Acetic acid production process

Abstract

The invention relates to the technical field of biology, and discloses an acetic acid production method which comprises the following steps: adding reinforced clostridium into fermentation liquor, introducing synthetic gas with specified flow, and stirring at specified rotating speed at specified temperature to generate acetic acid, the enhanced clostridium comprises clostridium which has silenced a thl gene and / or an aor gene. The acetic acid production method provided by the invention can improve the selectivity of an acetic acid product and increase the yield of acetic acid.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for producing acetic acid. Background Art

[0002] Under anaerobic fermentation conditions, some anaerobic Clostridium (e.g., Clostridium thermoacetica) use carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2) as raw gas to produce compounds such as ethanol, acetic acid, acetone, and butanol. For example, the above compounds can be generated through the Wood-Ljungdahl pathway (acetyl-CoA pathway). Among them, CO and H2 produce electrons through the action of hydrogen reductase. Electrons are transferred through the electron transfer chain and eventually combined with CO2 to generate acetyl-CoA (Acetyl-CoA) through a reduction reaction. Acetyl-CoA is further converted into acetic acid, or other organic acids and organic alcohols. It can also be converted into products such as ethanol, acetone, butanol, and butyric acid through further metabolism.

[0003] Due to the existence of multiple conversion pathways, the purity of acetic acid produced by anaerobic Clostridium is low. Summary of the invention

[0004] In view of the problem of low purity of acetic acid in the prior art, the present invention provides a method for producing acetic acid to improve the yield of acetic acid.

[0005] A method for producing acetic acid, comprising:

[0006] Adding enhanced Clostridium to the fermentation broth, introducing a synthetic gas at a specified flow rate, and stirring at a specified temperature and a specified speed to generate acetic acid;

[0007] The enhanced Clostridium includes Clostridium in which the thl gene and / or the aor gene have been silenced.

[0008] Optionally, the th1 gene is used to encode acetyl-CoA acetyltransferase.

[0009] Optionally, the aor gene is used to encode aldehyde:chlorophyll iron-sulfur protein oxidoreductase.

[0010] Optionally, the enhanced Clostridium is a Clostridium in which the thl gene and / or the aor gene are silenced based on CRISPRi technology.

[0011] Optionally, the gRNA sequence for silencing the th1 gene includes:

[0012] 5'-CACCGTGTGAAAGTGCCCGTGTTCAC-3';

[0013] 5'-AAACGTGAACACGGCACTTTCACAC-3'.

[0014] Optionally, the gRNA sequence for silencing the aor gene includes:

[0015] 5'-CACCGTGGCCCAGCGTACTCTGCAGC-3';

[0016] 5'-AAACTGCTGCAGAGTACGCTGGCCA-3'.

[0017] Optionally, the enhanced Clostridium includes strain CT-dCas9-thl, strain CT-dCas9-aor, and strain CT-dCas9-thl-aor.

[0018] Optionally, the source strain of the enhanced Clostridium is Clostridium thermoacetica.

[0019] Optionally, the synthesis gas includes CO, and / or a mixed gas containing CO2 and H2.

[0020] Optionally, the specified temperature includes 58°C.

[0021] The acetic acid production method provided by the present invention can improve the selectivity of the acetic acid product and increase the yield of acetic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 This is a schematic diagram of the production of acetic acid and other organic substances from synthesis gas via the Wood-Ljungdahl pathway in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] This embodiment provides a method for producing acetic acid, comprising:

[0026] Adding enhanced Clostridium to the fermentation broth, introducing a synthetic gas at a specified flow rate, and stirring at a specified temperature and a specified speed to generate acetic acid;

[0027] The enhanced Clostridium includes Clostridium in which the thl gene and / or the aor gene have been silenced.

[0028] Understandably, if Figure 1 As shown, the bacterial flora (including enhanced Clostridium) provided in the embodiment of the present invention generates acetic acid and other organic substances through the Wood-Ljungdahl pathway. CO and H2 produce electrons through the action of hydrogen reductase. The electrons are transferred through the electron transfer chain and finally combined with CO2 to generate acetyl-CoA (Acetyl-CoA) through a reduction reaction, and acetyl-CoA is further converted into acetic acid, or other organic acids and organic alcohols. It is further metabolized into products such as ethanol, acetone, butanol, butyric acid, etc.

