Moorella thermoacetica strain as well as preparation method and application thereof

Abstract

The invention relates to a Moorella thermoaceticus strain as well as a preparation method and application thereof, the invention provides the Moorella thermoaceticus strain for synthesizing n-butyl alcohol, the Moorella thermoaceticus strain is Moorelh thermoaceticus GTLB-iB2312 and is preserved in the China General Microbiological Culture Collection Center on September 19, 2024, and the preservation number of the Moorelh thermoaceticus strain is CGMCC No: 46146. The invention further provides a preparation method and application of the Moorelh thermoaceticus strain for synthesizing n-butyl alcohol. Through high expression of the thl gene and simultaneous knockout of silencing pta and ptb genes, the yield of n-butyl alcohol is increased. Through the editing, the concentration and the product conversion rate of the n-butyl alcohol produced by using the synthesis gas, especially hydrogen, as the raw material are greatly increased, more efficient production is realized, and the synthesis efficiency is improved.

Description

Technical Field

[0001] This application relates to the field of fermentation strain technology, and in particular to a strain of *Heat acetamipridae*, its preparation method, and its application. Background Technology

[0002] n-Butanol, also known as butanol, is a colorless liquid alcohol compound with the chemical formula C4H9OH and the structural formula [not provided in the original text]. It is a four-carbon straight-chain alcohol with a wide range of industrial uses, including as a solvent, a raw material for organic synthesis, and applications in the pharmaceutical, fragrance, and plastics industries. In recent years, demand for n-butanol has increased due to its potential as a biofuel additive. n-Butanol has several applications, including: 1. Chemical Raw Material: n-Butanol can be used as a chemical raw material to produce compounds such as butane esters, butyric acid, and butane. These products are widely used in industries such as coatings, plastics, solvents, lubricants, and plasticizers. 2. Solvent: As a solvent, n-Butanol can be used in industries such as paints, inks, cleaning agents, and coatings. Its solubility and volatility make it an important component in many applications. 3. Biofuels and Energy: n-Butanol can be used as a raw material for biofuels. It has high energy density and combustion performance, and may be used to replace some traditional fuels. 4. Pharmaceutical and Medical Applications: n-Butanol also has applications in the pharmaceutical field, serving as an intermediate in drug synthesis or as a solvent for certain drugs. 5. Biomanufacturing: With the increasing demand for sustainable development and green chemicals, the research and application of n-butanol production using microbial fermentation are receiving growing attention. The field of biomanufacturing is exploring the production of chemicals through biosynthesis, and n-butanol, as one of the target compounds, has considerable market potential. Overall, n-butanol has a variety of industrial applications, and the rise of sustainable development and green chemistry has created new market opportunities for it due to the demand for biomanufacturing. Although other alternatives exist on the market today, n-butanol, as an important chemical, retains its significance and value in industry due to its wide range of applications and potential market prospects.

[0003] Currently, the main methods for producing n-butanol include chemical synthesis and microbial fermentation. Chemical synthesis involves producing n-butanol through chemical reactions. Common methods include the gas-phase oxidation of butane and the aqueous-phase hydration of butene. These methods typically rely on petrochemical feedstocks, require high temperatures and pressures, and generate significant byproducts and waste, resulting in substantial environmental impact. Microbial fermentation utilizes the characteristics of microbial metabolites to produce n-butanol through the fermentation of sugars or syngas. In particular, using syngas (mainly composed of CO2, H2, and CO) as a feedstock can significantly improve the sustainability and economic efficiency of n-butanol production.

[0004] *Moorella thermoacetica* is a microorganism capable of surviving and reproducing under strictly anaerobic conditions. It possesses considerable metabolic diversity and potential, making it suitable for biosynthesis using syngas. The primary metabolic pathway for this microorganism's syngas metabolism is the Wood-Ljungdahl Pathway (WL Pathway, also known as the Wood-Ljungdahl pathway or Acetyl-CoA Pathway). This is an important metabolic pathway, such as... Figure 1 As shown, this metabolic pathway is a carbon fixation pathway that allows microorganisms to synthesize organic matter from inorganic compounds such as carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2), thereby enabling autotrophic growth. This pathway has been found in some anaerobic microorganisms, including certain archaea, anaerobic bacteria, and some methanophilic archaea. The key steps of this pathway include the fixation of CO2, its reduction to carbon monoxide (CO), and the final binding and reduction of CO and CO2 to acetyl-CoA. Acetyl-CoA is an important intermediate product that can serve as a precursor to other organic compounds, such as acetic acid and ethanol, and can also be used for the growth of the organism itself.

[0005] Some microorganisms, such as *Moorella thermoacetica*, *Clostridium tyrobutyricum*, *Clostridium acetobutylicum*, or *Butyribacterium methylotrophicum*, can synthesize acetoacetyl-CoA from two acetyl-CoA molecules, and then further metabolize it to produce the four-carbon compounds butyrate or butanol. This metabolic pathway is crucial for some anaerobic microorganisms or microorganisms in other special environments because they can utilize excess acetyl-CoA to produce organic substances such as butyrate or butanol. This is significant for understanding the metabolic pathways and adaptations of microorganisms in different environments. Figure 2 As shown, this is the metabolic pathway for the production of two-carbon (acetic acid, ethanol) and four-carbon (butyric acid, butanol) compounds following the Wood-Ljungdahl Pathway. This diagram illustrates the metabolic pathway from carbon dioxide (CO2) to... )The process begins with hydrogen (H2) and carbon monoxide (CO), and through a series of enzymatic reactions, ultimately synthesizes butyrate and butanol. In this process, CO2, H2, and CO serve as starting materials, entering the WL pathway (a specific microbial metabolic pathway in Clostridium), where a series of complex enzymatic reactions generate intermediates such as acetic acid or acetyl-CoA. These intermediates are key precursors for the subsequent synthesis of butyrate and butanol. Multiple enzymes play crucial roles in this metabolic pathway. For example, ACK and PTA enzymes are involved in the synthesis of acetyl groups, while adhE1 and bdhAB are related to the production of ethanol. These enzymatic reactions often require energy; ATP, mentioned in the diagram, serves as the primary energy source, driving these reactions. If the metabolic pathway extends further, it can generate four-carbon products such as acetoacetate and acetoacetyl-CoA as intermediates. These substances undergo a series of transformations to produce acetone and 3-hydroxybutyryl-CoA. Finally, the metabolic pathway branches to produce butyrate and butanol. Butyrate synthesis involves intermediates such as Butyryl-P and Butyryl-CoA, while butanol synthesis is closely related to Butyraldehyde. Enzymes such as buk, ptb, adhE2, and bdhAB play key roles in this process, catalyzing the final formation of butyrate and butanol. Furthermore, the diagram also involves several cofactors such as NADH and NAD. + They play a role in electron transfer in redox reactions, helping to drive the oxidation and reduction steps in metabolic pathways.

