene (which encodes an enzyme that converts dihydroxyacetone phosphate into glycerol-3-phosphate), the CYB2 gene (which encodes an enzyme that converts lactate into pyruvate), the ADH gene (which encodes alcohol dehydrogenase) and the PDC gene (which encodes pyruvate decarboxylase) from the acid-tolerant yeast YBC strain (KCTC13508BP), and introducing the gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain, wherein the gene encoding lactate dehydrogenase is introduced into the position of the deleted ADH gene, the position of the deleted PDC gene and the position of the deleted GPD1 gene.

[0053] On the other hand, the present invention relates to a recombinant strain #26-5 (accession number: KCTC 14215BP), which is constructed by adaptive evolution of a recombinant strain having the ability to produce lactic acid under high lactic acid concentration, wherein the recombinant strain having the ability to produce lactic acid is constructed by deleting the GPD1 gene (which encodes an enzyme that converts dihydroxyacetone phosphate into glycerol-3-phosphate), the CYB2 gene (which encodes an enzyme that converts lactate into pyruvate), the ADH gene (which encodes alcohol dehydrogenase), and the PDC gene (which encodes pyruvate decarboxylase) from the acid-tolerant yeast YBC strain (KCTC13508BP), and introducing a gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain.

[0054] On the other hand, the present invention relates to a recombinant yeast YBC6 strain, which is constructed by introducing a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis at the position of the PDC gene in the genome of the recombinant strain #26-5 (accession number: KCTC 14215BP), wherein the recombinant yeast YBC6 strain has improved lactate production ability and inhibited ethanol and glycerol production ability under high lactate concentration compared with the YBC strain (KCTC13508BP) or the YBC5 strain.

[0055] Adaptive evolution is a very powerful tool, but it can produce side effects. The adaptive evolution carried out in the present invention is to select a bacterial strain that is suitable for growing well in the presence of high concentrations of lactic acid. Even if the adaptive microorganism selected in this process is high in the concentration of free lactic acid, it can also grow, and even if obtained in the presence of lactic acid, it also has a defense mechanism for lactic acid concentration and promotes sugar metabolism, because the bacterial strain with high lactic acid production ability is selected in the selection process. Even when the concentration of lactic acid in the reactor increases during the fermentation culture, this feature of the selected bacterial strain can also increase the concentration of microorganisms in the reactor, and this high-concentration microorganism can increase the total lactic acid production rate (production rate). In particular, the parent strain used in the present invention is a bacterial strain that grows at a very fast speed under high concentrations of lactic acid and has a high glucose consumption rate, which is a feature before genetic manipulation. Therefore, adaptive evolution can, in the process of converting carbon flux from ethanol to lactic acid by genetic manipulation, in the presence of lactic acid, restore the lactic acid tolerance and the growth rate that decreases therewith. However, considering this adaptive evolution process from the perspective of microorganisms, microorganisms will inevitably select several directions. From the perspective of microorganism, under the acidic pH lower than pKa, when the external concentration of lactic acid increases, uncombined hydrated lactic acid is transferred and penetrates into the cell, which reduces the pH in the cell. Under the very large state of the effect of this lactic acid, when selecting the fermentation product (carbon flow) that can produce ATP and NADH and convert the sugar as growth substrate simultaneously, while carrying out DNA replication and protein production, promote growth and accelerate sugar metabolism to improve the evolutionary direction of lactic acid production performance (for example, production rate), it has the effect identical with external coercion, can not be the direction of natural selection, but for organism, it is very natural to select a direction, inducing the fermentation product that the influence of current stressor is less than the influence of lactic acid on this direction. In addition, the lactic acid production pathway in the parent strain used in the present invention is a pathway introduced from the outside, and wild-type microorganism is initially a microorganism grown while producing the ethanol as main by-product, so the carbon pathway that is strengthened to promote growth and increase lactic acid tolerance is obviously ethanol, and has determined to have increased the by-product ethanol of lactic acid production process.

[0056] To analyze the genetic factors that promote increased lactate tolerance and byproduct production induced by adaptive evolution, qPCR analysis (transcriptome analysis) was performed on microorganisms before and after adaptive evolution. In addition to qPCR analysis, the entire genome can be analyzed to detect differences between them. However, many genes in the genome have been modified by mutations, but in fact, only some of these genes are factors that appear as phenotypes through protein expression or their regulatory mechanisms. For this reason, it is believed that qPCR analysis is more suitable for discovering phenotypic genetic factors such as increased tolerance and increased growth rate.

[0057] To identify genes with differential expression in transcriptome analysis, all analyzed RNA sequences were screened for genes whose expression fold change in the adaptively evolved strains was less than or equal to twofold, or more than twofold, compared to the expression rate in the wild-type strain, and the corresponding genes were then analyzed using annotations (see Table 6).

[0058] In the gene pool (gene pool) that expression reduces, a specific gene is the LDH gene of encoding lactate dehydrogenase.As mentioned above, selection increases the microorganism of lactic acid concentration, the fastest growth rate and lactic acid production rate (in some cases, the highest lactic acid production concentration), but the preferred response of the microorganism that can respond to the oxidative stress caused by lactic acid or the inhibition of acidic pH is to reduce the lactic acid amount produced inside.In the adaptive evolution process, the minimizing of expression accumulates in microorganism.Certainly, selected bacterial strain also has excellent lactic acid production ability, but this is mainly considered to be the rapid increase of the microorganism concentration that causes due to the increase of sugar consumption rate or sugar transfer rate in fast growth rate and cell.Think, due to the minimizing of this expression, the lactic acid production ability of each cell reduces.

[0059] As a result of analyzing the gene pool with increased expression, it was observed that the expression of several gene groups performing the same function increased together. These genes were classified as follows.

[0060] Category A, related to fermentation products, is a group of genes involved in ethanol production, with the goal of increasing carbon flux to achieve growth rate, in addition to reducing lactate dehydrogenase expression. In particular, when examining the activities of conventional PDC (the gene encoding pyruvate decarboxylase) and ADH (alcohol dehydrogenase), genes that were tested as candidate genes but failed to show the expected activity were found to have been enhanced during the current adaptive evolution. Category E is related to hexose transporters, a group of genes associated with proteins that primarily act on C6 sugars (i.e., sugars such as glucose and mannose) and transport them into cells. Category B is a group of zinc finger proteins. Studies have reported that lactate-induced oxidative stress affects functional groups associated with zinc finger proteins, or that the enhancement associated with zinc finger proteins involves a reduction in reactive oxygen species (ROS) (Derek A. Abbott et al., Applied and Environmental Microbiology, 2320-2325, 2009). The relationship between oxidative stress, including lactate, and zinc finger proteins is very high (Xixi Zhou et al, The journal of biological chemistry, 290: 18361-18369, 2015; B Gao et al., Cell Death and Disease, 5: e1334, 2014; Ananda S. Prasad and Bin Bao, Antioxidants 8: 164, 2019). It is predicted that the acid resistance of the selected strain is partially related to these zinc finger proteins. Category D is a group of genes related to sulfate / sulfite. Specifically, increased expression of sulfate / sulfite reductase was observed, which is believed to be a mechanism that mitigates sulfate / sulfite-mediated oxidative stress through lactate-mediated oxidative stress. Furthermore, gene expression supporting structural and cellular responses to external stress was identified and collectively referred to as category D stress responses.

[0061] Through transcriptome analysis, various characteristics of the strain of the present invention were determined, and additional development methods can be designed when the characteristics of each gene are studied in further studies including reverse engineering in the future.

[0062] Transcriptome analysis did not detect an increase in the expression of genes related to glycerol production, but from the phenotype identified by fermentation culture, it was detected that the strain selected by adaptive evolution also showed increased glycerol production compared to the parent strain. Therefore, it is believed that the increase in glycerol is caused by the regeneration of NADH through the glycerol production pathway due to LDH inactivation, rather than insufficient NADH regeneration caused by the expression of related genes. Therefore, when LDH is activated (enhanced), glycerol will decrease again.

[0063] In short, due to the increase in lactic acid tolerance, more microorganisms can be obtained in the fermentation tank within the same time period, and due to the increase in sugar metabolism rate, the fermentation rate increases. However, at the same time, due to the increase in lactic acid tolerance, an increase in the by-products of the lactic acid process is observed. This phenomenon is due to the reduction in the expression of LDH, thereby increasing the expression of glycerol and ethanol production-related genes, resulting in increased tolerance. As a method to solve this problem, it is possible to consider enhancing LDH and removing ethanol production-related genes. In the present invention, first, by further expressing LDH, the production rate in each cell increases to further increase the total production rate, and by reducing the coenzyme NADH with LDH, the reduction of NADH responsible for the glycerol pathway is reduced, so that the level of glycerol increase is reduced, and by improving the lactic acid production ability, which directly competes with the ethanol production ability using pyruvic acid as a precursor node in the metabolic pathway, the production of ethanol is reduced. Thereafter, if necessary, the extra expressed ethanologenic genes can be removed.

[0064] In one aspect of the present invention, the bacterial strain undergoing adaptive evolution is the YBC5 bacterial strain, wherein the LDH gene derived from Lactobacillus plantarum is replaced at the g4423 (ADH), g3002-1 (PDC) and g2947 (CYB2) sites of the YBC5 bacterial strain genome, and due to the diploid characteristics of the YBC bacterial strain, a total of 6 gene copies have been inserted. In many cases, when multiple copies of the same gene are inserted, due to the feedback inhibition of the cell, the expression of the same gene is suppressed, and therefore the effect of increasing in proportion to the copy number is not brought into play, and the presence of the same gene may affect the stability of the genome. For this reason, the present invention has found a new method for enhancing LDH. As described in the inventors' prior patent applications (Korean Patent Application No. 2018-0044509 and Korean Patent Application No. 2019-0124701), LDH inserted at the g4423 (ADH) site of the YBC5 strain exhibited very high activity, and due to the influence of the g2947 promoter, LDH inserted at the g2947 (CYB2) site also exhibited sustained activity in the late fermentation period. However, LDH inserted at the g3002-1 (PDC) site phenotypically exhibited a relatively low effect of increasing lactic acid production capacity. In particular, qPCR showed that the PDC inherent to YBC exhibited a high expression rate through the highly active promoter of g3002-1 (PDC). Therefore, further research was conducted on the related phenomena.

[0065] First, the lactic acid production capacity was determined by removing g3002-1 from the wild-type YBC strain and introducing an LDH derived from plantarum. It was observed that this strain showed a good phenotype by removing the PDC, but produced a very small amount of lactic acid. Considering that the LDH derived from plantarum was strongly expressed due to g4423, the extreme differences in expression of the same gene at different genomic locations within the same strain are not a common phenomenon. When LDH is expressed in RNA, it is expected that RNA translation into protein will proceed smoothly. In short, it is assumed that the same gene is not transcribed at the corresponding PDC site. In order to solve this phenomenon, the inventors attempted to establish various hypotheses, but did not find a hypothesis to solve this problem. Therefore, the inventors hypothesized that the smooth expression was suppressed depending on the genomic site and gene structure problem of the LDH derived from plantarum. To solve this problem, an LDH derived from another strain was introduced to change the genomic structure, thereby promoting the expression of LDH.

[0066] For the target LDH to be introduced, selection is to have the gene that is suitable for acid-resistant bacterial strain feature with optimal acid pH and excellent expression in yeast. Considering necessary resources, it is almost impossible to find these genes from the numerous genes existing in nature using screening alone, and is a task that needs genome mining and experimental verification and many hypotheses. The present inventor first attempts to select the gene with similar characteristics from the literature, rather than carrying out such genome mining, injecting it into the g3002-1 site of the corresponding acid-resistant bacterial strain, and then measuring its activity. The present inventor attempts to find the gene verified under similar conditions before by literature search, and targets the LDH derived from Staphylococcus epidermidis and the LDH derived from cattle (Bos taurus), which is found to be effective in the gene tested in the subject document (Jae Won Lee et al, J.Biotecho 241, 2017).

[0067] After the three target genes were introduced into the g3002-1 site of the wild-type YBC strain (KCTC13508BP), the lactic acid production capacity was compared between them. As a result, a very interesting fact was discovered. The activity differences between the three genes were very large. Among them, the LDH (SeLDH) gene derived from Staphylococcus epidermidis showed a 39-fold increase in activity based on lactic acid production compared to the LDH (LpLDH) derived from Lactobacillus plantarum. As a result, the SeLDH gene alone obtained a yield of 0.39g / g at the g3002-1 site, and its activity was comparable to that of the LpLDH previously inserted at the g4423 site. As a result, the inventors obtained a method that can ensure high activity at the g3002-1 site and restore the LDH activity of adaptively evolved strains.

[0068] However, among various LDHs, the LDH gene of Staphylococcus epidermidis exists in the form of FDP-activated LDH, which requires FDP as a coenzyme (EI Garvie, Bacterial Lactate Dehydrogenases, Microbiological Reviews, 1980), and this FDP is an intermediate product of glycolysis. Therefore, compared with using common sugars such as glucose, fructose and sucrose as substrates, when other sugars are used, the activity of the LpLDH gene is likely to be affected by the availability of FDP, and its activity is likely to be affected. However, because the substrate mainly used in the commercial process is corn starch, which is mainly composed of glucose, fructose and sucrose and their saccharification products, sugarcane juice and its by-products, this limitation is minimized.

[0069] In one aspect of the present invention, the SeLDH gene was inserted into a strain selected by adaptive evolution, and the results showed that the strain showed a further increase in yield from 0.67 g / g to 0.75 g / g, and showed a substantial reduction in by-products as expected while achieving a further increase in productivity from 2.54 g / L / hour to 2.56 g / L / hour and an increase in concentration from 123 g / L to 130 g / L.

[0070] The ATP-related requirements of acid-resistant strain fermentation were found in the culture of the strain obtained by the present invention, as described in the existing literature (Antonius JA van Maris et al., Appl. Environ. Microbiol., 70; 2898, 2004). In other words, as the extracellular lactic acid concentration increases, energy is required to transfer the intracellular lactic acid to the outside, and due to the consumption of ATP, the fermentation requires an ATP supply. The consumption of ATP is due to the production of acid-resistant lactic acid (acid-resistant lactic acid) when 2ATP / glucose is added, which can be ensured by forcing the production strain to produce lactic acid while blocking the oxygen therein in general fermentation. Therefore, under the anaerobic conditions of general fermentation, the energy required for cell maintenance is insufficient. Oxygen supply is needed to supplement energy, and the supplied oxygen completely oxidizes the substrates in the TCA pathway to supply energy ATP, but at the same time, some substrates are converted into CO2 instead of lactic acid, resulting in a decrease in lactic acid production. Therefore, it is necessary to set optimal ventilation conditions to minimize the loss of lactic acid production while maintaining cell activity.

[0071] In addition, bacterial strain of the present invention is both yeast and Gram-positive bacterial strain, so this should be considered when setting fermentation method. As is well known, under high sugar concentration, even under aerobic conditions, Gram-positive bacterial strains also can experience anaerobic fermentation reaction, such as ethanol fermentation or lactic acid fermentation, rather than TCA fermentation. By contrast, Gram-negative bacterial strains can suppress the generation of fermentation products under aerobic conditions, so can only increase cells, and cell growth phase and fermentation product production phase can be carried out separately. However, these Gram-negative bacterial strains cause cell growth under aerobic conditions, and can ensure the high cell concentration in the fermentor tank, but cells can not be infinitely increased to improve fermentation rate, because many substrates consumed under aerobic conditions are converted into CO along with cell growth (in addition, it is natural that cell concentration is limited according to the nutrients and limiting substrates (limitsubstrate) of culture medium). On the other hand, Gram-positive bacterial strains grow cells under aerobic conditions and also produce fermentation products. Therefore, when the fermentation product is lactic acid, the NAD required for the glycolytic process can be provided by the NADH consumed by LDH, and the gram-positive strain is relatively advantageous in terms of lactic acid production, because compared with the gram-negative strain that substrate is converted (oxidized) into CO by respiration to provide NAD, reducing carbon loss is CO while lactic acid can increase. However, the rapid accumulation of the fermentation product in the reactor may quickly reach the lactic acid concentration at which growth inhibition occurs, and may have limitations in terms of proliferation to required cell concentration. Therefore, in order to overcome this, it is necessary to regulate the optimal oxygen supply rate, to maximize the optimal seed concentration and the initial growth rate of fermentation, and to prevent substrate from being excessively converted into CO due to excess oxygen. In addition, once the lactic acid concentration when growth stops is reached, it is necessary to regulate the oxygen supply rate by reducing the oxygen supply rate, to keep the microaerobic state where excessive CO loss does not occur, while supplying ATP, which is the energy lactic acid is discharged to the outside of the cell as described above. Therefore, for the addition of the neutralizing compound and the mixed compound, the fermentation of the present invention does not require a high mixing rate in the fermentor, but it is necessary to control the minimum oxygen supply rate at which sufficient cell growth occurs in the early stages of fermentation and sufficient ATP supply occurs in the later stages of fermentation while reducing CO2 loss due to excess oxygen, which is an important scale-up factor. Based on the optimization of the oxygen supply rate, the appropriate aeration rate and mixing rate can be found through many experiments, and the optimal value can also be found using parameters known in the art such as OUR and OTR.

[0072] In one aspect of the invention, compared to the parent strain YBC strain (KCTC13508BP) or mutant strains derived from the parent strain (YBC1, YBC2 / YBC3 / YBC4 / YBC5), the strain selected by adaptive evolution can achieve high cell concentrations and faster lactic acid production rates due to increased tolerance to lactic acid. In another aspect of the invention, the LDH in the strain selected by adaptive evolution is enhanced to achieve a reduction in ethanol and glycerol production.

[0073] In another aspect of the present invention, compared with the lactic acid production capacity obtained by introducing the LDH gene derived from Lactobacillus plantarum at the PDC (g3002-1) gene position of the genome of the parent strain YBC strain, it was found that the lactic acid production capacity obtained by introducing the LDH gene derived from Staphylococcus epidermidis at this position was increased by more than 30 times in terms of yield.

[0074] In the present invention, the gene encoding lactate dehydrogenase introduced is preferably an LDH gene derived from Lactobacillus helveticus (L. helveticus), an LDH gene derived from Rhizopus oryzae (R. oryzae), an LDH gene derived from Lactobacillus plantarum, an LDH gene derived from B. taurus, and an LDH gene derived from Staphylococcus epidermidis. More preferably, the LDH gene derived from Lactobacillus plantarum is introduced into the g4423 (ADH) site, and the LDH gene derived from Staphylococcus epidermidis is introduced into the g3002-1 (PDC) site.

[0075] In one aspect of the invention, the #26-5 bacterial strain with significantly increased lactic acid tolerance obtained by the adaptive evolution of the YBC5 bacterial strain (Δ g4423::ldh / Δ g3002-1::ldh / Δ g2947::ldh / Δ g1544) showed high lactic acid productivity and high lactic acid production concentration, thus significantly improving the economic efficiency of the method. In addition, the YBC6 bacterial strain (Δ g4423::LpLDH / Δ g3002-1::SeLDH / Δ g2947::LpLDH / Δ g1544) constructed by replacing the LDH of the #26-5 bacterial strain g3002-1 with the LDH derived from Staphylococcus epidermidis showed increased lactic acid production rate and production concentration, as well as the generation of suppressed ethanol and glycerol, thereby increasing output. The fermentation characteristics of YBC6 showed that output, production rate and production concentration had reached the level that can be used as acid-resistant bacterial strain commercialization.

[0076] Therefore, in another aspect, the present invention relates to a method for producing lactic acid, comprising (a) culturing a recombinant strain to produce lactic acid, and (b) collecting the produced lactic acid.

[0077] The present invention enables the realization of an excellent acid-tolerant strain having greatly increased lactic acid productivity, concentration and yield reaching commercial levels, greatly reduced ethanol yield, and greatly reduced glycerol by-product.

[0078] As used herein, the term "acid-tolerant yeast" is defined as a yeast that is capable of maintaining a biomass consumption rate (e.g., sugar consumption rate) of at least 10% or a specific growth rate of at least 10% at a pH below the pKa value of the organic acid (particularly lactic acid) when the culture medium contains an organic acid (particularly lactic acid) at a concentration of at least 1 M, as compared to when the culture medium does not contain the organic acid. More specifically, the term "acid-tolerant yeast" is defined as a yeast that is capable of maintaining a biomass consumption rate (e.g., sugar consumption rate) of at least 10% or a specific growth rate of at least 10% at a pH of 2 to 4, as compared to a pH of 5 or above.

[0079] The recombinant yeast according to the present invention can be produced by inserting the gene into the chromosome of the host yeast according to a conventional method, or by introducing a vector containing the gene into the host yeast.

[0080] As host yeast, host cells with high DNA introduction efficiency and high expression efficiency of introduced DNA are generally used. In one embodiment of the present invention, acid-resistant yeast is used, but the present invention is not limited thereto and any type of yeast can be used as long as it can fully express the target DNA.

[0081] Recombinant yeast can be prepared using any transformation method. The term "transformation" refers to the introduction of DNA into a host, causing the DNA to replicate as a chromosomal factor or through chromosomal integration. It also refers to the artificial induction of genetic changes by introducing exogenous DNA into cells. Common transformation methods include electroporation and lithium acetate-polyethylene glycol.

[0082] In addition, in the present invention, any known genetic engineering method can be used as a method for inserting a gene into the chromosome of a host microorganism. For example, there are methods using retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, poxvirus vectors, lentiviral vectors, non-viral vectors, etc. "Vector" means a DNA product containing a DNA sequence that is operably linked to a suitable regulatory sequence that can express the DNA in a suitable host. The vector can be a plasmid, a phage particle or a simple potential genome insert. When transformed into a suitable host, the vector can be replicated or perform a function independent of the host genome, or some of them can be integrated with the genome. Plasmid is the most commonly used vector form at present, but linear DNA is also a common form for yeast genome integration.

[0083] A typical plasmid vector includes (a) an origin of replication that is effective for replication, so that a predetermined number of plasmid vectors are included in each host cell; (b) an antibiotic resistance gene or an auxotrophic marker gene to screen host cells transformed with the plasmid vector; and (c) a restriction enzyme site for inserting exogenous DNA fragments. Even if there are no suitable restriction enzyme sites, the vector and exogenous DNA can be easily connected using synthetic oligonucleotide adapters or linkers according to conventional methods (Gibson assembly). If necessary, a method of synthesizing the entire desired sequence and using it is also commonly used.

[0084] In addition, when a nucleic acid sequence is aligned with another nucleic acid sequence based on a functional relationship between them, it is said to be "operably linked" to it. This can be a gene and a control sequence connected in such a way that gene expression can occur when a suitable molecule (e.g., a transcriptional activator) is connected to the control sequence. For example, when expressed as a preprotein involved in the secretion of a polypeptide, the DNA for a presequence or secretory leader is operably linked to the DNA of the polypeptide; when affecting the transcription of the sequence, a promoter or enhancer is operably linked to the coding sequence; when affecting the transcription of the sequence, a ribosome binding site is operably linked to the coding sequence; or when positioned to promote translation, a ribosome binding site is operably linked to the coding sequence.

[0085] Typically, the term "operably linked" means that the DNA sequence being linked is in contact with it, or that the secretory leader is in contact with it and present in the reading frame. However, the enhancer need not be in contact with it. The linkage of these sequences is achieved by ligation (connection) at convenient restriction enzyme sites. When such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0086] It should be understood that, when expressing DNA sequence dna of the present invention, not all vectors function the same. Similarly, for the same expression system, not all hosts function the same. However, those skilled in the art will be able to make appropriate selections from various vectors, expression control sequences and hosts, without the need for excessive experimental burden, and without departing from the scope of the present invention. For example, the host should be considered for selection of vectors, because the vector should be replicated therein. The number of times the vector is replicated, the ability to control the vector replication number, and the expression of other proteins encoded by the corresponding vector, such as the expression of antibiotic markers, should also be considered.

[0087] In the present invention, the carbon source may include, but is not limited to, one or more selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, cellulose, galactose, glucose oligomers, and glycerol.

[0088] In the present invention, the culture can be carried out under conditions such that microorganisms, such as Escherichia coli (E. coli), are no longer functional (e.g., cannot produce metabolites). For example, the culture can be carried out at 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.

[0089] Hereinafter, the present invention will be described in more detail with reference to Examples. However, it will be apparent to those skilled in the art that these Examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0090] Example 1: Adaptive evolution of acid-resistant strain YBC #1

[0091] In previous studies, the present inventors selected strains with acid resistance by testing various yeast strains, and determined the strain with the best acid resistance, i.e., the YBC strain, by adding lactic acid to the culture medium at the beginning of yeast strain cultivation and monitoring the growth and sugar consumption rate of the microorganisms. The strain was deposited in the Korean Type Culture Collection under the accession number KCTC13508BP.

[0092] Phylogenetic analysis showed that the YBC strain (KCTC13508BP) was similar to S. cerevisiae, was diploid, and was Crabtree positive.

[0093] A commercially viable yield was achieved by suppressing lactate consumption and inhibiting glycerol production while minimizing the inhibition of ethanol production in the YBC5 strain genetically modified from the corresponding YBC strain (Korean Patent Application No. 10-2020-0046779).

[0094] The YBC5 strain was obtained as follows: ADH (alcohol dehydrogenase) was deleted from the YBC strain and the LDH gene was introduced into the strain to construct the YBC1 strain, the g3002-1 gene (PDC gene) was removed from the YBC1 strain and LDH was expressed therein to construct the YBC2 strain capable of efficiently producing lactic acid and having inhibited ethanol production, the LDH gene was introduced into the YBC2 strain and g2947 (which is a gene that consumes lactic acid) was removed to construct the YBC4 strain in which the lactic acid consumption ability was removed, and the GPD1 (g1544) gene was removed from the YBC4 strain (allele 1 and allele 2 were removed to make it a diploid strain) to construct the YBC5 strain.

[0095] The method for constructing this strain is as follows:

[0096] The YBC1 strain is obtained by removing the g4423 gene, which is the main ADH gene of the YBC strain, from the YBC strain and introducing the LDH gene of SEQ ID NO.3 derived from plantarum at the position of g4423. Based on the information of g4423 and its UTR, the ORF of each gene has been removed and a 5'UTR and 3'UTR gene cassette have been included, and used as donor DNA. For each allele of g4423, the corresponding 5'UTR is represented by SEQ ID NO.4 and SEQ ID NO.5, and the 3'UTR is represented by SEQ ID NO.6 and SEQ ID NO.7. The donor DNA is produced by the cloning method using restriction enzymes as described above, Gibson assembly, and the method using gene synthesis. The LDH of SEQ ID NO.3 is synthesized and then introduced into the ORF site of g4423 to produce donor DNA, and the donor DNA is introduced into YBC to construct the recombinant strain YBC1.

[0097] In addition, the g3002-1 gene is a gene located at backbone 72 in the genome sequencing of the YBC strain and serves as a PDC gene. The g3002-1 gene (a gene located at backbone 72) was removed from the YBC1 strain and the LDH gene of SEQ ID NO: 3 was introduced therein to construct the recombinant strain YBC2.