[0029] In the Wood-Ljungdahl pathway, several key enzymes are essential for gas conversion and metabolic pathways to proceed, including:

[0030] Hydrogen reductase: converts H2 into hydrogen ions and generates electrons.

[0031] Methyltetrahydrofolate reductase: reduces methyltetrahydrofolate to tetrahydrofolate, providing reducing equivalents.

[0032] Ethanol dehydrogenase: reduces acetic acid to ethanol.

[0033] Aldehyde:ferredoxin oxidoreductase (AOR): Participates in the carbon metabolism process in syngas fermentation and regulates the ratio of acetic acid and ethanol in syngas fermentation products.

[0034] Acetyl-CoA acetyltransferase (thl or thiolase), also referred to as thiolase, through the catalytic action of thl enzyme, two-carbon intermediate acetyl-CoA can be converted into products of longer carbon chains, such as acetone of three carbons, butyric acid and butanol of four carbons, etc. These products are important products in the synthesis gas fermentation process and can be used as chemical raw materials or fuels. The activity and selectivity of regulating thl enzyme have an important impact on the efficiency of product generation and synthesis gas fermentation. Through gene editing, gene expression regulation or metabolic engineering methods, the expression amount of thl enzyme can be adjusted or its catalytic properties can be changed to achieve the regulation of product selectivity and output.

[0035] The enhanced Clostridium used in this embodiment silences the thl gene and / or the aor gene. Among them, silencing the expression of the thl gene can inhibit the metabolic pathway from Acetyl-CoA to Acetoacetyl-CoA, and minimize the production of three-carbon or four-carbon products. Silencing the expression of the aor gene can also inhibit the conversion of acetic acid (Acetate) to acetaldehyde (Acetaldehyde) and the rear-end end product ethanol, thereby achieving the purpose of reducing acetaldehyde production and increasing acetic acid production.

[0036] The acetic acid production method provided in this embodiment can improve the selectivity of the acetic acid product and increase the yield of acetic acid.

[0037] Optionally, the th1 gene is used to encode acetyl-CoA acetyltransferase.

[0038] Understandably, the th1 gene is a gene fragment for strengthening Clostridium, which is used to encode acetyl-CoA acetyltransferase. By silencing the expression of the th1 gene, the production of acetyl-CoA acetyltransferase can be reduced, which greatly reduces the conversion of acetyl-CoA to acetoacetyl-CoA and reduces the production of three-carbon or four-carbon products.

[0039] Optionally, the aor gene is used to encode aldehyde:chlorophyll iron-sulfur protein oxidoreductase.

[0040] It can be understood that the aor gene is a gene fragment on the fortified Clostridium, which is used to encode aldehyde:chlorophyll iron-sulfur protein oxidoreductase. By silencing the expression of the aor gene, acetic acid is converted to acetaldehyde, thereby achieving the purpose of reducing acetaldehyde production and increasing acetic acid production.

[0041] Optionally, the enhanced Clostridium is a Clostridium in which the thl gene and / or the aor gene are silenced based on CRISPRi technology.

[0042] Understandably, CRISPRi (CRISPR interference) technology is a method of regulating gene expression using the CRISPR-Cas9 system. The principle of CRISPRi technology is to achieve gene silencing by introducing specific gRNA and Cas9 fusion protein. Unlike double-strand breaks in gene editing, CRISPRi uses an inactivated Cas9 protein that cannot cut DNA. Instead, it binds to gRNA through inactivating mutations on the Cas9 protein (such as dCas9) and precisely locates to the promoter region of the target gene through the guiding action of gRNA. By designing a suitable gRNA, the promoter region of the target gene can be precisely located, thereby achieving highly specific gene inhibition.

[0043] Optionally, the gRNA sequence for silencing the th1 gene includes:

[0044] 5'-CACCGTGTGAAAGTGCCCGTGTTCAC-3';

[0045] 5'-AAACGTGAACACGGCACTTTCACAC-3'.