[0006] In syngas fermentation, hydrogen (H2) and carbon monoxide (CO) serve as energy and electron sources, playing a crucial role in the entire metabolic process. Hydrogenase and carbon monoxide dehydrogenase (CODH) are two key enzymes involved in converting H2 and CO into cellularly usable forms. The role of hydrogenase and CODH is to convert H2 and CO into cellularly usable electrons, which can then be used to synthesize acetyl-CoA, leading to ethanol, acetic acid, or other organic compounds, as well as ATP synthesis. Hydrogenase and CODH themselves can utilize hydrogen, or carbon monoxide (CO) and water (H2O), to generate carbon dioxide (CO2) and electrons, which can be used to synthesize ATP. Therefore, the activity of these two enzymes is critical to the efficiency of the entire fermentation process. The efficiency of hydrogenase and CODH directly affects the efficiency of the electron transport chain, and consequently, the efficiency of syngas conversion into the target products. In optimizing syngas fermentation, in addition to considering the activities of hydrogenases and CODH, it is also necessary to consider the regulation of the entire metabolic network, including enzyme expression levels, the concentration of metabolic intermediates, the regeneration and balance of NADH / NAD+ cofactors, and the conditions of the intracellular and external environments. Through metabolic engineering and fermentation process optimization, the efficiency of these enzymes can be improved, thereby increasing the overall production efficiency and product selectivity of the fermentation process.

[0007] While the production of n-butanol from syngas offers numerous potential advantages, several challenges also exist. *Moorella thermoacetica*, a microorganism capable of surviving and reproducing under strictly anaerobic conditions, possesses considerable metabolic diversity and potential, making it suitable for biosynthesis using syngas. To enable *Moorella thermoacetica* to effectively utilize CO2, CO, and H2 from syngas and produce the target product n-butanol, optimization of the microbial metabolic pathways is necessary, including at the genetic level and the control of NADH and NAD+ cofactor balance. Summary of the Invention

[0008] Therefore, it is necessary to provide a strain of *Heat-Muller's bacterium* that can be used for fermentation to produce n-butanol and to increase the yield of n-butanol, as well as its preparation method and application.

[0009] Firstly, this application provides a strain of *Moorella thermoacetica*, GTLB-iB2312, which was deposited on September 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No:46146. This strain activates the expression of the thl gene and knocks out the pta and ptb genes. The taxonomic name of the strain with accession number CGMCC No:46146 is *Moorella thermoacetica*, and the address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Secondly, this application also provides a strain of *Heat-acetylcholine* for synthesizing n-butanol, obtained by activating the expression of the thl gene of *Heat-acetylcholine* using CRISPR-Cas9 technology and knocking out the pta and ptb genes using CRISPRi technology.

[0011] Thirdly, this application also provides a *Moorella thermoacetica* strain for synthesizing n-butanol. The *Moorella thermoacetica* strain is *Moorella thermoacetica* GTLB-iB2403, which was deposited on September 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:46145. This strain activates the expression of the thl gene and hydrogenase gene and knocks out the pta and ptb genes. The taxonomic name of the strain with accession number CGMCC No:46145 is *Moorella thermoacetica*, and the address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] Fourthly, this application also provides a strain of *Heterocarpus thermophilus* for synthesizing n-butanol, obtained by activating the expression of the thl gene and hydrogenase gene of *Heterocarpus thermophilus* using CRISPR-Cas9 technology, and knocking out the pta and ptb genes using CRISPRi technology.

[0013] Fifthly, this application also provides a method for preparing the *Hormone thermophilus* strain for synthesizing n-butanol as described in any of the above embodiments, comprising the following steps:

[0014] The gRNA sequence for activating the thl gene was designed; the synthesized Cas9, gRNA sequence, and amplified thl gene were cloned into a linearized plasmid using polymerase chain reaction to form the pET-22b-Cas9-gRNA-thl complex vector; the pET-22b-Cas9-gRNA-thl complex vector was introduced into *Thermus thermophilus* cells by electroporation; after electroporation, the bacterial culture was transferred to YPD medium and cultured on a medium containing selective antibiotics to screen for positive strains, thus obtaining *Thermus thermophilus* + thl strain containing the overexpression of the thl gene;

[0015] GRNA sequences for silencing the pta and ptb genes were designed and cloned into the CRISPRi vector. dCas9-KRAB was selected as the vector and linearized. The vector was then ligated to the gRNA using DNA ligation to construct the CRISPRi-KRAB vector. The CRISPRi-KRAB plasmid was amplified and extracted. The CRISPRi-KRAB vector was introduced into the target cells *Moorelh themoaceca* + THL strain using electroporation. After electroporation, the bacterial culture was transferred to MRS medium and cultured on a medium containing selective antibiotics to screen for positive strains. The *Moorelh themoaceca* + THLΔptaΔptb strain with silenced pta and ptb genes was obtained, which is *Moorelh themoaceca* GTLB-iB2312.

[0016] Design a gRNA sequence to activate the hydrogenase gene; clone the gRNA sequence into the CRISPRa vector, wherein pcDNA-dCas9-VP64 was selected as the vector and linearized, and the vector was ligated with the gRNA using a DNA ligation reaction to construct the CRISPRa-VP64 vector; amplify the vector and extract the CRISPRa-VP64 plasmid; use electroporation to introduce the CRISPRa-VP64 vector into the target cell strain *Moorelh themoaceca* + thlΔptaΔptb, and after electroporation, transfer the bacterial culture to YPD medium for culture, and culture on a medium containing selective antibiotics to screen for positive strains to obtain *Moorelh themoaceca* + thlΔptaΔptb+H2 strain with activated hydrogenase gene; this is the *Moorelh themoaceca* strain *Moorelh themoaceca* GTLB-iB2403 used for the synthesis of n-butanol.

[0017] In one embodiment, the step of activating the th1 gene involves activating the forward sequence of the th1 gene as: 5'-GGCCGGCGAGGCTGCCGGTG-3'; and the reverse sequence as: 5'-CACCGGCAGCCTCGCCGGCC-3'.

[0018] And / or, in the step of activating the hydrogenase gene, the gRNA sequence for activating the hydrogenase gene is: positive strand gRNA: 5'-AGGCTGGAGTGGTGTCCTGC-3'; negative strand gRNA: 5'-GCAGACACCCTCCAGCCT-3'.

[0019] In one embodiment, the electroconversion conditions in the step of activating the hydrogenase gene are: voltage: 1.8kV, capacitance: 25uF, resistance: 200Ω, and time constant: 8.5ms.

[0020] In one embodiment, the gRNA sequence for silencing the pta and ptb genes is as follows:

[0021] Positive chain forward sequence: 5'-GGCCGGCGAGGCTGCCGGTG-3';

[0022] Reverse sequence: 5'-CACCGGCAGCCTCGCCGGCC-3';

[0023] gRNA sequence for silencing the ptb gene:

[0024] Forward sequence: 5'-GGGCTGCCGGCGGCGCCGGC-3'

[0025] Reverse sequence: 5'-GCCGGCGCCGCCGGCAGCCC-3'.