[0098] Corresponding UTR is used as recombination site to construct the box for replacing the g3002 gene.Similar to the above-mentioned method that LDH is introduced into the g4423 gene (ADH) site of YBC1, the UTR of g3002-1 is used to build this box.However, in order to simplify the process of gene replacement, when not considering allelic variation, a donor box (cassette) for an allele is prepared, but a donor box (cassette) can also be prepared for each allele.In addition, for the primer for gene replacement, except for the primer for producing the deletion strain, use respectively the following pair of primers that can detect the UTR of LDH and g3002-1, to increase the accuracy of gene replacement verification.

[0099] g3002-1 UTR-LDH-Forward Primer: GCAGGATATCAGTTGTTTG (SEQ ID NO: 8)

[0100] g3002-1 UTR-LDH-Reverse Primer: AATACCTTGTTGAGCCATAG (SEQ ID NO: 9)

[0101] In addition, the YBC4 bacterial strain is by the main CYB2 gene of YBC2 bacterial strain, i.e. g2947 gene, deleted from the YBC2 bacterial strain, and the LDH gene of SEQ ID NO:3 that is derived from plant lactobacillus is introduced at the position of g2947 gene and is constructed. In the genome sequencing of YBC bacterial strain, g2947 gene is the gene positioned at 41 places of skeleton. Based on the information of g2947 and UTR thereof, the ORF of each gene is removed and comprises 5 ' UTR and 3 ' UTR gene box (gene cassette), and it is used as donor DNA. For each allele of g2947, corresponding 5 ' UTR is represented by SEQ ID NO:10 and SEQ ID NO:11, and 3 ' UTR is represented by SEQ ID NO:12 and SEQ ID NO:13. Adopt the cloning method of restriction enzyme as mentioned above, Gibson assembly and the method for using gene synthesis to produce donor DNA.

[0102] However, in order to simplify the process of gene replacement, a donor cassette is prepared for one allele regardless of allelic variation, but one donor cassette may be prepared for each allele.

[0103] The YBC5 strain was constructed by deleting the g1544 gene, which serves as the GPD1 gene of the YBC4 strain, from the YBC4 strain. The g1544 gene is located at position 19 in the YBC strain's genome sequencing. Based on the information about g1544 and its UTRs, a gene cassette was constructed that removed the ORFs of each gene and contained the 5' UTR, 3' UTR, and an antibiotic marker, and used as donor DNA. For each allele of g1544, the corresponding 5' UTR is represented by SEQ ID NO:14 and SEQ ID NO:15, while the 3' UTR is represented by SEQ ID NO:16 and SEQ ID NO:17. Donor DNA was prepared using the aforementioned cloning method using restriction enzymes, Gibson assembly, and gene synthesis.

[0104] To simplify the gene replacement process, a donor cassette is prepared for one allele regardless of allelic variation, but a donor cassette can also be prepared for each allele. In addition, when using currently commercialized genetic engineering technology (CRISPR), donor cassettes can be prepared and applied without the use of antibiotic markers.

[0105] The genotypes of the prepared recombinant strains are as follows:

[0106] YBC2: Δg4423::ldh / Δg3002-1::ldh

[0107] YBC4: Δg4423::ldh / Δg3002-1::ldh / Δg2947::ldh

[0108] YBC5: Δg4423::ldh / Δg3002-1::ldh / Δg2947::ldh / Δg1544

[0109] However, to ensure economic feasibility for commercialization, the recombinant strain must achieve a production rate of 2.5 g / L / hour or higher and a lactic acid concentration of 120 g / L or higher at a pH below 3.7. Therefore, in the following examples, treatment to increase lactic acid tolerance was performed to improve the lactic acid production rate of the YBC5 strain.

[0110] As shown in Table 1, the YBC5 bacterial strain is subcultured, and lactic acid concentration is gradually increased to 80g / L from 10g / L. During subculture, mutants occur in the cell by natural mutation, and the bacterial strain growth of high-concentration lactic acid is relatively fast, and gradually becomes the dominant species in the whole bacterial strain flora. While increasing lactic acid concentration, this process is repeated, and the growth rate of the whole bacterial strain flora is detected. In addition, at the appropriate time point, the bacterial strain flora is placed on the agar plate containing lactic acid, and the bacterium colony produced is separated. At this moment, the bacterium colony selected is the bacterium colony that is the largest on the solid medium containing lactic acid because of fast growth. By this process, 42 bacterium colonies have been selected from the bacterium colonies grown under the liquid concentration of 40g / L, 50g / L, 60g / L, 70g / L and 80g / L.

[0111] [Table 1]

[0112]

[0113] During this process, changes in the concentration of lactic acid produced by the bacterial consortium in each lactic acid-containing culture medium are shown in Table 2 .

[0114] [Table 2]

[0115] LA0 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 1，14:00 0 0.20 4.34 54.5 1.0 0.0 0.0 10 / 2，15:00 25 6.72 2.52 9.2 38.0 0.8 0.0 LA10 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 1，14:00 0 0.20 3.10 54.2 11.4 0.0 0.6 10 / 2，15:00 25 5.72 2.56 13.9 42.5 0.8 0.6 LA20 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 1，14:00 0 0.17 2.94 55.2 21.7 0.0 0.9 10 / 2，15:00 42 6.50 2.53 6.8 58.0 0.0 1.3 LA30 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 4，9:00 0 0.21 2.83 52.0 32.6 0.0 1.5 10 / 5，10:00 25 4.06 2.75 43.4 37.3 0.0 1.6 LA40 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 5，10:00 0 0.10 2.78 54.5 41.9 0.0 2.6 10 / 7，9:00 47 5.40 2.56 21.6 64.4 1.1 3.4 LA50 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 7，11:00 0 0.22 2.75 54.6 51.9 0.0 2.5 10 / 9，8:00 45 1.34 2.75 53.6 55.4 0.0 2.8 10 / 10，9:00 70 5.92 2.64 29.5 65.7 0.0 3.3 LA60 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 9，8:00 0 0.07 2.73 54.6 61.6 0.0 3.1 3d 72 0.25 2.72 51.0 60.6 0.0 3.4 5d 120 6.23 3.43 18.1 81.2 0.0 3.7 LA70 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 12，09:00 0 0.05 2.73 51.7 68.7 0.0 3.8 6d 144 4.70 3.52 30.9 83.1 0.0 5.5 LA80_1 Cultivation time (hours) OD(A600) pH glucose lactic acid ethanol glycerin 10 / 18，09:00 0 NA 3.55 53.7 79.2 0.0 5.9 10 / 22，15:00 102 6.14 3.37 5.4 105.6 1.0 8.5

[0116] 42 selected colonies were inoculated into 5 ml conical tubes. Because this was a small-scale culture, the most uniform amount obtained from the colonies was inoculated to obtain a uniform inoculation OD. The culture medium used herein was mYP medium (5 g / L peptone, 4 g / L yeast extract, 5 g / L KH2PO4, 2 g / L MgSO4·7H2O, 0.15 g / L uracil) supplemented with 6% (primary) glucose or 12% (secondary) glucose and cultured at 30°C and 150 rpm for 96 hours.

[0117] The results of the 5 ml culture are shown in Table 3. From this culture, 15 colonies having a high lactic acid production concentration, a high cell concentration, or a high lactic acid production amount were selected and subjected to the following flask culture evaluation.

[0118] The selected colonies are as follows: 3, 5, 6, 8, 10, 22, 24, 26, 27, 31, 32, 35, 37, 38, 41.

[0119] [Table 3]

[0120]

[0121]

[0122] Selected colonies were evaluated by flask culture under the following conditions: 10% glucose (primary) was added to m-YP medium (5 g / L peptone, 4 g / L yeast extract, 5 g / L KH2PO4, 2 g / L MgSO4·7H2O, 0.15 g / L uracil) to adjust the total volume to 50 ml. The microorganisms were inoculated into the medium and cultured at 30°C and 150 rpm for 72 hours. In addition, one day after inoculation, a CaCO3 solution was added at 20% of the sugar injection concentration.

[0123] The results of the analysis of the flask cultures are shown in Table 4.

[0124] The results of flask cultures were analyzed based on the evaluation logic introduced for comprehensive judgment. First, the first five bacterium colonies were selected for each of production rate, lactic acid output, growth rate, ethanol concentration (by low order) and glycerol concentration (by low order), and the scores and weights in each were assigned to the selected bacterium colonies and summarized. As for weight, for the purpose of adaptive evolution, lactic acid production rate was given priority, while bacterium colonies with faster growth rate but lower lactic acid production capacity were excluded. Table 5 shows the evaluation process and its results.

[0125] [Table 4]

[0126]

[0127] [Table 5]

[0128]

[0129] As shown in Table 5, colony No. 26 (hereinafter referred to as "strain #26") was selected primarily because it minimized the increase in byproducts compared to the increase in lactic acid production capacity. When only lactic acid production capacity and lactic acid yield were considered without considering the increase in byproducts, colony No. 3 showed better results, but overall performance was prioritized in the first round. The trend of increasing these byproducts was also observed in subsequent adaptive evolution.

[0130] Example 2: Adaptive evolution of acid-tolerant yeast strain YBC #2

[0131] The performance of strain #26 selected in the first round of adaptive evolution in Example 1 was improved compared with YBC5 (see the comparison results with YBC5 in Table 4), but it did not reach the performance suitable for commercialization, so further improvement operations were carried out.

[0132] Because strain #26 has enhanced tolerance to lactic acid concentrations, cultivation was initiated at high lactic acid concentrations, and the number of subcultures at each concentration was increased. To improve growth characteristics under extreme conditions, cultivation was performed without the addition of the neutralizing agent CaCO3.

[0133] The lactic acid used was prepared as follows: impurities were removed from the culture medium produced by actual fermentation using a 0.2 μm filter, followed by concentration to prepare a 40-50% solution, which was then mixed with YP medium (20 g / L peptone, 10 g / L yeast extract) according to the desired lactic acid concentration. The sugar concentration was 10%, and the subculture medium was inoculated at 10% of the total volume of the fresh culture medium and cultured.

[0134] Figure 1 (a) shows the results of the subculture of strain #26. Even at a lactic acid concentration of 60 g / L, steady growth of the strain colony was observed. The strain colony was cultured in YP medium diluted with the culture medium on day 23, and 12 colonies were isolated. Colonies were selected based on size.

[0135] Under the same conditions as in Example 1, the selected colonies were cultured in flasks. The reference performance at this time was that of strain #26. Colony #5 was selected through a selection process similar to that in Example 1 and named strain #26-5 to distinguish it from the first round of results. The culture results of #26-5 are shown in Figure 1. Figure 2 shown.

[0136] In order to further increase the tolerance compared to strain #26-5, a third round of adaptive evolution was performed. The target strains used in this article were strain #26-5 and the strain colony cultured in the second round. The growth in the two flasks was compared at the beginning of the third round. As a result, the growth of the strain colony that continued to grow from the second round was more excellent. Due to the high possibility that there were mutants with stronger tolerance than the selected strain #26-5 in the strain colony, the adaptive evolution using strain #26-5 as the starting culture strain was stopped. The results of the third round are shown in Figure 2. Figure 1 (b) Shown. Lactic acid concentration was increased to 80g / L, and the growth of cells was observed, but it was observed that the growth rate was significantly reduced compared with the lactic acid concentration of 60g / L. Therefore, subculture was carried out under the reduced lactic acid concentration of 70g / L, which provided a steady growth rate. After the third round was completed, the corresponding strain flora was placed on an agar plate, and then bacterium colonies were selected. At this time, a YPDU agar plate containing the lactic acid of 45g / L and a plate with a lactic acid concentration of 50g / L and 60g / L were prepared, and the strain flora was inoculated thereon, and bacterium colonies were also produced on the agar plate containing lactic acid.

[0137] Although the colonies produced in the additional third round have a higher lactic acid tolerance than #26-5 and are therefore able to grow more at high lactic acid concentrations, the proportion of lactic acid in the fermentation product is further reduced, and the yields of ethanol and glycerol as by-products are further increased. Here, any one of the #26-5 strain and the third round of isolated strains should be selected as the target strain for commercialization, which will be further developed in the future. It is necessary to make a decision to choose to further develop the strain with high lactic acid tolerance in the medium and long term (research on restoring lactic acid production capacity and high reduction in by-products), or to select #26-5 with relatively low lactic acid tolerance but still good lactic acid tolerance. In the present invention, #26-5 has been further studied and is expected to be developed in a shorter time.

[0138] Example 3: Comparison of gene expression before and after adaptive evolution

[0139] In this example, changes in gene expression in YBC5 and #26-5 due to adaptive evolution were observed based on qPCR. Total RNA was extracted from each sample cultured in YPDU medium at 30°C and 200 rpm for 24 hours, and the RNA was analyzed by NGS, and then the changes in the expression levels of the same genes were analyzed, as shown in Table 6.

[0140] [Table 6]

[0141] Changes in gene expression in strains YBC5 and #26-5 and the corresponding genes, Category A: product-related, Category B: zinc finger proteins, Category C: sulfate / sulfite-related proteins, Category D: stress response, Category E: hexose transporters

[0142]

[0143]

[0144]

[0145]

[0146] *Fold change of - between the evolved strain and the original strain indicates decreased expression, and fold change of + between the evolved strain and the original strain indicates increased expression.

[0147] Through adaptive evolution, the expression levels of various genes increase or decrease in the bacterial strains of selected bacterium colonies, but the most significant difference is the weakening part of LpLDH (LDH derived from Lactobacillus plantarum), which is responsible for the production of lactic acid. It has been clearly shown that LDH enhancement research is necessary. However, as a result of the inventors' selection of bacterial strains that only produce lactic acid quickly, although LDH weakens, it is found that in bacterial strain #26-5, the expression of several transporter genes that transport sugar from the outside to the cell is enhanced. This is considered to be the main reason for obtaining faster lactic acid production ability, although LpLDH weakens.

[0148] The recombinant acid-resistant strain #26-5 was deposited in KCTC on June 15, 2020 (accession number KCTC 14215BP).

[0149] Example 4: Fermenter operation using selected adaptively evolved strains

[0150] In this example, #26-5 selected as the adaptively evolved strain in Example 3 was cultured in a bioreactor, and its lactic acid fermentation performance was measured.

[0151] The #26-5 strain was initially inoculated in 40 ml of mYP medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L KH2PO4, 2 g / L MgSO4·7H2O, 0.3 g / L uracil) and then secondary inoculated in 380 ml of mYP medium. The cells were cultured at 30°C and 200 rpm for 2 days, and all cells were harvested. The cells were then inoculated into 1.18 L of mYP medium and cultured at 30°C. At this time, the concentration of each component in the mYP medium was adjusted relative to the volume of 1.7 L, including the additional sugar solution and CaCO3 solution. Cultivation was started when the inoculation OD was 1.73, 100 ml of 42.33% CaCO was mixed with 450 ml of 62.5% sugar solution in a separate feed bottle, and the mixture of sugar and CaCO was injected into the bioreactor while continuously mixing in the bottle with a magnetic stirrer at a speed of 400 rpm so that the CaCO was uniform in the solution. In addition to this method of injecting a mixture of CaCO and sugar solution, in some fermentations, CaCO can be injected once every 2 hours in a predetermined amount (5-10 ml) separated from the sugar solution. However, considering that the increase in CO concentration caused by the introduction of CaCO can be minimized when a small amount is injected as evenly as possible, thereby improving fermentation performance, a mixture of CaCO and sugar solution is injected. In commercial fermentations, CaCO can be injected directly without mixing with water, so the reduction in lactic acid concentration caused by additional water can be avoided. However, in laboratory scale, sterilized CaCO is injected as the solution phase. In addition, in whole fermentation process, can be with sugar and CaCO consistent mixing ratio inject CaCO .In some cases, most of CaCO in 24 hours after inoculation, adding, this moment, initial bacterial strain growth is active, after this only injects sugar solution (based on fermentation ID 60).Sugar and CaCO The injection rate and the aeration rate of mixture are different in each batch, but based on the fermentation ID F60 in the table 7, the injection rate of mixed solution is 13.5ml / hour during initial 2 hours, is 15.3ml / hour after this.Based on F60, cultivate with the aeration rate of 0.7lpm and the stir speed (S.S.) of 700rpm in the cell growth stage.After 20 hours, changing culture condition to aeration rate gradually thus is 0.35lpm, and stir speed (S.S.) is 600rpm.

[0152] The fermentation results using strain #26-5 are shown in Figure 3 middle.

[0153] The results of the fermentation of strain #26-5 show that the production rate of lactic acid is 2.54g / L / hour, the output is 0.67g / g, and the lactic acid concentration is 123g / L. That is, the production rate and concentration of lactic acid are excellent, but there is a problem of reduced output compared to YBC5. This problem is caused by the production of 7g / L of ethanol and glycerol each during the fermentation process. This problem is due to the weakening of LDH and the expression of ethanologenic genes that occur as side effects of adaptive evolution (which has the effect of increasing fermentation rate and increasing tolerance to lactic acid). The method for offsetting these side effects is to enhance LDH and remove the corresponding ethanologenic genes, wherein the method of enhancing LDH is carried out in Examples 5 to 7. Figure 3 The sugar concentrations shown are those in the reactor during the injection of a mixture of sugar and CaCO in fed-batch mode. Commercial fermentations can be performed in batch mode, with the entire amount of sugar injected in the initial stage and CaCO injected separately. In the same way, it is possible to operate and optimize fermentations in fed-batch or semi-fed-batch mode, with a portion of the sugar injected during the fermentation as an appropriate combination with fed-batch.

[0154] Figure 4 The results of the comparison of only lactic acid concentration between strain #26-5 and strain YBC5 during fermentation are shown. Compared to the YBC5 strain, strain #26-5 showed an increase in fermentation rate and lactic acid production concentration, which is believed to be due to an increase in lactic acid tolerance. However, as mentioned above, the lactic acid production of the YBC5 strain ranged from 0.81 g / g to 0.83 g / g, while the lactic acid production of the #26-5 strain ranged from 0.63 g / g to 0.72 g / g, and the production at around pH 3 was 0.67 g / g to 0.68 g / g (see F59 and F60 in Table 6).

[0155] The effect and importance of ventilation in acid-resistant fermentation have been described above, and ventilation should be maximized to 24 hours, and this is the cell growth phase, and after this remains on minimum.Yet, even in the cell growth phase, excessive ventilation also can cause lactic acid production to reduce (referring to the F57 fermentation in Table 7).The aeration rate that maintains cell activity after 24 hours is very sensitive to the impact of output, therefore when being reduced to below 0.3lpm from the optimum aeration rate of 0.35lpm, based on 2L cultivation, the lactic acid production rate significantly reduces, or in serious case, lactic acid production stops, and when aeration rate is more than 0.4lpm, output reduces inversely with aeration rate.Aeration rate (aeration rate) can be expressed as the speed of oxygen transfer rate or oxygen flowing into cell.Yet, aeration rate is subject to the influence of the air discharge form of the structure of reactor, the shape of agitator and distributor, so when these factors change, should reoptimize.For those with microbial cell culture relevant knowledge, reoptimizing is not very difficult.

[0156] The fermentation results under various conditions are shown in Table 7 below.

[0157] [Table 7]

[0158] Results of optimizing culture conditions (including sugar injection and aeration) using strain #26-5

[0159]

[0160] Example 5: Comparison of the effects of introducing LDH at the g-3002 site of the YBC strain

[0161] Two copies each of LpLDH derived from Lactobacillus plantarum (SEQ ID NO: 3), LDH derived from bovine (BtLDH) (SEQ ID NO: 58), or LDH derived from Staphylococcus epidermidis (SeLDH) (SEQ ID NO: 1) were introduced into the PDC (g3002-1) site of the YBC strain.

[0162] The method for constructing this strain is as follows:

[0163] The major PDC gene of the YBC strain, g3002-1, was removed, and the yeast codon-optimized LDH gene of SEQ ID NO: 3 from Lactobacillus plantarum was introduced into the site of g3002-1 to obtain a strain. Based on the information of g3002-1 and its UTR, the ORF of each gene was removed, and LpLDH was introduced into its site to generate a gene cassette containing the 5'UTR and 3'UTR of g3002-1, and used as donor DNA (see Figure 5). In addition, the g3002-1 gene is a gene located at backbone 72 in the genome sequencing of the YBC strain and serves as a PDC gene. In order to simplify the gene replacement process, the donor cassette is generated for one allele without considering allelic variation, but can be generated for each allele. For each allele of g3002-1, the corresponding 5'UTR is shown in SEQ ID NO:59 and SEQ ID NO:60, and the 3'UTR is shown in SEQ ID NO:61 and SEQ ID NO:62. As described above, cloning methods using restriction enzymes and methods using Gibson assembly can be used to produce donor DNA, but the entire gene sequence can be synthesized and used. The recombinant strain was named "YBC1p".

[0164] To verify the correct execution of the genetic manipulation, transformants were identified using the following primers and, if necessary, correct transformants were identified by sequencing of genomic sections.

[0165] Likewise, the strain into which the BtLDH gene was introduced was named "YBCbt", and the strain into which the SeLDH gene was introduced was named "YBCse". The primers used for genome identification are as follows.

[0166] Forward primer used to identify 3002-1 ORF: GCAGGATATCAGTTGTTTG (SEQ ID NO: 63)

[0167] Reverse primer used to identify 3002-1 ORF: ATAGAGAAGCTGGAACAG (SEQ ID NO: 64)

[0168] Forward primer used to identify 3002-1UTR: GCAGGATATCAGTTGTTTG (SEQ ID NO: 65)

[0169] Reverse primer used to identify 3002-1UTR: CAGAATCTTAGAAAGGAGG (SEQ ID NO: 66)

[0170] Introduced forward primer for identification of LpLDH, BtLDH and SeLDH: GCAGGATATCAGTTGTTTG (SEQ ID NO: 67)

[0171] Reverse primer for identification of LpLDH: AATACCTTGTTGAGCCATAG (SEQ ID NO: 68)

[0172] Forward primer used to identify the introduction of BtLDH: ACCTTCTTGTTGTCTAGC (SEQ ID NO: 69)

[0173] Reverse primer for identifying the introduction of SeLDH: ATAACTCTTTCAGCTGGC (SEQ ID NO: 70)

[0174] The transformants were cultured in 50 ml flasks at 30°C and 150 rpm to identify their genotypes. The inoculum size was 0.1 OD and the culture medium used was YP medium (20 g / L peptone, 10 g / L yeast extract) with 6% glucose and 150 mg / L uracil.

[0175] The results are shown in Table 8.

[0176] [Table 8]

[0177] Comparison of the effects of introducing LDH at the g3002-1 (PDC) site

[0178]

[0179] As shown in Table 8, lactate production differed significantly due to variations in LDH within the same genome. Specifically, LpLDH substituted at the g4423 genomic locus in YBC showed very strong expression, corresponding to a yield exceeding 0.5 g / g, but was barely expressed at the g3002-1 locus. Furthermore, the LDH activity obtained at the g3002-1 locus by changing the LDH source to SeLDH was comparable to that in the case of the g4423 locus. These two results are novel phenomena that have not been reported to date.

[0180] Example 6: Effect of SeLDH substitution at the g3002-1 site in YBC1 and YBC5 strains

[0181] To verify the high activity of SeLDH at the g3002-1 site identified in the above example, the same genetic manipulation was performed on the YBC1 strain and the #26-5 strain (derived from YBC5).

[0182] The genotypes of the target strains YBC1 and YBC5 are as follows:

[0183] YBC1:Δg4423::LpLDH

[0184] #26-5 (from YBC5): Δg4423::LpLDH, Δg3002-72::LpLDH, Δg2947::LpLDH, Δg1544

[0185] The cassette and method used here were similar to those in Example 5. In the case of YBC5, LpLDH at the target position should be replaced with SeLDH, but in order to amplify a portion with low similarity between the two LDH sequences and identify the correct transformant based on this, the primers were changed as follows.

[0186] Forward primer used to identify the presence of LpLDH: GCAGGATATCAGTTGTTTG (SEQ ID NO: 71)

[0187] Reverse primer used to identify the presence of LpLDH: TTTCAAACCAGTACCACCA (SEQ ID NO: 72)

[0188] Forward primer 1 for identifying the presence of SeLDH: GCAGGATATCAGTTGTTTG (SEQ ID NO: 73)

[0189] Reverse primer 1 for identifying the presence of SeLDH: GAAGAAGAA TACAAAGCACC (SEQ ID NO: 74)

[0190] Forward primer 2 for identifying the presence of SeLDH: GCAGGATATCAGTTGTTTG (SEQ ID NO: 75)

[0191] Reverse primer 2 for identifying the presence of SeLDH: CACCAGCTTTAACAGTAAC (SEQ ID NO: 76)

[0192] The strain in which SeLDH was introduced at position g3002 of the YBC1 strain was named “YBC2se”, and the strain in which SeLDH was introduced at position g3002 of the YBC5 strain was named “YBC6”, and their genotypes are as follows.

[0193] YBC2se:Δg4423::LpLDH,Δg3002-72::SeLDH

[0194] YBC6:Δg4423::LpLDH,Δg3002-72::SeLDH,Δg2947::LpLDH, Δg1544

[0195] Transformants were tested and their genotypes were identified by culturing in 50 ml flasks at 30°C and 150 rpm. The inoculation OD was 0.1. The culture medium used was YP medium (20 g / L peptone, 10 g / L yeast extract), 5% glucose for YBC2 and YBC2se, and 10% glucose for YBC5 and YBC6, with 150 mg / L uracil added.

[0196] The results are shown in Tables 9 and 10 below.

[0197] [Table 9]

[0198] Identification of the effect of SeLDH on YBC1 (with or without pH control)

[0199]

[0200]

[0201] [Table 10]

[0202] Identification of the effect of SeLDH in YBC5 (without pH control)

[0203] ID Yield (g / g) #26-5 0.55 YBC6 0.76

[0204] As shown in Table 9 and Table 10, compared with YBC2 replaced with LpLDH at the same position, the YBC2se at the PDC gene of the YBC1 bacterial strain showed high yield under similar conditions. Considering the reduction of ATP output required for lactic acid transport to the extracellular space under acid-resistant conditions, in addition to PDC blocking, the strong LDH expression of SeLDH causes lactic acid production to be greatly improved compared with ethanol production, causing output to be basically similar to theoretical output. In addition, the bacterial strain #26-5, which is endowed with improved productivity but reduced output through conventional adaptive evolution, also shows a significant increase in output under the same conditions. This means that the activity of the LDH reduced by adaptive evolution is greatly improved by the SeLDH strongly expressed at the g3002-1 site.

[0205] Example 7: Fermentation tank operation using YBC6 strain

[0206] In this example, the YBC6 strain was cultured in a bioreactor and its lactic acid fermentation performance was determined.

