KR102324008B1 - Microorganisms fermenting galacturonic acid and pentose simultaneously - Google Patents

Abstract

The present invention relates to microorganisms that simultaneously ferment galacturonic acid and pentose, and pectin, which occupies the largest amount of pericarp, and its main components, galacturonic acid and xylose, in order to convert fruit processing by-products into raw materials for biological processes. , We have developed a recombinant microorganism that can efficiently metabolize the three carbon sources of arabinose, so wastes generated during fruit processing can be effectively treated.

Description

Microorganisms fermenting galacturonic acid and pentose simultaneously

The present invention relates to a microorganism that simultaneously ferments galacturonic acid and pentose.

Pectin-rich biomass refers to a by-product of the sugar and juice industry, sugar beet pulp and citrus peel waste, especially citrus fruits with a high pectin content up to 35% by dry weight. Annual citrus juice production generates the same amount of citrus peel waste, which is about 4.6 million tonnes of fruit waste in the United States alone. Also, beet pulp from sugar processing produces only 7% of the initial biomass of the product, but in the European Union alone the annual production is around 14 million tonnes.

Contrary to the damage caused by climate change, the frequency of hurricanes in the United States and Mexico has decreased. As of 2019, 51.8 million tons of oranges were produced, an increase of 4.2 million tons from the previous year, and there is also a positive forecast that it will steadily increase every year.

However, the amount of non-commodities that are generated due to the rapid increase in fruit production and the amount discarded due to the failure of supply and demand is also increasing. Worldwide, fruit-based biomass such as citrus gourd is rapidly emerging as the next-generation biomass.

Korea produces about 500,000 tons of citrus per year, about 60,000 tons of citrus peel waste is generated through the processing process, and about 50,000 tons of citrus fruits are thrown away as non-commodities, which causes environmental and economic problems. However, effective measures to utilize next-generation biomass have not yet been prepared.

Therefore, in order to convert the next-generation biomass, which has emerged rapidly due to climate change, to new and renewable energy, the 'development of a platform microorganism capable of biodegrading fruit processing by-products', which is intended to be developed through the present invention, is absolutely necessary.

The most abundant polysaccharide in pectin-rich biomass such as citrus fruits is pectin. D-galacturonic acid is composed of a single chain bond, and glucose, xylose, arabi It is characterized in that various monosaccharides such as L-arabinose and L-rhamnose are connected in a branched form.

When this pectin is subjected to acid and enzymatic (cellulase, hemicellulase, pectinase) hydrolysis treatment, a large amount of carbon sources such as glucose, frictose, galactose, xylose, arabinose and galacturonic acid can be obtained, so bioenergy production is reduced. It is a suitable resource as biomass for

1. Edwards, M.C., Doran-Peterson, J., 2012. Pectin-rich biomass as feedstock for fuel ethanol production. Appl. Microbiol. Biotechnol. 95 (3), 565-575. 2. Huisjes, E.H., de Hulster, E., van Dam, J.C., Pronk, J.T., van Maris, A.J., 2012. Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose. Appl. Environ. Microbiol. 78 (15), 5052-5059. 3. Protzko RJ, Latimer LN, Martinho Z, de Reus E, Seibert T, Benz JP, et al. 2018. Engineering Saccharomyces cerevisiae for co-utilization of D-galacturonic acid and d-glucose from citrus peel waste. Nat. Commun. 9:5059.

In the present invention, a fruit-based biotechnology that has rapidly emerged as a sustainable alternative resource due to climate change using gene scissors technology to construct a strain capable of simultaneously fermenting three unconventional carbon sources (xylose, arabinose, and galacturonic acid) The present invention was completed by confirming that it is possible to produce new and renewable energy derived from mass.

In order to solve the above problems, the present invention is ALD6 (acetaldehyde dehydrogenase gene) and PHO13 (haloacid dehalogenase Type IIA phosphatase gene) knock-out (knock-out), XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene), XYL3 (xylulokinase gene), lad1 (L-arabitol dehydrogenase gene), alx1 (L-xylulose reductase gene), gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) and Provided is a recombinant microorganism having the ability to simultaneously consume galacturonic acid and pentose (xylose, arabinose) in which lgd1 (L-galactonate dehydratase gene) is knocked-in.

Acetate production may be inhibited by knockout of the ALD6 (acetaldehyde dehydrogenase gene).

The pentose phosphorylation cycle may be promoted by knockout of the PHO13 (haloacid dehalogenase Type IIA phosphatase gene).

The xylose metabolic pathway may be generated by knock-in of the XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene) and XYL3 (xylulokinase gene).

The arabinose metabolic pathway may be generated by knock-in of the lad1 (L-arabitol dehydrogenase gene) and alx1 (L-xylulose reductase gene).

The galacturonic acid metabolic pathway may be generated by the knock -in of the gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) and lgd1 (L-galactonate dehydratase gene). have.

The strain may be Saccharomyces cerevisiae.

The strain may metabolize xylose, arabinose or galacturonic acid.

In order to convert fruit processing by-products into raw materials for bioprocessing, recombinant microorganisms that can efficiently metabolize three carbon sources: pectin, which occupies the largest amount of pericarp, and its main components, galacturonic acid, xylose, and arabinose. Since it has been developed, it is possible to effectively treat wastes generated during fruit processing.

1 is a metabolic engineering strategy for introducing metabolic pathways of three unconventional carbon sources (xylose, arabinose, galacturonic acid) to develop S. cerevisiae YE9 strain (a) and a primer set for confirming gene introduction It is a schematic diagram showing (b).
Figure 2 is a schematic diagram showing the intracellular metabolic pathways of the S. cerevisiae YE9 strain in which the metabolic pathways of three unconventional carbon sources (xylose, arabinose, and galacturonic acid) are constructed.
Figure 3 is the production of glycerol (glycerol) (b) and ethanol (ethanol) (c) through xylose (D-xylose) consumption (a) of the parent strain S. cerevisiae D452-2 and the improved strain S. cerevisiae YE9 This is the confirmed graph.
Figure 4 is the production of glycerol (glycerol) (b) and ethanol (ethanol) (c) through arabinose (L-arabinose) consumption (a) of the parent strain S. cerevisiae D452-2 and the improved strain S. cerevisiae YE9 This is the confirmed graph.
Figure 5 is glycerol (glycerol) (b) and ethanol (ethanol) (c) through galacturonic acid (D-galacturonic acid) consumption (a) of the parent strain S. cerevisiae D452-2 and the improved strain S. cerevisiae YE9 It is a graph confirming production.
6 is an improved strain S. cerevisiae YE9 glucose (D-glucose) (a) and fructose (D-fructose) (b), galactose (D-galactose) (c) through consumption of glycerol (glycerol) and ethanol It is a graph confirming the production of (ethanol).
7 is a graph showing the results of fermentation of galacturonic acid (D-galacturonic acid) of S. cerevisiae YE6 strain expressing two different L-glyceraldehyde reductase genes ( gaaD , YPR1 ) am.
8 shows S. cerevi siae YE9 in a mixed sugar (Glc, glucose; Fru, frictose; Gal, galactose; Xyl, xylose; Ara, arabinose) containing galacturonic acid (galUA, D-galacturonic acid). It is a graph showing the fermentation result of the strain.
9 is a galacturonic acid (galUA)-containing mixed sugar (Glc, glucose; Fru, frictose; Gal, galactose; Xyl, xylose; Ara, arabinose) conditions of the improved strain S. cerevisiae YE9 fermentation results. The graph shown (accumulated product results in FIG. 8).
10 is a carbon source consumption rate (g / L / h) results of the S. cerevisiae YE9 strain in xylose (D-xylose) mixing conditions containing various concentrations of galacturonic acid (D-galacturonic acid). This is the graph shown.
11 is a graph showing the results of intracellular metabolites of S. cerevisiae YE9 strains in which xylose (Xyl, D-xylose) and galacturonic acid (galUA, D-galacturonic acid) are alone or mixed. It is a schematic diagram
12 is a graph showing the result of fermentation the parental strain S. cereivisiae D452-2 and improved strain S. cerevisiae in terms YE9 citrus foil hydrolyzate (citrus peel hydrolysate).

In the present invention, the improved strain to decompose fruit-based biomass effectively produces carbon sources of glucose and fructose as bioethanol, and is resistant to various stresses such as organic acids and heat. The GRAS type yeast, Saccharomyces cerevisiae , will be used. However, natively S. cerevisiae does not metabolize xylose, arabinose, and galacturonic acid, so strain improvement is required.

Accordingly, the present inventors have established a Saccharomyces cerevisiae YE9 strain.

The present invention is a recombinant microorganism that can efficiently metabolize three carbon sources: pectin, which occupies the largest amount of fruit skin, and its main components, galacturonic acid, xylose, and arabinose, and effectively treats waste generated during fruit processing. can do.

In addition, the recombinant microorganism S. cerevisiae YE9 developed by the present invention does not require acidification (pH 3.5) of fruit waste to ferment fruit waste, unlike previous studies (Protzko et al., 2018), and exogenous galacturonic acid transporter gene It is possible to effectively ferment galacturonic acid without the need to additionally introduce.

The strain of the present invention has a high consumption of galacturonic acid without detoxification of the galacturonic acid transporter gene (D-galacturonic acid transporter gene).

The present invention is ALD6 (acetaldehyde dehydrogenase gene; accession no. NM_001180296; SEQ ID NO: 1) and PHO13 (haloacid dehalogenase Type IIA phosphatase gene; accession no. NM_001180296.1; SEQ ID NO: 2) are knocked out, XYL1 ( xylose reductase gene; accession no. XM_001385144.1; SEQ ID NO: 3), XYL2 (xylitol dehydrogenase gene; accession no. (XM_001386945.1); SEQ ID NO: 4, XYL3 (xylulokinase gene; accession no. XM_001387288.1; SEQ ID NO: 5) ), lad1 (L-arabitol dehydrogenase gene; accession no. MT321096; SEQ ID NO: 6), alx1 (L-xylulose reductase gene; accession no. AJ583159.1; SEQ ID NO: 7), gaaA (D-galacturonic acid reductase gene; accession no. MT309494; SEQ ID NO: 8), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene; accession no. MT321095; SEQ ID NO: 9) and lgd1 (L-galactonate dehydratase gene; accession no. MT321097; SEQ ID NO: 9) 10) provides a recombinant microorganism having the ability to simultaneously consume galacturonic acid and pentose (xylose, arabinose) that is knock-in.

Specifically, wild type S. cerevisiae D452-2 is used as the parent strain. Genetic modification was performed using Cas9 gene editing technology. In order to suppress acetate production, ALD6 (acetaldehyde dehydrogenase gene) was deleted. PHO13 was deleted to promote the pentose phosphate pathway. Pichia stipitis derived XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene), XYL3 (xylulokinase gene) was introduced for the xylose metabolic pathway (D-xylose pathway). Trichoderma reseyi (Trichoderma reesei) not come when M bromo-derived lad1 (L-arabitol dehydrogenase gene) , a mono spokes La (Ambrosiozyma monospo r a) derived alx1 (L-xylulose reductase gene) the arabinose metabolic pathway (L-arabinose pathway) introduced for Aspergillus niger ( Aspergillus niger ) derived gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene), T. reesei- derived lgd1 (L-galactonate dehydratase gene) It was introduced for the galacturonic acid pathway (D-galacturonic acid pathway).