[0026] Sixthly, this application also provides a method for producing n-butanol by syngas fermentation, wherein n-butanol is prepared by fermentation using the *Heterotrophic acetamipridae* strain used for n-butanol synthesis as described in any of the above embodiments.

[0027] In one embodiment, the syngas includes hydrogen, and also includes at least one of carbon dioxide and carbon monoxide.

[0028] In one embodiment, the culture pH is controlled at 6.8; and / or the culture temperature is controlled at 58°C ± 0.5°C.

[0029] Seventhly, this application provides a method for producing n-butanol by fermentation using syngas. The syngas includes at least two of hydrogen, carbon dioxide, and carbon monoxide. The fermentation strain includes at least one of Moorella thermoacetica, Clostridium tyrobutyricum, Clostridium acetobutylicum, or Butyribacterium methylotrophicum. The method involves overexpressing the thl gene in the fermentation strain using CRISPR-Cas9 technology and knocking out the pta and ptb genes using CRISPRi technology. In other words, the method for editing the Moorella thermoacetica strain of this application can also be applied to other syngas-producing strains.

[0030] The Moorelh themoaceca GTLB-iB2312 strain for synthesizing n-butanol provided in this application increases the yield of n-butanol by overexpressing the thl gene and simultaneously knocking out and silencing the pta and ptb genes.

[0031] The *Moorelh themoaceca* strain GTLB-iB2403 provided in this application for the synthesis of n-butanol, by overexpressing the *thl* gene and the hydrogenase gene, while simultaneously knocking out and silencing the *pta* and *ptb* genes, and by overexpressing the hydrogenase gene, increases the ability to produce four-carbon products, thereby improving the yield of n-butanol. These modifications significantly increase the concentration and product conversion rate of n-butanol produced using syngas, especially hydrogen, as a feedstock, achieving more efficient production and improving synthesis efficiency.

[0032] This application presents a promising, sustainable, and innovative method for producing n-butanol using syngas (a mixture of CO2, CO, and H2) as a feedstock. This method utilizes gases, rather than traditional sugar compounds, as the feedstock for microbial fermentation, offering several advantages and potential benefits. Interestingly, in experiments involving syngas fermentation to produce butanol, it was found that when the system contained only H2 and CO2, it could successfully utilize hydrogen, achieving an OD of approximately 6.8 and a butanol concentration of approximately 21 g / L. When the system contained only CO and CO2, it could successfully utilize CO, achieving an OD of approximately 12 and a butanol concentration of approximately 38 g / L. This demonstrates that the *Heterotrophic acetamipridae* strain used in this application for n-butanol synthesis has significant industrial value. Attached Figure Description

[0033] Figure 1This is a schematic diagram of the Wood-Ljungdahl Pathway (also known as the Acetyl-CoA Pathway) metabolic pathway.

[0034] Figure 2 A schematic diagram of the metabolic pathway for the production of two-carbon (acetic acid, ethanol) and four-carbon (butyric acid, butanol) compounds following the Wood-Ljungdahl Pathway;

[0035] Figure 3 This is a comparative schematic diagram of OD600nm for Examples 1 to 3;

[0036] Figure 4 This is a comparative schematic diagram of the n-butanol yield in Examples 1 to 3;

[0037] Figure 5 This is a comparative diagram of the NADH / NAD+ ratios in Examples 1 to 3. Detailed Implementation

[0038] To facilitate understanding of this application and to make the aforementioned objectives, features, and advantages of this application more apparent, a detailed description of specific embodiments of this application is provided below in conjunction with the accompanying drawings. Numerous specific details are set forth in the following description to provide a thorough understanding of this application, and preferred embodiments are shown in the accompanying drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application. This application can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application; therefore, this application is not limited to the specific embodiments disclosed below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0039] The n-butanol involved in this application, also known as butanol, is a colorless liquid alcohol compound with the chemical formula C4H9OH. It is a four-carbon straight-chain alcohol with a wide range of industrial uses, including as a solvent, a raw material for organic synthesis, and applications in the pharmaceutical, fragrance, and plastics industries. In recent years, the demand for n-butanol has increased due to its potential as a biofuel additive.

[0040] The hydrogenase involved in this application functions as follows: A hydrogenase is an enzyme that catalyzes the oxidation of hydrogen molecules (H2), thereby releasing electrons (e-) and protons (H+). These electrons and protons can be used in other reactions during cellular respiration. Reaction equation:

[0041] H2→2H + +2e

[0042] The carbon monoxide dehydrogenase (CODH) involved in this application functions as follows: CODH catalyzes the oxidation of carbon monoxide (CO) to produce carbon dioxide (CO2), releasing electrons and protons in the process. In this process, CO acts as an electron donor, which can further participate in the synthesis of acetyl-CoA during syngas fermentation. Reaction equation:

[0043] CO + H₂O → CO₂ + 2H₂O + +2e

[0044] Considering that in real biological metabolic environments, hydrogen and carbon monoxide almost never react alone as described above, but rather are accompanied by a series of more efficient cofactor reactions, such as coupling reactions with NADH / NAD+. Reaction equation:

[0045]

[0046] During syngas fermentation, hydrogenases and CODH (hydrogenase-co-dihydrogenase) convert H2 and CO into cellularly usable electrons. These electrons can then be used to synthesize acetyl-CoA, which in turn produces ethanol, acetic acid, or other organic compounds, along with ATP synthesis. Hydrogenases and CODH themselves can utilize hydrogen, or carbon monoxide (CO) and water (H2O), to generate carbon dioxide (CO2) and electrons, which can then be used to synthesize ATP.

[0047] Therefore, to increase the yield of n-butanol, it is necessary to overexpress the thl gene involved in the metabolic pathway and knock out or silence the pta and ptb genes to improve the strain's method of producing n-butanol from syngas. It should be noted that PTA stands for Phosphotransacetylase, and PTB stands for Phosphotransferase Butyrate.

[0048] Acetyl-CoA acetyltransferase (THL or thiolase), also known as a thiolytic enzyme, catalyzes the conversion of the two-carbon intermediate acetyl-CoA into the longer-chain product acetoacetyl-CoA, which is then further converted into four-carbon compounds such as butyrate and butanol. These products are important byproducts of syngas fermentation and can be used as chemical feedstocks or fuels. Regulating the activity and selectivity of the THL enzyme has a significant impact on product generation and the efficiency of syngas fermentation. Through gene editing, gene expression regulation, or metabolic engineering, the expression level of the THL enzyme or its catalytic properties can be adjusted to control product selectivity and yield. The THL gene is used to regulate the expression level of the THL enzyme. It should be noted that unmodified wild-type *Moorella thermoacetica* can use syngas to produce two-carbon compounds such as acetic acid or ethanol. However, its ability to synthesize higher-carbon-chain compounds, such as four-carbon butanol and butyrate, is insufficient. The main issue is the lack of the key enzyme (gene) for the conversion of two-carbon compounds to four-carbon compounds, specifically the gene for synthesizing acetyl-CoA. Other microorganisms, such as *Clostridium tyrobutyricum*, *Clostridium acetobutylicum*, or *Butyribacterium methylotrophicum*, can synthesize acetyl-CoA from two acetyl-CoA molecules, and then further metabolize it to produce the four-carbon compounds butyrate or butanol. This metabolic pathway is crucial for some anaerobic microorganisms or microorganisms in other special environments, as they can utilize excess acetyl-CoA to produce organic substances like butyrate or butanol.