[0046] Understandably, the above gRNA sequence can be used to silence the th1 gene. Wherein, 5'-CACCGTGTGAAAGTGCCGTGTTCAC-3' is the forward sequence.

[0047] 5'-AAACGTGAACACGGCACTTTCACAC-3' is the reverse sequence.

[0048] Optionally, the gRNA sequence for silencing the aor gene includes:

[0049] 5'-CACCGTGGCCCAGCGTACTCTGCAGC-3';

[0050] 5'-AAACTGCTGCAGAGTACGCTGGCCA-3'.

[0051] Understandably, the above gRNA sequence can be used to silence the aor gene. Among them, 5'-CACCGTGGCCAGCGTACTCTGCAGC-3' is the forward sequence.

[0052] 5'-AAACTGCTGCAGAGTACGCTGGCCA-3' is the reverse sequence.

[0053] Optionally, the enhanced Clostridium includes strain CT-dCas9-thl, strain CT-dCas9-aor, and strain CT-dCas9-thl-aor.

[0054] Understandably, the dCas9 protein is a variant of the Cas9 protein (CRISPR-associated protein 9), where d stands for "dead" or "disabled." Compared with the ordinary Cas9 protein, the nuclease activity of the dCas9 protein is inactivated, that is, it cannot cause double-strand breaks in DNA.

[0055] The dCas9 protein (or Sp-dCas9 for short) from Streptococcus pyogenes is selected in this embodiment. This protein is a commonly used dCas9 protein variant. It is obtained by transforming the Cas9 protein in the Streptococcus pyogenes strain and is used for CRISPRi (CRISPR interference) and other gene regulation applications. The Sp-dCas9 protein retains the DNA binding ability of the Cas9 protein and can be directed to identify and locate the target DNA sequence by binding to a specific gRNA sequence, but its nuclease activity has been inactivated. By combining an appropriate gRNA with the Sp-dCas9 protein, a dCas9-gRNA complex can be formed, which can bind to the target DNA sequence and regulate gene expression by blocking transcription or other mechanisms. Therefore, the dCas9 protein can be used to achieve applications such as gene silencing, transcriptional regulation, and genome positioning.

[0056] Optionally, the source strain of the enhanced Clostridium is Clostridium thermoacetica.

[0057] Understandably, Clostridium thermoacetica is an anaerobic bacterium belonging to the Clostridiaceae family, a strain having a fusiform cell morphology. It is a weakly aerobic strain that survives and reproduces mainly through anaerobic metabolism.

[0058] Optionally, the synthesis gas includes CO, and / or a mixed gas containing CO2 and H2.

[0059] Understandably, the synthesis gas can be CO, and / or, a mixed gas containing CO2 and H2. When the enhanced Clostridium is undergoing metabolic reactions, hydrogen can provide reducing power, help the enhanced Clostridium complete the metabolic process, and promote the growth and reproduction of the enhanced Clostridium. On the other hand, hydrogen can be combined with some acidic organic acids or other intermediates to form a relatively stable gas, reduce the pH fluctuation of the reaction solution, and maintain the stability of the reaction solution environment. The production and consumption of hydrogen will have an impact on the concentration of some anaerobic enhanced Clostridium metabolites and the balance of products. Carbon dioxide can provide an important metabolic substrate for enhanced Clostridium, promote the growth and reproduction of enhanced Clostridium, accelerate the fermentation rate, and increase product yield. In some cases, some acid-producing bacteria (such as lactobacillus) release a large amount of organic acid, causing the reaction solution to become very acidic. Carbon dioxide can release an alkaline ion in the reaction, thereby neutralizing the acidic substances produced, maintaining the neutrality or weak alkalinity of the reaction environment, and ensuring the normal growth of enhanced Clostridium. Carbon monoxide can be used as a carbon source, energy source and electron donor for strains, and participate in the metabolism and energy conversion of organic matter.

[0060] Optionally, the specified temperature includes 58°C.