In the present invention, ALD6 (acetaldehyde dehydrogenase gene) to inhibit acetate production and PHO13 (haloacid dehalogenase Type IIA phosphatase gene) to promote pentose phosphorylation cycle are deleted,

XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene) and XYL3 (xylulokinase gene) for the xylose metabolic pathway, lad1 (L-arabitol dehydrogenase gene) and alx1 (L-xylulose reductase gene) for the arabinose pathway, For galacturonic acid metabolism pathway, gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) and lgd1 (L-galactonate dehydratase gene) are introduced. It is a recombinant microorganism having the ability to simultaneously consume pentose (xylose, arabinose).

The XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene) and XYL3 (xylulokinase gene) are derived from Pichia stipitis.

The alx1 (L-xylulose reductase gene) has not come when M is a mono-bromo spokes La (Ambrosiozyma monospo r a) is derived.

The gaaA (D-galacturonic acid reductase gene) and gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) are derived from Aspergillus niger.

The lad1 (L-arabitol dehydrogenase gene) and lgd1 (L-galactonate dehydratase gene) are Trichoderma reesei ( Trichoderma reesei ) is the origin

<Example 1> Experimental method

<1-1> Strain and Fermentation

All strains are summarized in Table 1. One colony of the S. cerevisiae strain was precultured in YP medium (10 g/Lyeast extract, 20 g/L peptone) containing 20 g/L of glucose (YPD) at 30° C. and 250 rpm for 36 hours. The pre-cultured yeast was washed once, and the initial cell amount was recovered at 0.5 g/L or 25 g/L and resuspended in 20 mL YP liquid medium containing various carbon sources. The inoculated culture medium was fermented in a 100-mL Erlenmeyer flask at 30° C. and 130 rpm for up to 72 hours. All fermentations were performed in biological triplicates.

strain Description of the relevant genotype* Citations D452-2 Wild type; Mata leu2 his3 ura3 (Hosaka et al., 1992) DY02 Expressing the heterologous D-xylose pathway; D452-2 ald6::TDH3 _P -XYL1-TDH3 _T -PGK1 _P -XYL2-PGK1 _T pho13::TEF1 _P -XYL3-TEF1 _T this study YE3 DY02 int#4::CCW12 _P -gaaA-CCW12 _T this study YE4 DY02 int#4::PGK1 _P -lgd1-PGK1 _T this study YE5 DY02 int#4::TDH3 _P -gaaC-TDH3 _T this study YE6 Expressing the heterologous D-xylose and D-galacturonic acid pathway; DY02 int#4::CCW12 _P -gaaA-CCW12 _T -PGK1 _P -lgd1-PGK1 _T -TDH3 _P -gaaC-TDH3 _T this study YE6
YPR1 YE6 CCW12 _P -YPR1 this study YE6
gaaD YE6 int#6::CCW12 _P -gaaD-CCW12 _T this study YE01 Expressing the heterologous D-xylose, and L-arabinose pathway
D452-2 ald6::TDH3 _P -XYL1-TDH3 _T -PGK1 _P -XYL2-PGK1 _T
int#1::TEF1 _P -XYL3-TEF1 _T
sor1::FBA1 _P -lad1-FBA1 _T -PGK1 _P -alx1-CYC1 _T (Ye et al., 2019) YE9 Expressing the heterologous D-xylose, L-arabinos, and D-galacturonic acid pathway; YE6
int#7::FBA1 _P -lad1-FBA1 _T - PGK1 _P -alx1-CYC1 _T this study

* XYL1, XYL2 and XYL3 are from Pichia stipitis . gaaA, gaaC gaaD and is derived from Aspergillus niger and, lad1 lgd1 and is derived from Trichoderma reesei and, alx1 stems from Ambrosiozyma monospora.

<1-2> Strain construction by Cas9-based genome editing

All strains were improved by in vivo assembly and chromosome integration technology by applying Cas9 gene editing technology. In summary, there are three components for strain improvement: 1) guide RNA (gRNA) plasmid development, 2) donor DNA preparation, and 3) yeast transformation. The nucleotide sequence of the gRNA was designed to be target cleavage site-specific and 20-bp in length as shown in Table 2 below.

gRNA Target cut site PAM sequence
(5'-3') Target sequences Plasmid name base sequence (5'-3') SEQ ID NO: ALD6.1 ALD6 TGG GTCAGATCACACTTCCAAA 11 pRS42H-ALD6.1 PHO13.1 PHO13 CGG TCCCTTATCTATTAACTTTC 12 pRS42H-PHO13.1 YPR1.1 YPR1 GGG CATGGTAGATTATTATCTGT 13 pRS42H-YPR1.1 INT#4 Intergenic region upstream ASF1 GGG CTCTCGAAGTGGTCACGTGC 14 pRS42H-INT#4 INT#6 Intergenic region upstream ATG33 CGG TTGTCACAGTGTCACATCAG 15 pRS42H-INT#6 INT#7 Intergenic region downstream YGR190C TGG GATACTTATCATTAAGAAAA 16 pRS42H-INT#7

A plasmid expressing each gRNA nucleotide sequence was constructed by a fast cloning method based on PCR. To construct the pRS42H-ALD6.1 plasmid, for example, the pRS42H-GND1.1 plasmid (template plasmid) was amplified with primers Kim044/Kim045 (Table 3). The PCR product was treated with Dpn I and transformed into E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) strain. For transformation, LBA (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 100 μg/mL ampicillin) solid medium was used as a selective medium. The gRNA nucleotide sequence of the generated plasmid was confirmed and selected by Sanger sequencing using a primer for the T3 promoter. All gRNA plasmids in Table 2 were constructed through the same procedure using the other primers listed in Table 3.

Plasmid name primer sequence (5'-3') SEQ ID NO: pRS42H-ALD6.1 Kim044 AAGATCACACTTCCAAA GTTTTAGAGCTAGAAATAGCAAG 17 Kim045 TTGGAAGTGTGATCTTGAC GATCATTTATTCTTTCACTGCG 18 pRS42H-PHO13.1 Kim624 CTTATCTATTAACTTTC GTTTTAGAGCTAGAAATAGCAAG 19 Kim625 AAAGTTAATAGATAAGGG AGATCATTTATTCTTTCACTGCG 20 pRS42H-YPR1.1 Kim535 GGTAGATTATTATCTGT GTTTTAGAGCTAGAAATAGCAAG 21 Kim536 CAGATAATAATCTACCATG GATCATTTATTCTTTCACTGCG 22 pRS42H-INT#4 Kim310 TCGAAGTGGTCACGTGC GTTTTAGAGCTAGAAATAGCAAG 23 Kim311 CACGTGACCACTTCGAGAG GATCATTTATTCTTTCACTGCG 24 pRS42H-INT#6 Kim314 TCACAGTGTCACATCAG GTTTTAGAGCTAGAAATAGCAAG 25 Kim315 TGATGTGACACTGTGACAA GATCATTTATTCTTTCACTGCG 26 pRS42H-INT#7 Kim486 AGGAATTATGTTCGCCC GTTTTAGAGCTAGAAATAGCAAG 27 Kim487 GGCGAACATAATTCCTTAC GATCATTTATTCTTTCACTGCG 28

Donor DNA fragments were prepared through polymerase chain reaction (PCR) using the primers listed in Table 4. Each donor DNA fragment was constructed as a chromosomal sequence of 40-50 bp in length on both sides of the donor DNA fragment for in vivo assembly and introduction into the chromosome, and homologous recombination was induced. The primers in Table 4 were used to construct an expression cassette for the expression of each foreign gene. The donor DNA of the xylose expression cassette was designed so that the target genes (ALD6, PHO13) were completely removed while being introduced into the chromosome. On the other hand, the expression cassette of the arabinose pathway and the galacturonic acid pathway was introduced at a position that does not interfere with the expression of surrounding genes.

Finally, yeast transformation using Cas9 gene editing technology was performed by partially modifying the lithium acetate/single-stranded carrier DNA-polyethylene glycol method (ref). The gRNA plasmid, donor DNA fragment, and Cas9 protein expression plasmid (pRS41N-Cas9 plasmid) developed above were introduced into S. cerevisiae, and in YPD solid medium containing 100 μg/mL nourseothricin sulfate and 300 μg/mL hygromysin B. Selected and cultured. It was confirmed by performing colony PCR using the primers in Table 5 to confirm the success of strain improvement. The improved strains including the final strain ( S. cerevisiae YE9) are listed in Table 1.