[0207] The YBC6 strain was initially inoculated in 40 ml of mYP medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L KH2PO4, 2 g / L MgSO4·7H2O, 0.3 g / L uracil) and then inoculated in 380 ml of mYP medium for a second time. The cells were cultured at 30°C and 200 rpm for 2 days, and all cells were harvested. The cells were then inoculated in 1.18 L of mYP medium and cultured at 30°C. At this time, the concentration of each component in the mYP medium was adjusted relative to the volume of 1.7 L, including the additional sugar solution and CaCO3 solution. Cultivation was started when the inoculation OD was 1.74, with 100ml of 42.33% CaCO mixed with 450ml of 62.5% sugar solution in a separate feed bottle, and the mixture of sugar and CaCO was injected into the bioreactor while continuously mixing in the bottle with a magnetic stirrer at a speed of 400 rpm so that the CaCO was uniform in the solution. However, considering that the increase in CO concentration (the hindrance of oxygen transfer) caused by the introduction of CaCO can be minimized when injected as uniformly as possible, thereby improving fermentation performance, the mixture of CaCO and sugar solution was injected. In commercial fermentation, CaCO can be injected directly without mixing with water. However, in laboratory scale, sterilized CaCO is injected as the solution phase. In addition, CaCO can be injected with a uniform mixing ratio of sugar and CaCO throughout the fermentation process. However, in this fermentation, most of the CaCO was added within 24 hours after inoculation, when the initial strain grew actively, and only the sugar solution was injected thereafter. During the first 8 hours, the injection rate of the sugar and CaCO mixture was increased from 4.5 ml / hour to 18 ml / hour, and thereafter to 22.5 ml / hour. During the cell growth phase, the culture was cultivated with an aeration rate of 0.5 lpm and a stirring rate of 600 rpm. After 12 hours, the culture was cultivated with an aeration rate of 0.35 lpm and a stirring rate of 600 rpm, and gradually changed therefrom. After 33 hours, the culture was cultivated with an aeration rate of 0.4 lpm and a stirring rate of 600 rpm.

[0208] The fermentation results using the YBC6 strain are shown in Figure 6 middle.

[0209] Figure 6 The sugar concentrations shown are those in the reactor during the fed-batch injection of a mixture of sugar and CaCO. Commercial fermentations can be performed in batch mode, where the total sugar amount is injected in the initial stage and the CaCO is injected separately. In the same way, fermentations can be operated and optimized in fed-batch or semi-fed-batch, where a portion of the sugar is injected during the fermentation as an appropriate combination with fed-batch.

[0210] When culture result shows pH 3.16, lactic acid output is 0.75g / g, fermentation rate is 2.56g / L / hour, lactic acid concentration is 130g / L, which is the best result in the acid-resistant bacterial strain performance published so far. The patent (U.S. Patent No. 7,232,664) of Cargill Inc. of the United States about acid-resistant bacterial strain cultivation suggests that the output of 0.75g / g, the fermentation rate of 2.5g product / L / hour and the lactic acid concentration of 120g / L are used as the standard of the commercial performance of acid-resistant lactic acid bacterial strain. The result of this embodiment has reached the above-mentioned standards on all indicators. The embodiment of U.S. patent No. 7,232,664 discloses the gross output of 0.67g / g, the average fermentation rate of 0.8g lactic acid / g cell / hour and the concentration of 114g / L. Fermentation result of the present invention shows the output and performance that are better than this embodiment.

[0211] Figure 7 The results of comparison of only lactate production capabilities between the YBC5 strain and the #26-5 strain and the YBC6 strain are shown, and the excellence of lactate production by YBC6 is demonstrated, which is due to the combined effects of adaptive evolution and LDH enhancement at the PDC position in the genome.