[0061] Understandably, in the anaerobic fermentation reaction, the growth and metabolic rate of the enhanced Clostridium are affected by temperature. Too high or too low a temperature will have an adverse effect on the growth and metabolism of the enhanced Clostridium, and ultimately affect the quality and yield of the fermentation product. Here, the anaerobic fermentation reaction is carried out at a specified temperature. The specified temperature is generally selected to be the most suitable growth temperature of the enhanced Clostridium. After testing, the enhanced Clostridium is 58°C.

[0062] Application Examples

[0063] 1. Strains and culture conditions

[0064] The wild type Clostridium thermoacetica was used as the original strain and cultured in enhanced Clostridium medium.

[0065] The formula of the Fortified Clostridium Medium is as follows (per liter):

[0066]

[0067]

[0068] 1L of water, adjust the pH to 6.2-7.4.

[0069] The pre-cultured Clostridium thermoacetica bacterial solution was inoculated into a sealed shake flask with a volume of 250 ml, 100 ml of sterilized reinforced Clostridium medium was added, and then cultured on a constant temperature shaker at 58°C and 200 rpm for 72 to 96 hours.

[0070] 2. Select dCas9 protein

[0071] The dCas9 protein is a variant of the Cas9 protein (CRISPR-associated protein 9), where d stands for "dead" or "disabled." Compared with the ordinary Cas9 protein, the nuclease activity of the dCas9 protein is inactivated, that is, it cannot cause double-strand breaks in DNA.

[0072] The dCas9 protein from Streptococcus pyogenes (or Sp-dCas9 for short) is selected here. This protein is a commonly used dCas9 protein variant. It is obtained by modifying the Cas9 protein in the Streptococcus pyogenes strain and is used for CRISPRi (CRISPR interference) and other gene regulation applications. The Sp-dCas9 protein retains the DNA binding ability of the Cas9 protein and can direct recognition and positioning of the target DNA sequence by binding to a specific gRNA sequence, but its nuclease activity has been inactivated. By combining the appropriate gRNA with the Sp-dCas9 protein, a dCas9-gRNA complex can be formed, which can bind to the target DNA sequence and regulate gene expression by blocking transcription or other mechanisms, thereby achieving gene silencing.

[0073] 3. Gene acquisition and editing

[0074] The sequences of thl gene and aor gene can be obtained through online gene library. And the obtained gene sequence is edited using gene editing tools. Among them, the gRNA sequence is designed and matched with the exon region of thl gene. The gRNA sequence used to silence thl gene can be:

[0075] 5'-CACCGTGTGAAAGTGCCGTGTTCAC-3'; reverse sequence: 5'-AAACGTGAACACGGCACTTTCACAC-3'. The above gRNA sequences were synthesized and amplified using PCR.

[0076] Design the gRNA sequence and make it match the exon region of the aor gene. The gRNA sequence used to silence the aor gene can be: Forward sequence:

[0077] 5'-CACCGTGGCCAGCGTACTCTGCAGC-3'; reverse sequence: 5'-AAACTGCTGCAGAGTACGCTGGCCA-3'; synthesize and amplify the above gRNA sequence using PCR;

[0078] 4. Make the basic carrier

[0079] The pMTL83151 vector can be selected as the basic vector for CRISPRi transformation. pMTL83151 is a broad-spectrum plasmid vector suitable for a variety of Clostridium strains, including Clostridium thermoacetica. The pMTL83151 vector has the following main features: 1. Initiator and selective marker: The pMTL83151 vector contains a series of promoters that can be used for expression, such as Ptet promoter, Pthl promoter, etc., to meet the expression requirements of different genes. In addition, the vector also has selective markers, such as chloramphenicol resistance gene, for screening successfully transformed colonies. 2. Multiple cloning site: The pMTL83151 vector has multiple cloning sites that can be used to insert target genes or gRNA sequences. These sites are usually multiple cloning sites (MCS), which can be inserted into exogenous sequences by restriction endonuclease digestion. 3. Origin of replication and maintenance: The pMTL83151 vector uses a replication origin suitable for Clostridium strains to ensure stable replication and maintenance of the plasmid in the host cell. 4. Gene mutation tools: The pMTL83151 vector also contains some tools for gene mutation, such as a locus replacement system for selective markers, which can facilitate genome editing and replacement. 5. By inserting the target gene sequence or gRNA sequence into the appropriate site of the pMTL83151 vector, a CRISPRi expression vector can be constructed to silence the thl gene or other target genes in Clostridium thermoacetica.