of the template genome
DNA* donate
DNA
piece pra
Emer SEQ ID NO: sequence (5'-3') XYL1 , XYL2 expression cassette for deleting ALD6 ( ald6::TDH3 _P -XYL1-TDH3 _T -PGK1 _P -XYL2-PGK1 _T ) S. cerevisiae TDH3 _P Kim626 29 TAACATACACAAACACATACTATCAGAATACA CTATTTTCGAGGACCTTGTC SOO384 30 TCAACTTAATAGAAGGCATTTTTTAGATCTCCTAGGTTTGTTTGTTTATGTGTGTTTAT TC P. stipitis XYL1 SOO385 31 ATAAACACACATAAACAAACAAACCTAGGAGATCTAAAAATGCCTTCTATTAAGTTGA AC SOO386 32 AAT GCAAGATTTAAAGTAAATTCACTGTTAACGCATGCTTAGACGAAGATAGGAATCTTG S. cerevisiae TDH3 _T SOO387 33 GGA CAAGATTCCTATCTTCGTCTAAGCATGCGTTAACAGTGAATTTACTTTAAATTCTTGC SOO388 34 ATTCTTTGAAGGTACTT CTTCGAAAAATTCGCGTCTGCTAGCTCCTGGCGGAAAAAATTC S. cerevisiae PGK1 _P SOO389 35 TTTTAAAGTTTACAAAT GAATTTTTTCCGCCAGGAGCTAGCAGACGCGAATTTTTTCGAAG SOO390 36 CACCAA GGAAGGGTTAGCAGTCATTTTTTCTAGATGTTTTATATTTGTTGTAAAAAGTAG P. stipitis XYL2 SOO391 37 AATTAT CTACTTTTTACAACAAATATAAAACATCTAGAAAAAATGACTGCTAACCCTTCC SOO392 38 AAAAAATTGAT CTATCGATTTCAATTCAATTCAATACTAGTTTACTCAGGGCCGTCAATG S. cerevisiae PGK1 _T SOO393 39 GTCAAGTGTCT CATTGACGGCCCTGAGTAAACTAGTATTGAATTGAATTGAAATCGATAG Kim627 40 GTATATGACGGAAAGAAATGCAGGTTGGTACA AAATAATATCCTTCTCGAAAG XYL3 expression cassette for deleting PHO13 ( pho13::TDH3 _P -XYL1-TDH3 _T -PGK1 _P -XYL2-PGK1 _T ) S. cerevisiae TDH3 _P Kim628 41 ATGTGACATCTTTACTATTCTCCAGCACGTTT CTTCATCGGTATCTTCGC SOO374 42 AA TGGGGTAGTGGTCATTTTTAAGCTTGAATTCTTTGTAATTAAAACTTAGATTAGATTG P. stipitis XYL3 SOO375 43 AT CTAATCTAAGTTTTAATTACAAAGAATTCAAGCTTAAAAATGACCACTACCCCATTTG SOO376 44 GCAACTA GAAAAGTCTTATCAATCTCCGTCGACATCGATTTAGTGTTTCAATTCACTTTC S. cerevisiae TDH3 _T SOO377 45 CAAGATG GAAAGTGAATTGAAACACTAAATCGATGTCGACGGAGATTGATAAGACTTTTC Kim629 46 CTATAACTCATTATTGGTTAAGGTGTAGATG AAGTTGGGTAACGCCAGG gaaA expression cassette ( int#4::CCW12 _P -gaaA-CCW12 _T ) S. cerevisiae CCW12 _P Kim379 47 TTCCTCGGGCAGAGAAACTCGCAGGCAACTTG CACGCAAAAAGAAAACCTT Kim380 48 TCAACA CAGCTGGGGGAGCCATTTTTTATTGATATAGTGTTTAAGCGAAT A. niger gaaA Kim381 49 TCTGTC ATTCGCTTAAACACTATATCAATAAAAAATGGCTCCCCCAGCTG Kim382 50 TAGA ATGTATAAATAATAATAAACTAAGTCTACTTCAGCTCCCACTTTCC S. cerevisiae CCW12 _T Kim383 51 GGAT GGAAAGTGGGAGCTGAAGTAGACTTAGTTTATTATTATTTATACAT Kim384 52 TGTGAGGGCCGATTATGCAGGCCTAGA TGTTCTAGTGTGTTTATATTATC lgd1 expression cassette ( int#4::PGK1 _P -lgd1-PGK1 _T ) S. cerevisiae PGK1 _P Kim385 53 CCTCGGGCAGAGAAACTCGCAGGCAACTTG GTGAGTAAGGAAAGAGTGAG Kim386 54 GTGATGGTGACTTCAGACATTTTTTGTTTTATATTTGTTGTAAAAAGTAG T. reesei lgd1 Kim387 55 CTACTTTTTTACAACAAATATAAAACAAAAAATGTCTGAAGTCACCATCAC Kim388 56 ATTGATCTAT CGATTTCAATTCAATTCAATTCAGATCTTCTCTCCGTTCA S. cerevisiae PGK1 _T Kim389 57 CTGCCCATCT TGAACGGAGAGAAGATCTGAATTGAATTGAATTGAAATCG Kim390 58 CTCTGTGAGGGCCGATTATGCAGGCCTAGA AAATAATATCCTTCTCGAAA gaaC expression cassette ( int#4::TDH3 _P -gaaC-TDH3 _T ) S. cerevisiae TDH3 _P Kim391 59 CTCGGGCAGAGAAACTCGCAGGCAACTTG GAATAAAAAACACGCTTTTTC Kim392 60 GACTCCGGGGCG GAGCGGGGTAAAAGGCATTTTTTTTGTTTGTTTATGTGTGTT A. niger gaaC Kim393 61 TTCGAATA AACACACATAAACAAACAAAAAAAATGCCTTTTACCCCGCTC Kim394 62 ATTTAAAT GCAAGATTTAAAGTAAATTCACCTAAGCAATATCCGGCAACG S. cerevisiae TDH3 _T Kim395 63 TGAGAAGT CGTTGCCGGATATTGCTTAGGTGAATTTACTTTAAATTCTTGC Kim396 64 CCTCTGTGAGGGCCGATTATGCAGGCCTAGA ATCCTGGCGGAAAAAATTC gaaA , lgd1 , and gaaC expression cassettes ( int#4::CCW12 _P -gaaA-CCW12 _T -PGK1 _P -lgd1-PGK1 _T -TDH3 _P -gaaC-TDH3 _T ) S. cerevisiae YE3 CCW12 _P -gaaA-CCW12 _T Kim410 65 TCTTTAGGTTAATTGTCGCTGTTATTGTCTA GATTTTTTCTCGGAGATGG Kim411 66 TAGTTC CTCACTCTTTCCTTACTCACTGTTCTAGTGTGTTTATATTATCC S. cerevisiae YE4 PGK1 _P -lgd1-PGK1 _T Kim412 67 AGCCAA GGATAATATAAACACACTAGAACA GTGAGTAAGGAAAGAGTGAG Kim413 68 AAACTCGAA CTGAAAAACGTGTTTTTTATTCCCGATTATGCAGGCCTAG S. cerevisiae YE5 TDH3 _P -gaaC-TDH3 _T Kim414 69 TATTATTTT CTAGGCCTGCATAATCGGGAATAAAAAACACGCTTTTTCAG Kim415 70 CTACTCTCTTCCTAGTCGCCCGGTTGTT GAAAGTTTAATTGTGGGTTTTC lad1, and alx1 expression cassettes ( int#7::FBA1 _P -lad1-FBA1 _T - PGK1 _P -alx1-CYC1 _T ) S. cerevisiae YE01 FBA1 _P -lad1-FBA1 _T - PGK1 _P -alx1-CYC1 _T Kim553 71 CTTACACTTGTGTAATGACAAATGTTTTT TGAACAACAATACCAGCCTTC Kim554 72 TGTTTCACGTTATCAAGATTATGTCATCTATT GGCCGCAAATTAAAGCCT Overexpression of YPR1 ( CCW12 _P -YPR1 ) S. cerevisiae CCW12 _P Kim537 73 GTAACTTTGCAATATAATCAGGTCGCAAATAT CACGCAAAAAGAAAACCTT Kim538 74 GAAGAATTCTTTAACGTAGCAGGCAT TATTGATATAGTGTTTAAGCGAAT gaaD expression cassette ( int#6::CCW12 _P -gaaD-CCW12 _T ) S. cerevisiae CCW12 _P Kim541 75 CGGAGGAGACCGCTATAACCGGTTTGAATTTA CACGCAAAAAGAAAACCTT Kim542 76 TA ACCTTCTTTCCGAGAGACATTTTTTATTGATATAGTGTTTAAGCGAAT A. niger gaaD Kim543 77 TC ATTCGCTTAAACACTATATCAATAAAAAATGTCTCTCGGAAAGAAGGT Kim544 78 GT ATAAATAATAATAAACTAAGTTTATTAAACAATCACCTTATGACCAGC S. cerevisiae CCW12 _T Kim545 79 TG GTCATAAGGTGATTGTTTAATAAACTTAGTTTATTATTATTTATACAT Kim546 80 CTTGCTTGCTGTCAAACTTCTGAGTTG TGTTCTAGTGTGTTTATATTATC

^a Saccharomyces cerevisiae D452-2; Pichia stipitis CBS 6054; Aspergillus niger CBS 120.49; Trichoderma reesei ATCC 5676.

Flanking region is underlined.

primer sequence (5'-3') SEQ ID NO: primer sequence (5'-3') order
number D-xylose pathway D-galacturonic acid pathway Kim049 GGAACGGTGAGTGCAACG 81 Kim322 GCGCATCTATTTGCCGTC 103 Kim427 AAACTGTTCACCCAGACACC 82 Kim397 GCTGGGGGAGCCATTTTTTATTG 104 Kim194 AGCGCAACTACAGAGAACAGG 83 Kim398 GTGGGAGCTGAAGTAGACTTAG 105 Kim100 CGGCACCGTCGAACAATCTG 84 Kim323 TCACGACACACCTCACTG 106 Kim101 CCGCTTACTCTTCGTTCGGTCC 85 Kim399 CCTGTGATGGTGACTTCAGAC 107 Kim193 CTCAGCATCCACAATGTATCAG 86 Kim401 GAACGGAGAGAAGATCTGAATTG 108 Kim426 GCGCTATTGCATTGTTCTTGTC 87 Kim400 ACAGCCTGTTTCCACACAC 109 Kim547 AGGTATGCGATAGTTCCTCAC 88 Kim402 GCGGGGTAAAAGGCATTTTTTTTG 110 Kim125 TGCAGCTTCCAATTTCGTCAC 89 Kim408 GCCGGATATTGCTTAGGTG 111 Kim630 GAGGTGACACCCTTACCAAC 90 Kim631 CTGCTACTCACACCTTCAACTC 91 Introduction of L-arabinose pathway Kim632 CGCTGAACCCGAACATAGAAATATC 92 Kim490 GGCACTAGGAGCATTTGTCG 112 Kim633 TCGATATTTCTATGTTCGGGTTCAG 93 Kim304 GCTTCGCTAATCCAGAGGTC 113 Kim078 GATTGGAATTGGTTCGCAGTG 94 Kim400 ACAGCCTGTTTCCACACAC 109 Kim048 GAGGAAGACGTTGAAGGTGG 95 Kim491 GTCCCTTAGGGTGCGTATAATG 114 Kim149 TTTGAAGTGGTACGGCGATG 96 Kim577 CACCCAAGCACAGCATAC 97 Overexpression of YPR1 Kim634 TGGCTCGATAACGAAGATTCAG 98 Kim539 CAATTCCGTGAAACCCTTTTCTT 115 Kim635 GTCTTGTAGATTGAGAACTGGTCC 99 Kim540 CTGCCAACTTCTTCTTCATTCAA 116 Kim636 TCTATGAGGCAAGTAAGAGGCAC 100 Kim492 AACAGGCGACAGTCCAAATG 101 Introduction of gaaD gene cassette Kim077 TTGGAGTTCAAAACTGGCGAG 102 Kim326 GGTTCTGACTCCTACTGAGC 117 Kim093 GCAAAGATAGCGGCGTAGGTG 118 Kim549 GCATCCTTTGCCTCCGTTC 119 Kim327 AGCATCGAGTACGGCAGTTC 120

<1-3> Component analysis using High Performance Liqui Chromatography (HPLC)

The concentrations of glucose, fructose, galactose, xylose, L-arabinose, galacturonic acid, glycerol and ethanol in the culture medium were analyzed using HPLC (Agilent Technologies, 1260 series, USA). Specifically, to analyze the above components, the supernatant of the culture medium was subjected to a Rezex-ROA Organic Acid H+ (8%) column and 0.005 NH ₂ SO ₄ mobile phase on HPLC equipped with an RI detector, 0.6 mL/min flow rate and column temperature. The analysis was performed by setting it to 50°C.

