CN116004488A - Recombinant strain and method for producing glycollic acid - Google Patents
Recombinant strain and method for producing glycollic acid Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a recombinant escherichia coli strain and a method for producing glycollic acid. The recombinant escherichia coli strain provided by the invention has the fucO and yqhD genes knocked out and expresses the Gox0313 and aldA genes. The strain can efficiently utilize EG, and can utilize 6.84g/LEG within 120h, and OD of strain in stationary phase 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant escherichia coli strain and a method for producing glycollic acid.
Background
Ethylene Glycol (EG) is one of the main products of complete degradation of PET and is also an important organic chemical raw material. For biodegradable PET, under ideal conditions, the microorganism can secrete PET degrading enzymes, so that EG is produced by complete degradation of PET, EG can be further metabolized by other microorganisms, growth is provided or used for producing high-value chemicals, and finally, the environment-friendly concept of green chemistry is realized. The laboratory has constructed artificial mixed bacteria system to degrade PET in early stage, and the experimental result shows that EG is used as one of PET degradation products and has certain inhibiting effect on PET hydrolase. Related studies have also reported that EG is identified as a competitive inhibitor of PET hydrolase. Therefore, the design and construction of the recombinant mode biological high-efficiency utilization EG can effectively promote PET enzymolysis, and the use of EG as a raw material for producing high-value chemicals such as glycollic acid and the like can also widen the application field of EG.
The biological method can avoid the problems of difficult separation and purification caused by poor selectivity and a plurality of byproducts of the chemical method. In addition, glycolic acid has been widely used in the preparation of biodegradable materials, and EG is converted into glycolic acid, so that the conversion of PET waste into biodegradable materials can be further realized, and a new scheme is provided for the development of recycling economy. The research aims at constructing a recombinant escherichia coli which can efficiently utilize EG and convert EG into glycolic acid which is a high value-added product, and further realizing the reutilization of EG which is a plastic degradation product.
At present, most of reported microorganisms with EG utilization capacity are not model organisms, the genetic background and the genetic modification method are not clear, the model organisms utilizing EG are used for producing glycollic acid, and meanwhile, the model organisms are applied to industrial application of degrading plastics and converting the plastics into glycollic acid, so that an effective solution can be provided for recycling waste plastics. Compared with other bacteria, the escherichia coli MG1655 contains an endogenous pathway of glycolaldehyde metabolism, the key for realizing the utilization of EG and the efficient conversion of EG is to construct an oxidation pathway of EG to glycolaldehyde, and the escherichia coli has a definite genetic background and a simple genetic operation method and is a potential chassis microorganism for converting EG into high-value chemicals.
Incomplete oxidation of EG can produce glycolic acid, which is one of the inexpensive raw materials for the production of glycolic acid. Glycolate is an alpha-hydroxy acid, both alcoholic and acidic in nature, and has been widely used in chemical cleaning, synthetic biodegradable materials, textile industry, food processing and pharmaceutical industry. In addition, it is a very important intermediate in organic synthesis, and participates in long chain polymerization, esterification, oxidation/reduction, and other reactions. Currently, glycolic acid is produced by a series of chemical reactions, mostly by means of fossil resources and toxic chemicals. It is known that most microorganisms capable of producing glycolic acid using EG are non-model organisms, and it is difficult to perform chassis modification. Therefore, development of model organisms to produce glycolic acid efficiently is of great industrial value.
Disclosure of Invention
In view of this, the present invention provides a recombinant E.coli strain and a method for producing glycolic acid. The strain can efficiently utilize EG, convert the EG into glycolic acid, and the glycolic acid yield reaches 5.02g/L within 120 h.
In order to achieve the above object, the present invention provides the following technical solutions:
a recombinant escherichia coli strain, which has fucO (aldehyde reductase) and yqhD genes (alcohol dehydrogenase genes) knocked out and expresses Gox0313 (alcohol dehydrogenase gene), aldA gene (aldehyde dehydrogenase gene).