[0049] It's important to note that CRISPR-Cas technology is a gene-editing technique that utilizes the natural immune system of bacteria and archaea. By designing appropriate primers (gRNA) and proteins (Cas9), it targets and cuts the target DNA, enabling gene insertion, modification, or knockout. Compared to traditional gene-editing techniques such as ZFN and TALEN, CRISPR-Cas technology offers advantages such as high efficiency, simplicity, and applicability to a wide range of organisms, thus finding widespread application in research. Developing new strains from wild-type fungi using CRISPR-Cas gene editing technology is a rapidly developing field, opening up limitless possibilities for novel strain development. The latest CRISPR technologies can activate or silence target genes without adding foreign genes; these are called CRISPRa and CRISPRi technologies, respectively.

[0050] CRISPRa (CRISPR Activation) is a gene editing technology based on the CRISPR-Cas system, but it differs from traditional CRISPR-Cas9 methods used for gene knockout or knock-in. CRISPRa utilizes the DNA-binding ability of the Cas9 protein, rather than its nuclease activity, to activate the expression of specific genes. By introducing a Cas9 protein with its nuclease activity removed (dead Cas9, or dCas9), it does not cut DNA and can be safely used for gene regulation. The gRNA bound to dCas9 guides the dCas9 protein to precisely bind to the target DNA sequence. The CRISPRa system incorporates transcription activation domains (such as p65 and VP64), which can recruit intracellular transcription machinery and enhance the transcriptional activity of the target gene. By guiding the dCas9 protein and transcription activation domains to specific gene promoter regions, CRISPRa can increase the expression level of specific genes. Furthermore, by designing different gRNAs, CRISPRa can be programmed to activate different genes, providing a flexible tool for gene expression regulation.

[0051] This application describes the editing of *Moorella thermoacetica* strains through three main steps: first, activating the th1 gene for high expression; second, knocking out and silencing the pta and ptb genes; and third, reactivating the hydrogenase gene. In previous work, CRISPR-Cas9 technology was used to introduce high expression of the th1 gene, and simultaneously, CRISPRi technology was used to precisely edit the genome of *Moorella thermoacetica*. By knocking out the pta and ptb genes, the flow of side branches in the metabolic pathway was reduced, resulting in an enhanced strain, *Moorella thermoacetica+th1ΔptaΔptb*, also known as strain *Moorella thermoacetica* GTLB-iB2312, achieving high production of n-butanol. Experiments show that microorganisms such as *Clostridium* can utilize hydrogen (H2), carbon monoxide (CO), and carbon dioxide (CO2) as carbon and energy sources for growth and butanol production. However, under different gas composition conditions, the growth of microorganisms and the yield of butanol vary significantly. CO is clearly superior to H2. The specific process described above is as follows:

[0052] Step 1: Activate the th1 gene for high expression

[0053] It's important to note that unmodified wild-type *Moorella thermoacetica* can use syngas to produce two-carbon compounds such as acetic acid or ethanol. However, it lacks the ability to synthesize higher-carbon compounds, such as four-carbon butanol and butyric acid. This is mainly due to a deficiency in the key enzyme (gene) for converting two-carbon compounds to four-carbon compounds—specifically, the gene for synthesizing acetyl-CoA. Other microorganisms, such as *Clostridium tyrobutyricum*, *Clostridium acetobutylicum*, or *Butyribacterium methylotrophicum*, can synthesize acetyl-CoA from two acetyl-CoA molecules, and then further metabolize it to produce four-carbon compounds like butyrate or butanol. This metabolic pathway is crucial for some anaerobic microorganisms or microorganisms in special environments, as they can utilize excess acetyl-CoA to produce organic substances like butyrate or butanol. Acetyl-CoA acetyltransferase (THL or thiolase), also known as a thiolytic enzyme, catalyzes the conversion of the two-carbon intermediate acetyl-CoA into the longer-chain product acetoacetyl-CoA, which is then further converted into four-carbon compounds such as butyrate and butanol. These products are important byproducts of syngas fermentation and can be used as chemical feedstocks or fuels. Regulating the activity and selectivity of the THL enzyme has a significant impact on product generation and the efficiency of syngas fermentation. Through gene editing, gene expression regulation, or metabolic engineering, the expression level of the THL enzyme or its catalytic properties can be adjusted to regulate product selectivity and yield. The THL gene is used to regulate the expression level of the THL enzyme.

[0054] CRISPR-Cas9 is a gene editing technology. Cas9 (CRISPR-associated protein 9) is a protein in the CRISPR system. CRISPR-Cas9 consists of a CRISPR sequence and the Cas9 protein, enabling precise gene editing targeting specific gene sequences. Based on CRISPR-Cas9 technology, it is possible to prepare Moorella thermoacetica containing overexpression of the thl gene.

[0055] To better activate the expression of the thl gene, in one embodiment, the thl gene derived from Clostridium tyrobutyricum can be inserted into Moorella thermoacetica to overexpress acetoacetyl-CoA. Specifically, the pET-22b plasmid (developed by Novagen, Inc.) is used as the base vector. This plasmid is approximately 5500 bp in size, exhibits high expression levels, and is cloned using a multiple cloning site and restriction endonucleases. The plasmid contains the T7 transcription promoter, an ampicillin resistance gene selection marker, and other expression elements and tags. Restriction endonucleases are used to linearize the plasmid vector. A suitable gRNA sequence for activating the thl gene can be designed, with the forward sequence being 5'-GGGTGTGAAATATCGCCCGG-3' and the reverse sequence being 5'-CCGGCGATATTTCACTACCC-3'.

[0056] The gRNA sequence was synthesized in vitro to match a specific sequence in the target region of the thl gene. Using polymerase chain reaction (PCR), the synthesized Cas9, gRNA sequence, and amplified thl gene were cloned into a linearized plasmid to form the pET-22b-Cas9-gRNA-thl complex vector. *Moorella thermoacetica* was strictly anaerobic cultured in YPD medium (a yeast culture medium) at 55°C until the OD (absorbance) of the strain reached approximately 0.5. Then, the constructed complex vector was introduced into bacterial cells using electroporation. Electroporation was performed using the following parameters: voltage: 1.8 kV; capacitance: 25 uF; resistance: 200 Ω; time constant: 8.5 ms. After electroporation, the bacterial culture was immediately transferred to YPD medium at 55°C and statically cultured for 2 hours, followed by expansion culture at an appropriate dilution. After culturing for another 72 hours, the bacteria were cultured on a medium containing selective antibiotics to screen for positive strains, resulting in Moorellathermoacetica strains that overexpress the thl gene, also known as Moorella thermoacetica+thl strains, or Moorella thermoacetica+thl strains.