[0212] Name of depository institution: Korea Type Culture Collection

[0213] Accession number: KCTC14215BP

[0214] Date of deposit: June 15, 2020

[0215] Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do, South Korea 56212

[0216] Although the specific configuration of the present invention has been described in detail, it will be understood by those skilled in the art that this description is provided for illustrative purposes to provide preferred embodiments and should not be interpreted as limiting the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. Sequence Listing <110> SK New Technology Co., Ltd. <120> Recombinant acid-tolerant yeast with improved lactic acid production capacity <130> KHP212110806.1 <150> 10‑2020‑0077331 <151> 2020-06-24 <160> 76 <170> PatentIn version 3.5 <210> 1 <211> 948 <212> DNA <213> Staphylococcus epidermidis <400> 1 atgaaaaaat ttggtaaaaa agttgttttg gttggtgatg gttctgttgg ttcttcttat 60 gcttttgcta tggttactca aggtattgct gatgaatttg ttattattga tattgctaaa 120 gataaagttg aagctgatgt taaagatttg aatcatggtg ctttgtattc ttcttctcca 180 gttactgtta aagctggtga atatgaagat tgtaaagatg ctgatttggt tgttattact 240 gctggtgctc cacaaaaacc aggtgaaact agattgcaat tggttgaaaa aatactaaa 300 attatgaaat ctattgttac ttctgttatg gattctggtt ttgatggtttt ttttttgatt 360 gctgctaatc cagttgatat tttgactaga tatgttaaag aagttactgg tttgccagct 420 gaaagagtta ttggttctgg tactgttttg gattctgcta gattagata tttgatttct 480 aaagaattgg gtgttacttc ttcttctgtt catgcttcta ttattggtga acatggtgat 540 tctgaattgg ctgtttggtc tcaagctaat gttggtggta tttctgttta tgatactttg 600 aaagaagaaa ctggttctga tgctaaagct aatgaaattt atattaatac tagagatgct 660 gcttatgata ttattcaagc taaaggttct acttattatg gtattgcttt ggctttgttg 720 agaatttcta aagctttgtt gaataatgaa aattctattt tgactgtttc ttctcaattg 780 aatggtcaat atggttttaa tgatgtttat ttgggtttgc caactttgat taatcaaaat 840 ggtgctgtta aaatttatga aactccattg aatgataatg aattgcaatt gttggaaaaa 900 tctgttaaaa ctttggaaga tacttatgat tctattaaac atttggtt 948 <210> 2 <211> 316 <212> PRT <213> Staphylococcus epidermidis <400> 2 Met Lys Lys Phe Gly Lys Lys Val Val Leu Val Gly Asp Gly Ser Val 1 5 10 15 Gly Ser Ser Tyr Ala Phe Ala Met Val Thr Gln Gly Ile Ala Asp Glu 20 25 30 Phe Val Ile Ile Asp Ile Ala Lys Asp Lys Val Glu Ala Asp Val Lys 35 40 45 Asp Leu Asn His Gly Ala Leu Tyr Ser Ser Ser Pro Val Thr Val Lys 50 55 60 Ala Gly Glu Tyr Glu Asp Cys Lys Asp Ala Asp Leu Val Val Ile Thr 65 70 75 80 Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Gln Leu Val Glu 85 90 95 Lys Asn Thr Lys Ile Met Lys Ser Ile Val Thr Ser Val Met Asp Ser 100 105 110 Gly Phe Asp Gly Phe Phe Leu Ile Ala Ala Asn Pro Val Asp Ile Leu 115 120 125 Thr Arg Tyr Val Lys Glu Val Thr Gly Leu Pro Ala Glu Arg Val Ile 130 135 140 Gly Ser Gly Thr Val Leu Asp Ser Ala Arg Phe Arg Tyr Leu Ile Ser 145 150 155 160 Lys Glu Leu Gly Val Thr Ser Ser Ser Val His Ala Ser Ile Ile Gly 165 170 175 Glu His Gly Asp Ser Glu Leu Ala Val Trp Ser Gln Ala Asn Val Gly 180 185 190 Gly Ile Ser Val Tyr Asp Thr Leu Lys Glu Glu Thr Gly Ser Asp Ala 195 200 205 Lys Ala Asn Glu Ile Tyr Ile Asn Thr Arg Asp Ala Ala Tyr Asp Ile 210 215 220 Ile Gln Ala Lys Gly Ser Thr Tyr Tyr Gly Ile Ala Leu Ala Leu Leu 225 230 235 240 Arg Ile Ser Lys Ala Leu Leu Asn Asn Glu Asn Ser Ile Leu Thr Val 245 250 255 Ser Ser Gln Leu Asn Gly Gln Tyr Gly Phe Asn Asp Val Tyr Leu Gly 260 265 270 Leu Pro Thr Leu Ile Asn Gln Asn Gly Ala Val Lys Ile Tyr Glu Thr 275 280 285 Pro Leu Asn Asp Asn Glu Leu Gln Leu Leu Glu Lys Ser Val Lys Thr 290 295 300 Leu Glu Asp Thr Tyr Asp Ser Ile Lys His Leu Val 305 310 315 <210> 3 <211> 963 <212> DNA <213> Lactobacillus plantarum <400> 3 atgtcttcta tgccaaatca tcaaaaagtt gttttggttg gtgatggtgc tgttggttct 60 tcttatgctt ttgctatggc tcaacaaggt attgctgaag aatttgttat tgttgatgtt 120 gttaaagata gaactaaagg tgatgctttg gatttggaag atgctcaagc ttttactgct 180 ccaaaaaaa tttattctgg tgaatattct gattgtaaag atgctgattt ggttgttatt 240 actgctggtg ctccacaaaa accaggtgaa tctagattgg atttggttaa taaaaatttg 300 aatattttgt cttctattgt taaaccagtt gttgattctg gttttgatgg tatttttttg 360 gttgctgcta atccagttga tattttgact tatgctactt ggaaattttc tggttttcca 420 aaagaaagag ttattggttc tggtacttct ttggattctt ctagattgag agttgctttg 480 ggtaaacaat ttaatgttga tccaagatct gttgatgctt atattatggg tgaacatggt 540 gattctgaat ttgctgctta ttctactgct actattggta ctagaccagt tagagatgtt 600 gctaaagaac aaggtgtttc tgatgatgat ttggctaaat tggaagatgg tgttagaaat 660 aaagcttatg atattattaa tttgaaaggt gctacttttt atggtattgg tactgctttg 720 atgagaattt ctaaagctat tttgagagat gaaaatgctg ttttgccagt tggtgcttat 780 atggatggtc aatatggttt gaatgatatt tatattggta ctccagctat tattggtggt 840 actggttga aacaaattat tgaatctcca ttgtctgctg atgaattgaa aaaaatgcaa 900 gattctgctg ctactttgaa aaaagttttg aatgatggtt tggctgaatt ggaaaataaa 960 taa 963 <210> 4 <211> 988 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR of g4423 allele 1 <400> 4 gttaactcag ttttctctct ttccctccac cccacgttac tctgcgaaca aaaatacgca 60 cagaatgaac atctgattga ttaatattta tatattactt agtggcaccc ctacaaacaa 120 accaattttg aatatttctc accatcatga tatttattta gggcaagaat ttcatgtaca 180 tacgtgcgtg tactgcatag ttttgttata tgtaaataac cagcaatata tcaccaatga 240 taaatgctca gtaatttatt tggaaccaaa atagtttcag taatcaaata atacaataac 300 taacaagtgc tgattataca acagctgtta acaacacaaa cacgctctct tctattctct 360 tccctgcttg ttcgtgtggt atattcccga atttgcaatt tagaaattat attttttaaa 420 agaattgttc tccattttct ggtagtcgta agtggcaaat tggatcataa gacacaatct 480 tgttagttcg actgctaaca ccagacaaga ccgaacgaaa acagaaaaaa aagataattt 540 tgttagttcg actgctaaca ccagacaaga ccgaacgaaa acagaaaaaa aagataattt 540 tgttattctg ttcaattctc tctctctttt taaggtatct ttacattaca ttacatatcc 600 tgttattctg ttcaattctc tctctctttt taaggtatct ttacattaca ttacatatcc 600 caaattacaa caagagcaag aaatgaagca caacaacacg ccatctttcg tgattatttt 660 caaattacaa caagagcaag aaatgaagca caacaacacg ccatctttcg tgattatttt 660 atcatttcta tatcgtaact aaattaacaa atgctatgtt tcttaatttt taatgataaa 720 atcatttcta tatcgtaact aaattaacaa atgctatgtt tcttaatttt taatgataaa 720 tctaactgct accttaattt ctcatggaaa gtggcaaata cagaaattat atattcttat 780 tctaactgct accttaattt ctcatggaaa gtggcaaata cagaaattat atattcttat 780 tcattttctt ataattttta tcaattacca aatatatata aatgcaatta attgattgtt 840 tcattttctt ataattttta tcaattacca aatatatata aatgcaatta attgattgtt 840 cctgtcacat aatttttttt gtttgttacc tttattcttt atccatttag tttagttctt 900 cctgtcacat aatttttttt gtttgttacc tttattcttt atccatttag tttagttctt 900 atatctttct tttctatttc tctttttcgt ttaatctcac cgtacacata tatatccata 960 atatctttct tttctatttc tctttttcgt ttaatctcac cgtacacata tatatccata 960 tatcaataca aataaaaatc atttaaaa 988 tatcaataca aataaaaatc atttaaaa 988 <210> 5<210> 5 <211> 961<211> 961 <212> DNA <212> DNA <213> 人工序列(Artificial Sequence) <213> Artificial Sequence <220> <220> <223> g4423 等位基因2的5' UTR <223> 5' UTR of g4423 allele 2 <400> 5 <400> 5 gttaactcag ttttctctct ttccctccac cccacgttac tctgcgaaca aaaaatacgc 60 gttaactcag ttttctctct ttccctccac cccacgttac tctgcgaaca aaaaatacgc 60 acagaatgaa catctgattg attaatattt atatattact cagtggcacc cctacaaaca 120 aaccaatttt gaatattgtt caccatcatg atatttattt agggcaagaa tttcatgtac 180 atacgtgcgt gtactgcata gttttgttat atgaaaataa ccagcaatat atcaccaatg 240 aataaattct caataattta tttggaacca aataatgcaa taactagcaa actaagtggt 300 gattatacaa cagctgttaa caacacaaac atacgctctc ttctattatc tcttccctgc 360 ttgttcgtgt ggtatattca cgaatttgca atttagaaat tatatttttt aaaagaattg 420 ttctccattt tctggtagtc gtaagtggca aattggatca taagacacaa tcttgttagt 480 tcgactgcta acaccagaca acaccgaacg aaaacaagaa aaaatta ttctctctct 540 ttttaaggta tcttacatta catatcccaa attacaacaa gagcaagaaa tgaggcacaa 600 caacacacca tcatctttcg tgattatttt tatcatttct atcatgtaat taaattaaca 660 aatgttaagt ttattaattt ttaatgataa atctagttgc taccttaatt tctcatggaa 720 agtggcaaat actgaaatta tttaattcta cttcatttt cttataattt tttcaatta 780 ccaaatatat ataaatgcaa ttaattgatt gttcctgtca cataattttt tttgtttgtt 840 acctttattc tttatccatt taatttattt cttgtatctt tcttttctat ttctcttttc 900 tgtttaatct caccgtacac atatatatcc atatatcaat acaaataaaa atcatttaaa 960 a 961 <210> 6 <211> 257 <212> DNA <213> Artificial Sequence <220> <223> g4423 allele 1 3' UTR <400> 6 taagtcattt aatttattct tttagaatat atttattttg tctttatttt tgaaatgtta 60 atagtctttt ttttttactt tgaacaaaaa aaagtaaaat taaaacttat cttatatacg 120 cttttaaaca ttaaactcgt taacgaatta tataatgatt ttatcgaact actttatgtt 180 tttttaatag aataatcttc tttattaata taacttacta cttcttaatc ttgttgtcct 240 ccattcgaaa ctcgagt 257 <210> 7 <211> 255 <212> DNA <213> Artificial Sequence <220> <223> g4423 allele 2 3' UTR <400> 7 taagtcattt aatttattct tttagaatat atttattttg tctttatttt tgaaatgtta 60 atagtcttttttttactttg aaaaaaaaaa aaagtaaaat taaacttatc ttatatacgc 120 ttttaaacat taaactcgtt aacgaattat ataatgattt tatcgaacta ctttatgttt 180 ttttaataga ataatcttct ttattaatat aacttactac ttcttaatct tgttgtcctc 240 cattcgaaac tcgag 255 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> g3002-1 UTR-LDH-Forward Primer <400> 8 gcaggatatc agttgtttg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> g3002-1 UTR-LDH-reverse primer <400> 9 aataccttgt tgagccatag 20 <210> 10 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> 5'UTR of g2947 allele 1 <400> 10 atatattttg gctgacattg taattagatg agatccacaa tttttctttt gtttgactgt 60 tcgatatgga gaaggtggga tgcactatta ttatattcag aagtttattt gtacagttta 120 aagaacaaat agtggctaat cctatcctcg gactaaaaaa aatcgttcac ttctatccta 180 ctgtaaatct tatgaaaatg atgtaattca tatagttact atattttctt tcttttagaa 240 actttatgat atatatatat atataaaagg actaatcacc caactctcaa attcattaaa 300 aagaaatatg tttctatcat cttcttttct tattatacct cgtctaataa taaaaccaaa 360 caattttctg taaag 375 <210> 11 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR of g2947 allele 2 <400> 11 atatattttg gctgacattg taattagatg agatccacaa tttttctttt gtttgactgt 60 tcgatatgga gaaggtggga tgcactatta ttatattcag aagtttattt gtacagcttg 120 aagaacaaat agtggctaat cctatcctcg gactaaaaaa aattgttcac ttttatccta 180 ctgtaaatct tatgaaaatg atgtaattca tatagttact atattttctt tcttttagaa 240 acttcatgat atatatatat atataaaagg actaatcacc caactctcaa atttattaaa 300 aagaaatatg tttctatcat cttcttttct tattatacct tctctaataa taaaaataaa 360 caactttctg taaag 375 <210> 12 <211> 997 <212> DNA <213> Artificial Sequence <220> <223> 3'UTR of g2947 allele 1 <400> 12 ttgtgactct atggagttta cctattttat ataccactat atcacaaaaa gtaataacaa 60 cttttcaaat ataatacaat attcaataaa tatatttata tattctaaaa tctacgtttt 120 tctctttctt aaaaaaataa acaaactgac cctttcaatc ttcaatgtga tactttactt 180 attttatttc attacacaga aaggtataaa tatatacata acttaatggt ttattcattt 240 cttcttatta gacaacgtgg ttagttgttg tttaacccat tccaataata aatcagtttg 300 taaataacct tcactgttaa atactttatt aatctctaat gaactagtta aagttttctt 360 cttattatct atcaaagtca tattgtaaat tggtttattt tcttcaaatt ctgtctttaa 420 tttaattatt tcagtaccat tcttaccact atatacgata gatttttcaa catatttctt 480 aaagaaccaa aatattacag atagtacaaa atatgtaccg actaaaattt gttgatattt 540 aacgatatta tcatgaacaa attttttatc aatgatgaaa ctgattgctg caacgatggc 600 agttgaataa ccaattaata atttctgatc aactaattca aaggtttctt catagcctaa 660 tcttttcatg acatcaggta gactttcatt tatagtttgt gatacttcag agatggaata 720 aacgttaacg ggcttactca ttgtgcttta aaggagaatg cggaattaat gagctcttta 780 ctatgtatca gaactcgaac taatgcaaag acaaatggaa taaactagtt acaatatata 840 tgaattttgt ctgttctttt ataatatatt ataatggatt tcccaaattg atgattattg 900 gttcactaag aaagctagaa agaagatgag atttctcgaa tagtaaaata ttacgttaac 960 atatctgaga ttaaaccgat agtcaatttg tacgtta 997 <210> 13 <211> 997 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR of allele 2 of g2947 <400> 13 ttgtgactct atggagttta cctattttat ataccactgt atcacaaaaa gtaataacaa 60 cttctcaaat ataatacaat atttaataaa tatatttata tattctaaaa tctacgtttt 120 tctctttctt aaaaaaataa acaaactgac cctttcaatc ttcaatgtga tactttactt 180 attttatttc attacacaga aaggtataaa tatatacata acttaatggt ttattcattt 240 cttcttatta gacagagtgg ttagttgttg tttaacccat tccaataata aatcagtttg 300 taaataacct tcactgttaa atactttatt aatctctaat gaactagtta aagttttctt 360 cttattatct atcaaagtca tattgtaaat tggtttattt tcttcaaatt ctgtctttaa 420 tttaattatt tcagtaccat tcttaccact atatacgata gatttttcaa catatttctt 480 aaagaaccaa aatattacag atagtacaaa atatgtaccg actaaaattt gttgatattt 540 aacgatatta tcatgaacaa attttttatc aatgatgaaa ctgattgctg caacgatggc 600 agttgaataa ccaattaata atttctgatc aactaattca aaggtttctt cataacctaa 660 tcttttcata acatcaggta gactttcatt tatagtttgt gatacttcag agatggaata 720 aacgttaaca ggtttactca ttgtgcttta aaggagaatg cggaattaat gagctcttta 780 ctatgtatca gaactcgaac taatgcaaag aaaaatggaa taaacttgtt acaatatgta 840 tgaattttgt ctattctttt ataataaatt ataatagatt tcccaaattg atgattattg 900 gttcactaag aaagctagaa agaagatgag atttctcgaa tagtaaaata ttaccttaac 960 atatctgaga ttaaaccgat agtcaatttg tacgtta 997 <210> 14 <211> 1328 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR of allele 1 of g1544 <400> 14 agaaaatagt ttctccgatt aaattttttt ttcaaatcaa atctttattt aagaattggt 60 agtgtatagt agtataatat tgcctaagaa attggagtag tccgtaaaaa atgggacaaa 120 attgttgaaa ttgagcaacc tgaaaatttt atgctggtct caagtagaga aacagacgta 180 gaaccaaaat tgacccaatt tcttgttgcc tttaattggg tcattcataa gaattcaaaa 240 tatttcttt tcccactcac gcgagagata tgcgcacacg atatagttaa taccgcttgt 300 aacaatacgt agatggccaa aaatgaacaa aaggggacac tcctcaaaag aaaaattgc 360 ttgtttggct gtcttctcca attgaaatat acacacacac cgcggtaaaa aaaaaattga 420 aatggaaatc gcggtgggac aaaagtagca accacaacaa gggaattttc cttactgctg 480 cggcagatcc ttactcatct ctcgaatata tatagcctct tgggtccacg ggcaaaaaag 540 aaataaaaaa aagagaagca acagaaccgc acgcaacgta cgcagtgatc catccatttt 600 ccacaaaatt tatctatttt cttgtctata ttttttacgt acaactaact gatcttcttg 660 tccccctccc cccatttacc cgttaaaatg aaagctgaac aacagaaaat aataattcgc 720 tctggtggac aaaaaataca agaacaagag agtatcataa tttgtgggt cacaaatgac 780 cctacaactg tcacctagtt ggtacaaaat ttgaccctca ttctcaaata attactacat 840 ttgggtctgt attaatgcta atatttcaat atatctctat ctatcagtca catacaaatt 900 tatcttcatc ttaaagggac tcacttactc aataatggtc tatctttata ttttttcat 960 acgtatgtat gtacgtagta aagggccatc aatgatccat cttactatta ttattcttta 1020 gttatttcta agcaacaaaa ggtctgtacc acagtttcag tgtcgtcata cctcttcttt 1080 taatttcttt tcggggaggg atgtcttaat gctaacttct gtctcactat taacggtaaa 1140 tcgtattaat ctcaatatat atataaaggg ttgatatttt ccaccgtttt aaaaattatt 1200 cccttgtttc tctattatta attttagact acttatttta attatttttc ccttttttac 1260 ttattatata tatataacta tatattacca ataataatat aagcaatcac atatatttat 1320 [[ID=​​​​​​​​​​​​​​​​​​​​​gaaccaaaat tgacccaatt tcttgttgcc tttaattggg tcattcataa gaattcaaaa 240 tattttcttt tcccactcac gcgagagata tgcgcacacg atataatttaa taccgtttgt 300 aacaatacgt agatggccaa aaatgaacaa aatgggacac tcctcaaaag gaaaaattgc 360 ttgtttggct gtcttctcca attgaaatat acacacacac cgcggtaaaa aaaaaattga 420 aattgaaatc gcggtgggac aaaagtagca accacaacaa gggaattttc cttactgctg 480 cggcagatcc ttactcatct cttgaatata tatagcctct tgggtccacg ggcaaaaaag 540 aaaaaaaaa aagagaagca acagaaccgc acacaacgta cgcagtgatc catccatttt 600 ccacaaaatt tatttatttt cttgtctgta ttattacgt acaactaact gatcttcttg 660 tccccccccc cccatttacc cgttaaaatg aaagctgaac aacagaaaat aataattcgc 720 tctgatggac aaaaaataca agaacaagag agtatcatca ctatgtgggt cacaaatgac 780 cctacaactg taatctagtt gatacaaaat ttgaccctca ttctcaaata attactacat 840 ttgggtctgt attaatacta atatctgtat atctctctat ctatcagtca catacaaatt 900 tatcttcatc ttaaagggac tcacttactc aataatggtc tatctttata tttttatcat 960 acgtatgtat gtacgtagta aagggccatc aatgatccat attattatta ttattcttta 1020 gttatttcta agcaacaaaa ggtctgtacc acagtttcag tgtcgtcata tctcttattt 1080 taatttcttt tcggggaggg atgtcttaat gctaacttct gtctcactat taacggtaaa 1140 tcttattaat ctcaatatat atataaaggg ttgatatttt ccaacgtttt aaaacttatt 1200 cccttgtttc tatattacta atttaacatt acttatttta attatttttc ccttttttac 1260 ttattatata tatataagta catattacca ataataatat aagcaatcac atatatttat 1320 cccattaa 1328 <210> 16 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> 3'UTR of allele 1 of g1544 <400> 16 tccatcatca agaatatata tatataataa agccatccct tttacgaacc tgcctgcatt 60 tgcttaagac cgagcaaaaa aaataaatta caacataacg aaaaaaacaa acaaacttaa 120 gggggagaaa aaaaaataat atcccataac ttacatacac aacatacata aaattaaaaa 180 aataaacatt ttatcaataa ttttttttta aagtatatag agctactaat attatagaaa 240 tacagacgca acttaaagaa ctttgttcaa tcttttcaat cttctcagtc ttttctagtc 300 ataataaatt atcaaatgcg aatatttaaa tcaaaattat ataaggggta tatcgtatat 360 atataaattt atcaaatgtg tatatgtatt ttattatgtt ta 402 <210> 17 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR of g1544 allele 2 <400> 17 tccatcatca aaaatatata tatataataa agccatccct tttacgaacc tgcctgcatt 60 tgcttaagac cgagcaaaaa aaataaatta caatataacg aaaaaaacaa acaaacttaa 120 gggggagaaa aaaaaataat atcccataac ttacatacac aacatacata aaattaaaaa 180 aataaacatt ttatcaataa ttttttttta aagtatatat agctactaat attatagaaa 240 tacaaatgca acttaaagaa ctttgttcaa tcttttcaat cttctcaatc ttttctagtc 300 ataataaatt atcaaatgcg aatatttaaa ttaaaattat ataaagggta tatcatatat 360 atataaattt atcaattgtg tatatgtatt ttattatgtt ta 402 <210> 18 <211> 4032 <212> DNA <213> Artificial Sequence <220> <223> C2862_g1_i1 <400> 18 tctaatattt taatcttttg accaaatatg tttttgtcgc ctattgataa tagaaaaatg 60 taaccttcac aaacaaccct aataccaaga gaacgaaaga tagggtatat atatatcatg 120 aatgaatatc actaacaacg aaatataata ctcactttct cgaggcggcg tccatccata 180 caccgcatac ccattacaag aagccaagtc tgcctgcatt ttttttcttt ttcaataaag 240 aaaagaaaac cggggttttt gcctatttca attatagtta attctccgta gcttaatatc 300 atgttctctc gaaaatgtct tttgtttgca aatacctgca ataagtacaa ataatccggt 360 atgttgaaaa gaacaataaa aaataataag ggccaccgtt acactgtatg gccacacaca 420 ataccgtttg tggtatttcc cgcgtggaac aacaacaact gatttgtttc aaggttgctc 480 tccctccatt ttcacagaat ccaggttctt ggtgggtggc gtgttctggg attcctgtaa 540 tgacaacgcg agacaaagcc aggagacag aaaggggacg gcttctcatc ccatcagtcg 600 cagcaaccgc ggcttctctct agcacgttcc acgcttttta tagtggttaa ctcagttttc 660 tctctttccc tccaccccac gttactctgc gaacaaaaaa tacgcacaga atgaacatct 720 gattgattaa tatttatata ttactcagtg gcacccctac aaacaaacca attttgaata 780 ttgttcacca tcatgatatt tatttagggc aagaatttca tgtacatacg tgcgtgtact 840 gcatagttt gttatatgaa aataaccagc aatatatcac caatgaataa attctcaata 900 atttatttgg aaccaaataa tgcaataact agcaaactaa gtggtgatta tacaacagct 960 gttaacaaca caaacatacg ctctcttcta ttatctcttc cctgcttgtt cgtgtggtat 1020 attcacgaat ttgcaattta gaaattatat tttttaaaag aattgttctc cattttctgg 1080 tagtcgtaag tggcaaattg gatcataaga cacaatcttg ttagttcgac tgctaacacc 1140 agacaacacc gaacgaaaac aagaaaaaat aattatctc tctcttttta aggtatcttt 1200 acatcatatt acatatccca aattacaaca agagcaagaa atgaagcaca acaacacgcc 1260 atctttcgtg atttttat catttctata tcgtaactaa attaacaaat gctatgtttc 1320 ttaattttta atgataaatc taactgctac cttaatttct catggaaagt ggcaaataca 1380 gaaattatat attcttattc attttcttat aatttttatc aattaccaaa tatatataaa 1440 tgcaattaat tgattgttcc tgtcacataa ttttttttgt ttgttacctt tattctttat 1500 ccatttagtt tagttcttat atctttcttt tctatttctc tttttcgttt aatctcaccg 1560 tacacatata tatccatata tcaatacaaa taaaaatcat ttaaaagggc ccaacaaaat 1620 gtcttctatg ccaaatcatc aaaaagttgt tttggttggt gatggtgctg ttggttcttc 1680 ttatgctttt gctatggctc aacaaggtat tgctgaagaa tttgttattg ttgatgttgt 1740 taaagataga actaaaggtg atgctttgga tttggaagat gctcaagctt ttactgctcc 1800 aaaaaaaatt tattctggtg aatattctga ttgtaaagat gctgatttgg ttgttattac 1860 tgctggtgct ccacaaaaac caggtgaatc tagattggat ttggttaata aaaatttgaa 1920 tattttgtct tctattgtta aaccagttgt tgattctggt tttgatggta tttttttggt 1980 tgctgctaat ccagttgata ttttgactta tgctacttgg aaattttctg gttttccaaa 2040 agaaagagtt attggttctg gtacttcttt ggattcttct agattgagag ttgctttggg 2100 taaacaattt aatgttgatc caagatctgt tgatgcttat attatgggtg aacatggtga 2160 ttctgaattt gctgcttatt ctactgctac tattggtact agaccagtta gagatgttgc 2220 taaagaacaa ggtgtttctg atgatgattt ggctaaattg gaagatggtg ttagaaataa 2280 agcttatgat attattaatt tgaaaggtgc tactttttat ggtattggta ctgctttgat 2340 gagaatttct aaagctattt tgagagatga aaatgctgtt ttgccagttg gtgcttatat 2400 ggatggtcaa tatggtttga atgatattta tattggtact ccagctatta ttggtggtac 2460 tggtttgaaa caaattattg aatctccatt gtctgctgat gaattgaaaa aaatgcaaga 2520 ttctgctgct actttgaaaa aagttttgaa tgatggtttg gctgaattgg aaaataaata 2580 agagctctac cgttcgtata atgtatgcta tacgaacggt agcgatcgct ttgtctttat 2640 ttttgaaatg ttaatagtct tttttttta ctttgaacaa aaaaaagtaa attaaaact 2700 tatcttatat acgcttttaa acattaaact cgttaacgaa ttatataatg attttatcga 2760 actactttat gtttttttaa tagaataatc ttctttatta atataactta ctacttctta 2820 atcttgttgt cctccattcg aaactcgaga ggaacaattt ctgagtctct ctcgcaccct 2880 ttcgtacgta ccgtttttcc aatttctttc gggaaacgga actggacgca tttatttga 2940 ctgttgaaag ggagatttaa tatttatata gagagatata acaactaact tataagttta 3000 tacaggctgt tatcacatat atatatat caacagagga ctagctcaat agaataacat 3060 tagatatgtc gatgctgaac cgtttgtttg gtgttagatc catttcacaa tgtgctactc 3120 gtttacaacg ttctacaggg acaaatatat cagaaggtcc actaagaatt attccacaat 3180 tacaaacttt ctattctgct aatccaatgc atgataacaa tatcgacaag ctagaaaatc 3240 ttctacgtaa atatatcaag ttaccaagta caaataactt attgaagaca catgggaata 3300 catctacaga aatcgatcca acaaaattat tacaatcaca aaattcttca cgtcctttat 3360 ggttatcatt caaggattat acagtgattg gaggtggttc acgtttaaaa cctactcaat 3480. acacagaact tttatttcta ttgaataac tacatagtat cgatccacaa ttaatgaatg atgatatta gacgatta gctcattatt ataagaatac ttcacagga actaataag tcaccatccc taaattggat gaattcggta gaagtattgg aatcggtaga aggaaatccg caactgcaaa agtctatgta gttagaggtg agggccaagt tcttgtaaat aatagacaaa ttaacgacta ttttgtcaaa ttaaggata gagaatctgt aatgtatcca ttacaagtaa tcaatgggat tgctaattat aatgtattta ttactacatc aggtggtggt tcaactggtc aagctgacgc cgcaggatta gctattggta aagctttaat tgcattcaat ccattgttaa agcaagact acatagccc ggatgtttga ctaccgatta cagacgtgtc gaaagaaga aacctggtaa agttaaagct agaaaatcac caacttgggt caaaagatag acgcacacga 4020. tttctttcgt tacatattct tacatatttt aaacatatac attcgtacca tgtaaatatt aatatcaaca is <210> 19 <211> 3067 <212> DNA <213> Artificial Sequence <220> <223> c4821_g17_i3 <400> 19 caattaactg atggtccatt ctttaacaaa taattttttt tttgttagaa gttattttaa 60 aaggaattaa cagaaaagca atgactgggg tcattaggat tgtcaatata aaagcaacta 120 aactcctaag agtttactgt acgaacggcg ataaggtagt tcatcatact tacttaataa 180 ttacaggaag tgacaataca aaaagaaatc tagttctgga gagaaaacca agggtaagga 240 atgaaacaga actgggaaga gaaggattct tcctgctgtc ctgcgctttt tcctagtgga 300 aatacatgca caaatttttt tttctgatgt atatttcctc tgtgtgaacc acgtagctct 360 gtgaaaaagt atcgtaggct agtttgaatg tggaaaatta gcgggggtgg ggccccgata 420 gaggctaaag ttatgttaaa attgtctacg ctagattcac tgaaattaca cgttgaactg 480 gaaaaataat ttccccgggt gaatgaaatt tgtcatgcag ctgtaaaacg ggacacagaa 540 aacggcgcat ggtgaaaatt tttcagttgc ttttttggtg gctagtattc aaataatttc 600 tccttgcagc cacatagatg aaaatgaaga agttaaagaa caaaaagatc ccctacaata 660 tagatttgca actacatgca accataatca tggtaacaat tgaacaaaat gcagcagcta 720 aaggtgcaaa ttagtttctt ttgtgcatta atttcgcctg aaataatttt cctttttttt 780 ttttttttta ttttttctgg aatcaacatt caaattatct aaagaacctc tgcagaattg 840 tttttttt cttaaagatc aaccaactta aggaaatttt tttcaaagtt ttgctagtgt 900 tttctctcct ttaacccact tcatccaatg gttatcttg tcgttatgct acgatatttt 960 ccaggcggaa ttgctttttc tgccttgttt tgattattaa atagtttctc cctttattaa 1020 taattattcc atgaacaaaa tctccccttc atttgattca gaaatcactg cagattaaag 1080 acactcatgc aagttgaaat tgaattaata aattactttt atttcatgca aagctcaaca 1140 acaaggacaa catgaatgat gaaaattcca aaaagtaact ctttcagaaa taggaaaaaa 1200 aaagatataa aaggtcaacg aatattccaa cttttacaga aataatttcc tttacaactt 1260 ttcctatttc atatttcatt tcttttgttt attttaaaaa taaaaaacca tacaactaaa 1320 gatttatatt atatctcttt aacaataaca attcagtaaa tatatacttc aatatgtctg 1380 ctgctcctgt tgaagaaaac attaataacg agtctcaaca attgactcca actgcctctg 1440 gctccaactc tgttctatct actccatcta acaaagctga cagagatgaa ctaaaagatg 1500 aagctgaaaa cgctgaagat aatgtcgctg cttttgacga tatgccatta aagccagctt 1560 ccgcttacgt caccgtctcc atcatgtgtg ttatgattgc tttcggtggt ttcgttttcg 1620 gttgggatac tggtaccatt tctggtttcg ttaaccaaac tgattttatt aacagattag 1680 gtcaaaagcg tcacgatggt tctcactact tatccaaggt cagaactggt ttaattgtct 1740 ctttttcaa cattggttgt gctatcggtg gtgttatctt atctaagatc ggtgatgtct 1800 acggtagaag aatcggttta attactgttg ttaccattta cgtcgtcggt ttaattattt 1860 ccattgctac ccaacatgct tggtaccaat atttcattgg tagaattatc tctggtctag 1920 gtgttggtgg tatttctgtt ttatccccaa tgttgatttc tgaagtttct ccaaagcatc 1980 taagaggtcc attagtttcc tgttatcaat tgatgattac tctaggtatt ttcttaggtt 2040 actgtactaa ctacggtacc aagaactact ctaacactgt ccaatggaga gttccattag 2100 gtctaggttt cgcttgggct ttattcatga ttggtggtat gatgtttgtt ccagaatctc 2160 cacgtttctt agtcgaagtt ggtagaaatg aagatgctaa gagatctatt gctgtctcta 2220 ataaggtttc catcgacgat ccatctgtac aagctgaatt agaattatta atggctgctt 2280 ccgaagctga aagattagct ggtaatgctt cctggggtga attattcgct accaagaaca 2340 agattttcca acgtttaatc atggcttgtg ttatccaatc tctacaacaa ttgactggtg 2400 ataactattt cttctactat ggtaccacta ttttcaacgc tgtcggtatg aatgattctt 2460 tcgaaacttc tattgtttta ggtattgtta actttgcttc cactttcgtc ggtatctggg 2520 ctgtttctag attcggtaga agaactctat tattatgggg ttccgcttcc atgactgctt 2580 gtatggttgt tttcgcttct gtcggtgtta ctagattatg gccagatggt gctaaccaca 2640 aggaaaactc ttctaagggt gctggtaact gtatgattgt tttcacatgt ttcttcattt 2700 tctgtttcgc tccaacctgg gctccattag ttttcgttgt ctgttctgaa tctttcccat 2760 tgagagttag atctaagtgt atggctttag ctcaagcttg taactggatc tggggtttct 2820 taattggttt cttcactcct ttcattactg gtgctattaa cttttactat ggttacgttt 2880 tcatgggttg tctatgtttc tcctggttct acgttttctt ctttatccca gaaaccaagg 2940 gtctatctct agaagaagtc gatcaaatgt ggctagaagg tgtcttacca tggaagtctg 3000 ctcaatgggt tccaccatct aagagaggtg ccgaatacga tgccgaagct atggctcatg 3060 atgataa 3067 <210> 20 <211> 5390 <212> DNA <213> Artificial Sequence <220> <223> c4795_g1_i1 <400> 20 gacaataata aaaaataaag gtgttgaact gtcaacaaaa tacagttaat tgtacggtat 60 gtaattttca tcattcacat cgacttatgt ttatgctgct cctcttcata atctgctaca 120 attaaattgc tctttttttt ttgttatcaa cagaatatat atttcctgag gggggaaaaa 18​​​ttgtcaactt ttgattgtat cgcataaaat atttactcca ttgcaaatag gaacttattg 300 actataacag taatttcctt tattaataat gatttattt ctctttatgt atttgcataa 360 taactgggac atttttgctc ttgttcagcg gtaaatcgtc tagacgaagc ctatgtatct 420 attaatctat tatagaggtg atgtccctc gtagtcaata aattctaagt acacatatac 480 atacgtaggg gcactcacac tattatattt atttctttc tttcttat ctggcaatac 540 gatacggaga ccggagaag aatgttcgtg ggaaaaaaaa actttttt ttgttcttag 600 aaggtttcat ttcaccag agtaactccg gataaaaag ggataccgta aaaccccgtg 660 cgagtgagat ttgaattcct atcatatcgc aatttgtcgc aattcatatg gttctttcat 720 780 acaatagcaa tgaatttaaa ccctgaaata attatattat tgattgatg agttttcaat 