In the invention, the chassis strain is escherichia coli K12 series strain, and in some implementations, the chassis strain is MG1655.
In the present invention, the Gox0313 gene is derived from Gluconobacter oxydans Gluconobacter oxydans. The gene is subjected to codon optimization aiming at escherichia coli MG1655, and an RBS sequence is added at the front end of the gene, and the amino acid sequence of the gene is shown as SEQ ID NO. 1. In some embodiments, the invention introduces BamHI and EcoRI cleavage sites at both ends of the Gox0313 gene, respectively, through which the Gox0313 gene is inserted into a plasmid vector.
In the invention, the aldA gene is an endogenous gene of escherichia coli MG1655, and in some specific embodiments, an RBS sequence is added at the 5' end of the aldA gene, and the nucleotide sequence of the aldA gene is shown as SEQ ID NO. 2. In some embodiments, the invention introduces XbaI and SalI cleavage sites at both ends of the aldA gene, respectively, through which the aldA gene is inserted into a plasmid vector.
In the present invention, the recombinant E.coli strain has a genome which integrates chloramphenicol tag and Arabidopsis resistance tag genes. The invention utilizes lambda-Red homologous recombination technology to integrate the chloramphenicol tag and the Arabidopsis resistance tag into the positions of the fucO and yqhD genes in the genome while knocking out the fucO and yqhD genes.
In the invention, knockout of fucO and yqhD genes and insertion of chloramphenicol and Arabidopsis resistance tags are realized by using lambda-Red homologous recombination technology.
The invention also provides application of the recombinant escherichia coli strain in production of glycolic acid.
The invention also provides a construction method of the recombinant escherichia coli strain, which comprises the following steps:
taking an escherichia coli K12 series strain as chassis bacteria, knocking out fucO and yqhD genes by adopting a lambda-Red homologous recombination technology, and integrating chloramphenicol tags and Arabic resistance tags into genome of the chassis bacteria to obtain intermediate strains;
connecting the optimized Gox0313 and aldA genes to a PTrc99a vector to obtain a recombinant plasmid;
and transforming the recombinant plasmid into the intermediate strain to obtain the recombinant escherichia coli strain.
The invention also provides a method for producing glycollic acid, which takes EG as a carbon source, ferments the recombinant escherichia coli strain disclosed by the invention, and marks the strain FYGA.
In the invention, the fucO and yqhD genes are knocked out by utilizing the lambda-Red homologous recombination technology. Firstly, constructing fragment 1 containing a fucO gene knockout fragment and a resistance tag sequence, wherein the nucleotide sequence of the fragment 1 is shown as SEQ ID NO. 3; fragment 2 containing yqhD gene knockout fragment and resistance tag sequence, its nucleotide sequence is shown in SEQ ID NO. 4. The kind of the resistance tag sequence is not particularly limited in the present invention, and a person skilled in the art may select the resistance tag according to practical situations, and in the specific embodiment of the present invention, the resistance tag is a chloramphenicol tag or an arabica resistance tag. The chloramphenicol tag and the Arabidopsis resistance tag are integrated into the genome of Chassis bacteria by lambda-Red homologous recombination technology, respectively replacing the fucO and yqhD genes.
The recombinant escherichia coli strain FYGA provided by the invention has the fucO and yqhD genes knocked out and expresses Gox0313 and aldA genes. The strain can efficiently utilize EG, can utilize 6.84g/L EG within 120h, and has OD at strain stabilization stage 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
Drawings
FIG. 1 shows a schematic diagram of recombinant E.coli producing glycolic acid using EG;
FIG. 2 shows an EG pathway map for E.coli;
FIG. 3 shows EG concentration changes during degradation of EG by recombinant strain FYGA;
FIG. 4 shows the change in concentration of glycolic acid produced by recombinant strain FYGA using EG;
FIG. 5 shows OD during EG degradation by recombinant strain FYGA 600 。
Detailed Description
The invention provides a recombinant escherichia coli strain and a method for producing glycollic acid. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
The lambda-Red homologous recombination technology is adopted in the escherichia coli MG1655 to knock out the fucO and yqhD genes in the genome, the fucO gene is knocked out to introduce an Arabic resistance tag for substitution, the yqhD gene is knocked out to introduce a chloramphenicol tag for substitution, and the resistance tag is not required to be knocked out. The final knockout fucO gene and yqhD gene strains were both arabidopsis and chloramphenicol resistant. The sequence of the knockdown fragment is shown in SEQ ID NO. 3-4.