[0057] Step 2: Knockout and silence PTA and PTB genes

[0058] After activating the thl gene as described above, the pta and ptb genes are then knocked out using CRISPRi technology. The specific process is as follows:

[0059] To increase the synthesis of the target product n-butanol, a "strong trunk, weak branches" strategy was adopted. The CRISPRi gene silencing technology was used to silence the key gene pta for acetic acid production and the key gene ptb for butyric acid production, resulting in a Moorella thermoacetica strain with the gene phenotype ΔptaΔptb. In this application, the dCas9 protein (or simply Sp-dCas9) from Streptococcus pyogenes was selected. This protein is a commonly used dCas9 protein variant. It is derived from the Cas9 protein in Streptococcus pyogenes strain through modification for CRISPRi (CRISPR interference) and other gene regulation applications. The Sp-dCas9 protein retains the DNA-binding ability of the Cas9 protein and can directionally recognize and locate target DNA sequences by binding to specific gRNA sequences, but its nuclease activity has been inactivated. By binding an appropriate gRNA to the Sp-dCas9 protein, a dCas9-gRNA complex can be formed. This complex can bind to the target DNA sequence and regulate gene expression by blocking transcription or other mechanisms, thereby achieving gene silencing.

[0060] In this application, the CRISPRi technology was used to silence the key pta and ptb genes. The pre-cultured Moorellathermoacetica+thl bacterial culture was inoculated into a 250 mL shake flask, 100 mL of sterilized YPD medium was added, and then the culture was carried out on a constant temperature shaker at 55 °C and 200 rpm for 48 to 72 hours.

[0061] Visit a professional genome website (such as https: / / www.ncbi.nlm.nih.gov / ) and search for "pta[Moorella thermoacetica]" and "ptb[Moorella thermoacetica]" to obtain detailed information about the corresponding genes.

[0062] gRNA sequences were designed for phosphotransacetylase (PTA) and phosphatebutyryltransferase (PTB), and matched to exon regions of the genes. The following two sets of optimal gRNA sequences were obtained, such as the gRNA sequence for PTA:

[0063] Forward sequence: 5'-GGCCGGCGAGGCTGCCGGTG-3'

[0064] Reverse sequence: 5'-CACCGGCAGCCTCGCCGGCC-3';

[0065] gRNA sequence of PTB:

[0066] Forward sequence: 5'-GGGCTGCCGGCGGCGCCGGC-3'

[0067] Reverse sequence: 5'-GCCGGCGCCGCCGGCAGCCC-3';

[0068] The above gRNA sequences were synthesized and amplified using PCR.

[0069] Using the dCas9-KRAB vector as the suitable CRISPRi vector for this project, the appropriate gRNA was cloned into the CRISPRi vector, and plasmid DNA was extracted. The PCR product was purified and digested with restriction endonucleases. The dCas9-KRAB vector was linearized and digested with the same restriction endonucleases. The gRNA DNA fragment was ligated into the linearized dCas9-KRAB vector using T4 DNA ligase. Amplification was performed, and plasmid DNA was extracted and named the CRISPRi-KRAB plasmid.

[0070] The CRISPRi-KRAB plasmid was introduced into the target cell strain *Moorella thermoacetica* + thl using electroporation. The target cells were pre-cultured in YPD medium to the logarithmic growth phase (OD600 approximately 0.6-0.8) and collected by centrifugation at 55°C. *Moorella thermoacetica* + thl cells were pretreated with 1M glycerol solution. The collected cells were then pretreated with an equal volume of 1M glycerol solution and incubated at room temperature for 10 minutes. The CRISPRi-KRAB plasmid was added and electroporated. 2 μg of the CRISPRi-KRAB plasmid was added to a cell-electrolyte mixture and transferred to an electroporation dish. Electroporation was performed using an electroporator: voltage: 1.8 kV, capacitance: 25 μF, resistance: 200 Ω, time constant: 8.5 ms. After electroporation, the bacterial culture was immediately transferred to MRS medium at 55°C and statically cultured for 2 hours, followed by expansion culture with appropriate dilutions. The strain Moorella thermoacetica+thlΔptaΔptb obtained by CRISPRi-KRAB electrotransfection, also known as Moorella thermoacetica+thlΔptaΔptb strain, is named Moorelh themoaceca GTLB-iB2312. The applicant deposited it with the China General Microbiological Culture Collection Center (CGMCC) on September 19, 2024, with accession number CGMCC No:46146.

[0071] Step 3: Activate the hydrogenase gene

[0072] It is worth noting that industrial applications often contain hydrogen, such as steel tail gas and coal chemical off-gas. Therefore, it is necessary to develop methods that enable microorganisms to achieve, or even exceed, cell concentrations and butanol concentrations when utilizing hydrogen as a substrate, while also utilizing CO. Thus, CRISPRa technology was used to overexpress hydrogenase to promote more efficient hydrogen utilization. Simultaneously, the NADH / NAD+ ratio of the system was measured to assess the impact of hydrogenase overexpression on cofactors and to discuss its relationship with butanol production. In other words, the syngas source in this application can be steel tail gas, coal chemical off-gas, etc.

[0073] This application selects *Moorella thermoacetica* strains because *Moorella thermoacetica* is a microorganism capable of surviving and reproducing under strictly anaerobic conditions, possessing certain metabolic diversity and potential, making it suitable for biosynthesis using syngas. The main metabolic pathway for this microorganism's syngas metabolism is the WL pathway. Previously, *Moorella thermoacetica* strains enhanced using a gene-editing technique were first created, resulting in strains with the gene phenotype +thlΔptaΔptb. These strains were found to synthesize highly selective n-butanol. Here, the dCas9 protein from *Streptococcus pyogenes* (or simply Sp-dCas9) is selected. This protein is a commonly used dCas9 protein variant. It is obtained by modifying the Cas9 protein from *Streptococcus pyogenes* strains for CRISPRa (CRISPR activation) and other gene regulation applications. The Sp-dCas9 protein retains the DNA-binding ability of the Cas9 protein, and can directionally recognize and locate target DNA sequences by binding to specific gRNA sequences, but its nuclease activity has been inactivated. By binding an appropriate gRNA to the Sp-dCas9 protein, a dCas9-gRNA complex can be formed. This complex can bind to the target DNA sequence and regulate gene expression by blocking transcription or other mechanisms, thereby achieving gene regulation.