840 aaagagag agaagaa aaagttagaa ttgatatcgt agatggcttt atccacctat 900 tcattcaagt ctgtcagcac ttcattcaggt tagagatag acctaatacg ctggttccac 960 aaattgaa ctaataaatt acactatatt cctttttgtc tctggataaa agatgtatta 1020 tagtttcaag atacatattt gaaacgtaca agtaataca agttgttaaa accataattt 1080 aaaaaaaa ttcttgc aacattgatt ggaacgacta cattaaggt tctatatccg 1140 atgcttcaat atgcgagat tttagaaag caatgcct acagagaat gttaagtaat 1200 ataatcaaga cattttttc ttacaaagc aaaaaaagt gaaagtcgg aaatgtctta 1260 agacccgaga atccaggac cgatgtgaaa aaagtttaat tatcaatga tattacagt 1320 tattcatacg tacattaat tcccaatcta atatatattc atatgagtgt agtgtatatata 1380 atcttaacta atgcatactt cactttaat gattacaaa tgaaacagca ttttaatct 1440 tattattagc attackaccaatc atttaaagta atttattatt tcgaggatag atagtatttc 1500 ttgtcgacat aacataa gcaaatttct tgtatctcta attaggtaac tcccgctccc 1560 cccccaaaga aaaaaaaac cactctgca agtttcagtt atttaataat atgggaagc 1620 gabacattcg gagttttatta attattacac acatatacg tcatatttat ctataagtgg 1680 taactaatat gccaattttt tacaagaaac aaacgaacac ccatatgtta cggtaatggg 1740 aaacaacata atttgtcaaa tatatggcat atatctatca agttttacct gatatcttca 1800 attcggaaag ttacttgtta tggtaaaaatgaattagctg gcccttaatt tatgacaaga 1860 atgagctatt atctggggta cgtttattta tcgtatacct acttataaga atgtaaat 1920 aataagtttt gaaagatttg attaagactt tgggaatggt aaaattgtta ataatgattt 1980 attaattact gtcagatatt aaaactccat cgttaccaga agttcattat aattttcaca 2040 tgcttctact aaaatatttt tgttgagctg ttatgcgtgt catttgtgac actgcgatta 2100 tgagtatgtc attcatttaa catcagtttc tccaagttat ttaatttttt tagtgtcata 2160 ttgttattac cccaatattg tcatacattt atccccaact aattataata ttctcaataa 2220 ttatagcttg gcgagtaaat ctttcaataa tctgttgaga aaaacctgtc ataaaatatt 2280 acgaatttct tttcaacagg tacaagcaca tgaataatct taactttatt ctctattggt 2340 ttaattaaac acttttaaac tgtggaaaca tactaatatg gtttatacag acatgtacgt 2400 atactccaat ttttattga aatacatacc ctaatttcag cccttcattt tacgcgtatc 2460 atcttgaaca gatacaagtt acctaattag gaaatgtaat atcttgaagc caaaaatcta 2520 ttttttctct ctcttctcg gaaaacgc gatcaatctc tttaccgaat gaggtaatct 2580 taattacacg aaaaatttc agatattttt ctctctttct cgaacagtgt ttggttaatc 2640 gaaacataat cgtaaaataa acacataaac cttccgtttg caataccttg ccgtcaattt 2700 aacacccttt tcatactttt tcaaatatt atattcaact aaaagttaaa aatcagttaa 2760 ctaacgtatt tttacaacat ttgttaaggg aataatagaa gctatcaaac gttaagttat 2820 cacacagtta tatcatcaaa caacaatgtc atttgataga ccagaaattt atagtgcgcc 2880 agttttacaa ggtgttacac caaacgatga tgataacaca gaaattatca aatcctttag 2940 aaattttatc cttgaattta gaatcgattc acaatttatt tacagagaac aattaagaaa 3000 tgcattatta gttaagaatt attcattaag tgttaatatg gaacacttaa ttggttataa 3060 tgaggatctt ttcaaaaaat tgtctgatga accatctgat attatcccat tatttgagaa 3120 tgcaatcact caagttgcta aaagaatcac tatcctaaat agatctcagg agtctaccac 3180 aggtaatgga caagcaacag gtgaggatat cgcatctttg attccaccat ttcaattaat 3240 cctaaattcg aaagctaatc aaattccaat gagagaatta ggttctgaac atgtctccaa 3300 agttgttaga tttcaggta ttgttatctc tgcatcagta ttaacatcca gagctacaca 3360 tttacgtcta atgtgtaaga attgtagaca tacaacatcg atcactgtaa atacattcaa 3420 ttccattact ggtactcaag tttctttacc acattcctgt ttatctaatg ttcaaactga 3480 atcaggtcaa gtaagttcca tggaggcaag tgctccacca aaaaattgtg gacctgatcc 3540 atatatgatt atccatgaag cctctacatt tattgatcaa caatttttga aattacaaga 3600 aatcccagaa atggtaccag ttggtgagat gccacgtcat ttaagattat catgtgatag 3660 atatttgaca aataaagttg ttccagggtc tcgtgttaca gtagtcggta tttattccat 3720 ctataccgct aaaggtgcag gaccaagttc aggtaacgaa ggtggtgtct ctattagaaa 3780 tccgtatatt aaagtattag gtttacaaac tgatatcgat acaaatactt tctataattc 3840 tgtttccatg ttttccgaag aagaagaaga agagttttta caactaagta gaaatccaaa 3900 tattatgat cttgtcgcta aatctatcgc tccttcaatt ttcggtaatg aggacattaa 3960 gaaagccatt gtttgttat tgatgggtgg ttccaagaaa ttattgcccg atgggatgag 4020 attaagaggt gatatcaacg tttactact gggtgatcca ggtactgcaa agtctcaatt 4080 attgaaattc gttgagaaag tctctccaat ctctgttat acatcaggta agggttcttc 4140 tgcagcaggt ttaactgcga gtgttcaaag agatccaaca acaagagaat tttatttaga 4200 aggtggtgct atggttcttg cagatggtgg tgttgtttgt attgatgaat tcgataaaat 4260 gagagatgaa gatcgtgttg cgatccatga agcgatggaa caacaaacca tttctattgc 4320 aaaagcaggt attactacag ttttgaattc aagaacaagt gttcttgcgg cagcaaatcc 4380 aatctatggt cgttatgatg aaatgaaatc tccaggtgaa aatattgatt tccaaacaac 4440 aatttgtct cgttttgata tgatttcat tgttaaggat gaacatgatg aagccccgtga 4500 tatctctatc gctaaccacg ttattaatat tcatacaggt cgtgtctcgc aagaacaaga 4560 agaaatggaa aacaatggtg aagaaataag tatggataaa ttgaagcgtt acattactta 4620 ttgtagaaga aaatgtgcac caagattatc tgttcaagct gctgaaagat tatcatccca 4680 attcgttacc attagaaaag aattattaat aaatgaattg aattctactg aacgttcttc 4740 aattccaatc actgttcgtc aattagaagc tattattcgt atcactgaat ctttagccaa 4800 attagagtta agtcccgtag ctcacgagag acacgttcaa gaagcaatca gattgtttca 4860 agcatctacc atggatgcag catctcagga tccaatcggt ggtatggatc caacagggaa 4920 ttcaagatct atacttgcag agattcgtga aattgaacaa gaactaaaga gaagattgcc 4980 gattggttgg tcaacatcaa tacaaacatt aagaagagag tttgttgaat ccaatagatt 5040 ttcacaacct gcattagata aagcattata tgcgctagag aaacatgata ctattcaatt 5100 aagacatcaa ggtcaaaatg tttatagaag tagtatatga tctggtgata acacatgggg 5160 taaagtaatc ttgaatcaaa agtttgtaat attaatacat caatttgtct agattgaaac 5220 atatatatac gtacatacaa ctaatgaata tattctaggc aagagcaaaa aataatacat 5280 agtatatgga tccgattctt atggagattg aagtaaactc aaccagccct atactcttca 5340 atttattctt gagttgtaag ttaaccgaag gacggtcgga acgaacgcag 5390 <210> 21 <211> 5128 <212> DNA <213> Artificial Sequence <220> <223> c4321_g4_i2 <400> 21 attgaaataa ctctttcttt gatacatcac ctaaagacta tcaaacacat ttattaatat 60 taagatttta aaatattaag aaacttttcg tcaaaagtat atttcaaagt tttttttaat 120 atattcattc agtttaaaaa tctcattaat tcattcatga taatataaga gattttctaa 180 atattttctc acatcatcgt tctacaaaaa taatattatt gcaaaactaa attaaagaag 240 gaattttata attttaagtt ctaactttca actgtttttt attgttcaat tttattttca 300 ttaacttttt cacaaaattc tacaagtatt tccttaatga atttaaccgc ttatcatcca 360 tatccaattt ctaaccaatc aacacaatat cataaaactc taatggaaaa tccatcgata 420 ccaaattcaa gtgtagcaac ctcatctatt acaactacgc ccacaaatga tttcaataat 480 gatttacctt caaactctag gaggaatct gaaacaggct ccacaaaggt aacattacca 540 ccaatctcta gtatcatcaa tgctccacaa gaacaacaaa ataaagtat tagatctgaa 600 atagttgaac ccgaaaactc tacttcttta aggacatctc ctttaacgca gacggatatt 660 caaaattcac agtcaatgaa taatagtact ataactcctg ctggtccagg tattagcact 720 tacgttcaac caatggtaaa ttccagaaga ccttccgcaa cacagcagca aatgaatatt 780 aattatgcta caccaagaaa tggtatggca gtactaggtg gggtttcttc ccaagcaact 840 ccaattggta ccccagggaa tagcccaaac ggtaattatt tagctaatca agcgattata 900 cagcaagaaa atgaagctgc tgcttatgca attcaacaga aacaacaatt gcaacagata 960 fạttcaac aacaagttca agctcaagct caagctccac aacaaggcta ttattatgtt 1020 gttgcaccat ttcaacaaca acaacaagtt caaacgcaag cacaacaaat gccacaaatg 1080 atgccaatgt ctatcactca acaacaaatt gctttccaaa aggctcaagc acaacaggtt 1140 gacgaacaac aggctcagca gcagcatatg cttcaacttg cacaacagca acaacaacaa 1200 gctatttcta catatcctgt tgttgttaat atgccacatc caaatgagat tcaacacag 1260 1320 ccaagacaac cagaaggtat aatgaatcaa ggtcacccaa taatggttcc aaccactgct 1380 attccaacaa gttctcaacc agttcaaaaa ccaacaatga ctgctggtta tgttacatca 1440 gaaggtttaa ttcctgttcc aacaagtatt caatctaatc taagcttagc tgttagatta 1500 cgtaaacaat gcccagtatg tggtaagatt tgttctagac catctacttt gaaaactcat 1560 tatttgatcc acactggtga tacaccattt aaatgtccat ggagaacatg taagaaatct 1620 tttaacgtta agagcaacat gctaagacat tttaaaatgcc atcaaaagaa atcaccaaag 1680 gttactaaag gtggttctaa ttctggtgat gaaaaaact ctagaagacaa tgaaaagaca 1740 attaaggcta ttgaaggggc agtatcatca tctgaaaaac aatcgaaagc tactgatgac 1800 1860 1920 aagcaaaaag ggctatctat samaaatttt actacatata aaaatgaata ttcatacta 1980 ttaaacacaa atagaattgt aaagtttcga aaacaggttt ccattaactg ggacaaggat 2040 acgtttctcg gatctgtttg gctgctatat tattaacaat ctatccagtt tccaaaaact 2100 gcacttccct attcataaac tagcatctga ttatttttga aagccgattt gagtttcaaa 2160 tcttacttaa tgaaacttta ataatccctt ctgtctctat ctttaagagg ttttgatacc 2220 samaaatct taacaaaagt ccctcatttt caattccgtc aaaattgatt ggttaataat 2280 attcaatgat tgatttgcat atatcgtctc gaatttgagt cttcaagctc actattgtga 2340 catagacaaa atattatctc aaaggataaa gcaaaaaatt aaattgaaat ttcagtaatt 2400 aatcgaatgg tttgttagta attaataatt atgaggtcaa atgaaaacag atcgatactc 2460 gtttcggaaa tgctaagagt aaaccaaaat aaggttttat ttccaaaaaa aggaaagtaa 2520 aaagaaacta tacattgcct attgtggaag gtttagtaaa tctccgaaga acctgcgggc 2580 gagcggatga attttgtttc ctgagaaata aaattttttg atatatctct gtaaatatcc 2640 gtagtactgc tgttgtttcc tagaatattt agaaacatcg aagagaaagg aacgcgggac 2700 aaagaaataa gatttctatg tttagcgtgg gtagtaaggt cacttgtacg tattgttctg 2760 acatcgcata gatatctaca aaattaagtc aatttagaaa agtgatcagc aggtgagaag 2820 atccagtagc caattcatta tttgtgcaca aatttactgc aaaaggttat gtatcttgca 2880 tatccatatc gagacctaat ttaggaattt taatatttta actgtcctca aacttattca 2940 attcatttac ccttctctga ctatttcaaa caaggcactg gaatttctta gataaagaaa 3000 aatataattg caacatttgt ccacattatt gctctgttta acagcgaaaa tcgtgtaaat 3060 tttagtggga aaacataata ttaactacta ccaaattctt tcatgggtac tatcagctta 3120 aggtggaaaa taccaagtgt tcttctatta tctaatagtc cctaagatat ggagggaccc 3180 aagtaagaga tattgaaatg ttccccaaag ctatgcccca cttgaatatg ctttcccttt 3240 caagcttcct aaacatgtaa cattcttagt attggataag tgctgactta tataacaagg 3300 tttttctttt aaatccagga tatataaaca aactctaagt aaaaaggtta gagcaccgaa 3360 ctaaacgaaa tcaagaa cttcgtttga agcgaattgt atagctcaac caatcaggaa 3420 chaatagatca atattagt atgatgtgat atattgatgc tactaaagt taacggaag 3480 acgaaaatga tatccgatta atcgatttac tcgaatacag taattatta gagatgaaa 3540 gataattaa ttgtgaaac attackataat ccaacacag atcaatatt agtatgccct 3600 taccacctct taactctttg ttaggttgtt tttagacaa gctataaatc ataagaggtc 3660 aggtgataag agccactgtg ccgaacctga accgtagat atagttgaa gtagtgactg 3720 gaatagcct ggcccgctat gatctgta acagaccag tacacactca acataggaa 3780 gttagatacg cacgtgataa gagtgtagc cttagaagct taagtagata acatgagcg 3840 acaatacaat agtatgtccg ataagagttc cgataagaga acacatgtcc tagttgttct 3900 aaatacaa tagcatatac atgctcgtca acgaggaggt ccctacgtta ggattccttc 3960 cgagtgatta tcacagtgt ggtatgagac acacgaccag caataataa agaagata 4020 fatheradaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaataaaaaaaaaaaaaaaaaaaaaaaaaaaataaaaaaaaaaaaaaaaaaaaaa / s                                                                                                                                             caagtgctta ataaaaata attack cttaaata attack attack windows 4140 agtgtacata agatacata tataata gattacata taaatactct tatcacaga 4200 aagcgtctaa aactaact attacka tgattgatt aactaacac aaagacac 4260 taagactcg aagacatcc aagggaatcg aagacatcta cttaatgtag atctgagaa 4320 tctaggaatc tgagaatcta agaatctgta gttaacatca acagataca gcttggcact 4380 atcaccaact gaacccatct tagctatgca cacaacatag tctactgtca actcttta 4440 ttggaacgaa gagaacta gaactagaa ctacttatga taacttcaga catcttatca 4500 actaagaatc tcgaattatt attackcatga tacagcatag cgtaatgagc aactggatcc 4560 ttatcgtaca agaactatct atcatagaca acgtctcttg taagttccct cgattaatga 4620 aaggaactaa ccaatgactc ttcattca taggacact caatacgac tctcgtgtat 4680 aatctatttc caaaaaaaaaaaca ccgtaaaaca ccagagccgt tacaaaaaat 4740 tcgaaagttt gattatttgc tattgttaa ttggaggt ttctacggc tgtttcttagg 4800 cagggagtat taaagataag attatcactt cctaaaacat ttcaaatgag agtaatgtat 4860 tgatatgcat ttgagaatgt acggataata ataatacatc ccatataagt ctaatatatc 4920 aaagaatgat accgtaataa ttcaatcaga ccacatacta aagcttattc tcggtttatt 4980 acgaaaagat atttgtctaa aataagtttt ctaagtattt tgattccatg ttattggcaa 5040 tatatatctc atgaatcaaa tcaagatgtt gcaaaatcca ttatgaggtt gttgcaaagt 5100 ccaatatgag gttctaagtt tctatatc 5128 <210> 22 <211> 2759 <212> DNA <213> Artificial Sequence <220> <223> c144_g1_i1 <400> 22 ccattgtcca acactaccaa caataacact aacaccgaag gttcaggggt tgagtccggt 60 gaacacactt tatcgagtat cccacctcaa cataatgcca atacgttctg tttttttggt 120 ttttattata ttatgtaaat tataaaatct taaatacatg aacgaactga aactcaaatg 180 tgagtccaga tttacccttt atacacgttg cttttactaa cttaagcttt gatcatcaaa 240 tgaattgtga tttcctaata ctttgtgttg tattttgt accatatttc tgtcacttat 300 gtaaaatgga atacaata ctactattca attagtgatc agtcagaact ctttgagaac 360 tgaagtatt ctttcaatc ttctattc tctcactct cttctacag ttctcctc 420 cttgatgggg gaacccaaga tgcagatttc catggttgta caccttctc atatagtaat 480 tggattctt ccaagacaa accaatcgtt tccggtagaa agagaaac atatagaaac 540 atagctacca aaaaccgac aaatacgtaa ccatagtaga agtggataga accggtaatg 600 aatggtgtga aaaaaccaat caaaaattgc cataaccagt tacatgcggt agaatcgac 660 atggctctgg acttgaacct cgaggggaat gattcggcaa ccacatata agcacagga 720 gcccatgatg ttgcgaagca aaataggtag aaacaggtaa atacaatcat cacattacca 780 gcacctttcg aggatggggc actgtcacca tgaggataaa gatattgac tccgacactt 840 gcgaatatga ccatacaggc catcatgcca gctgctccaa atatagaca tttacgacgg 900 ccgattttgt ccacaactat aacagcaata atagtggaga agaaattcac cgtacccaga 960 atgatagaag tctcaatcc gtcagtaaga cccactgatt tgaaaatagt tgtaccgtaa 1020 aagaaaaagt agtttcacc agtaagttgt aaaaacgttt gcactagaat acctgtaatc 1080 aaacgctgaa ggatatttga ctcaggtgaa aaagttcct tccatgaagc ttcaccttgt 1140 tccctttggg caagcacacc ggcgatatt tcttactt ctccatgtac ccatgcatcc 1200 tctggtgaaa tcttgttgat cttggcaata gaagcgcgtg cctcatcatg tctttcctgt 1260 tcaccaagt atcttgggga ttctggaacc atagcatac caatgatgat aattaggggcc 1320 cacaaaaagc aaagtccaac agggatccctc cattgtgcag tattata ctttctggtg 1380 ccataacac tacaatacc taggaaata ccaacgtca tgttcaatg atacaatgaa 1440 acaagcccac ctctcatgtc tttaggagct atttcagaca aaagcattgg accacacc 1500 gaacatccac cagcaccgag accataatg atcttaccga taaagtattg gtaccacttg 1560 tgatttgaac taatctgaat aattgcacca atcatatata ccaataccac gatgacaatt 1620 gctaaccttc tacctaagt atctgcaaaa cgggcaaaa gagaccctc tatagcacaa 1680 ccaacactga acattgccac tagaagaccc atacgcacat tactcaagta atattctcca 1740 gtactgtgtt tgtaagaacc gaaattcatt ttaaagttgt ccatgttaat gaacccagcc 1800 gtaataccac tatcccaacc aggtaggaac cccccaaagg agataggaat acaaagcaga 1860 tagatagtaa gataacctag atatcccctc tttggtggtt caatggaatt tccgttatg 1920 acttcattgt cataaacccc atccgaccac tctttttcta caggtggcga gacataaact 1980 tcaatattag aggcatcttg aatgtctata ttactatgaa aagatgattg tgaactagac 2040 attttttta ttttaattttt taaagccttt cttttcttt tttttagtta tattattata 2100 gaataaaagt ataagaataa atgaatgatg cccattgacc ttgcccttta tatatgatgg 2160 tcgaattgca attctatgat agatgatgat aaattcaata gcttatatca tctctttaac 2220 ataggttcaa gggaagtaca ctcagggata tgatctgcat tcaaacggtt cggacggtcc 2280 agacaattca gacggttcag agtattcgtg gaaagtgaac cggctgctat cattagctaa 2340 tactatcctc caaaataata cgtcattact ttgggtgaaa tgcattatta gctaaactgc 2400 attcattgta cgtcactctc tgtggtacta acaaaccact gtaaaaaaga aaaaaaaacg 2460 ccgaattctt tgtgcctgac agattgttgg gatctccacg gatcattttc agcgccccat 2520 gttttgccga atgcataccc cgcccttacg ttgagctttc ataactttaa atttcagtcc 2580 ggaggtattt gttttgagag tgggcctcgc agtgaaaatt ttggaaaagt ttcacacata 2640 gttaagttac atttggacct attttcacaa tacaatggaa ctttttttcc aacttttaat 2700 atcccttccc ctatacaagg gtaaaatatt ttcatttttt actttccctt ccctttcac 2759 <210> 23 <211> 1217 <212> DNA <213> Artificial Sequence <220> <223> c2309_g1_i1 <400> 23 cgcaaatttg acaaatggtc attttactcc aatttttttt cttattttga aaaatttccc 60 tgataaacaa aaaaaaaaat tgaattactt ctaaaaacgt taatcattta tcctcattgg 120 aagttacttt ttttttcctt ctctgaaacg tgcggagatg atgaggggta aatattttga 180 atttctcttg tttttctttc ttgctgctga catctcacgt ttgacgaaat ggagaacatc 240 agttgccgcg gatccgaaaa gacgattaac taaaaacgcc tcttcattta gttaatcttc 300 ttgctggttt cgcgtctcct tattaccggt tcagctgatt gatattatct cggagatgag 360 caaaacac cagttgagtt catgattcta tatttgtaaa ctagttttac aatgacatca 420 taaattaaag ggaaagac aagagttact taagaatctc gagttctgtt tgtttgtttg 480 tttttttaat taggataatat cgctgagaga tagatttaag attacataa aaacaactga 540 tacaagaata attaacatta acgacctctc aaccataaag tgaacgtagt ccacttatat 600 tttcatattg cttaactgga tttcattta gaaattgtac agctcattga atagtcgagt 660 caataattca aattccgatt atttaattac cacacaccct tatatttgat caactgacaa 720 aggacatttc cttagtgaaa cagacataat agccagcaaa tcattccatt gcattttgat 780 taagactcat tttttatcat atattgttac tgtttcagaa aatgtcaact tttccatcaa 840 ttttaatgat ggatattaca gattctaata tatcgatacc tgattcaaac gaccccacaa 900 gaaggaagt tggtcttaat aaagacattt atgactcgtt tgacaatgaa ccatggttac 960 ataattcatc acaagatatc acacaattga agatgatgaa agttgataga gatctattat 1020 ttcaaattat tatgattgaa gatatttcaa aatctaagca atctcaattc gatgaattaa 1080 gtacacgtat agatcctaaa aatcaacgtg ttgatacttt aagaaattca aagggtccta 1140 aaaaatttga tattgttact caagttgatt tagataatga taatgatact tcaacaacaa 1200 caacaaataa taataat 1217 <210> 24 <211> 2127 <212> DNA <213> Artificial Sequence <220> <223> c3558_g1_i1 <四百> 24 gttttattta ataataataa tatttaatta ttattcactt ctacttcaaa taatattcaa 60 ctagtttcat ttttttaatt tattgaaaac atactttttc cctttggtac aggtacattg 120 caataataat taataaatta acccttagaa tttttatttt gtcgatctaa ataaaaaaag 180 aaattaaatt atacgatata tataaatcga ctatcaattt agatcatttt taattcgctg 240 ttaatttatt aaaaaaatcc ttcaacctgt tgtaaaatta gacaaacata tcatactaat 300 It should be noted that the content "<四百>" in the translation of is an approximation as the original "<400>" might have been intended to be "400" in a more standard context. Also, the translation of some tags like <210>, <211>, <212>, <213>, <220>, <223> etc. might need to be adjusted according to specific patent-related terminologies and conventions in the English language within the patent field, but the literal translation as shown above is provided based on the rules.CAaaaataat ttctattaaa atggagaat tgtattacta tataccact aatcaacgtc 360 caaataatgg tcaaata tcacatgtac aacaaca ggtacaaca gtacaacctc 420 aattagttgc atattatcct tctacaata ttatattcc ashaaaaaaacaac 480 aacaactaca gaacagcga aaaaaatt cacaatcgca tcctcaatta tatccatacc 540 ctcaattaat gtatactaat cattggtaa atccaaggta tactacacta tacagtccaa 600 ttatttctca accaggtaca tcgacagcaa ttcctattac aagtacttct gctacatcaa 660 tatataatga acgttctaat caataata cacctattcc tacaatgaca agtaatcaa 720 taactggttc aattcattat aaacgaaa taaatgtcag tcctacagca gtgacacata 780 ataatccacc aggtgtacaa ttaccaccat tgtcaagctt ggtgtcacag attaaatcaa 840 catcccaatc atgtcctgat atttctacat tgacatcaca atcgagttct tcattaata 900 caatgaatgc aaacaacgca agagaattta agtctgcagc tacatctac tcatcagctt 960 caagttttcaa tgacataaaaacactg ctactactac tactaataac catagaata 1020 gtatacctta tatacttacc tttcatcgc agaagaga caatctcac tcggtcacta 1080 atcataatca atctacaca ttaccaatta cgaatttccc agtaagagaa atactccac 1140 caatctatac aatttcacca aaaaaaata atgcaataat taaatta atagttaca 1200 atagaaactc aaatgaaat ttaaatcatt tactactag aaatgatatc actgtattg 1260 aaccagtag atgaatat acaatatta atgatga atttaag ggcaaaatg 1320 gtaaaattcc atcacaaca agaaaacaat gtccaatttg tgtaaaatt tgttcaagac 1380 cttctacttt aagactcat ttttaattc atacaggtga taatccatttt aaatgttcct 1440 gggttggttg tagaaagt tttaatgtta agtaatat gttaagacat ttaaatcac 1500 atcaagaaa actagaaaaa tagctaaga aacaagctga tttattaaaaaaaaaaaat 1560 tgaaacaac aaccaacaat agaaatagat agaaacagt atggctaaa 1620 nations cattcatt taaatcaat caattttt cattttactg tctcactt cttactccat 1680 agaataagc atacctaca attaatatt ttgaatatt ccttgaataat ataatcaa 1740 agaaaataac gaaaatattc ttcattgctc taatagcatt cattattctt tattcacttc 1800 atctatagat attctaaatt taaataatat gataatctct tttttttttt tttgcctttt 1860 cagatctctt tatgtaaacg acacactcgc catctttcaa caagacaacg cggccagccc 1920 aaaattttta ttgtcctatt taagcaaagt gtaaactttt cagaagtgac aatgttgaat 1980 taaataaaaa aaaaggaatg caatttctta aatgaataat ttacacatta attagaaaaa 2040 aatcttaaat attttacaaa aaccgaaata aaacttagtt tcaggaataa aagcatagaa 2100 caatgtaaaa aaatcgtggt tttaatg 2127 <210> 25 <211> 5413 <212> DNA <213> Artificial Sequence <220> <223> c1715_g1_i1 <400> 25 ggcgtattca aagtatagtc aagaacataa tatattgagg gtgtgtcaat accacattat 60 agatttagga tatactcgat taagaataat aacaatgggt gcgagttgta aggatcaaaa 120 gaaagctgtt gctatctgtc ttcagagatc tccttgtgta atgatagaaa gaaatagtcc 180 tcaaaaatgc atagacgatc caaacttaag caaggatttg ccagaacttt gtatagcaca 240 aatgaaagca tttctagatt gtaaacgagg gatggttgat atgaccaaaa gaatgagagg 300 taacgctcct ttatctacag gtaagtacga tgaacaatat gataatttat gcaaagggaa 360 gttcgatccc agggaagaaa tgcataaatt acaagtttta aattctcaag agaaagaata 420 aaaaggagaa tgaatttttg taaataaaat gacaacaacg aaagagatta atattccata 480 cgtttcataa ttcaaaaatt aatttaagaa tatggtttt atgttataca agatgtatat 540 aagaatgtgt acataagtac cggtataaat cacaatatct ataaacattg atgtaggatt 600 atcctgagtt ttaagaagtt aacttctgtg taaaaatatg ggcctagagt ataggatgat 660 tataccggtt aatcaatata tccttcttaaa ttattaatat gcaactgtat aagtctgtaa 720 tgaccttggt gattaatatt attttcattt gttatagccg attgcgtaat agtgaaaaaa 780 aaatcttaat agcggatcta tgtcttcctt gctgtcagct caagttctta aagaactagg 840 acaaatacac ttgaaaatcc aactttaatc aaattagatg gaagtaatag ttatatcaat 900 agctagctat agttatattc aaaccaactc tataaaaag cactaatatt gcaaatgga 960 tgacgatctg caaatact taaatgtggt gatatttgtc tctcacaa gtctggagca 1020 aaaaattggt gatatgttg aaactaac aaaacagaaaaaacagaaaaaaactac 1080 agattagaa agagctcaga atctatttga cctcattag gggcagctat cctcattaag 1140 aagaaggctt aaatacga accgaataatc gataaagatc aaatttagga aagagatcca 1200 acagttacag gataaagata ttcgaggc acggaagat atcaggga tatatgatcg 1260 acttagagat ctgaaaaagt cagatgatac aaataccaga gagcagatg atggtggtag 1320 aagagaaggg gagtcagaaa gagattactt agtgaggaca ggtaagcta ctgcattgg 1380 ttcaaatca ggatttataa tcgatgataa agtaaattct ccagctacaa aaagataaa 1440 agtggagat gacgcaatac tcgaatctcc cactactgat gactactgaga tggcgaatga 1500 acaatggtt gaaacaata ctgataactc ttcagaaagc gattacaac gaataata 1560 tgggatata tccgaaatg aggattataa tgaagcgac ataatactg aggatgagga 1620 daddy gaggaaagt agggaagct aatgatgagt gtgatgaggtt 1680 aacatatcag aaagattaaaagtggat agcccaaga tctagggga gaaaaaata 1740 caatgaagct ccattacctg agtggcgtaa atcacatcct gaaatttcctg atgcagact 1800 tgatgatatt ttaaaatttc ctggtgatat acacccttta ttattcact atcagaaac 1860 ttgcgtacaa tggttatacg aattatacca acaggtgca ggtggaataa tcggagatga 1920 gatgggtctg gggaaaaaaa ttcaagtgat agcattctt gcagcgctac accattctgg 1980 gctattaaat ggcccagttt taattttg cccgcaaca gtcatgaaac aatgggtca 2040 tgaactccac cattggtggc ctccattccg tctgtcatt tgcattca taggtcggg 2100 tatgtcagat aaagcaaaa tgaagaaac agaattcgaa gattgatga tgaattcaaa 2160 cccggatgaa ttttcctacg acgatttca gattctaa aaggcaaat ctgccttgga 2220 atcgtctctg catttagaca attackacg aagagtggtt gaaaagggtc attackctaat 2280 tacacatat gttggtctca ggatacattc agaaagctg ttaaagtag actgggatta 2340 tgttgtctta gacgaaggcc ataagattag aaatccggat tctgaaatat cattaaccac 2400 aaagaaatta agaactccaa ataggataat tttatcaggt actccaattc aaaacaatct 2460 gaatgaatta tggtctctgt ttgacttcat atatccaggt aagctaggaa cattaccagt 2520 atttcaacaa cagtttgtta tcccaataaa taccggtggc tatgcaaatg ccaccaatat 2580 tcaagttcag actgggtata aatgtgctgt tgcgttgagg gatctaattt ccccatatct 2640 actgcgaagg gtcaaaagtg acgtagcaaa ggacttacct caagaaag aaatggtact 2700 attttgtaaa ttgacacagt atcaaagaaa tagtaccta gaattcctga actcaaacga 2760 attgaaacaa attaaaggtg gaagagaca tgttctatac ggtatcgaca tcttgaggaa 2820 aatatgtaat caccctgata ttctggagag agaggagaag caaaacgaac tcgactatgg 2880 2940 agatgggaac aaaaccttgc ttttcaccca atctagacaa atgttggata ttctggaaaa 3000 atttgtagca agtggagatc ctgatttgag taatatcagt tatctaagaa tggatggtac 3060 aactaatatt tcaagcgac aagctttagt agacaggtttt aacaatgagg atttgacct 3120 gttttatta actaccagg ttggtggcct ggggataaat ttaacaggtg siaacaggat 3180 tatcatttt gatccagact ggaatccatc tacagattta caagctcgtg aacgtgcgtg 3240 gagaattggc caaagagag aagttcaat ttatagatta atggtgtcag gctcgataga 3300 ggagagata tatcacaggc aaatctttaa acaatctta actataaaa tcttactga 3360 tccaaaacag aagattct tcaaatgaa tgaactaca gatttatta gtttaggagg 3420 agatgatgga ttagctcag agagcttgc gaacgaggtc gagacacata cgcagacact 3480 gaaggaatct aaactaac aaagtgatga ctttgaacag gttgccaata tagcaggtgt 3540 ctcgaaatta gaaggttttct ttcaaaga agaaaagaa gccagtaaaa atgaagatga 3600 aagattaata gcagggttaa tcagcgaaag tggtaactta gaaaatgcca gtactcatga 3660 acaggttgtt ggatctcata tgacatctaa acatctacc aaatttattg caagagaagc 3720 tgaaaaaatt gctgggcagg ctgtcaatgc tattcgtgaa tctagagaa agacccagaa 3780 atatgatatt ggtaccccaa catggacagg taaatttggt caagctggta aggtcataaa 3840 gaaaaaaaata aagccgtcaa agaaaaatgc tctggcatca tcagatatct tgaagaactat 3900 tcgtgatcgt caaatagaat cgaaaaagaa tgaatcgttg aatgacttgg ctgatccaaa 3960 ccgtaaatta atgagaga tcgtaaatct tttaaatgaa tcatctcagt ataccttacc 4020 atctgcttct atcattgagg atcttaacat agatgtaaag gataaaaatg ttattatcaa 4080 tgtcagagct ttactaagag ctgttgctaa atttgataaa gtgaaaaaaa tgtggacatt 4140 gaacaatgaa tttgttaata attgagcaaa cttttttccc caaggacaat taaatactag 4200 aggaagaaa gttagccaca gaagagaat atatatggat ttgcattatg aatatataaa 4260 tatttaaacc ttaacggaaa ccaaaccctt cagattcggt tacagctaac atcaatttat 4320 tctctagctt ttcttttgag gaatattccc atatacacag ttcgttaaaa catgtgtgtg 4380 caattggcag atcattacta tccctagctc ccaatctgct tatcttaaaa gtcaaggttg 4440 atatacctgt agctggtact ctattagaac tagtaatgaa ttgtaacact ttaccttgca 4500 ttttataatc ccaattttcc aaaatctccc aaaaccaatt tacaacagaa gtttcattgg taaaaccgcc ttgatattta gtaacagaac gtaacatttg aaaatcatat tttgtatgct catcatcccc acataataa cgttccaatt cttcagaatt aaataggcct atggatttac aatttgaaaa gactctgctg aatccatcca taaatctttc aaaagacgct gctaccgatt ttgttagata gaaatcaatc cacaatttta cataatcgga tttatttgac tgagttaccg ggatattaga gccattcttg caaagttcta ccgtcacagt attcgaacct ttggatttt tactaccttt attattcgct gttagtttat tatgatatgt ggtttcaaat gtaaggcaga aaacgtcatt aaaatcatcc ttcgaatatt ctaacatctt taataatt gaagctgtct 5040. ctggatatag ctctgtgtaa tctgcaaaag ttaacgtttc attgcacatc tttttataaa gtgcctttgg ttgcctttg ttccgtta cattgccat gctatgacaa cacccaacag gtaatatat tcttcttgtg attgaatttt ttcttttgat ttggaagatg gtacaattgg aaaccaacat aatctactat cctttatatg atcaaacaaa ccagttgtcg gactgaataa agattttgtt aaaaggataa accattcctt tctcaaacca cccgcatcga 5280 taccaggttc tttaataaat tcaattctta gagatttcaa taaatcacct tgatgctctt 5340 tgataacctt taatgaatca tgggtaatat gatctcttcg tattttaatc ttaaaataaa 5400 cttctatagt ctt 5413 <210> 26 <211> 2985 <212> DNA <213> Artificial Sequence <220> <223> c4733_g1_i1 <400> 26 tcagctacta atactggtgc cacctctgtt cataaaccag taggtatcgt cactccaggt 60 tatggtttgc cttattttgc ctcttcttta acagagacta aaaataactt cttattcaat 120 gttgctgctt tatcttatca agataaaaaa aatagattag gtagcgatta cattactcca 180 ttatcaatcg ctaaacaatt aggctttaac gttattacgc ctgtttcaaa gaaagaatta 240 gagttaactt ctttattatc tgttgcttta gctactttat ccaattccaa ttccactatt 300 catttattcg atggtttaac ttctactcgt tcattttcaa ctttaaatag taacattgtt 360 aactccgaat cttaattgc taatttagct aagactctag gtaatgaacc atcctttgat 420 gctatcttaa agggttcaa tgaacaaata ggtagtcaat tgacaaagtt ccaatattct 480 ggtccttcaa atccagaggt tttattcgtt acttatggta ctaccgaatc tgaactattt 540 agttccgttg taccaacttt