<1-4> Analysis of intracellular metabolites using Gas Chromatography/Mass Spectrometry (GC/MS)

In order to analyze intracellular metabolites under single or mixed sugar conditions of xylose and galacturonic acid, metabolite extraction and GC/MS analysis were performed as follows.

First, at fermentation times of 0, 24, 48 and 72 h, the metabolites of cells contained in 5 mL of culture medium were extracted and vacuum dried, and then derivatized by methoxyamination and trimethylsilylation. To perform methoxyamination, 10 μL of pyridine containing methoxyamine hydrochloride was added to the vacuum-dried sample, and then treated at 30° C. for 90 minutes. Then, 40 μL of N-methyl-N-(trimethicilyl)trifluoroacetamide was added, followed by treatment at 37° C. for 30 minutes to perform trimethylsilylation. GC/MS analysis was performed using an Agilent 6890 gas chromatography equipped with an Agilent 5973 mass screening detector and an RTX-5Sil MS column. 1 μL of the derivatized sample was injected into the GC instrument in splitless mode. The temperature of the initial oven was maintained at 50°C for 1 minute, then improved at a rate of 20°C/min, and finally the conditions were set to maintain at 330°C for 5 minutes, and the ion source and transfer line were The temperatures were set at 250°C and 290°C, respectively. Helium gas was used as the carrier gas and flowed at a rate of 1.5 mL/min. The isolated metabolites were adjusted to be ionized at 70 eV, and intracellular metabolite analysis was performed to detect them in a scan mode in a mass range of 50-550 m/z.

<1-5> Fermentation of Citrus peel hydrolysate

In order to confirm that the improved strain S. cerevisiae YE9 can produce bioenergy using actual waste biomass as a substrate, citrus peel, one of the fruit-based waste biomass, is subjected to heat treatment and enzymatic treatment to increase citrus fruit juice. A lysate was prepared. Specifically, the tangerine peels of tangerines harvested in Jeju in January 2019 were washed once, dried with hot air at 60°C for about 24 hours, and then ground with a grinder to make tangerine gourd powder. The powdered citrus peel was heat-treated at 121° C. for 1 hour using a high-pressure steam sterilizer, and then cooled at room temperature to prepare a citrus peel hydrolysate. For simultaneous Saccharification and Fermentation (SSF), the improved yeast S. cerevisiae YE9 with an initial cell concentration of 25 g/L and two complex hydrolases (60U/g biomass pectinase, 80FPU) were added to the hydrolyzate of citrus peel. Cellic CTec2) of /g biomass was added, and fermentation was carried out at 30° C. at 130 rpm for 240 hours. All fermentations were performed in three biological replicates, and the fermentation results of the improved yeast S. cerevisiae YE9 were compared with those of the parent strain S. cerevisiae D452-2.

<Example 2> Fermentation of three unconventional carbon sources derived from fruit-based biomass S. cerevisiae Development of YE9 strain

Prior to strain development, a foreign gene was introduced into yeast S. cerevisiae D452-2 using CRISPR/Cas9 gene editing technology to develop a strain that simultaneously ferments three unconventional carbon sources derived from fruit-based biomass, and the recombinant yeast S. cerevisiae YE9 was developed.

First, the acetaldehyde dehydrogenase gene ALD6 was removed to suppress acetate, which is a major by-product of yeast and causes inhibition of growth. The PHO13 gene was removed to improve metabolic flux.

Next, to use xylose, one of the lignocellulosic biomass, xylose reductase ( XYL1 ), xylitol dehydrogenase ( XYL2 ), and xylulokinase ( XYL3 ) genes derived from Pichia stipitis were introduced. To use phosphorus arabinose (L-arabinose), the L-arabitol dehydrogenase gene lad1 derived from Trichoderma reesei and the L-xylulose reductase gene alx1 derived from Ambrosiozyma monospo ra were introduced. Lastly, in order to use D-galacturonic acid (galUA ), which is a major component of pectin, D-galacturonic acid reductase ( gaaA ) derived from Aspergillus niger , 2-keto-3-deoxy-L-galactonate aldolase ( gaaC ) and Trichoderma reesei- derived L-galactonate dehydratase gene lgd1 was introduced.

<Example 3> S. cerevisiae Fermentation results of YE9 strain

Wild yeast D452-2 and improved strain S. cerevisiae YE9 were compared with three unconventional carbon sources (xylose, arabinose, and galacturonic acid) as the sole carbon source.

Specifically, for fermentation experiments using flasks, preculture was first performed in YP liquid medium containing 2% (w/v) glucose, and 4% (w/v) xylose or 4% (w/v) v) Initial cells were inoculated at 25 g/L in YP broth containing arabinose and 2% (w/v) galacturonic acid, and fermentation was performed at 30° C. and microaerobic conditions (130 rpm). All experiments were performed in three biological replicates, and the mean and standard deviation were shown.

As a result, the parent strain S. cerevisiae D452-2 did not consume all three unconventional carbon sources (xylose, arabinose, and galacturonic acid), but the recombinant yeast YE9 consumed glycerol or ethanol. ) was produced as an energy source ( FIGS. 3 to 5 ).

As a result of fermenting xylose as a substrate, the recombinant yeast YE9 consumed all of the xylose at 9 hours of fermentation and produced about 0.6 g/L glycerol and 11.3 g/L ethanol (FIG. 3). As a result, the recombinant yeast YE9 had a slower fermentation rate than xylose, but consumed 30.2 g/L of arabinose for 72 hours of fermentation and accumulated 1.9 g/L of ethanol (FIG. 4).

As a result of fermenting galacturonic acid as a substrate, recombinant yeast YE9 consumed 6.7 g/L of galacturonic acid during 72 hours of fermentation and accumulated 0.3 g/L glycerol and 0.3 g/L ethanol, but after 12 hours of fermentation, It was confirmed that the consumption of lacturonic acid stopped ( FIG. 5 ).

Previously reported studies showed that the recombinant S. cerevisiae strain consumed little or less than 1 g/L without transporter expression and pH control (Protzko et al., 2018), whereas the Recombinant strain S. cerevisiae YE9 proved effective for fruit pectin fermentation by consuming 6.7 g/L of galUA without pH adjustment.

The existing parent strain S. cerevisiae D452-2 can consume glucose, fructose, and galactose. In order to confirm that the recombinant yeast YE9 can consume three kinds of conventional carbon sources (glucose, fructose, galactose), fermentation was carried out for three types of 40 g/L conventional carbon sources, respectively, and the fermentation conditions are the same as the fermentation conditions.

As a result of fermenting glucose as a substrate, it was consumed within 1 hour of fermentation, and glycerol and ethanol were respectively 2.0 g/L and 16.5 g/L (Fig. 6a). All were consumed to produce 1.9 g/L and 15.1 g/L of glycerol and ethanol, respectively (FIG. 6b).

In addition, as a result of fermenting galactose as a substrate, it was consumed within about 30 hours of fermentation and produced 8.2 g/L of glycerol (FIG. 6c).

In addition, two types of L-glyceraldehyde reductase ( Aspergillus niger- derived gene gaaA and endogenous gene YPR1 ) were compared to finally complete the galacturonic acid metabolic pathway. Here, the endogenous gene YPR1 is known as a gene presumed to have the function of L-glyceraldehyde reductase.

As a result, despite the expression of the two L-glyceraldehyde reductase genes, there was no effect on the improvement of galacturonic acid fermentation (FIG. 7).

<Example 4> S. cerevisiae Mixed sugar fermentation result of galacturonic acid and fermentable sugar of YE9 strain

Through the single sugar fermentation result, the YE9 strain was able to rapidly consume the two types of pentose sugar, but it was confirmed that the galacturonic acid was not completely consumed. Since galacturonic acid requires 2 moles of NADPH cofactor during the metabolic process, mixed sugar fermentation was performed by adding fermentable sugar as a source of cofactor to galacturonic acid fermentation in order to solve the redox imbalance. Five types of monosaccharides (glucose, fructose, galactose, xylose, L-arabinose) abundantly present in fruit-based biomass were used as cofactor sources for galacturonic acid metabolism.

Fermentation conditions for carrying out the mixed fermentation are as follows. Pre-culture was performed in YP broth containing 2% (w/v) glucose, and initial cells were cultured in YP broth containing 2% (w/v) galacturonic acid and 4% (w/v) monosaccharides. It was inoculated at 25 g/L and fermented at 30° C. and microaerobic conditions (130 rpm). All experiments were performed in three biological replicates, and the mean and standard deviation were shown.

The addition of glucose among the five monosaccharides for supplying the cofactor suppressed the consumption of galacturonic acid by glucose repression, a characteristic characteristic of S. cerevisiae , and consumed 3.3 g/L of galacturonic acid (Fig. 8a), and frictose. The addition of (Fig. 8b) and galactose (Fig. 8c) also improved the consumption of some galacturonic acid by consuming 4.5 g/L and 4.6 g/L of galacturonic acid, respectively, during 72 hours of fermentation, but rather than fermentation of galacturonic acid alone. consumed less. On the other hand, the addition of pentose (Fig. 8d) xylose and (Fig. 8e) arabinose consumed 13.1 g/L and 11.9 g/L of galacturonic acid within 72 hours of fermentation, respectively, resulting in simultaneous consumption with galacturonic acid. At the same time, it was confirmed that not only consumed 1.96- and 1.78-fold more than single sugar fermentation, respectively (FIG. 8g), but also improved the consumption rate of galacturonic acid (FIG. 8h).

The addition of xylose improves the consumption rate of galacturonic acid, and arabinose serves to induce galacturonic acid to be metabolized continuously. , L-arabinose, and galacturonic acid) were fermented.

As a result of simultaneous fermentation by mixing three types of carbon sources (xylose, L-arabinose, and galacturonic acid), xylose was consumed in 36 hours and consumed about 3 times slower than fermentation of two types of mixed sugar, but xylose was depleted Since galacturonic acid is continuously consumed together with arabinose, it is possible to obtain a result of consuming a higher amount of galacturonic acid (15.3 g/L) per unit time than the result of single or mixed fermentation of galacturonic acid. There was (Fig. 8f, g).

Although the previously reported results emphasized that a transporter is required for efficient consumption of galacturonic acid, the improved strain S. cereiviae YE9 in the present invention was able to consume galacturonic acid without additional introduction of the galacturonic acid transporter. However, it is possible to increase the consumption efficiency of galacturonic acid under the mixed sugar condition of pentose, and the fermentation ability to simultaneously consume galacturonic acid and pentose was also verified.

9 shows the results of two products (glycerol, ethanol) accumulated through the fermentation of mixed sugars of galacturonic acid and monosaccharides of FIG. 8 . When glucose was added in galacturonic acid fermentation, 2.4 g/L and 16.9 g/L of glycerol and 16.9 g/L of ethanol were respectively accumulated during 72 hours of fermentation (Fig. L accumulated ( FIG. 9b ). In the galactose addition, glycerol and ethanol were accumulated at 1.6 g/L and 2.4 g/L, respectively, so that almost no by-products were accumulated (FIG. 9c).