The exogenous gene Gox0313 is synthesized by Nanjing Jinsri biotechnology Co., ltd, codon optimization is carried out for chassis cell escherichia coli MG1655, RBS sequence is added in front of synthesizing exogenous gene, bamHI and EcoRI restriction sites are respectively introduced at two ends of synthesized fragment, gox0313 gene and plasmid vector pTrc99a are assembled through BamHI and EcoRI double restriction and T4 connection, thus obtaining recombinant plasmid pEc01004. The method comprises the steps of amplifying an aldA gene fragment in an escherichia coli genome by using primers aldA-F and aldA-R through PCR, adding a RBS sequence and enzyme digestion sites XbaI and SalI, wherein the used primer sequence is shown in table 1, assembling the aldA gene fragment and a plasmid vector pEc01004 through double enzyme digestion of XbaI and SalI and T4 connection, obtaining a recombinant plasmid pEc01005, taking escherichia coli MG1655 with fucO and yqhD genes knocked out as chassis cells, transforming the plasmid pEc01005 into the chassis cells to collect recombinant strain FYGA, wherein the recombinant strain has Arabic resistance, chloramphenicol resistance and ampicillin resistance, and the recombinant escherichia coli has a glycolic acid production schematic diagram by using EG and an escherichia coli path diagram shown in figures 1 and 2 respectively.
TABLE 1 primer list required for constructing recombinant plasmid pEc01005
Shake flask fermentation experiments were performed on recombinant strains in M9 medium with 10g/L EG as the sole carbon source. Determination of EG concentration Change during fermentation, glycolic acid concentration Change and OD during FYGA degradation of EG 600 The values and results are shown in FIGS. 3 to 5.
At the 5 th hour of shake flask fermentation, total RNA of the strain FYGA is extracted, cDNA is subjected to reverse transcription, qPCR reaction is performed, and exogenous genes and endogenous gene transcription levels in a path for producing glycollic acid by using EG in the recombinant strain are determined by taking an endogenous gene gapA of escherichia coli MG1655 as a reference gene.
Wherein, the culture medium comprises the following components:
m9 medium: 200mL of 5 XM 9 salt solution, 2mL of 1M MgSO4 solution, 100 mu L of CaCl2 solution, 1mL of trace metal solution and 700mL of 2g/L yeast extract powder solution are measured, 10g/L EG solution is added as a carbon source, the mixture is fully and uniformly mixed and dissolved after ultrasonic treatment, the volume is fixed to 1L, and the mixture is sub-packaged and sterilized.
5×m9 salt solution: weigh 6.78g Na 2 HPO 4 、3g KH 2 PO 4 、0.5g NaCl、1g NH 4 Cl was dissolved well and fixed to 200mL.
Trace metal solution: 1.6g FeCl was weighed out 3 、0.2g CoCl 2 ·H 2 O、0.1gCuCl 2 、0.13g ZnCl 2 、0.2g Na 2 MoO 4 、0.05g H 3 BO 3 Ultrasonic dissolving in 0.1M HCl, and fixing volume to 1L.