gtcagttaga gttccattac catttgacac taacgaattc 600 gttaattcaa ttccatcaag tgttaagaag attgtcatca tcggtcaatc attgaatgaa 660 aatcatgctg tcccatcttc cctaagatta gatgtctctt cagctttatt cttccatggt 720 cgtaaaaata tttcaattca agaacatatc tatcaacctg atttgcttg gactactcgt 780 gaagtatcca acatcgcaaa ccaattcgat gtcaagacaa tcagtaccgc tgctcaaact 840 ggtaagcatg cttatttta tctaccagat gattccaaat ttattaatat cccagctact 900 ttagtcaaga ctttagcttc tactactaac gatattcaat tctctactaa attcaataac 960 tctgttcata gtggtgcatt tgaagctgat attgcagttg gtaatgttga aacaggtact 1020 gcttctgccg atttcatctt ggttcaagat atcaatctat taaaccattt agatatcgtt 1080 aatgctatta agggaaatgg taccattgtt tatttagcta atcgtgatat tactaaatat 1140 ccacaacaat tcatcgctga tttaatcacc aagaaaatta ctttagttat tgttgaccct 1200 actgaatacg aagatgatat cgattcctta gttgctttga ttcaaggtca attctatcaa 1260 tctggtttac aattagctaa taaccaaatt caatcaaaaa ttgtatctaa tttatctcaa 1320 gaacaaattc atgatatttt gaacgctaat gaggattcag aagaatatca attctcaatc 1380 tttactgttt caaacttacc tgaacctgaa ttctccgaag aagttcgtga acagttacct 1440 tctttcttcc aagctgattc attcaaacca aataatatta aacaacaaca agctattgtt 1500 aatgacccac cttcaattac ttcaacaatt actgaattga ctaaaagatt agctttcaag 1560 gaagcatacc acgttgaaaa gaaattaaga ccagatttac cactaattaa gaaccacata 1620 atcaaggtta aagaaaacag acgtttgact ccagcagact acgatagaaa cattttccat 1680 atcgaattcg atatctctgg tactgattta acttacgata ttggtgaagc tcttggtatc 1740 catgcaagaa ataacgaaca acaagttctg gaattcttac aatcttatgg tgtagatcca 1800 gaacaaatcg ttcaagtacc aaacaaggat caaccacaat atattgaatc aagaactgta 1860 ttacaagtat ttgttgaaaa tctagatcta tttggtaaac cacctaagaa attctacgaa 1920 tccctaatcc cattcgctga agatgaagat gaaaagaaat ttttgcagga tttaattact 1980 ccaggtggtg cattggaatt gaaaaatttc caagaagtcg aattttattc atatgctgac 2040 atctttgctc gtttcccatc agtgagacca gaattagctg atttgattaa tatcattgct 2100 ccattgaaga gaagagaata ttctattgca tcctcacaaa agatgcatcc aaatgaaatt 2160 catttgttaa tcgtcgttgt tgattgggtc gacaaacagg gtagaaagag atatggtcaa 2220 gcctctaaat atatctctga tttacaaatc ggtcaagaat tagtcgtcag tgttaaacca 2280 tcagttatga aattacctgc tgatccaaag gctcctgtca ttatgagtgg tctaggtact 2340 ggtttggcac catttaaagc aattgtcgaa gaaaaattat ggcaaaagca acaaggttac 2400 gagattggtg atatcttctt atacttgggt tccagacatt gtagacaaga atacttatat 2460 ggtgaagttt gggaagctta taaagatgct ggtatcatta gtcatatcgg ggctgctttc 2520 tcaagagatc aaactcaaaa gatttatatc caagatcgta tcagagagaa tttagacgat 2580 ttgaaggtcg ctatgattga tcaaaacggt tctttcttct tatgtggtcc aacttggcca 2640 gtaccagata ttacttctgc tttggaagat atcattgcag ctgatgctaa ggaaagaaac 2700 gttaaggttg acttgaatga agccatcgaa gaattgaagg aaacttcaag atatatctta 2760 gaagtttact aattcgttac atatatattt atttatgata catttattta ataaactttt 2820 ttttagtaaa tatttctttt tttttgttgt taaaatatag tacgaatatt ttttttttac 2880 ataagactga ctacagatgt accatcttgg aatcttgttc tgaacactct gttggcatta 2940 gtgaacatac cttccttcaa agaaacaaca ataaactgag cccca 2985 <210> 27 <211> 2512 <212> DNA <213> Artificial Sequence <220> <223> c4679_g6_i1 <400> 27 taataataat aataataatg atgaaattcc aatattgatc gattcaaata acaatgaaac 60 taggaatatc gatacttcta aacaattacc tttaacacat catgaaattg attttgaaaa 120 tgatctaact ttggaagata gtgatacaga tatcgatatg atgatgtg atgaatga 180 tgatgacatt gaaaata attackatta storm gtgtttcaag gtaataattc 240 ttccaggaga agaatgcgtg attattttaa attcaattta ttcaatctt caatcacc 300 aaggttaca acgaatctg aaatttatt gagtcagag gaaaggata spiders 360 actcccacaa ttcaatgatc aatcaattt aagaaaaaa tcccatttt ggaaccctaa 420 gacttcatct ttctgaaa ggtacaatag taaaaaa gatggcatta atacactaa 480 tattcctgat atagatgaca tctcactag agatttcgaa tattctgat tattcgatat 540 tgaaaatcat ttagttcagg actcatctc atcctcatta ttattacca tacaatccgt 600 agaaccaatc ttcaaaata cgttgaatcc aatggtcact atgaccaag ttactacaca 660 attackcacaaa ttacacaac tgcacaat acaactagt catcactcgt 720 ttcaccttct tctccagtaa tgacaattgc tccctcattg gtaactacag atgatatttc 780 aaatacttc gtcgaaccaa ttaatcaat gatccattt gaacagata tgaatttgag 840 ttatttcgat atttattca ataatgttct taacgacaac acagttgaag aggaccact 900 taaatcagtc cttgaggaac caccattagt agaaacgag actccagaa aagtcacccc 960 aactacgccg gctccttcct tgactccttc cttgactcct cacaaccta tcagagaag 1020 aggttcaaat actctgccaa agacaagagg acgtaaacca tctttaatcc cagatgccag 1080 caacaatc tgttgtgact actgtgatag aagattcaa agacaagagc atctaagag 1140 acacattaga tcgctgcata tctgcgagaa accattcacc tgtcacatat gtcagaaaaa 1200 ttcagtagg agtgatatt tgaatcagca tatcagact cattctcacg atgaacaaa 1260 ttgatctggt tcctctaagc tccttctg cgctcatata tagagataca tacatataga 1320 tagatatata gactcgtgtt ttactgata taatgataa tgataaatca acctttttaa 1380 atttaatgtt tctgaataga gtcatctaaa cgtggttgtg acttctggtc tctgagtc 1440 tgccgatttt cgctgcaca gaacaatgag ctaccaaaaa agagaaagt atgggcgtta 1500 ttattagaat agaacatgt cgatatctca agtacaacat atgtggga gatctaat 1560 atccaatac aagaccaagt gctatcaaaa ttgcacattt tatcccaacc caatgtctgg ttacaacagc aataccgctt atcaattgca actagataga tttctgaat ctccatcaca father tgttcagatt gtaagaattc mother tggtgttcta catcctttaa tgtatttcta tgttccagat gtgcatcctt gcataagcaa ctactaaata aggaccctta ttattccaat atcaaatcga tcaaattgga tacgtggtct gatgatgaat tgttcaattt catacataaa ccaaatcaat caatcaatag agacatatat actacttcag acaatgcata tgacttgga cattle aaagaaaata tatggatcct ggactagag tcggattagc taaagaaga gccaatagaa aatatcctct attack aggagaccaa gagattatga attack tattgtagac attack aatcgaatca attack gattcactaa tgaagataat attgtggaag cattatccat ggctcatggt aatattgata acgccattga aatcttaaga fatheratgatg agtaccttaa ctcaagagac tacagagatg attatgatag tcgaaacagt buycat cgctatcaga aaacagatat cgcaaccgtc cggacagcaa ccgcacccca agtttgccaa gaagaccaga taatagtgga ccaaaagatg ctgtatttga 2340 tgggtcattt ggtaacgcta ctacaactac aacaaaagct cccaaggcag ctgtattcga 2400 tggtttatca cctgatgcct tatctaatct ccaagcatct gaatatcaag tacaacagaa 2460 tgaattgatg aaacaacaaa tgttgcaaca acagcaacag caacagcaac aa 2512 <210> 28 <211> 3243 <212> DNA <213> Artificial Sequence <220> <223> c4955_g2_i1 <400> 28 taataataat aataataata atacaacaat cttaccatct actcatatac ccaattcagc 60 cattaatgat attaatagaa ctgctaccgc tgctacaaca acaacaatta ctaccgctaa 120 gattactact tcaaaatatg atcaatcaaa aattcataat ttaccaagtc caacttgttc 180 cgtatcaggt aataacaaca atgctaatat caaaagcaat agtaataaca acagtaatac 240 taatagtgga gtctctactc cacctgaaga tgtagaacca atgaatttag tttgtaaatg 300 ggataattgt aacaaaatct tcgttcaacc ggaattatta tatcatcatt tatgtcaaga 360 tcatgttggt agaaaatctc aaagaaattt acaattagat tgtcattggg ataaatgtca 420 aacaaagaca gagaaaaagag atcatatcac atcacatatt agagttcata tcccattgaa 480 accatttgct tgttcatctt gttcaaaaaa atttaaaaga ccacaggatt taaagaaaca 540 tttaaagatt catttagatt ctggtaacat tatgaaaagg aaaaggggtc caaaagtcgg 600 ttctaaaaga attaataaga atggtattaa atcaatagat aataaacata ttcaaacagg 660 tattgatcaa agatcaagaa gtttaccttc aacaagcttc actaatttac ctcatttaag 720 taatggtttc agaaattca ttactaatga tattcaatct tatcaacctg tattgactca 780 tagattagat acaagattac aaaatataat gggtcaaact cttactgctg ctcaattaca 840 agaacaacca catttatatc atccaatcga taaaaattta caaggtccta acatgtctag 900 tgaaagagta tctgtttcat cagttatgga tacattacca cgtcatgtag cagctaacgc 960 agcaggtttc ttttcagaat tatctaacaa catggctaat aacacagcat tatatcaaca 1020 tcatcaacaa cagcaacaac aacaacaagc acactcaagt attcaattgc aatcacatcc 1080 acagacttta atcggtaact attctaaatt gccaccattg atgtgtta catcataccc 1140 aacacaacaa catacaaca tgattgaaag ttcaatgaat gtcccaacta acaaatgac 1200 tatgttacca tcaatggcag aagttactgg tctacaacct agataccac aacaacagca 1260 gcaagcacaa siactagaa gtagtccaaa tgcaactatc atctcatcat atccactat 1320 ccaaaacatg cctcacaac aagttccatt acaggcaca gttatggcaa gacctatgcc 1380 aggtactcaa ttgccatata atttggttgt taatgcaatg cctgttgctg gtagtacaat 1440 gatatggtt gagaatagat atagtacatt aagatca actggtcatt ctagtggttc 1500 tgatgattca gatctgatt cagaatctga aagagatt gagagaag atttcgaaga 1560 aagcttggat ttgttaatg ttattagaga ttattgatg tgtacattat ttagagaga 1620 atatgatgaa tccgtcgatg aaaattga ggatttgatt atgataaat tttggaagga 1680 atcaaaggt ttgatatcta atatccaac tattagagtt tgaaagaat attagaa 1740 dattaaaaaattaattt atgtaatgta tatcaaaaaatacatg aaaaataa 1800 atataaatct gccacaatat ggaatggaat atgatgtgat gtgtgtacag atgtatatcg 1860 cacaaatgaa ttaatgctaa tgttgtaaaa ttgtgacttt tttactagtg ctttttttta 1920 ttgcgggaag gaatgaatat tgttttatgg ttgatgatag aatgtaatgc ttgagttcaa 1980 ctatgaaccg aatgattcat tagactctta ggtaaagaag atgtaactga tctagaattt 2040 gtagcggaac ttgaagatct tacaattttt tcatttgtat tatttgcttc atcattattt 2100 gatgctattg atggaattcc tgtattagaa tcacagtctg attgttcatg ttcatgttct 2160 tctaatggag gtccaacata caattgagat ggtacttgtt caacattatt tggcattgaa 2220 aaattgacga tatgtgtgga atcatttgta ttaactttaa atataaaact attatctctg 2280 ataactcttt catatattct atttcttact acgattactg caactgcaca tggagtagta 2340 ataaaaccaa caactgcacc atatgaaagt ttcattaatt gtggctctgg ataattatta 2400 aaatacctat catgattatt tattttatta ccaattgatg caaagatacc catacaaatt 2460 ggccaaacga agataaataa tgcaacagcg aataacattg ctctaataac tttcctaatt 2520 aaccattcaa taatattaaa atattatttt tcattttggat atttacaac atgatttctc 2580 CAaattttta aaagggacat cttaggtaaa tcaatctct tattattact atatttatta 2640 2700 aatttattc ttttacaat gattgaatta taccaaaacca tgaggaat 2760 tcaatgtat tgtttaaata atcatcataa cctaccatta attctcaac aaccaagta 2820 acaccaactt gatgataa agttaatgca caatcacctg ataatgtatt agggaatgcc 2880 cataatgtaa ctaaatgagg agatttctga tacataccat aagcgatacc aaattcagaa 2940 ccaccaccta taatggcaga acctaatcct tgatataaga atagataaac tattgaaaat 3000 atcaacgggt attttataaa tagtgtaatc atctatcgta agttatgtgt gtctgtgtgt 3060 gagtgtaatt ttgtgatgat ctgggtttga attaatata attgtaattt gtttagttta 3120 tagtagtacg tatatgtgag cttatctttt taccaaatca atgaacagtt ttataacgta 3180 tcttctttcc ttctctctta aaaaaaaaataat atagaattt aaatatgcag 3240 cag 3243 <210> 29 <211> 5008 <212> DNA <213> Artificial Sequence <220> <223> c8855_g1_i1 <400> 29 cttaagaatt agatatataa ctaaaattaa gttgaaaagt tacgttacat gatgcggata 60 ctacttcaag ctagtgaata attgaattca caatacttgg gggggtacta gaatgggata 120 atacattata gtgtgtggta gttcaaaaat gaattatacc agacggtatg gcatgctata 180 ataggttatc tataaactat ataaatcaaa aaaagagatt tttacatgat gatacaaaat 240 acattgctcg aaggtcttgt atcgatgtga gcaatacgat ttctcaatag actacaacgg 300 tatctgattt ttatcttttt tatgtaataa gtacttgttt tacatacttt tttcacttga 360 tattctataa tgaaagttag atgtctattt aaattggcca ttttcgttat tttaaagtac 420 aactgagttt tgattcatag tcattatagt tgtgaatcgg tttccatttc ttgaatgtcc 480 cgatgagctt cttcatattt gcacacagat gcattgtaaa agtacgtttt tatattatag 540 taattacgaa acttttgtga ctagtattta attatttta aaaaaaaaca gtaagaagct 600 gaattctta cgtacataa gaattctta aaaaga actagtcta aagagttag 660 caatactgta cggtataaac acccactacc aatccatcca ctagatctac ctcattatt 720 tgttcataat ccgatatcat ggacctactg gttgtatagt tacatcacaa gttataatgc 780 atcatcgaca aagatacatg tagatatcac aaaacacac aacttcatac atagaagt 840 tagagaacta accgatatga atatttgtg ggataatga ttctttggca caggccaatt 900 gtccagaagt gaacctacgt ggtatgaaca aacaagcaag aagttccaaa gcagtggcga 960 tgaaaagagc aatggtatca gtttagagcg agtcactaaa ttaagac aaaagagt 1020 tgagttcaag aaacaacgtg aaatagtaga ggagaattg ttacactta ggagagaagg 1080 taatttgaca cctgaacaag aagcagagat tcttgaaca gaagagaca aactacgtaa 1140 multiply multipliedxtt multiplied multiplied multiplied multiplied 1200 agagactaaa cgactgctat tacagcaatc tgaaatatttt gatgaaaatg ataatttatt 1260 gaatttggaa tcactcgaat taatgcctgt agagacgata ttcttaagtt ttgcattacc 1320 tattttagat atatcaccag tagatttcat tttgaagtgt tgctttactg atatatctcg 1380 gtattcagag gaattacaca cattattgat ccaatatgct gcatatcatc attacagatc 1440 tcatggatgg tgtgtacgct ctggtataaa atttggtagt gattatattt tatacaagag 1500 aggtccacca tttcaacacg cagacttttg tataatggta ttagattcta attgttcgaa 1560 accttataca tggtattcta ccattgctag agtatgcggc acggcaaata agacattagt 1620 cctttgctat gtagaacgtc tagagactga agaacaaata ttagaatggt tacaaggggg 1680 acagttaaca aaagttttta acagctttaa ggtcggtgag gttatataca gaagatgggt 1740 agcaggaaga aaccgtgact aataatgtca gatcgatttg aacgactgaa tgaagagata 1800 atttattact acttggtatt tactatgaat gtttaattat ataagttcaa aggtatataa 1860 ttgttttttt ttccattata tgaatgcatg catgtgactg tttattctag tctataataa 1920 cctatttacc attgtatgta tctagaaccc aaccacaatc tctcattccc ttgatgtgtg 1980 cttttggtcc ttctaccaga tcgtcataaa ttttgtttat cttctgatca tgggtagctg 2040 gttttgatat acaaacaacc tcttcgtttg gtaggatgca ttttagttgt gattttggaa 2100 atggacatgg acaatcggac ttcgataata cacattctag cgtaacaggg cataagtagt 2160 ttttacaatc actgtctaat actctgtctt catatggttc taattttgga ggttgttgct 2220 gttgctgttg ttggttgaag ttaaacataa acccttgaac atccagcaat aaaaaaaatg 2280 ctgctactag tatgacttta ttaaatgatc ttaaatagga catattattt ccttctctct 2340 cctggactat tcgataacga cctaatgttc atatatctac ccgtatatac gataaatatg 2400 ataccttgca atcataataa actgtgttat ataaatatat atgtttgtat cctggaacta 2460 gatggttggt tagttaacac agtggcatct cttcgctcca tgttgacatc aaagtggtga 2520 agttcgtggt tagtgatatt ctacgcgtta attttttcga tttcaacaaa cgcgaaattc 2580 tgttttgatg aaacttctct tttcaataac aacaacaaca aaggttgaaa gtctggtcat 2640 tccatctttg tttcgttttg acattgtata tatcaatatg tgtcaagtcg tgttgtgcag 2700 aggaagaaac aaacgacgat ttgggtaat ttcagtagct gcagaagacg ttgaggagga 2760 tgtattacag gatcgatcaa tataccaacg catataataa gattctcaga gaatcttcaa 2820 atccgtcgac atgccaacta gtcatttttg tgtcatgttt gaatatcgat gcactgtgtg 2880 caactaggat gttatccacc cttttcaaga aacaactagt ccaattacaa attgtacctg 2940 tgtttggtta ttctgaatta aaaacacatt ataagaaatt agatgaaaac attaatagca 3000 tagttttggt cggttttggt agttacattg atattgagac atttctagaa attgaccctc 3060 aagaatatgt gttggatacc tcatatagtg aatcgttaat tcaaaaacca gagaataata 3120 catacaaaag atacatttac gtattggata gtcataggcc gtggaatcta gataatttat 3180 ttggttctga cattgtacaa tgttcgatg atggcacagt ggaagattca ttaggggaac 3240 agaaagaggc atattttaaa ttgatagggc tagaaacagc agcaggagat gataactcgg 3300 aagaagaatc agatgatgag gaaaacacag atgacgatga taacgacgat gatgaggatg 3360 ataatgactc cttagaaaat ggtaagagac tacaccctga tagtataaaa tataagaaac 3420 aagctcgaaa aaaaggagg aaaaata gtcgatatga aaatgtactg gagaatact 3480 actcccaagg tactacagtt gttaattca tatcatctca actctattca ttgatttccg 3540 ctattggtga aactaattta actcaattat ggctagccat cctaggtgca acttcattag 3600 attackacata ctcctcagtt tacaatact atacccaat tatgcaggac gaagttaaaa 3660 ggttatcacc tgggaatagt tttctcgtat ctgcacacg ttcaatggt acagggtctt 3720 cttcaaaaac accagatact ttatcttg aagttcagcc agattactat ctatttttat 3780 tgagacactc ttcattatac jagagtttct attattctaa tttgttaac gctaaattat 3840 cactatggaa tgaaaatggt aggaacgac tgcataaaat gtttgcaaga atgggtatac 3900 cattaagtac tgcacatgaa acgtggcttt atatggataa ctccattaaa agagaattag 3960 gaaatatttt ccataaaaat ttagatagat acggttaca agacatcata agagatggtt 4020 ttgttcgaac atttgggtac aggggatcta taagtgcaag tgaatatgtt gatcattag 4080 cggcattatt agaagctgga tcaacggtga acagctcaa tcatagtaat acatctatt 4140 cccctgggaa atctagtagt atgataata atgcaatg tatgacgat attgataatg 4200 gtgcacaaga agaggatgat gagcaggatg tagcagcagt taaccgtag aaagcgttgt 4260 cttccatgga aaatatcaga aaacaatggg tttctaattt ctggttaagt tgggatgcat 4320 tagacgaaaa gatatagat atattactc gaggtattaa gcatgcaca tttctcaaa 4380 aggcaatatt taacaccggt gttactgtcc ttgaaagaa atgattaa catttaagaa 4440 tatacagatt atgtgtctta cagatgtc cagatctgtc atttatcag aacccattaa 4500 cattattgag attagggaac tggttaatag atgttgtgc cgaggcagaa gataagcaat 4560 tattacccat gttgctggct tgtttagatg aggatactga cacatactta gttgctgggc 4620 tttcaccaag atatccaaga ggtttagata atttgagaa gaagaacct atattaaata 4680 actttagtat ggcattca caatcactg cccaactgg tgcaaggtt aaaattgaca 4740 acttcgaaag ttctataatc gaatcagaa aagatgatt gtcaccgttc ttagagaggt 4800 taacattaag tggattgtta tgagatagtt ctgattataa catgatagt agaatgaaaa 4860 gagggaaggt tattaaaata atataaaaaa ttaaataaga tagtcatatt cacattacat 4920 agtcatatac atatttacaa taatttaata tgttagatat tagaacacat cattggagtt 4980 ttgatgatta taaatattct tttgttaa 5008 <210> 30 <211> 3479 <212> DNA <213> Artificial Sequence <220> <223> c7636_g1_i1 <400> 30 gggaaagaag gataatgatg agttccgcca agagaccact tcaagaagtg gataatgagt 60 tgcttgattt tgcagctcag aatgaggcta atattgaaca tgataaagaa caagccccta 120 agagaaggaa acgtatttat gaagcgatta cccaatactc catgaacact caagatgagg 180 caggttctaa ttcaaattta tcttatcctg gttatatcaa gaaggtgaaa ttacgtaact 240 tcatgtcgca tgaaaatttt gaattagaat tgggtccaca actaaatttc attgtaggta 300 ataacggtag tggtaagagt gccattctta cggccattac aattggatta ggtggtaaaa 360 ccagtgatac aaacagaggt actaaattaa cggatttaat aagagaagga accgcgtcaa 420 ctaagattac attgtattta gataaccgtg gtccaggatc ctacgatccct gagaaatttg 480 gtgataccat tattattgag agaacatta gacgtgatag ttccaatgtg tttagtgtta 540 agaccgaaaa tgtaacgaa gttgggaaca aaaagaaaga tgtccagctt attgttgatt 600 ttttctccat cccaattata aacccaatgt gtttcttatc tcaggatgct gcaagaagat 660 ttctgacagc cagtacctca caagataaat accatcattt tatgaaggt actctttag 720 aagatactaa aaaattta gataacgca gttctattgt tgtaagct cagagata 780 tgaggttaca tgccggttca tgaggtac tgaacagga atataggac tccagaac 840 tagcgcgtga gttcaataaa acagtgatc tgaacgaaaa gaaatgcta ctatgtgcca 900 agatcttatc tctcgatatc gaggctata ctaaatccag taatgccgtg gaacaggaaa 960 ttgtcaataa tgggaaccag aaaaaatt ttgacaaag atcgagaaa cggaagcag 1020 atttgaaag atttgttttcg gatcaaag aagctgaag gggtattgaa atcaatga 1080 father tashaaagac cagattga gagctaaaaa agatgagtt gcaaattga 1140 gggctttgta taatgctgaa gaacgtaatc aaactcaaac aaaacaaagt attactgatt 1200 gtaaaaacag aattcagctt ttcaataaga aaattgcaaa gttcgaacag aaaatcaatg 1260 aagaaatggg tggggataga gaagcaatga aggagcaact aaagacactg gaaaaggaaa 1320 gagacgtagc ccaaggaaat ctttcagcta tgcaaactac attaagggat ctgcaaaata 1380 gggaaaagag cgaatgtgac caacgtaatg ttgaagttcg cactttagaa gatggtattg 1440 cggcaaaaac gtctgaatac aataaaatca agactggtaa taacgatttt ctacttaact 1500 tcgacagaaa aattaatcag ttatttgctg aaattgagcg caataaaaat catttccact 1560 ctatgccaat aggtccactt gggaggttg taagtataaa acgtgagtat aatcaatgga 1620 ccaaaatat ccagaaattt ctgtcatcga cagtcagctc tttccttgtt acagacttaa 1680 atgacgatcg attattgaga aggataatga aaaaatgtaa tattagaaat attggtgtat 1740 taatttacaa aatgaaaagg ctcgatgttt cttcgtttct agttcgagca tcatatccaa 1800 ccatttacga tgccctcgtc tttgataccc cagaaatgga aagtttattc attgatgtaa 1860 catatttgga aaagtggtt ttgatagaa attaaaga ggctaggaat ttcctacaag 1920 gaaatcctgg gagaattcga attgcattat ctttgaggga tcgtaatggt ggttatcagc 1980 tacgtggtgc aaccagtta gattctgtca atatgaatc ccagataaaa attaaagttg 2040 gttcttcaa cgaggataat cttgcatatc tgaacaac tattgatgaa gaaggaaag 2100 agatagaaaaatcaagaat aagtacgaaa cagttatatt tatacaga magagaatga 2160 atacgaccaa ccaacaatg aaacgttttgt cggagatat taagagaag ggacatgaga 2220 tcacgcaatt aactgttaaa gctaatgcaa ttgtggatac tgggttactg acatcgatga 2280 acgagaaag agataagcaa gaggggcag ttgcagtata tgaggccaca gtaagggaga 2340 tagatgctaa attgtgcg ctccgtgaaa agatacagcc aaaagata agctatgaca 2400 atgcaaaca cagtcttcgt gaagcaata aacattaga tgaacttaaa gcagctgtta 2460 atagccgttc agacaagta gaagatata atgctgatat ccagaattgt gagcatgaaa 2520 taggaaagct atctcaaag aaaatcgc tggaaaaaa taagaagtt cttgttaatg 2580 2640 ctaatctacc tgataaaaaa gatgagttga aacgtgagat tgataagatt agtaaaagaca 2700 taagaagggc agaaaattct attggtatat cagaaaaaa ggtggtccag ctatttaacg 2760 2820 tacaacttca acaatccatc aaagagcgtg taatcaaata taacttaaat gtcaacgaaa 2880 cgttcttaaa ggccaatttg gacttcatcg gatctctgaa aatgagaaag ttaaccggga 2940 aattattctt taagaaggaa gaacatagtt tagagatata tgtttcaaca ccgggtgata 3000 caacggaaag aagtgtagat accttatcag gtggtgaaaa atcgtattca cagatggcac 3060 ttctattagc tacttgggaa ccaatgcgtt cgagaattat tgcccttgat gagttcgatg 3120 tttatatgga tcaagttaat agaaaaattg gtaccggttt aattgtaaat aaattgaaag 3180 acaaggttag aactcaaact atcatcatca cacctcagga tattggtaaa attacggaca 3240 tcaacgattc tggtgttatg attcatagga taaaagatcc aaaaagacaa aataattccg 3300 ataatcgtgc aaattagtgt ctttttttag atatttttat gacacggtcg ccaatatggc 3360 gtgcatttac ttgtttgttg atgcgacttt ttcgttggaa tttgaatgta atattaaact 3420 atataaattt tataaacggt aaaatatata tatatatcaa attatgagat aaaacaaat 3479 <210> 31 <211> 3024 <212> DNA <213> Artificial Sequence <220> <223> c4682_g2_i1 <400> 31 aattaactat ttatcaggtt attattatta ttatttaacc cgaataactt tattgaacaa 60 tataaaagat agtatattca actccagtta gtatctcctc tgacatttaa atcattacgg 120 agttttcttc tcttttacct ttttttttac ttttgctttt cctgaccata ttaaacataa 180 cggtgaaaac atacttttac aatattaaac attccttttt gatccaagga gaatataaat 240 aaatacaaaa aaaataccga aaagaaataa atcatttcag agttaaagta aatacattat 300 accgattaaa ttgtaagaac atatagatta ataaaacaaa ataaactact aataattata 360 atattccaaa ctccgtcgtt ttgaaatata cactttcttt ctttttttta cacagcctac 420 tttgacagta taatttaaga atattaaaat taatcattat ctttttattg tattatatatt 480 tttgcacctt atttcccac catccccacccaattatttta accatact ctattaaaag 540 ttttgatata aattaaaaaaaaatagtc atttgattattttattatta taaaaaga 600 ataatacccc aaatacataa caataatgca agctaataa gaacctgtaa ttgatattac 660 tatacaagt ggtaacgatg aagtaataat attaaaggga ccacctgaaa ctgctccacc 720 ggtattatta tcaggtatca taacattatc tacatgtgaa actgtaaag ttaaatctgt 780 ttcattaaga ttacaggta gatgactta taatgtaccc attaataata aagataaatc 840 gaaaaggt aaatgaa ctatagatca aaaaagtt aaacgtttat ctgctgatag 900 atggttatat catcataaat gggatgatt tacaatagat aattatttta aaggttatata 960 taaaaattt siaaaaga cacctattat ggattctaaa atgttagac atactgtcgt 1020 accgtcacat ccaatagcac aaggtactc agctccattt aagaatggtt tagcagacc 1080 aagatcaact acttctttat tatcattaaa gataca atattctt caccttttcca 1140 aagaagaaaa tctcatactt tattaaaggg gaatatgaa tcccttta catcaatatt 1200 accaggtgat attaacgaa ctattgatgg tttacctgat acaaatgtaa attattatt 1260 agaagcaatt attgaagaa ctaatggtaa atctgatctt tattgtaga atatgttag 1320 aattgtaaga acaattactc cagatatcgc agaatttct gaaacggtaa atgttacaga 1380 tacatggatt gatagaattt ttttcgat ttctgttggt gcaagactt tagccattgg 1440 ttcaaagtt ccataaata ttccgtgat tcctttaca tcagggatta gactaggaac 1500 gattcgaata tcattatatg aaactgcaga atattgcttt aaagtaca gaacaaaat 1560 tgatcgtgtc gtatcagat tgaaattga aaatccggaa aattattag ttaattaat 1620 taaagacgat aaatttcaag agaatggga attagatttg ccattagaa tcccagcaag 1680 tttatctaaa tgtactcaag attgtcaagt tatchagaa attagagtaa cacataatt 1740 taaatgttca atcaattttt ataatgcaga tggacacgta tcaaattta aggctaattt 1800 acctgtttgt attattcattt cagaatttgt accattgaaa gttagacgaa tggaatctac 1860 aacagatttc acttgtatca caaaggatat atcaaaccag attcgtaacg aagatgcaaa ggaacaatt tttgaagcag gtcatacagg attagtatct ccacaatatg attagtatc aatgttaccc gtaagagcat taaatgaatt attggctcct ccagaattg aaaaccatgt ctttgataga agattttgta atgacatgga tgtggaatcc tcaatgttac ctcctccatc agatgtcgcc ccagatttac taccatacga accaatgaa gagttgtctg catcaaagat cttaaaggat attgatctag ataattctac tcatagaaga atgagtgatg catcatgtca aacaatgcct gaatttgcat ttgctagtat tgatacggca gtagaggaag gactacctaa tgaaacaact aatgaacaac aacttattcc cacctacaa aattatacat ttggccaaag tcaaacaaac aatagtaatt atagggattc tcgtcataat agtaacgcaa gtaataacat tcaaacaaat gatatagata tggaaccatt aaatggtagt gaatgccac ctccatccta 2520. ccgatttttg gttctattca 2520. ccgatttttg gttctattca agacagacct agacggttta gatctacttc actaacaaac acattcacat gatctgattt aggaccaatt gctattccag tgccagctca tacaataaat tcatcccaaa atattataag 2640 atcggtacca caagtctcca gaatgagaaa ttcttctgtt tcttcaaaca atccttttct 2700 aaatgatgtc attgtatcgg acgcatttgc aactatggta tcagagacaa ttcaaaggtt 2760 ttcaccaatt gctaactcaa gtagcaattc aagtgaatcg agagatattt ttaactctcc 2820 aacaaacaat caattatacc acacattttc agagccatat gaaaagttaa ctgcacaaaa 2880 cgaacgcaga tattcagtca cagagacaat gaagaataat aacaacgatt ttatatcaag 2940 aaaggatatt ctaactattg aatccagaat ggatcaagta gctatgggtc cagaaaacta 3000 attaaattca tatttggcaa ttat 3024 <210> 32 <211> 2232 <212> DNA <213> Artificial Sequence <220> <223> c4154_g1_i1 <400> 32 ctgacaataa taaacctttg ttttaaactc accgaagtat ctctttcctt tggttcccgc 60 cttcctttaa ttgatgtcaa tacgaatgtt atacaagagc ataatttcac gtgcagcaga 120 caatttcaag aaagtccgtt tataaaaga aacaacac agggcactga ttaaagcatt 180 tgcacaaat gaaattgtgt gatctttct tagatggaca gttgcaaca ctctcaactc 240 aacgtataac tacattcag atcattactg gataaaatat cataagttta tgaaccaag 300 atattgtcta atataatga ctaagcagac taccgtcatt cagaagact tatatatcta 360 tataaaga tatacatatc gattcatcca agatttcccc caatattga attctattaa 420 cactttcctc atagcatac cgaactataa cacaaatgt ctaagtata tgaattaac 480 aacaatatca gtgtaacaca aacagtta gaccactgga ataatttct tggcacatta 540 tcacaccac agagattct aagatgggcc 600 actgctttg gtttacagg tttagctacc attgatatgt tatctagat tcattctcag 660 gttgaacaat atccgttagt tccattgatt ttcatcgata cattacataa tttcccacaa 720 actttagatc ttcacaagt tgtacaagt aaatattata aaccattgaa tcaatccatt 780 aatgtgttca aaccagtaaa ttgttccgat gaacagagt ttgctaataa gtatggcgat 840 ttattatgg aaaccgatga ggataaatat gatttcttag caaaagttga acctgctagt 900 agagcatata aagaattagg tgttaccgct gtgttcacag gtagagaaa atcacaaggt 960 gcagctagat ctgaattaaa atttgttgaa attgatgagt tgaataaaat tattaagatc 1020 aatccattag ctaattggac attcaatgaa gtccagtcat atattcaaga aaataatgtt 1080 ccgactaatg aattgttgaa actaggttat aagtctattg gtgactacca ctctacccag 1140 ccagttaagg aaggtgaaga cgaaagaagt ggtagatgga agggcaagac aaaaaccgaa 1200 tgtggtatcc atgaaacaag taggtttgct caatttttaa aagataagaa tgaatcaact 1260 aacgaatcaa ctaccactaa agcctagcga agatgacatc ttaaacagag caatgtttat 1320 ataaacctta catattttat agaataattc atgttacaat tactcaagta aactttctgc 1380 caatctgtat acttcccagg atatatatat ttaagtttgt actagggttc atattgacaa 1440 tccgttaggt ctaattttat tttcttcat tatatccgct ccgtccttag cggaaggtcc 1500 tatctcgctg aagcaacaaa ttaatagaat ataaaagaaat atattggaag aagatcttga 1560 accacgagga tgatatatac tgccatacc tggttaagag aaggtgttaa caacaatct 1620 atcacaacta cgttatattt aattgctact ataccacaat gggtaacttt agatttccag 1680 taaagacgaa actaccacct ggtttcttta atgcaagaat cattagggat aattttaaaa 1740 gaacaagc agcagagaat gaagtcacca ttaagcatt aaaatatt gctagaata 1800 ctgtacttcc accaaggca cgtttgcaag cacaattgca acttaatc atgcctact 1860 attackaaat gacaagtt agatagat gtattgcttc tggtacagcc agagctgtaa 1920 taagtgattt tagattatgc agaacacaat tcagagaaaa ggcaagggct ggtgagctac 1980 caggtgtgaa gaaagtgtg tggtaaatcc attattact gtctagaat attattttac 2040 accaacattc catgaaaga aaagtatacc agattgattt tcttatattt taatctaaaa 2100 ttttatatag gatactggt gtaccatgta catacatata atacctgtc ataaattttt 2160 gtatattcta ataacaac attcgtaaat aacaggattg caatagcagg ctcaagaat 2220 complete cg 2232 <210> 33 <211> 7626 <212> DNA <213> Artificial Sequence <220> <223> c422_g1_i1 <400> 33 ataagacata acagtgtagg aaaatgttag gaacataatg aagttctatt tattcataag 60 gttcttcaaa atgttggtat gtagaataga actgtttcgg tcgcctaaga tagacctcta 120 gaaaacgtat gaacttttct gttaaaattg gttgctaatg caccccccac attcattttc 180 ttcattagtc aattgacaga aattgtgtat agccacataa atatgatatt gctataacac 240 agaattacgc cgatatttat cgattcctac gttaaatgag tgactcggaa aatatcgtta 300 cttttcagat tcgtttagtt cagtcattcc gagaactttt tatataccag ataaaaaggt 360 gggatgtcta gatcattaga aattcagaaa aagtagattt attgttggct caacaagtgg 