On the other hand, in the xylose condition, 4.5 g/L and 12.8 g/L of glycerol and 12.8 g/L were accumulated, respectively (FIG. 9d), and in the arabinose condition, glycerol and ethanol were accumulated at 4.2 g/L and 4.1 g/L, respectively. Although it did not have a significant effect on the production, it is thought that it can serve as a carbon source for cofactor supply and cell growth through simultaneous consumption of galacturonic acid (FIG. 9d). In addition, when arabinose is fermented alone, glycerol is hardly accumulated, whereas in mixed fermentation with galacturonic acid, glycerol is accumulated. could

Finally, as a result of fermentation by mixing two types of pentose sugars (xylose, L-arabinose) and galacturonic acid, glycerol and ethanol were accumulated at 5.3 g/L and 16.5 g/L, respectively, so that not only the consumption of galacturonic acid but also the accumulation of by-products It also showed the possibility of renewable energy production by increasing it (FIG. 9d).

From the previous mixed sugar results, it was confirmed that the addition of xylose and arabinose was the optimal sugar for improving galacturonic acid consumption. However, according to the results of previous studies, there was a report that the presence of 10 g/L galacturonic acid in an acidic medium (pH 3.5) inhibited the consumption of galactose, xylose, and arabinose, while not affecting the consumption of glucose ( Huisjes et al., 2012). In order to check whether the YE9 strain fermenting three kinds of non-conventional carbon sources developed from this study shows the same results as in the previous study, xylose and various galacturonic acids (1-100 g/L) mixed sugar conditions And the consumption rate of galacturonic acid was confirmed (FIG. 10).

In order to confirm the consumption rates of xylose and galacturonic acid, the recombinant strain YE9 was first pre-cultured in YP broth containing 20 g/L glucose, containing 1-100 g/L galacturonic acid and 40 g/L xylose. One YP liquid medium was inoculated with an initial cell concentration of 0.05 g/L, and fermentation was performed at 30° C. and microaerobic conditions (130 rpm). All experiments were performed in three biological replicates, and the mean and standard deviation were shown.

As a result of checking the xylose fermentation rate at various galacturonic acid concentrations (1-100 g/L), it was confirmed that the xylose consumption rate decreased as the galacturonic acid concentration increased. It was confirmed that Xylose was not consumed at all. Also, as in the previous study results, the presence of galacturonic acid can affect the consumption rate of xylose, but since the recombinant strain YE9 can use galacturonic acid, an inhibitor of xylose, as a substrate, eventually xylose is consumed. could get the results.

<Example 5> S. cerevisiae Profiling of intracellular metabolites during galacturonic acid metabolism in YE9 strain

Through the results of previous experiments, it was confirmed whether the addition of xylose to the culture medium affects the consumption of galacturonic acid. In order to understand how xylose helps the consumption of galacturonic acid and which limiting-step pathway is being resolved, intracellular xylose and galacturonic acid in monosaccharide and mixed sugar conditions using GC/MS equipment Metabolite (intracellular metabolite) analysis was performed (FIG. 11).

The target material for intracellular metabolite analysis was an intermediate metabolite generated in the metabolic process of two carbon sources. The intermediate metabolites of the intracellular xylose metabolic pathway are xylose, xylitol, xylulose (D-xylulose), and xylulose-5P, and the intermediate metabolites of the galacturonic acid metabolic pathway are D-galacturonic acid, L-galactonate, 2-keto-3-deoxy-L-glyceraldehyde, L -Glyceraldehyde (L-glyceraldehyde). Common intermediate metabolites of both carbon sources are glycerol and pyruvate.

For intracellular metabolite analysis, preculture was first performed in YP broth containing 2% (w/v) glucose, 2% (w/v) galacturonic acid and 4% (w/v) xylose. was inoculated at an initial cell concentration of 0.05 g/L in YP broth alone or mixed with After 24 hours (24 h), 48 hours (48 h), and 72 hours (72 h), 5 mL of the culture medium was collected and analyzed.

Thereafter, intracellular metabolites were extracted and GC/MS analysis was performed using an Agilent 6890 GC instrument with an Agilent 5973 MSD. The analyzed result was expressed as a value normalized by cell biomass (Signal abundance). All experiments were performed in three biological replicates, and the mean and standard deviation were shown.

As a result, when galacturonic acid is fermented as a single sugar, galacturonic acid accumulates in the cell immediately after strain inoculation and is rapidly converted to L-galactonate, the first intermediate metabolite, but the second metabolite 2-keto-3-deoxy-L-glyceraldehyde did not accumulate at all. On the other hand, in the case of mixed fermentation of xylose and galacturonic acid, as fermentation proceeds, intracellular galacturonic acid and L-galactonate are rapidly depleted, and at the same time, 2-keto-3-deoxy- The result of accumulating L-glyceraldehyde (2-keto-3-deoxy-L-glyceraldehyde) can be confirmed (FIG. 11).

These results suggest that the uptake of galacturonic acid into cells is sufficiently possible, and the intermediate metabolite, 2-keto-3-deoxy-L-glyceraldehyde, It was confirmed that lacturonic acid did not accumulate in single fermentation, whereas in xylose and galacturonic acid mixed fermentation, it was accumulated, thereby accelerating the metabolism of galacturonic acid.

<Example 6> S. cerevisiae Fermentation of citrus fruit hydrolyzate of YE9 strain

To confirm that the improved strain S. cerevisiae YE9, which can simultaneously consume three types of carbon sources rich in pectin (xylose, arabinose, and galacturonic acid), can produce bioenergy by fermenting the actual pectin hydrolyzate, Simultaneous Saccharification and Fermentation (SSF) was performed using citrus peel hydrolysate (FIG. 12).