[0074] In this patent application, the applicant used a modified Moorella thermoacetica strain (an earlier-formed strain of Moorella thermoacetica + thlΔptaΔptb) as a basis to search for the intrinsic hydrogenase gene sequence within it. For the selected Cas9 protein (SpCas9) derived from Streptococcus pyogenes, the recognized PAM sequence is 5′-NGG-3′.

[0075] Designing gRNA sequences to activate hydrogenase: From the hydrogenase RNA sequence, select a gRNA sequence located upstream of the transcription start site for design. A set of gRNA sequences is obtained, and the most suitable sequence is selected, such as: positive strand gRNA: 5'-AGGCTGGAGTGGTGTCCTGC-3'; negative strand gRNA: 5'-GCAGACACCCTCCAGCCT-3';

[0076] Synthesize and amplify the above gRNA sequences using PCR;

[0077] Cloning gRNA into the CRISPRa vector: pcDNA-dCas9-VP64 was selected as the vector and linearized. The vector was then ligated to the gRNA using a DNA ligation reaction to construct the CRISPRa-VP64 vector. pcDNA-dCas9-VP64 is a commonly used CRISPRa gene editing tool. It is a plasmid vector that fuses the receptor protein dCas9 (deactivated Cas9) with the transcription activator VP64 (viral protein 64). dCas9 is the non-functional form of the Cas9 protein, having lost its endonuclease activity, but it can still interact with the gRNA targeting the DNA sequence, forming a dCas9-gRNA complex. This brings the VP64 activator closer to the promoter region of the target gene, thereby enhancing the expression of the target gene. The pcDNA-dCas9-VP64 vector is commonly used for promoter activation experiments and high-throughput screening and can be used for transfection in various mammalian cell lines.

[0078] CRISPRa-VP64 plasmid extraction in this application: CRISPRa plasmid was extracted using a plasmid extraction kit;

[0079] Transfection with CRISPRa-VP64 vector: The CRISPRa-VP64 vector was introduced into the target cells Moorella thermoacetica+thl using electroporation to obtain a new strain overexpressing Hydrogenase, Moorella thermoacetica+thlΔptaΔptb+H2, which is the Moorella thermoacetica+thlΔptaΔptb+H2 strain used for the synthesis of n-butanol.

[0080] In other words, overexpressing the Clostridium hydrogenase gene using CRISPR-Cas9 technology, i.e., overexpressing the hydrogenase gene using CRISPR-Cas9 technology, is carried out as follows: a) Using the pcDNA-dCas9-VP64 plasmid as the base vector, this plasmid is widely used in protein expression and purification. Restriction endonucleases are used to linearize the plasmid vector; b) Designing suitable gRNA sequences. Positive strand gRNA: 5'-AGGCTGGAGTGGTGTCCTGC-3'; Negative strand gRNA: 5'-GCAGACACCCTCCAGCCT-3'; This gRNA sequence is synthesized in vitro to match the PAM-specific sequence of the target region of the hydrogenase gene. c) Using polymerase chain reaction (PCR), the synthesized Cas9, gRNA sequence, and amplified hydrogenase gene are cloned into the linearized plasmid to form the pcDNA-dCas9-VP64-Cas9-gRNA-hydrogenase complex vector. d) *Moorellathermoacetica* was cultured at 55°C in YPD medium under strictly anaerobic conditions until the OD reached approximately 0.5. The constructed plasmid was then introduced into the bacterial cells using electroporation. Electroporation was performed using a microplate reader with the following settings: voltage: 1.8 kV, capacitance: 25 uF, resistance: 200 Ω, time constant: 8.5 ms. Immediately after electroporation, the bacterial culture was transferred to YPD medium at 55°C and statically cultured for 2 hours, followed by expansion culture at an appropriate dilution. After culturing for another 72 hours, the bacteria were cultured on a medium containing selective antibiotics to screen for positive strains. The strain Moorella thermoacetica+thlΔptaΔptb+H2, which overexpresses the hydrogenase gene, was obtained. This strain is the Moorella thermoacetica+thlΔptaΔptb+H2 strain, which is the Moorella thermoacetica strain used to synthesize n-butanol, namely Moorelhthemoaceca GTLB-iB2403. The applicant deposited it with the China General Microbiological Culture Collection Center (CGMCC) on September 19, 2024, with accession number CGMCC No:46145.

[0081] Subsequently, recombinant strains can be cultured from the above-mentioned strains, growth curves can be measured, and butanol production and NADH / NAD+ ratio can be detected.

[0082] The following specific embodiments illustrate the basic strains that highly express the thl gene in this application. The Moorella thermoacetica strains in the following three embodiments are all Moorella thermoacetica+thlΔptaΔptb, that is, Moorella thermoacetica+thlΔptaΔptb strains, which are the Moorella thermoacetica strains used to synthesize n-butanol.

[0083] Example 1:

[0084] Anaerobic growth and production experiment of the basic strain Moorella thermoacetica GTLB-iB2312 in H2 and CO2 environments

[0085] Prepare the culture medium: Prepare a Clostridium perfringens culture medium and adjust the pH of the Clostridium perfringens culture medium to 6.8.

[0086] Pretreatment of anaerobic Clostridium: Take out the basic strain, Moorella thermoacetica+thlΔptaΔptb, inoculate it into the culture medium, and incubate it in a constant temperature shaker at 58℃ for 72 hours.

[0087] Prepare a 1L fermenter: Sterilize the fermenter using an automatic sterilizer and fill it with 500ml of enhanced Clostridium culture medium.

[0088] Inoculation: The prepared bacterial culture was transferred into the fermenter using a sterile pipette, so that the initial cell density was OD600 = 0.05.

[0089] Cultivation process: The fermenter was set to a constant temperature of 58℃, and the rotation speed inside the fermenter was controlled at 100 rpm. The growth of the microbial community was monitored during the cultivation process.

[0090] pH adjustment: The pH value is controlled at around 6.8 by adding hydrochloric acid and sodium hydroxide. The amount and time of addition can be adjusted as needed.

[0091] Feeding: Add hydrogen and carbon dioxide (volume ratio 2:1) at a rate of 30L / hr;

[0092] Detection: After 72 hours of incubation, OD600nm, n-butanol, NADH / NAD+, etc. are measured every 12 hours to assess bacterial growth and product production.