420 ctgtaaagaa tatgatatcg acgattatat aatgagttac tgttaaatgt gaccgtatct 480 cttttcttgt ttgactccgg taattgggct ataatttaat tataatatat ttagtcggca 540 ttgtattatg gtatagaatg gagggttaag agaaaaattg tgaacatata tagctaagcc 600 aacagaattt aaatgatata gaaaaatgaa tattctcgtc aacgacataa attgtagcag 660 tcttccaccc caagtcattg ccaaatgtga aatgtatttc tttttacttt caaaattaca 720 gtataaaaaag gcaatttaat ataacttaag tttaattgat gaaatcatca gctaacattt 780 ggctagtttg aaacctgaaa ataaccaatc tttcaaagat atataatggc attgattctg 840 aaaaacgact atatcacttt atggtgttga ctttcaaata ttttgggaa aagaattttc 900 tgagtaacgc aaagtagtgt caaattttga gagtgtatta ccaactacga ttagtctctg 960 aaaatggata taaattgtga agaagtcggt tatttacgcc taagtgaagt cgataacatt 1020 tcgaagtcgt gtctttcttg gttatggtt atattaacac gatttctgtc ctctatggta 1080 caagcccata ttatacgtat taagtatttc gatttatcag gtccaagaag atttcagcag 1140 agcttgtctg atataatagt tgttgttaac catgttactt aaccatatcg gtctcttcta 1200 tcttaaaaat aatagtctta aatacattat agcgttccaa acatatccat atcttattat 1260 gaattattgt ttacatatta tttagtacaa caataatatg taattcggga ttttcagaag 1320 taaacatatt tgtaaaaatt atgaataagt cacagctaaa aaaaaaaac atttattca 1380 cttcgaggaa aacaacgaat agcgctcgct caacttgtt tatgaatttc atatggtaaa 1440 cgtacgcaat gtctttcatc tcttgataa gtatatctt taattaactt ccaacaatca 1500 aacagttgtg ataattatac tttttgaca atatatataa acctaggtca tagtcattgt 1560 tgttgtataa cactttgtaa cataggtagg gtcgaacatt cctattttgt tgtcacattc 1620 gcatagttca tgagaaatg aaaactgacc atcagacga gagatatta aactcacaa 1680 aatgtattac taagcacagc aacgatcgga aaaattaga aaaatattc cagttgtgta 1740 attcttgcta gcgcaccctct tttgttgat gtcatgcata ttgcataaa gatgaaaat 1800 attttccagc ggcgttccttg ttttttaa tatatatata taaaggcgat ctttttggtt 1860 tactgctaac tactattca ttcattcctt cagttatgag tttagaaaa tttgctattt 1920 aatttcagaa caagttataa tcatagcacc atatttagat aaacttttct tctgatattt 1980 taccttctga catgatatag ttctaatagg tagaggtttt cacaagatag taatatttga 2040 gatatatttt atttatacaa gcaaggtgaa gtcggtatgc tatttataca atttggtgga 2100 tttatattag acatttattg caacaatgaa gaatcataat ccgatctgct ttataccttt 2160 agaatcgatg caaatctata gttgtggttt accgatgaat gttttggtct cttttagtta 2220 attcatatgt tcgtaatagt gaacatctca taacgataaa atgctcgaac catacaaata 2280 ttaagatagt acgagtatga accatatatt tcgagagtac attatctacc atgtatccca 2340 ttaggctatt ttgtcgatta ttgttatgct gattttgcct gttaatagcg attcttcact 2400 tttcactatg taaaatccgt aaaatcacca tatcaaaaga ttagcaatca ggcacgcggt 2460 atgattgctc agtaaatcca attattattc agtataacaa gagcgatctc gttccttcca 2520 ataggatgat attgtcctct ctaaaattaa tgaatacctc gatgattgtg ctgtcatgtg 2580 ccatttcaat tttgtctttc gaggcattat aatttccgac aacaagttag gaaggatcgt 2640 tgttagtcgc accgggaaaa aagtgtgggt aaaacagatg gccaaaaaaa aaaaattgg 2700 taagcacttc gtagtaagac ctttgcggaa ctcagtgtat atgtatacgt caacagatgt 2760 aaatagctat gacgaaga taatggcaac tgggaatact ggacggcaag taaccgaaat ttgctcacgt ttgctcacgt ttgctcacgt ttgctcacgt ttgctcacgt actgaaatca agaattcaca to attack ttacagtatc to act acaaccacac tttcatgtta ttgaagagtt cgaatcgcaa tgattgtaac ccagaatgat gactgttgca aatagagta cggcatgtta aatacga agtttataa caatatatat atataccgaa aagaaagaca gttttgttca actaaatacg cttatgcatg ctctcattca tatttcaata ccaagttcgt gttttttttt ggttgactaa taattttctg agtatacca gaagtaatca tcaactcaat gtcgaaat ctattccaat tgctcttgaa gcatacagag tctgatatag attgcaacgt ttttaccaca gctgacctta aggatgactt accacaaaca gtttctcaat tactgaatag aaatgatcct ttcgctcaag ttaaagaaaa tatcaaacca aataactga atgtcgtatt cacagatgaa ttgacattat tagggcatt gcctcatttg accagtttaa aagaacaacg tttagtcatc aatgttaaca tcggtcataa cgattactca gtcgtgtcta cattaaaaga tctaaatatt gttactttga tatctaacga ttataactct gctctcaaga 3540 atattaacgt cgctaattct gttgccttta actcttctac tacagtattg cactttatca 3600 actacagaaa gtgcaccaat gatttgcagg atatcaaaga aaatgaatta ctaccggtta 3660 gtttaatcaa taatggccaa ttacaatctg aagacaatat gctatccgaa ttaaacaatt 3720 tttcattgtc tcctacttca tcagaagatg catctgttgc tgttatcaac ttatctcctt 3780 atggaaagga attttctaga tatttgccat ctcaagtctc acttattgat atcaacattt 3840 atagaccatg ggatattgat caacttttaa cctttttgt cccttctatc agaaagattg 3900 tgattgttca aggcgctagt gcagatgatg atggtgaaca atctcatgct tttgatccat 3960 tgttgttaga ttttttcagt gattttaata aattggtcga aagaaagatt gaccaattga 4020 tttatcaaa agtaggctta atttccatct gtgatataaa agactcctta gaaattattg 4080 tttccaacgc tgttaaggat gctccaaatg ctcatctatt tgtaggtaaa ccggtcgatg 4140 gtattaatgg taaatattcc agttctattc tttcttcgat tgatcaccaa cgtactttcg 4200 aaagttctta tatcagggtt ctgcaacaat tattttcttc aaatttgaat attttaaacg 4260 aatttcagag tgattctatt gtggctaatt cccggaata cggctttggt tatctgttga 4320 attcagataa cattcgtgaa aagttggttg aaaatgctag aagcttactt gacttcactt 4380 ccttcaaaga tataccagct gctgatgcca ctaatttggt taagcttttg tcaaaatgga 4440 ttgattgcaa ccgctcttca aagagtacca ccgaagagtc gaacgaaatt tctactgcta 4500 ttttaatat ttttaagagt tatccggaat gtcaatcaat caagacattc ttagaaatat 4560 ctgatgatat cgaagattac ttatttaaat caaactggtt aattggttct gatgcttggt 4620 catatgatgt aggcaactcg ggtgttcatc aagttttaag ttcaaagaag aataaata 4680 tgttaatcat tgattcagaa acttcctcca ctataaaacg aaacaagtct cactcaaaga 4740 aaaatattgg tttgtatgct atgaatttcc acactgtata tgtcgcttc gttgctgtct 4800 attcatctta tactcaactg ttaacttcat tattagaagc tgcgaaattc aatggtcctt 4860 ctgttgtcgt tgcctatcta ccttatgaaa ctgaaaagta caccccagtc gatattttaa 4920 aggaaaccaa aattgccgtt aattctggat attggccatt gtacagatac gatccatcta 4980 ttgaagatga taatgaagct ttccaacttg actcttctgt tattagaaag gaactccaag 5040 acttcctaga ccgggaaaac aagttgacat tattaacgaa gaaagaacct ggaattgaga 5100 ccactgttga acaatctgtt tcagatgcta ttgctaaaaa aatggaatta agaaacaagg 5160 ctgccttaca tcaactactt aatggtttgt caggacctcc gttacatatt tattatgcat 5220 ccgatggtgg taatgcttca tctcttgcca atcgtttggc tatagagct actgcaagaa 5280 acttaaacgc tacgtctcta tcaatggaca ctattgttat ggatgaatta tcaggcgaag 5340 aaaatgttgt ttttattacc tccacggctg gtcaaggtga atttccacag gatggtaaaa 5400 cattctggca agaactaaaa atggcaggac aagctgatct atcaagtatt aggttctcgg 5460 tatttggttt aggtgattcg aatattggc caagaaagga agattctcgt tacttcaata 5520 aaccatcaaa agatttattt tccaaattac aatcactcgg tgccgatcca tttgttccac 5580 tgggcctagg tgatgaccaa gaggataatg gttatgaaac tgcgtattcc atatgggagc 5640 aacaattgtg ggttgaactt ggtgtggata agattgaagt tgccgacgaa ccaagagaac 5700 tgactgccga agatgtcaaa ttacaatcca atttcttacg tggtacatta gcagccgatt 5760 tagtaaatga agaaactggc aacattacta atgaaaatac acaaattgcg aagttccatg 5820 gtttgtacat gcaagatgat agagatatta gagccactcg caaagaacaa ggtttagaac 5880 cattatatgc attcatggct agagttagaa cccctcatgg tactgcatct cctgagcagt 5940 ggttattact tgataaatta tctgacgaaa ctggtactgg tactattaaa ttaactaaca 6000 gggctacttt ccaactgcat ggtgtattaa agaaagatat taagcataca atcagatcga 6060 tgaactcgtt actaatggat actctagctg gttctggtga tgttaataga gatgttatga 6120 tatctgcaat tccggaaaat aagaaagtac atgatcaatt ggtttctatt ggtaaacaga 6180 tatctgagta ttttttgcca aagacgactg cttatcacga aatttggtta catggtgttg 6240 acgaacgtga cgatgaccca acctggccta ccatttacga gaatagaaaa gaaggtccaa 6300 gaaagaagaa gacaatggtt agtggtaatg ctctggtaga cgttgaacca atatattccc 6360 cggtttatct tccaagaaaa ttcaaagtca atattgccgc acctccatat aacgatgtcg 6420 atgtttggtc aagtgatgtt ggtttaatat caattattaa tcaagatact caagaaattg 6480 aaggtttcaa tctattagcg ggtggtggta tgggtacaac tcacaacaac ataaagacat 6540 ggcctgatac tggtaaaatg ctaggttttg ttactccaga caatgttatt aaagccattg 6600 agagtgtatt aatttttcaa agagataatg gtgaccgtac aaaccgtaaa cacgcacgtt 6660 taagatacac tatcgacaca gttgggtttg aaaactttaa aaatattgtt gaagaaagac 6720 tagtttttga tttcaaacca ccaagagatt atactatcga ttccaatgtt gacaaatttg 6780 gttgggtcaa agacgaaagt ggtctgaacc atttcacaac ctttattgaa aatggtagag 6840 ttctagatgc tcctggtatg aatcaaaaga caggtctgag ggaaattgcg aagtatatgc 6900 aattgaagaa atgcggtgag tttagattaa ccggtaacca acatatcatt attagtaaga 6960 tacaagataa ctatctacca gaattaaaag aattgctaaa acagtttcaa ttggataacc 7020 ttcaattgtc aggattgaag ttatcatcct catcgtgtgt cggtttccca acttgtggtt 7080 tagctatggc tgaatcagaa agatttttcc ccattattat tacagaaata gaagaaacat 7140 tggaagattt tggtttacgt cacgattctg ttgtattaag aataactggt tgtggtaatg 7200 ggtgttcacg cccatggtta gctgaagttg ctttgatagg taaagcccca aatgtttaca 7260 atattatgct aggtggtggt tatcacggta acagattgaa caaactgtac agatcaaatg 7320 tcaatgataa agacatctgt ggtattctaa agcctttgtt taagcagtgg gcacttgaaa 7380 gatatgaagc tgaacacttt ggtgactttc taattagaaa agatataatc aaggaaacta 7440 cagaaggaaa atatttccat gataatgttg cccaggaagc ctattaatta ttttttttat 7500 ctatttgtat atttatattt atttatttat ctatttacta aaaagaatat tattattcta 7560 aagtacgtga tcttcggtat atagtttaaa atcagttgaa tgactacatc attttttttt 7620 aagagg 7626 <210> 34 <211> 1418[[ID=2*]] <212> DNA <213> Artificial Sequence <22*> <223> c3775_g2_i1 <400> 34 tttcagtat atgctgttaa taagcaaaga tatatcggtt gtgtgtgaac tacacataat 60 tatacgttga tttcaatgg aatagaatg agacttatcc gacatcggtt ttccagtgaa 120 aatattgc cccttagtgc ctctccgata tcatagatat gttgcctaaa gccagtgatt 180 gttgactcac atacagacac accctattct tgaattgcct atcgcttatc atttacaata 240 tacccctgga cctcatttcc tatcctttt tgccccacta ttagccagga aaaaatgatt 300 tactgtttga acaatcccgt cttcgcaaat catatattca cccacattcc cgaaaactcg 360 gaaaactatt tcttgcggta aggcaatgaa aatgaggaac agaaccactt cggacttcga 420 cggccgttcc ttcggccggt ttggcaccaa atttttcgag aagcaaaaaa gaaacgaagg 480 aaacagaaag agaggggcag agacgaaaa tggaagaaaa aaaagcaatt ccacgcacat 540 tctacgaggg ggtctctgtg aaggagtgtc cgatgatttc cgattactta cataagtggc 600 attatgagat tcctactgat tttaattcaa tagcaaaaa aaaatttcg tcgagaataa 660 tgaggaggga tagatatgtg ccaaatttgt tgttttgaag ataagtattt gagacatata 720 taaaccgagt taaaattggt atatgattag atagttcct gctgttcctg ttccttctg 780 gtaatgaggc gaaccaaaga atcatagctt ttaaaata aaaaaaaaaacaacagc 840 gaggaactg ataagacttc cgaaatccgt gaaattttat tattgaacgg ttctaaaaa 900 taatgactaa ggtggactga caccaaattt tgcataagta atattatta taacaagtt 960 ttagagaca tgtatatct cataactaca taacatttac acatatata tctaatacat 1020 aacatata ctggaatatt cgtgttctt taattatttt ttatcctttt attaatttat 1080 tatgtatggt ctcttaaaa tatatcat gatgatgcat taattaatgt tattcatttc 1140 cccgcgttta gttgttcaga taacttgcct tgagcgatta gtgccgcagg agcatccatt 1200 acaggtaaca tgatttcaat cattctaatg gtagagtttt tctggaaatt tttacgtta 1260 gttaactttc tccattctcc tactgtggat actctataag ttcataatc tgtggccccg 1320 aagacaggca atagttgtaa atgatccccaa ctttggactg cattatatgc agcatcgggt 1380 ccgtggatta atctttcaat agtataccca tcattatt 1418 <210> 35 <211> 2172 <212> DNA <213> Artificial Sequence <220> <223> c4997_g1_i1 <400> 35 agacagacag tcacatttgg taactgttct ttaaccgttt gagatatgta cacatgtatg 60 tgaacattat taagattcct gtcctcttag atacataata tataaatagg gatacgtata 120 ctctgtaaag tgggatctta caagagagtg agagagagag caacagaata aaaaaaaaag 180 gtacacacac agtatcagtt gagcataacg atgggggcta tcacacagaa accaagagac 240 atatatgaga agaaagatgg tgatgatcat ttaaatacga tgaaccatga gcgttatgag 300 tgtccgcatc cggattgtaa taaaagtttc tcaaggcagg aacatttagg tagacataaa 360 ttaaatcatt ggccaaaaga aattttcact tgtaattatt tatttcctga gactaaatta 420 ttatgtggta aaacatttgt taggaaagat ttattagtaa ggcatgaaaa gagacatact 480 aaggagaaaa atagattaca tgcaaaggag attacaccaa ctgctacaaa tgtaactacg 540 gaaaagaaag ttgtcaagaa aagacaatcc aagaagaaac agcaacagca agggaaagat 600 attackaatg ttattccaga gtcaatgcg gatgattcag gtatgggaac atcaaaata 660 aagaataga aaaaaatat aactaaaga ttagcacta aattgatag agtcgcttcc 720 gttccaataa gtttgaaaaa tgatgaat actacaaca caagtaatgg atgaatt 780 ccaacaagta gtaatttgaa tcaaaaaaaaattcaa agagtttatt aaatatgact 840 agtttgaatg ataatactt agcacagct acattttcg gtaatggacc aggacaatct 900 tcaataatct tcgattggtt atgggctcct gaacctaaa gcaaattaaa cgatacaaca 960 agtatgaata ttcagaaca ttctggtttg aatcaccat ttatgttat gtcgcagcaa 1020 cagatgcctc aacagcaaca acaacaa cagatattgc ttccgaatgg tactttacca 1080 ttacattac aacagtattc ttacattagat actacgaata ttaatcata taataatac 1140 aatttaccat taagtccaga atgataaaacatataatg ttggtaacaa ttccaatatt 1200 gttgtattcc cagtacaaga ggtacaatg gattattcag atgaagaag attcccaata 1260 acacaatcaa tgaataata tagtattt gtacatactg ttacaatgag tccaactca 1320 caatattatg atacaagtca tacagggact ttaccacac atcattcaac tgtcaatac 1380 aacaacggta taactactat tggaactaa aaatttggtc gtagacgtaa attackaca 1440 atcgataaaa attcaccacc aaaggattta tccactgtga ttaacgccga aaagaaatta 1500 ttattaccat tgacacaac aacattagca attctaaaa taaatactgt aattaataat 1560 cctttaccaa aagatcg taggatatca atagatgcag aaattgttaa tggtaatgaa 1620 atattaaaa atgacgacac taggggtaat father gtashaacga tattaacgag 1680 gaaacaaaga atgttactga tcgtctaagt gtcaattata ttctctatg aatgttgtgt 1740 attggaagat ctgcattatt atccatctgt aaaaaatttg gatttaagct tcccttttt 1800 1860 caatatatag atatatcaa ttttttcat aaaacgaaaa gcaaatgat aactacatcg 1920 ctatttacat tgatcact cacaattt tggattaac tttcacgaca tcattacatc 1980 attgttttgc attgtcacc ttccacttg cttgttgaca ccattaaacg gtaactcac 2040 attgatgaaa tgaaatttgg accaacagaa ctagcctcgc ctcattattc aaaggaacag 2100 ggaaaggcat ggaaggagcg gaggggaaca gcaagagaga actcctcttc ctcttcctct 2160 tcctcttcct ct 2172 <210> 36 <211> 4720 <212> DNA <213> Artificial Sequence <220> <223> c7526_g1_i1 <400> 36 aatttttgac ttgtaattaa acatggtcag atctctggat gttatgtcat cttttttcac 60 cttgagtata tatattatat aaagaaaaca taaaacgtat caaggacttt agtagtttgt 120 ctcttttcac tggtttctat atttgaaaca ctttttattt cacttaatat tgccctcatt 180 ttattttttt attgtgtgct ttgagttaaa gtaaataaga aaccatttca acggaatctc 240 cgaactttcg aaaaacaaag acagaagaag taacaatgac tgcaacttct gatgcatcat 300 tgaaacaaac tctctatggg tttgctgcaa gagatccatt aaataaatta tactacacta 360 cattaaacaa aggtcaaaat actaaggatt caattccaga tgttgctgtt caattattaa 420 atgataacga tccatttgct acaattttgg aaaatgtctc tgaaacattg actacagtat 480 tcactgacga acaaactttg ttgaaaagtt tacctcatct atttcaattg gaacagaaac 540 caatcatcat taatatcgat ttatccttac aagattattc tattatttct gctatcaagg 600 atttgaatat tgttacttta gtatcaaatg acggttcttc tgctattgct catgctcaat 660 tagctagtaa cattgcttta caaagacaaa tcccagtatt ccatttcatt aactattcaa 720 taatcgataa atcaggtgaa atcattccag atttccaaga aatagaacaa tcccaagaaa 780 taaccaaaga cgacaacgaa gaagagaag aagaagaaat ttccttggaa caatatttaa 840 ctgaacaaaa aatacaatct tttgatctat tagcacaagg ttctaatcca tctgtcgcaa 900 ttgtcaatct ttctcaatac tcaaaggaca ttgcctcagt attaccaaat accgcctctt 960 taattgatgt taagatttac aggccatgga acattcaaga actgttacaa ttgattgcac 1020 cttccgtctc taagattgtt gttattcaag gttcctacaa ggataattat actacagttt 1080 ctcaatcatt cgatcctttc ttattagatt tcttctccga ttttcaaaaa ctggtcgaaa 1140 gaaacatcga tcatgtaatc ttaactaaag taggtgaatt accaattgat gctattcatg 1200 actcattaga tatcatcatt aataacgctc ataaagaaaa tccagatcaa aacttatatc 1260 taggtaagcc ataccacgaa caaattcaaa ataaagaata catcgatttg attcattcct 1320 ctgttaagaa tgttctaaaa ttagagaaag cttatttgaa ggttctaaga caattattct 1380 ctagtaattt acaaatttta aatgaatatt caaatgatac tgttaatggt aacactcctg 1440 aatatggttt tggttactat ttaaaacaag atcaaactcg tgaacgatta attaatttaa 1500 tcaaatcttc attagatgtc tctcttttcg ctggtgtttc taatggatca gcagtagttg 1560 aaaacttatc caaatggtta aaattcaatg aatctcttga tgatcaacaa gttgaagaag 1620 ctaacgttat tgctcatgat atctttgaaa ctttactagc taataaatct aacgacacaa 1680 ttgctaaatt cttatccgtt gcttctactg aggacgcttt cactttcaaa tcacattggt 1740 tagtcggttc ggatgcttgg tcttatgatt taggcaactc tggtgtacat aacgttttat 1800 catcaaagaa aaacattaac atgttattaa ttgattctga accatacact gctaagaaca 1860 aaattgctca taagaaaaat gttggtctat atgctatgaa ctatcataac gtctatgttg 1920 cttctgttgc tgtctattcc tcttacactc agttattaac tgcaatgctt gaagctaaca 1980 aattcaatgg tccttcttta attctagcct atttaccata ttcagaagaa tcaaatacac 2040 cattagatgt tctaaaggaa actaaagttg ctgttgaatc tggttattgg ccattataca 2100 gatatgatcc aagtaaagag gatgaagatg atgaaactca tggtttcaca ctagattctt 2160 ccgtcattaa gaaagaattg caagacttct tagaccgtga aaacaaattg actctattga 2220 ttaagaaata cccaatcgtt gctgacaata ttaagaattc tgcaagtgat accattacaa 2280 gaaaacatga tgctagaaat aaagctgctt tagatgaatt gcttgatggt ttatctggtc 2340 cgccattaca catctattat tcttcagatg gtagtaattc tatcaattta gctactcgtc 2400 tatgcaaacg tgccgtcgct agaggtttaa aagctaccgt attatcaatg gaacaagtta 2460 tcgtcgacga attaccaggt gaagaaaatg ttatcttctt tacatctacc gctggtcaag 2520 gtgaattccc acaagacggt aaatcattct gggatgaatt aaaggcttct accatagatt 2580 tggctggttt gaatgtatct gttttggtt taggtgactc caaatattgg ccacgtaagg 2640 aagatgctcg ttactacaac aaaccttcta aggatttagc tgctaaatta gaggttcttg 2700 gtgctaactt tattgtccct ctaggtttag gtgatgatca agatgctgat ggtttccaag 2760 aaggttatca agcttgggaa ccaaaattat gggaggctct aggtgttgac aacgtcgatg 2820 ttccagatga accaagacca tggaacaatg aagatatgaa actcaactca gatttcttaa 2880 gaggtaccat tgttgaaggt ttaaacgacg agtccacttt agcaattcat ccatacgatc 2940 aacaattgac taaattccat ggttgtata tgcaagatga tcgtgatatc agagatatcc 3000 gtaaggctca aggtttagaa cctttattta gtttcatgtc aagagttaga ttaccaggtg 3060 gtaaagccac tccagaacaa tggttggctt tagataaaat tgcaagtgaa gtcggtaatg 3120 gtactatgaa gatttctaca agagcaactt tccaattaca tggtattcta aagaaggatc 3180 tgaaacatgc tatcagaggt atgaattcta ctttaatgga cactttagct gcctgtggtg 3240 atgttaacag aaacgttgtg gttactgctc ttccaaccaa tgctaaggtt ttcaaccaag 3300 tatctcagat gggtactgat atttctgaat atttcttacc aaagacaact gcttatcatg 3360 aaatttggtt acaaggtacc gacgaacgtg atgatgatct aaactggcca caaatttcg 3420 agaatagaaa ggaaggtcca accaagaaga agactttagt aagtggtaat gcattagtcg 3480 acgtcgaacc aatttacagt aatgtttatt taccaagaaa gtttaaggtt aatattgcag 3540 ttccaccata caacgacgtt gatgttttct ctattgattt aggtttaatt gctattgtta 3600 atccagatac acagattatt gaaggttaca acttatatgc tggtggtggt atgggttcta 3660 ctcacaacaa tactaagaca tatccaagaa ctggttctga ttttggtttt gttaaaccag 3720 aagatgttat tcctgctatt caagctgtta tgattatgca aagagataat ggtgatcgtc 3780 aagatcgtaa acatgcccgt ttaaagtata ctattgatga tattggcgtt cctcaattca 3840 aggctatggt tgaagaagaa tggggtaaga agtttgaacc atctagacca tacgaacaat 3900 ttatttctaa ccacgattac ttcgggtggg ctaaggatga gactggtcta aaccattata 3960 cttgtttcat tgaaaatggt agagttgttg atactcctga attacctcaa aagactggtt 4020 tagttaagat tgctaaatta ctacaaaaga ataaatctgg tcattttaga ttaactgcta 4080 ctcaacatgt tttgatttct gatattgaag ataaggactt ggatgaagtc aagaagatct 4140 taaagcaata caaattagat attacagaat tgagtggtat tagaattgct tcttcatcat 4200 gtgtcggttt accaacttgt ggtttagcta tggcagaatc tgaacgttac ttacctgtct 4260 taattgatga aatcgaagag gtcctagaag aatttggtct acgtcatgat tctattgtta 4320 tgagaatgac aggttgtccg aatggttgtt ctcgtccatg gttagcagaa attgctttaa 4380 tcggtaaagc tccacatact tacaacttaa tgctaggtgg tggttactat ggtcaaagat 4440 tgaacaaatt gtacagagca tcagtcaagg atgatgatgt tattggtatc ttaaagccaa 4500 tatttaagag atgggcttta gaaagagaag aaggtgagca tttcggtgat tttgttgttc 4560 gtgttggtat tatcaagcca actttagaag gtaaatattt ccacgatgat atcgctgaag 4620 acgcttatta gaggaacggc gtctattatt ttcatgtata tgtatattta taatattta 4680 tttcacaact tatttatttt aactattaat tctttaaaat 4720 <210> 37 <211> 953 <212> DNA <213> Artificial Sequence <220> <223> c10116_g1_i1 <400> 37 caaatatttt gaaaagtgtt agttaatact tattcaaact aactcatata catccctaac 60 aatcaaagac tcacttacat aatgaagatc ctaacatccg aagaaattaa tgctcatagt 120 gcctatactt taaaaggtgg tgcattaggt gccgttatag gtttagctgg ttcagctgca 180 ttatttaaat tcttaccaaa aagattccca ggttttaaac caagtcaaat ggcatggtct 240 gctaagactg cattattt tactcctcca actttattta cagctatttg tgcagaagaa 300 gcatctaata gatttgatgc tttgaaatat tccggttcat atatgtcaga tgaagctcta 360 gagagacaag cagcttggga taaattatca aagaaggagc aaatggttga aactttaaat 420 aataataat ataaattat tacaggttta tgggctgctt cattatatgc atcatgggaa 480 attattaata gagataaaat tatgaatgct actcaaaaag ctgttcaagc aagaatgtat 540 gcacaattta ttactgtaat attattatta tgttcagttg gtttaagtac ttatgaaaag 600 aaattaaatc cagataaagc taaacattta gagagtcaac gttgggctaa tgctttaaaa 660 gctgctgcag aacaagaaaa gatggcagat gcacaaacta ctttctctaa tgaagaaaga 720 agagatgcaa agattttcaa atatgattaa tctgttttgt ttgtttgttt gcataatatt 780 attattacca ttttactatc acgcaatgcc atattttata cattttatac acaatccaac 840 tttcctccca aatttatttg aacatctata tttatgaact tctatttttt ttaattcctg 900 tttcacttac tcaatcttat ttaatgactt tattctcgta taaaaaaata caa 953 <210> 38 <211> 5517 <212> DNA <213> Artificial Sequence <220> <223> c2453_g4_i1 <400> 38 cacagctttg gataatgtta ccagtgatga tctgtataaa aatacgtaat ttgaaaagat 60 gagcttacat gttgtgagaa gagctgctct gcccttaaac taagaaacaa caattgtcga 120 caacagtatt cctttgatcg aaacaagaaa atgtcagttg aaactttgat atactggtca 180 attgaattaa taatataaca caaagaagag tattcctatc gtaaaccatt agttagatcc 240 caaaggaga ttgattca tgcccaactt taagatgtac atttagtttc aattaagtta 300 tattaagagt tgtaatttca agatattcac cctatgccga attackacatat tcggagtagt 360 cgttaagatc atactactc tgtaggtgac taaataatc tgttctacgt aattaactct 420 cccagaaattt ccggagtaat cacaagaag gaagatatgg ttatcggaat ttcattgac 480 ctgtcagatg gttgaatat tagtagta aaccaaaaca atctcacaca tacatcaatt 540 aaatctcaat tgagtattaa tagttaatc atagataaa ttcaaaata tgtcccttat 600 aatcaaactt ttcgagatca agttttcat haacctttga atacgactcg gcgagcggct 660 aatttctatt cgcggatcgg taaggaacgg attacktca agttcacata aagaaataa 720 gaaagtgtct tgtttacaga acgatcagtt atatacaac taaatggttg caaaaatac 780 tagattaaaaaaaaaaaaagaaaactggggca agccgggaat cgaacccggg 840 acctcccgca ccccgagcgg aaatcatacc tcgaccac gtgcccttga aatttcaaat 900 cttgaaattt tgttgggtca ttatatacgg gagctgaaat tcggtaata ccgaaatatt 960 ttaaatgta ttatccgatt tattcattag tattcttaatc tgataatgg tctagtatttt 1020 tctcgagcta ttattgtatc ttagtattca aagatgatt agatatatga ggaaattatg 1080 gaaaaaag gaaatttca aacgtttcac taatatgtaa ataatttgt vakaccgtttt 1140 cttctaataa ttttaaataa agaaacatct gatatgtttt gatgcaaag tagtggacat 1200 cgtcctttga cacactgtg aactgatat tcttaaaaat tgtcaatag acccaatca 1260 aatttatcat gattaaatcg ctggatcaat acacagggtc atattag tctgatttga 1320 attcaatct gcttcatgtc ttatctcgc aaaccaaaat ttagttatta atggattaaa 1380 actgtatatt tataccatct aacaacagaa tgttttggttt aaactatagg gtatcattaa 1440 actcaattat father aaaaagagaa tttagtatct taaatgtatt attaataatc 1500 ttagatcggt aacattgtc aaaaggt gattgcatt aaaattag gtcaacact 1560 tagcttat taatagtcg atagtgag aaggacaata tagtttaat attttagtat 1620 attttgaatt gaaatgga aactggatgg gatgatatt gatgtgggt acatcctaca 1680 gtaatggtaa tattatcaga attaccacaa gttgaacaaa tcacgtagac tatttctagc 1740 gctatacc tcacaatctg tatataagt ctacaagtgt tcgtctatgt catgattcga 1800 taccacggaa aagaatagat caatagaaag tggtaacaat tatctgggac tgtcttggag 1860 atatcagacg tcccattcat cttgaatttg aaacatatat ctctccgtaa caattcgaaa 1920 ttgtcactaa tggaagcaga aactcagata ccaaacacat cttttgagta acacaattat 1980 ggttctttga cggaagattt attattccaa attaagagg ggattttgat ttaaaaatgt 2040 caacattatt tataagaac atatccaatt caaatcctta acaatagcta atagaaactg 2100 ctctaagacg tgtattatat aaacccagag aaagttaaaa tatgtccttt tacaacttag 2160 aagttctcga aacgctaata attacctgtt cttctgaagc tgctaaacga atctagaatt 2220 gatttgttaa ccaaaaaagg aacacaaaca cttgtgaaac ttgtataata tgtgtaatta 2280 tccggaattt tacagtttga attagctctt tatgttaaga acaacattaa ccctttttat 2340 tctgtattcc ccactaattc cccagacaat aaggttagtt ctccattggc ctgcatctcc 2400 cacagaatat aacacaggag attaaataat ccgcgtaata tatcaagata tccatatgga 2460 aaaatatgac aagacagtct cttcagcgtg cttctttggt tgataaatgt caacaggctt 2520 ttgttcaaat aaggaaaccg gagtaaacaa cacgaattag gtattgaact aaaacaggaa 2580 cttcaaaata aaaccgaacc ccttcatcta cccacgttaa tgaacagagt tgaacgatca 2640 tatattcatt tggaataaat ttctccacat ctattaagtt acggattaaa taaatggaac 2700 atctccaatt aataacattt ccttaattaa tgccagggag tcggaattat gtttacttat 2760 ttcaggagtt aacagaatta ttttcaatc gtggaaataa gaaagctcgg aacatttatt 2820 tctcaattgc atattagcta agaaaaagta atggcgaaaa gaaaaaaacg gagaatttt 2880 tttattgaag tcttggatag gcggaatgat ttgtaaagtt gtcatagaaa actaattaag 2940 ttttgagaag gtatctagca ttgctcaact tatagggtac gctttggaag aattaaaaca 3000 aattaacttt acgtagtagc tccgacattg ttctggcatt gactttatca aaatcgcata 3060 ttggaacaat caattagggt cttctgtgcg cttagacat taccaacaaa ttccaacgga 3120 accctacaaa gtgtggggga agaagagtaa ttgttgcctt ttcctctctc aagcaagtgt gtaatcaaac acacctttta gctaattcac tctccgcgaa gtttaaat gaagtttttc cacctatttt caaattcaat tcccaacatc caaatttt tcctttacaa gaatcacaca ggttattaca ttaggaattg cttttttttt ttttttttct tctttccccc ccaagattgg tgatttgcct tccagattgg aaattattta cgcaaggaat agctgcagaa gcaaggagta 3420. 3480. aaatgtcgca gtcaatagtt ttcccgcccc gcgttttttc tccggcgatt ttatctccgc ttgtactatg ttatctttgg agaatgctat ttccaagagt ttcggaaaaa catttatgaa aagaataat atcaaaaact gtatgcgaag ataacgttgt aggatatatt tctacgatgg aacactgtgc cccgcgaatt ttaaagatgc aaaacaataa ctacagatct atctgagaat atatcatggc acgaccccca ccccataaca ttgcttctgc attagtttct cttttccccg 3720 3780. tctcggaaaa atatattcgt ttttccgaac aaacaaaaca tttcatgtac gtatattcct tcatgttcaa tagtagctta tgtaacaatt tgttcggtat cctattgata ctttagcgaa atatattca ttgtgtgtat ggtatgacag atgtcagat atatatatat atatataaaa 3900 ggaagaaact ttcccattct agactaaga cattcattta atggtttggt tctttcgttt 3960 tccaccttc cccttcgttt gtcaatctgt cattgaatt tcaacaatc tcattagta 4020 acactagtat acaatcgttc acaatatta ttgtactgta cattattat tattatttt 4080 ttaagaagg tcaccagat taatataat ttcagtttg aaggtactc aattgtaaga 4140 aaaagtataata taatataata accaaatga gtgttaatcc ccaactaaa ttccagctg 4200 atacaatga tagaccattt agatgtgagc ttgtcatcg cggctttcac agactcgaac 4260 aaaaaaag acacgttaga acacataccg gagaaaaacc gcacggatgt cagttccccg 4320 gatgtaacaa atttttcagt agaactgat aattaagag acatcgaga acacatattg 4380 gtacatctca aagaaagact aggaata ttccaaagaa tatagtgag actcaaattt 4440 catcaaacc attackatc gctgcttca agaagtgat taaaaaggg atagtacac 4500 ctccaaagac atatactgtt ccatcattaa catcattatt accatgag actacaccaa 4560 tgtcttcacg taattatta gctggttcca catcacttga aagaccgatt tcaagaaca 4620 tgtttcctcc aagtatacag aaagtttccc ctatgagtga aaatagttct gctgaatcat 4680 ccataccgaa ttctccaatt tctcaaata attctatatc gatactagt agttcattgt 4740 ccttaaatttc attactac agtaatgtta acaataca taatcaatg tcatctgtat 4800 catctgtatc atcatattca catggtagtt tcaatttt agatacatca ttaaattat 4860 caagtaaag acgtgcagat ttccaattg ttcagaga aaatgatgca gatagcactg 4920 gtagtagtaa tatagattt atagtaatc aaccgaattc tattcaatta ccaccatta 4980 aatctatatt agctatatc ataatttca atatgat gatatcatcc space 5040 ctcgtgcatc tacataccaa cacaaccat atttcatcg ataggtaacg atgaatca 5100 caatcataac cacatactag father catacataca cacatacata cattcataca 5160 ttcattcata actaatcaac gaacattaaa cattcatcat ttgacaaaa tcacttgtcc 5220 tcttattcaa atagttccca gatcctgctt acatataat atctgtcggc atatctttatt 5280 atgaaattca cttaccgaca tggaaaccgc ccaccaacaa acaatactgg aaaagtccgt 5340 ccttattata aaataaaaaa attgtttgcc gtttcattta ttaatttatg cgcatctttt 5400 ttttcttttt tttttgtttc gtacgtaaag aatttcggga agatcctacc attccgtccg 5460 acgtgatgat ccgggtaacg tgtgttttgt tctattcgcc cgcgcgcccc ccacttc 5517 <210> 39 <211> 2495 <212> DNA <213> Artificial Sequence <220> <223> c8818_g1_i1 <400> 39 gaaaaattta ttaaatatta ataagtcttc tgtgtttgtt tttttttggc ttttctaata 60 cttatagttt tatttgcctc atgattgatt gattccgtac ttttctctat ttctcctgag 120