As a result of fermenting the citrus fruit hydrolyzate, the parent strain, S. cerevisiae D452-2, consumed all glucose and fructose within 2 hours of fermentation, but consumed no galacturonic acid, xylose, or arabinose. On the other hand, the improved strain S. cerevisiae YE9 in which the metabolic pathway of xylose, arabinose and galacturonic acid was introduced consumed glucose, fractose and xylose within 2 hours of fermentation, and consumed galacturonic acid and arabinose. I was able to check the results. In addition, the result of converting the actual pectin hydrolyzate into biofuel using the improved strain S. cerevisiae YE9 was confirmed by producing up to 4.5 g/L and 18.7 g/L of glycerol and ethanol.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Microorganisms fermenting galacturonic acid and pentose simultaneously <130> PN2004-158 <160> 120 <170> KoPatentIn 3.0 <210> 1 <211> 1503 <212> DNA <213> Unknown <220> <223> ALD6 (acetaldehyde dehydrogenase gene) <400> 1 atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact taccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taa 1503 <210> 2 <211> 939 <212> DNA <213> Unknown <220> <223> PHO13 (haloacid dehalogenase Type IIA phosphatase gene) <400> 2 atgactgctc aacaaggtgt accaataaag ataaccaata aggagattgc tcaagaattc 60 ttggacaaat atgacacgtt tctgttcgat tgtgatggtg tattatggtt aggttctcaa 120 gcattaccat acaccctgga aattctaaac cttttgaagc aattgggcaa acaactgatc 180 ttcgttacga ataactctac caagtcccgt ttagcataca cgaaaaagtt tgcttcgttt 240 ggtattgatg tcaaagaaga acagattttc acctctggtt atgcgtcagc tgtttatatt 300 cgtgactttc tgaaattgca gcctggcaaa gataaggtat gggtatttgg agaaagcggt 360 attggtgaag aattgaaact aatggggtac gaatctctag gaggtgccga ttccagattg 420 gatacgccgt tcgatgcagc taaatcacca tttttggtga acggccttga taaggatgtt 480 agttgtgtta ttgctgggtt agacacgaag gtaaattacc accgtttggc tgttacactg 540 cagtatttgc agaaggattc tgttcacttt gttggtacaa atgttgattc tactttcccg 600 caaaagggtt atacatttcc cggtgcaggc tccatgattg aatcattggc attctcatct 660 aataggaggc catcgtactg tggtaagcca aatcaaaata tgctaaacag cattatatcg 720 gcattcaacc tggatagatc aaagtgctgt atggttggtg acagattaaa caccgatatg 780 aaattcggtg ttgaaggtgg gttaggtggc acactactcg ttttgagtgg tattgaaacc 840 gaagagagag ccttgaagat ttcgcacgat tatccaagac ctaaatttta cattgataaa 900 cttggtgaca tctacacctt aaccaataat gagttatag 939 <210> 3 <211> 957 <212> DNA <213> Unknown <220> <223> XYL1 (xylose reductase gene) <400> 3 atgccttcta ttaagttgaa ctctggttac gacatgccag ccgtcggttt cggctgttgg 60 aaagtcgacg tcgacacctg ttctgaacag atctaccgtg ctatcaagac cggttacaga 120 ttgttcgacg gtgccgaaga ttacgccaac gaaaagttag ttggtgccgg tgtcaagaag 180 gccattgacg aaggtatcgt caagcgtgaa gacttgttcc ttacctccaa gttgtggaac 240 aactaccacc acccagacaa cgtcgaaaag gccttgaaca gaaccctttc tgacttgcaa 300 gttgactacg ttgacttgtt cttgatccac ttcccagtca ccttcaagtt cgttccatta 360 gaagaaaagt acccaccagg attctactgt ggtaagggtg acaacttcga ctacgaagat 420 gttccaattt tagagacctg gaaggctctt gaaaagttgg tcaaggccgg taagatcaga 480 tctatcggtg tttctaactt cccaggtgct ttgctcttgg acttgttgag aggtgctacc 540 atcaagccat ctgtcttgca agttgaacac cacccatact tgcaacaacc aagattgatc 600 gaattcgctc aatcccgtgg tattgctgtc accgcttact cttcgttcgg tcctcaatct 660 ttcgttgaat tgaaccaagg tagagctttg aacacttctc cattgttcga gaacgaaact 720 atcaaggcta tcgctgctaa gcacggtaag tctccagctc aagtcttgtt gagatggtct 780 tcccaaagag gcattgccat cattccaaag tccaacactg tcccaagatt gttggaaaac 840 aaggacgtca acagcttcga cttggacgaa caagatttcg ctgacattgc caagttggac 900 atcaacttga gattcaacga cccatgggac tgggacaaga ttcctatctt cgtctaa 957 <210> 4 <211> 1092 <212> DNA <213> Unknown <220> <223> XYL2 (xylitol dehydrogenase gene) <400> 4 atgactgcta acccttcctt ggtgttgaac aagatcgacg acattcgtt cgaaacttac 60 gatgccccag aaatctctga acctaccgat gtcctcgtcc aggtcaagaa aaccggtatc 120 tgtggttccg acatccactt ctacgcccat ggtagaatcg gtaacttcgt tttgaccaag 180 ccaatggtct tgggtcacga atccgccggt actgttgtcc aggttggtaa gggtgtcacc 240 tctcttaagg ttggtgacaa cgtcgctatc gaaccaggta ttccatccag attctccgac 300 gaatacaaga gcggtcacta caacttgtgt cctcacatgg ccttcgccgc tactcctaac 360 tccaaggaag gcgaaccaaa cccaccaggt accttatgta agtacttcaa gtcgccagaa 420 gacttcttgg tcaagttgcc agaccacgtc agcttggaac tcggtgctct tgttgagcca 480 ttgtctgttg gtgtccacgc ctctaagttg ggttccgttg ctttcggcga ctacgttgcc 540 gtctttggtg ctggtcctgt tggtcttttg gctgctgctg tcgccaagac cttcggtgct 600 aagggtgtca tcgtcgttga cattttcgac aacaagttga agatggccaa ggacattggt 660 gctgctactc acaccttcaa ctccaagacc ggtggttctg aagaattgat caaggctttc 720 ggtggtaacg tgccaaacgt cgttttggaa tgtactggtg ctgaaccttg tatcaagttg 780 ggtgttgacg ccattgcccc aggtggtcgt ttcgttcaag tcggtaacgc tgctggtcca 840 gtcagcttcc caatcaccgt tttcgccatg aaggaattga ctttgttcgg ttctttcaga 900 tacggattca acgactacaa gactgctgtt ggaatctttg acactaacta ccaaaacggt 960 agagaaaatg ctccaattga ctttgaacaa ttgatcaccc acagatacaa gttcaaggac 1020 gctattgaag cctacgactt ggtcagagcc ggtaagggtg ctgtcaagtg tctcattgac 1080 ggccctgagt aa 1092 <210> 5 <211> 1872 <212> DNA <213> Unknown <220> <223> XYL3 (xylulokinase gene) <400> 5 atgaccacta ccccatttga tgctccagat aagctcttcc tcgggttcga tctttcgact 60 cagcagttga agatcatcgt caccgatgaa aacctcgctg ctctcaaaac ctacaatgtc 120 gagttcgata gcatcaacag ctctgtccag aagggtgtca ttgctatcaa cgacgaaatc 180 agcaagggtg ccattatttc ccccgtttac atgtggttgg atgcccttga ccatgttttt 240 gaagacatga agaaggacgg attccccttc aacaaggttg ttggtatttc cggttcttgt 300 caacagcacg gttcggtata ctggtctaga acggccgaga aggtcttgtc cgaattggac 360 gctgaatctt cgttatcgag ccagatgaga tctgctttca ccttcaagca cgctccaaac 420 tggcaggatc actctaccgg taaagagctt gaagagttcg aaagagtgat tggtgctgat 480 gccttggctg atatctctgg ttccagagcc cattacagat tcacagggct ccagattaga 540 aagttgtcta ccagattcaa gcccgaaaag tacaacagaa ctgctcgtat ctctttagtt 600 tcgtcatttg ttgccagtgt gttgcttggt agaatcacct ccattgaaga ggccgatgct 660 tgtggaatga acttgtacga tatcgaaaag cgcgagttca acgaagagct cttggccatc 720 gctgctggtg tccaccctga gttggatggt gtagaacaag acggtgaaat ttacagagct 780 ggtatcaatg agttgaagag aaagttgggt cctgtcaaac ctataacata cgaaagcgaa 840 ggtgacattg cctcttactt tgtcaccaga tacggcttca accccgactg taaaatctac 900 tcgttcaccg gagacaattt ggccacgatt atctcgttgc ctttggctcc aaatgatgct 960 ttgatctcat tgggtacttc tactacagtt ttaattatca ccaagaacta cgctccttct 1020 tctcaatacc atttgtttaa acatccaacc atgcctgacc actacatggg catgatctgc 1080 tactgtaacg gttccttggc cagagaaaag gttagagacg aagtcaacga aaagttcaat 1140 gtagaagaca agaagtcgtg ggacaagttc aatgaaatct tggacaaatc cacagacttc 1200 aacaacaagt tgggtattta cttcccactt ggcgaaattg tccctaatgc cgctgctcag 1260 atcaagagat cggtgttgaa cagcaagaac gaaattgtag acgttgagtt gggcgacaag 1320 aactggcaac ctgaagatga tgtttcttca attgtagaat cacagacttt gtcttgtaga 1380 ttgagaactg gtccaatgtt gagcaagagt ggagattctt ctgcttccag ctctgcctca 1440 cctcaaccag aaggtgatgg tacagatttg cacaaggtct accaagactt ggttaaaaag 1500 tttggtgact tgtacactga tggaaagaag caaacctttg agtctttgac cgccagacct 1560 aaccgttgtt actacgtcgg tggtgcttcc aacaacggca gcattatccg caagatgggt 1620 tccatcttgg ctcccgtcaa cggaaactac aaggttgaca ttcctaacgc ctgtgcattg 1680 ggtggtgctt acaaggccag ttggagttac gagtgtgaag ccaagaagga atggatcgga 1740 tacgatcagt atatcaacag attgtttgaa gtaagtgacg agatgaatct gttcgaagtc 1800 aaggataaat ggctcgaata tgccaacggg gttggaatgt tggccaagat ggaaagtgaa 1860 ttgaaacact aa 1872 <210> 6 <211> 1134 <212> DNA <213> Unknown <220> <223> lad1 (L-arabitol dehydrogenase gene) <400> 6 atgtcgcctt ccgcagtcga tgacgctccc aaggccacag gggcagccat ctcagtcaag 60 cccaacattg gcgtcttcac aaatccaaaa catgacctct ggattagcga agctgaaccc 120 agcgccgatg ccgtcaaatc tggcgctgat ctgaagcccg gcgaggtgac cattgctgtc 180 cgcagcactg gtatctgtgg ttcagatgtc catttctggc acgccggctg cattgggccc 240 atgatcgtcg agggcgacca catcctcggc cacgagtctg ccggcgaggt catcgccgtc 300 cacccgactg tcagtagcct ccaaatcggc gatcgggttg ccatcgagcc caacatcatc 360 tgcaacgcat gcgagccctg cctgacaggt cgatacaacg gctgcgaaaa ggtcgagttc 420 ctatccacgc cgccagtgcc cggactgctg cgacgctacg tcaaccaccc agccgtttgg 480 tgccacaaga ttggcaacat gtcgtgggag aacggcgcgc tgctggagcc cctgagcgtg 540 gctctggccg gcatgcagag ggccaaggtt cagctcggtg accccgtgct ggtctgcggc 600 gctggtccga ttggattggt gtcaatgctg tgcgctgctg ccgccggtgc ttgcccgctt 660 gtcatcacag acatttcaga gagccgtctg gcgtttgcaa aggagatctg cccccgcgtc 720 accacgcacc gcatcgagat tggcaagtcg gctgaggaaa cggccaaaag catcgtcagc 780 tcttttgggg gcgtcgagcc agccgtgacc ctggagtgca ccggtgtgga gagcagcatt 840 gcagcggcca tctgggccag caagtttgga ggaaaggtct ttgtgatcgg cgtcggcaag 900 aatgaaatca gcattccctt tatgagggcc agtgtacgcg aggtcgatat ccagctgcag 960 tatcgctaca gcaacacctg gcctcgtgcc atccggctca tcgagagcgg tgtcatcgat 1020 ctatccaaat ttgtgacgca tcgcttcccg ctggaggatg ccgtcaaggc atttgagacg 1080 tcagcagatc ccaagagcgg cgccattaag gtcatgattc agagcctgga ttga 1134 <210> 7 <211> 816 <212> DNA <213> Unknown <220> <223> alx1 (L-xylulose reductase gene) <400> 7 atgactgact acatccaac ttttagattc gatggccact taaccattgt cacaggtgcc 60 tgtggtggtt tagctgaagc tttaatcaag ggtttgttgg cctacggttc tgacattgct 120 ttgcttgata tcgaccaaga aaagactgct gccaaacaag ccgaatacca caaatacgct 180 actgaagaat tgaagttgaa agaagttcca aagatgggtt catatgcctg tgatatttct 240 gattctgata ccgttcacaa ggtgtttgct caagttgcta aggattttgg taagttgcca 300 ttgcacttgg ttaacacagc tggttactgt gaaaacttcc catgtgaaga ttacccagcc 360 aagaacgctg agaagatggt gaaggttaac ttgttgggtt ctttgtatgt ttctcaagcc 420 tttgctaagc cattgatcaa agaaggtatc aagggtgctt ctgttgtttt gattggttct 480 atgtctggtg ccattgtcaa cgatcctcaa aaccaagttg tctacaacat gtccaaggct 540 ggtgttatcc atttggctaa gactttggct tgtgaatggg ctaagtacaa catcagagtt 600 aattctttaa acccaggtta catctacggt cctttgacca agaatgttat caatggtaac 660 gaagaattgt acaacagatg gatctctggt atcccacaac aaagaatgtc cgaaccaaag 720 gaatacattg gtgctgtttt gtacttgctt tctgaatctg ctgcttcata cactactggt 780 gccagcttac tggttgatgg tggtttcact tcttgg 816 <210> 8 <211> 1239 <212> DNA <213> Unknown <220> <223> gaaA (D-galacturonic acid reductase gene) <400> 8 atggctcccc cagctgtgtt gatggtagga acaggcgagt acacgaccgg ctacgtcggt 60 ggtacagcct cgacctccga caagaaagtg ggtgtcgtgg gcctaacgct cttcgacttg 120 cgtcgtcgcg gcaaagttgg cgatttgagc atggtgggcg tatctggatc caaattccct 180 ggaatccgcg cacacttgca gaagaatatc tccgaagtct acaacggcct tgatgtctcc 240 ttcacctcct ttcccgccga caacacctcc gacccagaag cctacaaagc tgccattgac 300 gcccttcccg ccggctctgc aatcaccatt ttcacacccg accccaccca ttaccctatc 360 gctctgtacg ccattcagcg caagatccac gttctcatca ctaagcccgc gaccaagctc 420 ctctctgacc acctcgattt gctcgctgag tctcgcaagc acaatgtcgt tgtgtacatt 480 gaacaccaca agcgcttcga cccggcctac agtgacgccc gcgctaaggc tgccaagctt 540 ggtgacttca actactttta cagctacat agtcagccca agagccagct ggagacgttc 600 aaggcctggg ctggtaagga ctcggatatc tcttattatt tgaacagcca ccacgtggat 660 gttaatgaga gcatggtgcc ggactatgtc cccgtgaagg tgacggctag tgcagcgacg 720 ggaactgctg tcgagctggg ctgtgcccat gagacggagg acacgattac tctacttgtg 780 gaatggaaga agaaggatgg atcaagaatg gctacgggtg tttacacatc tagttggacc 840 gcaccaaaga gggccggtgt acactctaac cagtacttcc attatatggg ctcgaagggt 900 gaaatccgtg tcaatcaggc gaagcgtggc tatgatgttg ccgaggatga ggctggattg 960 tcttggatta acccgttcta tatgaagtac gcaccagacg aggagggtaa cttcggtggt 1020 cagacgggct acggatacat cagtttcgag aagttcattg atgccgttac ggctgttaat 1080 gaggggcggt tgacgctcga tcagctggat gccaggccga tcccgacgct gaagaacact 1140 attgccacga cggcaatcct gcatgcagga cgcatttcct tggatgagaa gcggtcggtg 1200 gagatcgtga ccgaggatgg aaagtgggag ctgaagtag 1239 <210> 9 <211> 1008 <212> DNA <213> Unknown <220> <223> gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) <400> 9 atgcctttta ccccgctccg ccccggagtc tacgctccaa ccatgacttt cttcgaccct 60 tcaaccgaag accttgacgt ccctaccatt cgcaagcacg ccgttcgcct cgcaaaagcc 120 ggtctcgtcg gtctcgtctg catgggctcc aacggcgaag ccgtacacct cacccgggca 180 gagcgcaaga ccgtgatcaa cgagacccgc tccgcactcg ttgaagccgg cttctccaac 240 gtccccgtca tcgcaggagc cagcgaacaa tccattcgcg gcaccatcga gctctgcaag 300 gaatcctacg aagccggagc tgaatatgcc ctgatcgttc cccccagcta ctaccgctac 360 gccaccggca acgaccaaac cctctatgaa ttcttcacca gcgtcgccga tggttccccc 420 atccctctca tcctctacaa ctaccccggt gccgtggcag gaattgacat ggactccgac 480 ctcatcatcc gcatctctca gcaccccaac atcgtaggca cgaagttcac ttgcgccaac 540 accggcaagt tgacccgtgt tgcttccgcc ctgcacgcca ttacccctcc ttcgccattg 600 gctccggcgc agcgcaagtt ccccagcaca aagacggagg caaaccaccc atacgttgcg 660 ttcggaggta ttgcagattt ctccctgcag acgctggcgt ccggaggttc cgcgatcctg 720 gcgggtggcg cgaatgtcat ccccaagctg tgtgtgcaga tcttcaacct ctggagcgcg 780 ggtcgcttca cggaggctat ggaggctcag gagttgttga gtagggctga ctgggtcctc 840 actaaggcgg ctatccccgg tacgaagagt gcaattcaga gctactatgg atatggtgga 900 ttcccgcgtc gcccgttggc tcgcttgagt gccgagcagg cggaggcggt ggctgagaag 960 atcaaggatg ccatggaggt tgagaagtcg ttgccggata ttgcttag 1008 <210> 10 <211> 1353 <212> DNA <213> Unknown <220> <223> lgd1 (L-galactonate dehydratase gene) <400> 10 atgtctgaag tcaccatcac aggcttcagg agccgcgatg tgcggttccc cacgtcccta 60 gacaagacgg gctcggatgc gatgaacgct gcgggcgact attcagcggc atactgcatc 120 ctcgagactg attcagcgca cagtggtcat ggcatgacat tcaccattgg acgcggaaac 180 gacatcgtct gcgccgccat caaccacgtc gcggaccgac tcaagggcaa gaaactgtca 240 tcactagtgg ccgactgggg caagacctgg cggtatctgg tcaacgacag ccagctgcgg 300 tggattggcc ccgaaaaggg cgtcatccat cttgcgctcg gagccgtcgt caacgccgtc 360 tgggacctgt gggcaaagac gctcaacaag ccggtttggc gcatcgttgc cgacatgacg 420 cccgaggagt atgtccgctg catcgacttc cgctacatta ccgacgcaat cacccccgag 480 gaagccgtgg cgatgctgcg cgagcaggag gccggcaagg ccaagcgcat cgaggaggct 540 ctccagaacc gagcggtgcc tgcatacaca acaagtgccg gttggctggg atacggagag 600 gacaagatga agcagctcct gagagagacg ctggctgccg gatacagaca cttcaaggtc 660 aaggttggcg gcagcgtcga ggaggaccga aggcgcctcg gcattgctcg cgaaattctt 720 ggtttcgaca agggcaacgt tctcatggtc gatgccaacc aggtctggtc cgttcccgaa 780 gcgatcgact acatgaagca gctcagcgag tacaagccct ggttcattga ggagcccacc 840 tcacccgacg acatcatggg ccacaaggcc attcgcgatg ccctcaagcc ctatggcatc 900 ggcgtcgcta ccggcgagat gtgccagaac cgcgtcatgt tcaagcagct gatcatgacg 960 ggcgccatcg acatctgcca gattgatgcc tgccgcctcg gcggcgtcaa cgaagtgctg 1020 gccgtcctgc tcatggccaa gaagtacggt gtgcccattg tgccgcattc cggcggcgtg 1080 ggccttcccg agtacaccca gcatctgagc accatcgact acgtggtcgt cagcggcaag 1140 ctttccgtct tggagtttgt agaccacctc cacgagcact tcttgcatcc ttcagtcatc 1200 aaggacggat actaccagac