As can be seen from FIGS. 3 to 5, recombinant strain FYGA was able to efficiently utilize EG as compared to the control group. The OD of the strain at the stationary phase can be utilized at 6.84g/LEG within 120h 600 Is maintained at about 6 to 7. The glycolic acid yield is rapidly increased within 24 hours before the culture, and reaches 5.02g/L within 120 hours.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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atttgacacg ctggaagatg ctatctcaat ggctaatgac agtgattacg gcctgacctc 1260
atcaatctat acccaaaatc tgaacgtcgc gatgaaagcc attaaagggc tgaagtttgg 1320
tgaaacttac atcaaccgtg aaaacttcga agctatgcaa ggcttccacg ccggatggcg 1380
taaatccggt attggcggcg cagatggtaa acatggcttg catgaatatc tgcagaccca 1440
ggtggtttat ttacagtctt aa 1462
<210> 3
<211> 2296
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgacttagc cgccacgctg gcggtcaaag tgacacaggc cgggcatcag gcaacgattg 60
tttcgacaga taaaggctac tgtcagttac tttcaccgac attacgtatt cgtgattact 120
tccagaaacg ttggctggat gcgccattta tcgataaaga atttggcgtt caaccgcagc 180
agttgcccga ttactgggga cttgcgggga tcagcagttc aaaggtaccg ggtgttgcgg 240
gaatcggacc aaaaagcgcc acgcagctgc tggtcgagtt tcagagtctg gaagggatat 300
atgagaatct ggatgcggtt gccgaaaagt ggcgcaaaaa attagaaacc cataaagaga 360
tggcgtttct gtgccgcgat attgcccgct tacaaaccga tttgcatatc gacggcaatt 420
tacagcaatt gcggttggta cggtaacggc gagccggata cgccgcaaac gtcgtatccg 480
gcattatcac atcagcgcat cgcggaaccc ctatttgttt atttttctaa atacattcaa 540
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 600
agagtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg 660
taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg 720
acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat 780
gcgcacccgt tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt 840
cgctacttgg agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggatcc 900
tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct 960
atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt 1020
gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct 1080
tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct 1140
tcctaatgca ggagtcgcat aagggagagc gtcgaccgat gcccttgaga gccttcaacc 1200
cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct 1260
tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg 1320
accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc 1380
acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg 1440
ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc 1500
gaggctggat ggccttcccc attatgattc ttctcgcctc cggcggcatc gggatgcccg 1560
cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag cttcaaggat 1620
cgctcgcggc tcttaccagc ctaacttcga tcactggacc gctgatcgtc acggcgattt 1680
atgccgcctc ggcgagcaca tggaacgggt tggcatggat tgtaggcgcc gccctatacc 1740
ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctaacatc 1800
cttctccttg ttgctttacg aaattactct tcaattcgta acccataggt tttgaatttc 1860
tccagcacta cggcaatctc ttcatcgctc agcactggca ccgggtccgt aatcgccagg 1920
gtcgtcaggt aaagttgcgc cagcacttca acttcatgcg ccagccataa cgctttttcc 1980
agattcacct cacaagcgat aagcccatga tgttgtaaca aagttgcctt acgatttttg 2040
agagccagcg caacatgttc agaaagttcg cgtgttccaa aggtcgcata aggcgcgcaa 2100
ggaatagaat taccgccagc cgccgcaatc atgtagtgaa tagcggggat cgatcggtta 2160
agaatggaaa ctgccgtgca atgaacggca tgattgtgaa caaccgcgtt ggcatccggt 2220
ctgctttgat aggctgccat atggaaacgc cattcgcttg aggggagctt tccttcctca 2280
tgtttaccgt tgccat 2296
<210> 4
<211> 1765
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cattgagacg caaccctgcc agcggcacct ctgacgtggc ataggtttcg cactcaaacg 60
gcaacggcac cgtcagcagc aggtattcat tggcatcata acgaaacacg cgttcattga 120
tataaccgat tttatgcccg gaaaagagaa ttatgatgcc aggctcgtac atcaccggtg 180
tacgtgcgaa aggcgtctcg ccatacaaca aacgcacatc gggcaacagt cctgacaaac 240
tattttcttt atttttcagt ttattaactt tatccgccag caagcggcaa atctcttcac 300