[0093] For detailed data, please see... Figures 3 to 5Group 1. The horizontal axis represents time (hours). In this process, OD600nm was measured using a UV-Vis spectrophotometer, n-butanol concentration was measured using HPLC, and the NADH / NAD+ ratio was measured using an enzymatic method. The enzymatic method for measuring NADH / NAD+ is a method that uses an enzyme-catalyzed reaction to determine the content and ratio of NAD+ and NADH in a sample. It involves the following steps: Sample preparation: After sampling, cells are lysed and tissue homogenized using a high-pressure homogenizer. Acidic and alkaline treatment: Utilizing the difference in stability of NAD+ and NADH at different pH values, the content of both can be determined separately by treating the sample under acidic or alkaline conditions. Equal proportions of 5N hydrochloric acid or 5N NaOH solution are added. Under alkaline conditions, NAD+ is destroyed by heating, while NADH is relatively stable; under acidic conditions, NADH is unstable, while NAD+ remains stable. Neutralization: The sample treated with acid or alkaline is neutralized to facilitate the next enzymatic determination. Enzyme cycling reaction: NAD+ / NADH-Glo TM The detection reagent contains NAD cycling enzyme, reductase, reductase substrate, and luciferin detection reagent. The NAD cycling enzyme converts NAD+ to NADH, and the reductase reduces proluciferin (the reductase substrate) to luciferin. Detection by fluorescence: Luciferin is detected via Ultra-Glo... TM Quantification of rLuciferase: The generated light signal is proportional to the amount of NAD+ and NADH in the sample. Ratio calculation: The NAD+ to NADH ratio can be calculated by measuring the luminescence values ​​of acid-treated and alkaline-treated samples. Standard curve preparation: A standard curve is prepared using purified NAD+ or NADH to quantify the concentrations of NAD+ and NADH in the sample. Data analysis: Based on the intensity of the luminescence signal, the concentrations of NAD+ and NADH in the sample are calculated using the standard curve, and the NADH / NAD+ ratio is obtained accordingly. Specific steps are as follows: The sample is compared with NAD / NADH-Glo... TM The test reagents were mixed. After incubation for a period of time, the luminescence signal was measured using a luminescence detector. Based on the intensity of the luminescence signal, the concentrations of NAD+ and NADH were calculated by comparing it with a standard curve. The NADH / NAD+ ratio was calculated by comparing the luminescence signals of acid-treated and alkaline-treated samples.

[0094] Example 2: Anaerobic growth and production experiment of the basic strain Moorella thermoacetica GTLB-iB2312 in CO and CO2 environments.

[0095] Prepare the culture medium: Prepare a Clostridium perfringens culture medium and adjust the pH of the Clostridium perfringens culture medium to 6.8.

[0096] Pretreatment of anaerobic Clostridium: Take out the basic strain, Moorella thermoacetica+thlΔptaΔptb, inoculate it into the culture medium, and incubate it in a constant temperature shaker at 58℃ for 72 hours.

[0097] Prepare a 1L fermenter: Sterilize the fermenter using an automatic sterilizer and fill it with 500ml of enhanced Clostridium culture medium.

[0098] Inoculation: The prepared bacterial culture was transferred into the fermenter using a sterile pipette, so that the initial cell density was OD600 = 0.05.

[0099] Cultivation process: The fermenter was set to a constant temperature of 58℃, and the rotation speed inside the fermenter was controlled at 100 rpm. The growth of the microbial community was monitored during the cultivation process.

[0100] pH adjustment: The pH value is controlled at around 6.8 by adding hydrochloric acid and sodium hydroxide. The amount and time of addition can be adjusted as needed.

[0101] Feeding: Add carbon monoxide and carbon dioxide (volume ratio 2:1) at a rate of 30L / hr;

[0102] Detection: After 72 hours of incubation, OD600nm, n-butanol, NADH / NAD+, etc. are measured every 12 hours to assess bacterial growth and product production.

[0103] For detailed data, please see... Figures 3 to 5 Group 2.

[0104] Example 3: Anaerobic growth and production experiment of the optimized strain Moorella thermoacetica GTLB-iB2403 in H2 and CO2 environments (In this application, strain Moorella thermoacetica GTLB-iB2403 is obtained by further activating and expressing the hydrogenase gene based on strain Moorella thermoacetica GTLB-iB2312)

[0105] Prepare the culture medium: Prepare a Clostridium perfringens culture medium and adjust the pH of the Clostridium perfringens culture medium to 6.8.

[0106] Pretreatment of anaerobic Clostridium: The optimized strain, Moorella thermoacetica + thlΔptaΔptb + H2, was taken out, inoculated into the culture medium, and cultured in a constant temperature shaker at 58°C for 72 hours.

[0107] Prepare a 1L fermenter: Sterilize the fermenter using an automatic sterilizer and fill it with 500ml of enhanced Clostridium culture medium.

[0108] Inoculation: The prepared bacterial culture was transferred into the fermenter using a sterile pipette, so that the initial cell density was OD600 = 0.05.

[0109] Cultivation process: The fermenter was set to a constant temperature of 58℃, and the rotation speed inside the fermenter was controlled at 100 rpm. The growth of the microbial community was monitored during the cultivation process.

[0110] pH adjustment: The pH value is controlled at around 6.8 by adding hydrochloric acid and sodium hydroxide. The amount and time of addition can be adjusted as needed.

[0111] Feeding: Add hydrogen and carbon dioxide (volume ratio 2:1) at a rate of 30L / hr;

[0112] Detection: After 72 hours of incubation, OD600nm, n-butanol, NADH / NAD+, etc. are measured every 12 hours to assess bacterial growth and product production.

[0113] For detailed data, please see... Figures 3 to 5 Group 3.

[0114] In the above three groups of experiments, Figure 3 The OD600nm value shows the change in cell concentration over time or other experimental conditions; Figure 4 The n-butanol concentration represents the change in n-butanol concentration during the experiment when H2 or CO is used as a raw material. Figure 5 The NADH / NAD+ ratio reflects the metabolic state and redox balance of different groups during the experiment. Among them, the cell concentration and butanol concentration of the basic bacterial species Moorella thermoacetica+thlΔptaΔptb were higher when using CO as a substrate than when using H2 as a substrate, and the NADH / NAD+ value was correspondingly higher.

[0115] The modified strain Moorella thermoacetica+thlΔptaΔptb is far superior to the original strain in terms of cell growth using H2 and production of n-butanol, and is comparable to the original strain in terms of CO utilization.

[0116] The main methods, steps, and conclusions of this patent application can be summarized as follows:

[0117] Methods and steps:

[0118] Constructing a recombinant anaerobic Clostridium moorella thermoacetica system;

[0119] Genetic engineering: The hydrogenase of the target microorganism Moorella thermoacetica+thlΔptaΔptb was activated using CRISPRa technology, thereby obtaining an enhanced strain of Moorella thermoacetica+thlΔptaΔptb+H2 that overexpresses hydrogenase.

[0120] Syngas fermentation to n-butanol experiment: Syngas fermentation experiments under three different conditions were conducted in a fermenter to confirm that the engineered bacteria have the ability to produce n-butanol using H2 or CO.

[0121] Among them, the cell concentration and butanol concentration of the basic bacterial strain Moorella thermoacetica+thlΔptaΔptb were higher when CO was used as the substrate than when H2 was used as the substrate, and the NADH / NAD+ ratio was correspondingly higher.

[0122] The modified strain Moorella thermoacetica+thlΔptaΔptb+H2 is far superior to the original strain in terms of cell growth using H2 and production of n-butanol, and is comparable to the original strain in terms of CO utilization.