accaaccgag gccggctaca gcgttgagat gaagccggag 1260 agcatggaca agtatgagta tcccggcaag aagggcgtaa gttggtggac gaccgacgag 1320 gctctgccca tcttgaacgg agagaagatc tga 1353 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALD6 Target cut site <400> 11 gtcaagatca cacttccaaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PHO13 Target cut site <400> 12 tcccttatct attaactttc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YPR1 Target cut site <400> 13 catggtagat tattatctgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Intergenic region upstream ASF1 <400> 14 ctctcgaagt ggtcacgtgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Intergenic region upstream ATG33 <400> 15 ttgtcacagt gtcacatcag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Intergenic region downstream YGR190C <400> 16 gatacttatc attaagaaaa 20 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-ALD6.1_Kim044 <400> 17 aagatcacac ttccaaagtt ttagagctag aaatagcaag 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-ALD6.1_Kim045 <400> 18 ttggaagtgt gatcttgacg atcatttatc tttcactgcg 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-PHO13.1_Kim624 <400> 19 cttatctatt aactttcgtt ttagagctag aaatagcaag 40 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-PHO13.1_Kim625 <400> 20 aaagttaata gataagggag atcatttatc tttcactgcg 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-YPR1.1_Kim535 <400> 21 ggtagattat tatctgtgtt ttagagctag aaatagcaag 40 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-YPR1.1_Kim536 <400> 22 cagataataa tctaccatgg atcatttatc tttcactgcg 40 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#4_Kim310 <400> 23 tcgaagtggt cacgtgcgtt ttagagctag aaatagcaag 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#4_Kim311 <400> 24 cacgtgacca cttcgagagg atcatttatc tttcactgcg 40 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#6_Kim314 <400> 25 tcacagtgtc acatcaggtt ttagagctag aaatagcaag 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#6_Kim315 <400> 26 tgatgtgaca ctgtgacaag atcatttatc tttcactgcg 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#7_Kim486 <400> 27 aggaattatg ttcgcccgtt ttagagctag aaatagcaag 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pRS42H-INT#7_Kim487 <400> 28 ggcgaacata attccttacg atcatttatc tttcactgcg 40 <210> 29 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_Kim626 <400> 29 taacatacac aaacacatac tatcagaata cactattttc gaggaccttg tc 52 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_SOO384 <400> 30 tcaacttaat agaaggcatt tttagatctc ctaggtttgt ttgtttatgt gtgtttattc 60 60 <210> 31 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL1_SOO385 <400> 31 ataaacacac ataaacaaac aaacctagga gatctaaaaa tgccttctat taagttgaac 60 60 <210> 32 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL1_SOO386 <400> 32 aatgcaagat ttaaagtaaa ttcactgtta acgcatgctt agacgaagat aggaatcttg 60 60 <210> 33 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> s. cerevisiae_TDH3T_SOO387 <400> 33 ggacaagatt cctatcttcg tctaagcatg cgttaacagt gaatttactt taaatcttgc 60 60 <210> 34 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> s. cerevisiae_TDH3T_SOO388 <400> 34 attctttgaa ggtacttctt cgaaaaattc gcgtctgcta gctcctggcg gaaaaaattc 60 60 <210> 35 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> s. cerevisiae_PGK1P_SOO389 <400> 35 ttttaaagtt tacaaatgaa ttttttccgc caggagctag cagacgcgaa tttttcgaag 60 60 <210> 36 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> s. cerevisiae_PGK1P_SOO390 <400> 36 caccaaggaa gggttagcag tcattttttc tagatgtttt atatttgttg taaaaagtag 60 60 <210> 37 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL2_SOO391 <400> 37 aattatctac tttttacaac aaatataaaa catctagaaa aaatgactgc taacccttcc 60 60 <210> 38 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL2_SOO392 <400> 38 aaaaaattga tctatcgatt tcaattcaat tcaatactag tttactcagg gccgtcaatg 60 60 <210> 39 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1T_SOO393 <400> 39 gtcaagtgtc tcattgacgg ccctgagtaa actagtattg aattgaattg aaatcgatag 60 60 <210> 40 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1T_Kim627 <400> 40 gtatatgacg gaaagaaatg caggttggta caaaataata tccttctcga aag 53 <210> 41 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_Kim628 <400> 41 atgtgacatc tttactattc tccagcacgt ttcttcatcg gtatcttcgc 50 <210> 42 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_SOO374 <400> 42 aatggggtag tggtcatttt taagcttgaa ttctttgtaa ttaaaactta gattagattg 60 60 <210> 43 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL3_SOO375 <400> 43 atctaatcta agttttaatt acaaagaatt caagcttaaa aatgaccact accccatttg 60 60 <210> 44 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> P. stipitis_XYL3_SOO376 <400> 44 gcaactagaa aagtcttatc aatctccgtc gacatcgatt tagtgtttca attcactttc 60 60 <210> 45 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3T_SOO377 <400> 45 caagatggaa agtgaattga aacactaaat cgatgtcgac ggagattgat aagacttttc 60 60 <210> 46 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3T_Kim629 <400> 46 ctataactca ttattggtta aggtgtagat gaagttgggt aacgccagg 49 <210> 47 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim379 <400> 47 ttcctcgggc agagaaactc gcaggcaact tgcacgcaaa agaaaacctt 50 <210> 48 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim380 <400> 48 tcaacacagc tgggggagcc attttttatt gatatagtgt ttaagcgaat 50 <210> 49 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaA_Kim381 <400> 49 tctgtcattc gcttaaacac tatatcaata aaaaatggct cccccagctg 50 <210> 50 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaA_Kim382 <400> 50 tagaatgtat aaataataat aaactaagtc tacttcagct cccactttcc 50 <210> 51 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12T_Kim383 <400> 51 ggatggaaag tgggagctga agtagactta gtttattatt atttatacat 50 <210> 52 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12T_Kim384 <400> 52 tgtgagggcc gattatgcag gcctagatgt tctagtgtgt ttatattatc 50 <210> 53 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1P_Kim385 <400> 53 cctcgggcag agaaactcgc aggcaacttg gtgagtaagg aaagagtgag 50 <210> 54 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1P_Kim386 <400> 54 gtgatggtga cttcagacat tttttgtttt atatttgttg taaaaagtag 50 <210> 55 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> T. reesei_lgd1_Kim387 <400> 55 ctacttttta caacaaatat aaaacaaaaa atgtctgaag tcaccatcac 50 <210> 56 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> T. reesei_lgd1_Kim388 <400> 56 attgatctat cgatttcaat tcaattcaat tcagatcttc tctccgttca 50 <210> 57 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1T_Kim389 <400> 57 ctgcccatct tgaacggaga gaagatctga attgaattga attgaaatcg 50 <210> 58 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_PGK1T_Kim390 <400> 58 ctctgtgagg gccgattatg caggcctaga aaataatatc cttctcgaaa 50 <210> 59 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_Kim391 <400> 59 ctcgggcaga gaaactcgca ggcaacttgg aataaaaaac acgctttttc 50 <210> 60 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3P_Kim392 <400> 60 gactccgggg cggagcgggg taaaaggcat tttttttgtt tgtttatgtg tgtt 54 <210> 61 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaC_Kim393 <400> 61 ttcgaataaa cacacataaa caaacaaaaa aaatgccttt taccccgctc 50 <210> 62 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaC_Kim394 <400> 62 atttaaatgc aagatttaaa gtaaattcac ctaagcaata tccggcaacg 50 <210> 63 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3T_Kim395 <400> 63 tgagaagtcg ttgccggata ttgcttaggt gaatttactt taaatcttgc 50 <210> 64 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_TDH3T_Kim396 <400> 64 cctctgtgag ggccgattat gcaggcctag aatcctggcg gaaaaaattc 50 <210> 65 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE3_CCW12P-gaaA-CCW12T_Kim410 <400> 65 tctttaggtt aattgtcgct gttattgtct agattttttc tcggagatgg 50 <210> 66 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE3_CCW12P-gaaA-CCW12T_Kim411 <400> 66 tagttcctca ctctttcctt actcactgtt ctagtgtgtt tatattatcc 50 <210> 67 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE4_PGK1P-lgd1-PGK1T_Kim412 <400> 67 agccaaggat aatataaaca cactagaaca gtgagtaagg aaagagtgag 50 <210> 68 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE4_PGK1P-lgd1-PGK1T_Kim413 <400> 68 aaactcgaac tgaaaaagcg tgttttttat tcccgattat gcaggcctag 50 <210> 69 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE5_TDH3P-gaaC-TDH3T_Kim414 <400> 69 tattattttc taggcctgca taatcgggaa taaaaaacac gctttttcag 50 <210> 70 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE5_TDH3P-gaaC-TDH3T_Kim415 <400> 70 ctactctctt cctagtcgcc cggttgttga aagtttaatt gtgggttttc 50 <210> 71 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE01_FBA1P-lad1-FBA1T-PGK1P-alx1-CYC1T_Kim553 <400> 71 cttacacttg tgtaatgaca aatgtttttt gaacaacaat accagccttc 50 <210> 72 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae YE01_FBA1P-lad1-FBA1T-PGK1P-alx1-CYC1T_Kim554 <400> 72 tgtttcacgt tatcaagatt atgtcatcta ttggccgcaa attaaagcct 50 <210> 73 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim537 <400> 73 gtaactttgc aatataatca ggtcgcaaat atcacgcaaa agaaaacctt 50 <210> 74 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim538 <400> 74 gaagaattct ttaacgtagc aggcattatt gatatagtgt ttaagcgaat 50 <210> 75 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim541 <400> 75 cggaggagac cgctataacc ggtttgaatt tacacgcaaa agaaaacctt 50 <210> 76 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12P_Kim542 <400> 76 taaccttctt tccgagagac attttttatt gatatagtgt ttaagcgaat 50 <210> 77 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaD_Kim543 <400> 77 tcattcgctt aaacactata tcaataaaaa atgtctctcg gaaagaaggt 50 <210> 78 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> A. niger_gaaD_Kim544 <400> 78 gtataaataa taataaacta agtttattaa acaatcacct tatgaccagc 50 <210> 79 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12T_Kim545 <400> 79 tggtcataag gtgattgttt aataaactta gtttattatt atttatacat 50 <210> 80 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae_CCW12T_Kim546 <400> 80 cttgcttgct gtcaaacttc tgagttgtgt tctagtgtgt ttatattatc 50 <210> 81 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Kim049 <400> 81 ggaacggtga gtgcaacg 18 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim427 <400> 82 aaactgttca cccagacacc 20 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim194 <400> 83 agcgcaacta cagagaacag g 21 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim100 <400> 84 cggcaccgtc gaacaatctg 20 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim101 <400> 85 ccgcttactc ttcgttcggt cc 22 <210> 86 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim193 <400> 86 ctcagcatcc acaatgtatc ag 22 <210> 87 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim426 <400> 87 gcgctattgc attgttcttg tc 22 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim547 <400> 88 aggtatgcga tagttcctca c 21 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim125 <400> 89 tgcagcttcc aatttcgtca c 21 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim630 <400> 90 gaggtgacac ccttaccaac 20 <210> 91 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim631 <400> 91 ctgctactca caccttcaac tc 22 <210> 92 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Kim632 <400> 92 cgctgaaccc gaacatagaa atatc 25 <210> 93 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Kim633 <400> 93 tcgatatttc tatgttcggg ttcag 25 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim078 <400> 94 gattggaatt ggttcgcagt g 21 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim048 <400> 95 gaggaagacg ttgaaggtgg 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim149 <400> 96 tttgaagtgg tacggcgatg 20 <210> 97 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Kim577 <400> 97 cacccaagca cagcatac 18 <210> 98 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim634 <400> 98 tggctcgata acgaagatc ag 22 <210> 99 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Kim635 <400> 99 gtcttgtaga ttgagaactg gtcc 24 <210> 100 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kim636 <400> 100 tctatgaggc aagtaagagg cac 23 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim492 <400> 101 aacaggcgac agtccaaatg 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim077 <400> 102 ttggagttca aactggcgag 20 <210> 103 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Kim322 <400> 103 gcgcatctat ttgccgtc 18 <210> 104 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kim397 <400> 104 gctgggggag ccatttttta ttg 23 <210> 105 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim398 <400> 105 gtgggagctg aagtagactt ag 22 <210> 106 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Kim323 <400> 106 tcaggacaca cctcactg 18 <210> 107 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim399 <400> 107 cctgtgatgg tgacttcaga c 21 <210> 108 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kim401 <400> 108 gaacggagag aagatctgaa ttg 23 <210> 109 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Kim400 <400> 109 acagcctgtt ctcacacac 19 <210> 110 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Kim402 <400> 110 gcggggtaaa aggcattttt tttg 24 <210> 111 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Kim408 <400> 111 gccggatatt gcttaggtg 19 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim490 <400> 112 ggcactagga gcatttgtcg 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim304 <400> 113 gcttcgctaa tccagaggtc 20 <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Kim491 <400> 114 gtcccttagg gtgcgtataa tg 22 <210> 115 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kim539 <400> 115 caattccgtg aaaccctttt ctt 23 <210> 116 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Kim540 <400> 116 ctgccaactt cttcttcatt caa 23 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim326 <400> 117 ggttctgact cctactgagc 20 <210> 118 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Kim093 <400> 118 gcaaagatag cggcgtaggt g 21 <210> 119 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Kim549 <400> 119 gcatcctttg cctccgttc 19 <210> 120 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kim327 <400> 120 agcatcgagt acggcagttc 20