[0080] 5. Clone gRNA and dCas9 into CRISPRi vector and extract plasmid DNA

[0081] The PCR product was purified and cut with restriction endonucleases. The pMTL83151 vector was linearized and digested with the same restriction endonucleases. The gRNA DNA fragment was ligated to the linearized pMTL83151 vector using T4 DNA ligase. Amplified, and plasmid DNA was extracted. Three different plasmids were obtained, named CRISPRi-dCas9-pMTL83151-thl plasmid, CRISPRi-dCas9-pMTL83151-aor plasmid, and CRISPRi-dCas9-pMTL83151-thl-aor plasmid.

[0082] 6. Introduction into target cells

[0083] Cultivate Clostridium thermoacetica in reinforced Clostridial medium until the logarithmic growth phase (OD600 is approximately 0.6-0.8) and collect the cells by centrifugation at 58°C. Pretreat Clostridium thermoacetica cells with 1M glycerol solution. Pretreat the collected cells with an equal volume of 1M glycerol solution and let stand at room temperature for 10 minutes. Add the different plasmids in step 1.7 and perform electroporation. Add 2μg of CRISPRi-dCas9-pMTL83151-thl plasmid, CRISPRi-dCas9-pMTL83151-aor plasmid, and CRISPRi-dCas9-pMTL83151-thl-aor plasmid to the cell-electrolyte solution mixture and transfer to the electroporation dish. Perform electroporation using an electroporator:

[0084] Voltage: 1.8kV

[0085] Capacitor: 25uF

[0086] Resistance: 200Ω

[0087] Time constant: 4.5ms

[0088] After electroporation, the bacterial solution was immediately transferred to a fortified Clostridium medium at 58°C and cultured statically for 2 hours, and then expanded with an appropriate dilution multiple.

[0089] The CRISPRi-transformed strains CT-dCas9-thl, CT-dCas9-aor, and CT-dCas9-thl-aor were obtained respectively.

[0090] 7. Genome extraction and positive clone identification

[0091] Take 2 mL of cells cultured to the early stationary phase of strains CT-dCas9-thl, CT-dCas9-aor, and CT-dCas9-thl-aor in enhanced Clostridium medium and wash once. Add 200 μL of ET buffer (500 mM EDTA-10 mM Tris) and 40 μL of mercaptoethanol and incubate at 30°C for 20 min. Centrifuge at 5000 g for 5 min, resuspend the cells in 300 μL of SECP (1 Msorbital-0.1 M EDTA-0.01 M citrate-phosphate, pH = 5.8) containing 20 mg / mL snail enzyme, and incubate at 58°C until lysis. Collect the cells by centrifugation and resuspend them in 0.05 M Tris-0.05 M EDTA buffer. Add 20 μL of 20% SDS to the suspension and incubate in a 60°C water bath for 30 min. After cooling to room temperature, add 160μL 5M potassium acetate solution, shake well, ice bath for 1h, centrifuge at 12000g for 5min, and transfer the supernatant to a new EP tube. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix vigorously, centrifuge at 12000g for 10min, and transfer the supernatant to a new EP tube. Repeat the extraction 1-2 times. Add 1 / 10 volume of 3M sodium acetate solution (pH=6.2) to the supernatant, mix well, add 0.54 volume of pre-cooled isopropanol dropwise, place at -20℃ for 10min, centrifuge at 12000g for 10min, and discard the supernatant. Add 100μL 70% ethanol to the precipitate and wash twice, 10min each time. After drying, dissolve in 30-100μL deionized water or TE buffer containing RNase.