此处似乎有误,原内容为 ,但翻译时应保持原样

此处似乎有误,原内容为 ,但翻译时应保持原样

Claims

1. A recombinant strain capable of producing lactic acid, the recombinant strain being constructed by deleting g3002-1 allele 1 and allele 2 encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain with a preservation number of KCTC13508BP, and introducing a gene encoding lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 2 derived from Staphylococcus epidermidis at the position of g3002-1 allele 1 and allele 2, The 5'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 59, and the 3'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 61; the 5'UTR sequence of the g3002-1 allele 2 is SEQ ID NO: 60, and the 3'UTR sequence of the g3002-1 allele 2 is SEQ ID NO:

62. 2 . The recombinant strain according to claim 1 , wherein the nucleotide sequence of the gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis is SEQ ID NO:

1.

3. The recombinant strain according to claim 1, wherein the g4423 allele 1 and allele 2 encoding alcohol dehydrogenase are further deleted or inactivated, The 5'UTR sequence of the g4423 allele 1 is SEQ ID NO: 4, and the 3'UTR sequence of the g4423 allele 1 is SEQ ID NO: 6; the 5'UTR sequence of the g4423 allele 2 is SEQ ID NO: 5, and the 3'UTR sequence of the g4423 allele 2 is SEQ ID NO:

7.

4. The recombinant strain according to claim 1, wherein the g1544 allele 1 and allele 2 encoding the enzyme converting dihydroxyacetone phosphate into glycerol-3-phosphate are further deleted or inactivated, The 5'UTR sequence of the g1544 allele 1 is SEQ ID NO: 14, and the 3'UTR sequence of the g1544 allele 1 is SEQ ID NO: 16; the 5'UTR sequence of the g1544 allele 2 is SEQ ID NO: 15, and the 3'UTR sequence of the g1544 allele 2 is SEQ ID NO:

17.

5. The recombinant strain according to claim 1, wherein g2947 allele 1 and allele 2 encoding an enzyme that converts lactate to pyruvate are further deleted or inactivated, The 5'UTR sequence of the g2947 allele 1 is SEQ ID NO: 10, and the 3'UTR sequence of the g2947 allele 1 is SEQ ID NO: 12; the 5'UTR sequence of the g2947 allele 2 is SEQ ID NO: 11, and the 3'UTR sequence of the g2947 allele 2 is SEQ ID NO:

13.

6. A recombinant strain capable of producing lactic acid, the recombinant strain being constructed by deleting g1544 allele 1 and allele 2 encoding an enzyme that converts dihydroxyacetone phosphate to glycerol-3-phosphate, g2947 allele 1 and allele 2 encoding an enzyme that converts lactate to pyruvate, g4423 allele 1 and allele 2 encoding alcohol dehydrogenase, and g3002-1 allele 1 and allele 2 encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain with a deposit number of KCTC13508BP, and introducing a gene encoding lactate dehydrogenase into the acid-tolerant yeast YBC strain. wherein the gene encoding lactate dehydrogenase is introduced into the positions of the deleted g4423 allele 1 and allele 2, the deleted g3002-1 allele 1 and allele 2, and the deleted g1544 allele 1 and allele 2, The gene encoding lactate dehydrogenase introduced at the position of the deleted g3002-1 allele 1 and allele 2 is a gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis with an amino acid sequence of SEQ ID NO: 2, The 5'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 59, and the 3'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 61; the 5'UTR sequence of the g3002-1 allele 2 is SEQ ID NO: 60, and the 3'UTR sequence of the g3002-1 allele 2 is SEQ ID NO:

62. The 5'UTR sequence of the g4423 allele 1 is SEQ ID NO: 4, and the 3'UTR sequence of the g4423 allele 1 is SEQ ID NO: 6; the 5'UTR sequence of the g4423 allele 2 is SEQ ID NO: 5, and the 3'UTR sequence of the g4423 allele 2 is SEQ ID NO: 7, The 5'UTR sequence of the g1544 allele 1 is SEQ ID NO: 14, and the 3'UTR sequence of the g1544 allele 1 is SEQ ID NO: 16; the 5'UTR sequence of the g1544 allele 2 is SEQ ID NO: 15, and the 3'UTR sequence of the g1544 allele 2 is SEQ ID NO:

17. The 5'UTR sequence of the g2947 allele 1 is SEQ ID NO: 10, and the 3'UTR sequence of the g2947 allele 1 is SEQ ID NO: 12; the 5'UTR sequence of the g2947 allele 2 is SEQ ID NO: 11, and the 3'UTR sequence of the g2947 allele 2 is SEQ ID NO:

13. 7 . The recombinant strain according to claim 6 , wherein the nucleotide sequence of the gene encoding lactate dehydrogenase derived from Staphylococcus epidermidis is SEQ ID NO:

1.

8. A method for producing a recombinant yeast strain having increased lactic acid tolerance and increased lactic acid production capacity, the method comprising: (a) inducing adaptive evolution of the recombinant yeast strain toward high lactic acid concentration by sequentially culturing the recombinant yeast strain having lactic acid production ability from a low concentration lactic acid medium to a high concentration lactic acid medium; (b) selecting a recombinant yeast strain having an improved lactic acid production ability in the high-concentration lactic acid medium; as well as (c) introducing a gene encoding lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 2 derived from Staphylococcus epidermidis into the genome of the selected strain at the positions of g3002-1 allele 1 and allele 2 to replace the gene encoding lactate dehydrogenase having a nucleotide sequence of SEQ ID NO: 3, The recombinant yeast strain having the ability to produce lactic acid in step (a) is YBC5 strain constructed by deleting g1544 allele 1 and allele 2 encoding an enzyme that converts dihydroxyacetone phosphate into glycerol-3-phosphate, g2947 allele 1 and allele 2 encoding an enzyme that converts lactate into pyruvate, g4423 allele 1 and allele 2 encoding alcohol dehydrogenase, and g3002-1 allele 1 and allele 2 encoding pyruvate decarboxylase from the acid-tolerant yeast YBC strain with a deposition number of KCTC13508BP, and introducing a gene encoding lactate dehydrogenase having a nucleotide sequence of SEQ ID NO: 3 at the positions of the deleted g4423 allele 1 and allele 2, the deleted g3002-1 allele 1 and allele 2, and the deleted g1544 allele 1 and allele 2. The 5'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 59, and the 3'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 61; the 5'UTR sequence of the g3002-1 allele 2 is SEQ ID NO: 60, and the 3'UTR sequence of the g3002-1 allele 2 is SEQ ID NO:

62. The 5'UTR sequence of the g4423 allele 1 is SEQ ID NO: 4, and the 3'UTR sequence of the g4423 allele 1 is SEQ ID NO: 6; the 5'UTR sequence of the g4423 allele 2 is SEQ ID NO: 5, and the 3'UTR sequence of the g4423 allele 2 is SEQ ID NO: 7, The 5'UTR sequence of the g1544 allele 1 is SEQ ID NO: 14, and the 3'UTR sequence of the g1544 allele 1 is SEQ ID NO: 16; the 5'UTR sequence of the g1544 allele 2 is SEQ ID NO: 15, and the 3'UTR sequence of the g1544 allele 2 is SEQ ID NO:

17. The 5'UTR sequence of the g2947 allele 1 is SEQ ID NO: 10, and the 3'UTR sequence of the g2947 allele 1 is SEQ ID NO: 12; the 5'UTR sequence of the g2947 allele 2 is SEQ ID NO: 11, and the 3'UTR sequence of the g2947 allele 2 is SEQ ID NO:

13.

9. A recombinant yeast strain YBC6, constructed by introducing a gene encoding a lactate dehydrogenase having an amino acid sequence of SEQ ID NO: 2 derived from Staphylococcus epidermidis into the genome of recombinant strain #26-5 with a deposit number of KCTC 14215BP, replacing the gene encoding a lactate dehydrogenase having a nucleotide sequence of SEQ ID NO: 3, at positions of g3002-1 allele 1 and allele 2. Compared to the YBC strain with a deposit number of KCTC 13508BP, the recombinant yeast strain YBC6 has enhanced lactate production under high lactate concentrations and suppressed ethanol and glycerol production. The 5'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 59, and the 3'UTR sequence of the g3002-1 allele 1 is SEQ ID NO: 61; the 5'UTR sequence of the g3002-1 allele 2 is SEQ ID NO: 60, and the 3'UTR sequence of the g3002-1 allele 2 is SEQ ID NO:

62.

10. A method for producing lactic acid, comprising: (a) cultivating the strain according to any one of claims 1 to 6 and 9 to produce lactic acid; and (b) collecting the produced lactic acid.

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