gtttcatatc gcgtaatttc ttaggaataa tgcggcaatt tgattgtgcg caattttgta 360
gcatttctcc agcactctgg agaaataggc aagacattgg cagaaatgag cattgagagc 420
cagggcgctg gcgatcacaa tgaaaaacat caggcagatc gttctctgcc ctcatattgg 480
cccagcaaag ggagcaagta cgcggaaccc ctatttgttt atttttctaa atacattcaa 540
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 600
agagtatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 660
aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 720
tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 780
ttattcacat tcttgcccgc ctgatgaacg ctcacccggg gtttcgtatg gccatgaaag 840
acggtgagct ggtgatctgg gatagtgttc acccttgtta caccgttttc catgagcaaa 900
ctgaaacgtt ttcgtccctc tggagtgaat accacgacga tttccggcag tttctccaca 960
tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 1020
ttgagaatat gttttttgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 1080
acgtggccaa tatggacaac ttcttcgccc ccgttttcac gatgggcaaa tattatacgc 1140
aaggcgacaa ggtgctgatg ccgctggcga tccaggttca tcatgccgtt tgtgatggct 1200
tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 1260
cgtaagcttt ttacgcctca aactttcgtt ttcgggcatt tcgtccagac ttaagttcac 1320
aacacctcac cggagcctgc tccggtgagt tcatataaag gaggaacgta tggctaatcc 1380
aaccgttatt aagctacagg atggcaatgt catgccccag ctgggactgg gcgtctggca 1440
agcaagtaat gaggaagtaa tcaccgccat tcaaaaagcg ttagaagtgg gttatcgctc 1500
gattgatacc gccgcggcct acaagaacga agaaggtgtc ggcaaagccc tgaaaaatgc 1560
ctcagtcaac agagaagaac tgttcatcac cactaagctg tggaacgacg accacaagcg 1620
cccccgcgaa gccctgctcg acagcctgaa aaaactccag cttgattata tcgacctcta 1680
cttaatgcac tggcccgttc ccgctatcga ccattatgtc gaagcatgga aaggcatgat 1740
cgaattgcaa aaagagggat taatc 1765
Claims (11)
1. A recombinant escherichia coli strain, characterized in that the fucO and yqhD genes are knocked out and the Gox0313, aldA genes are expressed.
2. The recombinant E.coli strain according to claim 1, wherein the chassis strain is E.coli K12 series strain.
3. The recombinant escherichia coli strain of claim 2, wherein the escherichia coli K12 series strain is MG1655.
4. The recombinant escherichia coli strain according to claim 1, wherein the Gox0313 gene is derived from gluconobacter oxydans.
5. The recombinant escherichia coli strain according to claim 1, wherein the nucleotide sequence of the Gox0313 gene is shown as SEQ ID No. 1.
6. The recombinant escherichia coli strain of claim 1, wherein the aldA gene has a nucleotide sequence as shown in SEQ ID No. 2.
7. Recombinant E.coli strain according to any one of claims 1 to 6, characterized in that its genome also integrates chloramphenicol-and Arabidopsis resistance-tagged genes.
8. The recombinant escherichia coli strain of claim 7, wherein the chloramphenicol tag and the arabidopsis resistance tag are integrated into the genome in place of the fucO and yqhD genes, respectively.
9. Use of a recombinant escherichia coli strain according to any one of claims 1-8 for the production of glycolic acid.
10. The method for constructing a recombinant E.coli strain according to any one of claims 1 to 8, comprising:
taking an escherichia coli K12 series strain as chassis bacteria, knocking out fucO and yqhD genes by adopting a lambda-Red homologous recombination technology, and integrating chloramphenicol tags and Arabic resistance tags into genome of the chassis bacteria to obtain intermediate strains;
connecting the optimized Gox0313 and aldA genes to a PTrc99a vector to obtain a recombinant plasmid;
and transforming the recombinant plasmid into the intermediate strain to obtain the recombinant escherichia coli strain.
11. A process for producing glycolic acid, characterized in that the recombinant escherichia coli strain according to any one of claims 1 to 8 is fermented with EG as a carbon source.
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