[0092] Using primer sets

[0093] 5'-CACCGTGTGAAAGTGCCCGTGTTCAC-3'(forward)

[0094] 5'-AAACGTGAACACGGCACTTTCACAC-3'(reverse)

[0095] as well as

[0096] 5'-CACCGTGGCAGCGTACTCTGCAGC-3'(forward)

[0097] 5'-AAACTGCTGCAGAGTACGCTGGCCA-3'(reverse)

[0098] Diagnostic PCR identification of positive clones was performed, combined with sequencing verification to confirm positive clones.

[0099] 8. Strain growth test

[0100] Prepare the culture medium: Prepare the Fortified Clostridium Medium and adjust the pH of the Fortified Clostridium Medium to 6.8.

[0101] Pretreatment of anaerobic Clostridium: Four desired strains, Wild (original strain), CT-dCas9-thl, CT-dCas9-aor, and CT-dCas9-thl-aor, were taken out, inoculated into the culture medium, and cultured in a constant temperature shaker at 58°C for 72 hours.

[0102] Prepare a 1L fermenter: sterilize the fermenter using an autoclave and fill it with 500 ml of fortified Clostridium medium.

[0103] Inoculation of bacterial strains: The prepared bacterial strain culture was transferred into the fermentation tank using a sterile pipette, and the final initial cell density was OD600 = 0.05.

[0104] Cultivation process: The fermentation tank was set at a constant temperature of 58°C, the rotation speed in the tank was controlled at 100 rpm, and the growth of the bacterial colony during the cultivation process was monitored.

[0105] pH adjustment: Control the pH value at around 6.8 by adding hydrochloric acid and sodium hydroxide. The amount and time of addition can be adjusted as needed.

[0106] Feed: Hydrogen and carbon dioxide (volume ratio 1:2) were added at a rate of 30 L / hr;

[0107] Detection: After 72 hours of cultivation, OD600nm, acetic acid, ethanol, acetone, butyric acid, butanol, etc. were measured to evaluate bacterial growth and acetic acid production. Specific data are shown in Table 1.

[0108] Table 1 Growth and product status of different strains after 72 hours of culture

[0109]

[0110] As can be seen from Table 1, the four different strains have different selectivities. Silencing the aor gene and the thl gene are both beneficial to increasing the selectivity of acetic acid, especially when both genes are silenced at the same time, the selectivity is the best, reaching 96.8%.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for producing acetic acid, characterized in that: include: Adding enhanced Clostridium to the fermentation broth, introducing a synthetic gas at a specified flow rate, and stirring at a specified temperature and a specified speed to generate acetic acid; The enhanced Clostridium includes Clostridium in which the thl gene and / or the aor gene have been silenced.

2. The method for producing acetic acid according to claim 1, wherein: The th1 gene is used to encode acetyl-CoA acetyltransferase.

3. The method for producing acetic acid according to claim 1, wherein: The aor gene is used to encode aldehyde:chlorophyll iron-sulfur protein oxidoreductase.

4. The method for producing acetic acid according to claim 1, wherein: The enhanced Clostridium is a Clostridium in which the thl gene and / or the aor gene are silenced based on the CRISPRi technology.

5. The method for producing acetic acid according to claim 4, wherein: The gRNA sequence used to silence the th1 gene includes: 5'-CACCGTGTGAAAGTGCCCGTGTTCAC-3'; 5'-AAACGTGAACACGGCACTTTCACAC-3'.

6. The method for producing acetic acid according to claim 1, wherein: The gRNA sequences used to silence the aor gene include: 5'-CACCGTGGCAGCGTACTCTGCAGC-3'; 5'-AAACTGCTGCAGAGTACGCTGGCCA-3'.

7. The method for producing acetic acid according to claim 1, wherein: The enhanced Clostridium includes strain CT-dCas9-thl, strain CT-dCas9-aor, and strain CT-dCas9-thl-aor.

8. The method for producing acetic acid according to claim 1, wherein: The source strain of the enhanced Clostridium is Clostridium thermoacetica.

9. The method for producing acetic acid according to claim 1, wherein: The synthesis gas includes CO, and / or a mixed gas containing CO2 and H2.

10. The method for producing acetic acid according to claim 1, wherein: The specified temperature includes 58°C.

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