Claims (12)

ALD6 (acetaldehyde dehydrogenase gene) and PHO13 (haloacid dehalogenase Type IIA phosphatase gene) are knocked out, XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene), XYL3 (xylulokinase gene), lad1 (L-arabitolase gene) dehydrogenase gene), alx1 (L-xylulose reductase gene), gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) and lgd1 (L-galactonate dehydratase gene) In the recombinant microorganisms having the consumption capacity of galacturonic acid, xylose and arabinose that are knock-in, the microorganism is Saccharomyces cerevisiae , which is galacturonic acid, xylose and a recombinant microorganism having the ability to consume arabinose. The recombinant microorganism according to claim 1, wherein the production of acetate is inhibited by knockout of the ALD6 (acetaldehyde dehydrogenase gene). The recombinant microorganism of claim 1, wherein the ALD6 (acetaldehyde dehydrogenase gene) is represented by SEQ ID NO: 1, galacturonic acid, xylose and arabinose. According to claim 1, wherein the PHO13 (haloacid dehalogenase Type IIA phosphatase gene) to promote the phosphorylation cycle of the pentose by knockout, galacturonic acid, xylose, and recombinant microorganisms having the ability to consume arabinose. The recombinant microorganism of claim 1, wherein the PHO13 (haloacid dehalogenase Type IIA phosphatase gene) is represented by SEQ ID NO: 2, galacturonic acid, xylose and arabinose. According to claim 1, The xylose reductase gene (XYL1), XYL2 (xylitol dehydrogenase gene) and XYL3 (xylulokinase gene) that the xylose metabolic pathway is generated by the knock-in, galacturonic acid, xylose and arabinose Recombinant microorganisms with consumption ability. According to claim 1, wherein the XYL1 (xylose reductase gene) is represented by SEQ ID NO: 3, XYL2 (xylitol dehydrogenase gene) is represented by SEQ ID NO: 4, and XYL3 (xylulokinase gene) is represented by SEQ ID NO: 5, A recombinant microorganism having the ability to consume galacturonic acid, xylose and arabinose. The consumption capacity of galacturonic acid, xylose and arabinose according to claim 1, wherein the arabinose metabolic pathway is generated by knock-in of the lad1 (L-arabitol dehydrogenase gene) and alx1 (L-xylulose reductase gene). Recombinant microorganisms having According to claim 1, wherein the lad1 (L-arabitol dehydrogenase gene) is represented by SEQ ID NO: 6 and alx1 (L-xylulose reductase gene) is represented by SEQ ID NO: 7, Galacturonic acid, xylose and arabinose Recombinant microorganisms having a consumption capacity of The galacturonic acid metabolism according to claim 1, wherein the gaaA (D-galacturonic acid reductase gene), gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) and lgd1 (L-galactonate dehydratase gene) are knocked-in. A recombinant microorganism having the ability to consume galacturonic acid, xylose and arabinose, wherein a pathway is generated. According to claim 1, wherein the gaaA (D-galacturonic acid reductase gene) is represented by SEQ ID NO: 8, gaaC (2-keto-3-deoxy-L-galactonate aldolase gene) is represented by SEQ ID NO: 9 and lgd1 ( L-galactonate dehydratase gene) is a recombinant microorganism having the ability to consume galacturonic acid, xylose and arabinose, which is represented by SEQ ID NO: 10.
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