[0123] Problems solved and advantages: Improved product selectivity: Overexpression of the hydrogenase gene increases the ability to produce four-carbon products, thus increasing the yield of n-butanol. Improved synthesis efficiency: These edits significantly increase the concentration and product conversion rate of n-butanol produced using syngas, especially hydrogen, as a feedstock, achieving more efficient production.

[0124] Advantages and potential applications: Increased product yield and improved purity: Optimized metabolic pathways and enhanced product selectivity help increase the yield of n-butanol and may improve product purity.

[0125] Environmentally friendly and sustainable: Using waste materials such as syngas as raw materials reduces dependence on traditional raw materials (such as sugar compounds), helps reduce production costs, and is beneficial to environmental protection.

[0126] Technological innovation and efficiency improvement: The application of CRISPR technology to precisely edit genes accelerates the speed and accuracy of traditional genetic engineering, and improves production efficiency and product quality.

[0127] Exploring new fields and applications: Optimizing the synthesis of n-butanol from microorganisms using gene editing technology demonstrates the broad application prospects of CRISPR in industrial production and provides new ideas for the development of novel biocatalysts and biotechnology.

[0128] Interestingly, in experiments on the production of butanol via syngas fermentation, it was found that when the system contained only H2 and CO2, it could successfully utilize hydrogen, achieving an OD of approximately 6.8 and a butanol concentration of approximately 21 g / L. If the system contained only CO and CO2, it could successfully utilize CO, achieving an OD of approximately 12 and a butanol concentration of approximately 38 g / L. See Example 1 for the results.

[0129] In the syngas fermentation experiment of Example 1, microorganisms such as Clostridium were able to utilize hydrogen (H2), carbon monoxide (CO), and carbon dioxide (CO2) as carbon and energy sources for growth and butanol production. Data showed that microbial growth and butanol yield differed significantly under different gas composition conditions.

[0130] Here are a few factors that may explain this phenomenon:

[0131] Substrate utilization efficiency: CO may be utilized more efficiently by microorganisms because it can be directly converted into carbon dioxide (CO2) by carbon monoxide dehydrogenase (CODH), and then into acetyl-CoA, a key intermediate in butanol synthesis.

[0132] Energy acquisition: Compared to H2, the oxidation of CO may provide microorganisms with more energy (in the form of ATP), thereby supporting faster growth and higher butanol production.

[0133] Electron transport chain: In some microorganisms, CO oxidation may be more directly associated with the electron transport chain, which may lead to higher energy efficiency and stronger growth vigor.

[0134] Enzyme activity and expression: Microorganisms may modulate the activity and expression of their enzymes based on available substrates. In the presence of CO, the expression of enzymes related to CO metabolism may be induced, thereby promoting growth and butanol production.

[0135] pH and redox potential: CO metabolism may affect the pH and redox potential of the fermentation medium, which may affect the metabolic pathways and enzyme activity of microorganisms, thereby affecting growth and product formation.

[0136] Growth inhibition: In the presence of only H2 and CO2, there may be limitations on the utilization of H2, or the utilization of H2 may have a growth-inhibiting effect on some microorganisms, which limits the increase in cell density.

[0137] Substrate concentration effect: CO concentration may be sufficient to support higher cell densities because it provides a richer carbon source, while H2 may play a supporting role in intracellular metabolic pathways.

[0138] Competition for metabolic pathways: In the presence of both H2 and CO, microorganisms may preferentially utilize CO because the CO metabolic pathway may be more advantageous in terms of energy production.

[0139] Regulation of product synthesis: In the presence of CO, certain metabolic pathways may be activated that are more likely to synthesize butanol than other metabolites.

[0140] To gain a deeper understanding of these phenomena, further experiments and analyses are needed, including detailed studies of metabolic pathways, determination of enzyme activity, and monitoring of the intracellular environment. These studies will allow for better optimization of fermentation conditions, improving butanol yield and efficiency. This method has the potential to address the challenges of using carbohydrates as substrates, such as resource competition and environmental pressures. Furthermore, the use of the strictly anaerobic microorganism *Moorella thermoacetica* to produce n-butanol from syngas holds promise for providing a more sustainable and environmentally friendly alternative for industrial production, representing a significant breakthrough in biosynthesis and playing a crucial role in sustainable chemical production.

[0141] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that the terms "in one embodiment," "for example," and "again," etc., in this application are intended to illustrate the application and not to limit it. The embodiments described above only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A strain of *Acetobacter thermophilus* for the synthesis of n-butanol, characterized in that, The aforementioned Moorelh themoaceca strain is GTLB-iB2312, which was deposited on September 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo:46146.

2. A strain of *Acetobacter thermophilus* for the synthesis of n-butanol, characterized in that, The thl gene was activated by CRISPR-Cas9 technology and the pta and ptb genes were knocked out by CRISPRi technology.

3. A strain of *Acetobacter thermophilus* for the synthesis of n-butanol, characterized in that, The aforementioned Moorelh themoaceca strain is GTLB-iB2403, which was deposited on September 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo:46145.

4. A strain of *Acetobacter thermophilus* for the synthesis of n-butanol, characterized in that, The thl gene and hydrogenase gene of *Heat muhrella* were activated using CRISPR-Cas9 technology, and the pta and ptb genes were knocked out using CRISPRi technology.

5. A method for preparing a *Hormone thermophilus* strain for synthesizing n-butanol as described in any one of claims 1 to 4, characterized in that, Includes the following steps: The gRNA sequence for activating the thl gene was designed; the synthesized Cas9, gRNA sequence, and amplified thl gene were cloned into a linearized plasmid using polymerase chain reaction to form the pET-22b-Cas9-gRNA-thl complex vector; the pET-22b-Cas9-gRNA-thl complex vector was introduced into *Thermus thermophilus* cells by electroporation; after electroporation, the bacterial culture was transferred to YPD medium and cultured on a medium containing selective antibiotics to screen for positive strains, thus obtaining *Thermus thermophilus* + thl strain containing the overexpression of the thl gene; GRNA sequences for silencing the pta and ptb genes were designed and cloned into the CRISPRi vector. dCas9-KRAB was selected as the vector and linearized. The vector was then ligated to the gRNA using DNA ligation to construct the CRISPRi-KRAB vector. The CRISPRi-KRAB plasmid was amplified and extracted. The CRISPRi-KRAB vector was introduced into the target cells *Moorelh themoaceca* + THL strain using electroporation. After electroporation, the bacterial culture was transferred to MRS medium and cultured on a medium containing selective antibiotics to screen for positive strains. The *Moorelh themoaceca* + THLΔptaΔptb strain with silenced pta and ptb genes was obtained, which is *Moorelh themoaceca* GTLB-iB2312. Design a gRNA sequence to activate the hydrogenase gene; clone the gRNA sequence into the CRISPRa vector, wherein pcDNA-dCas9-VP64 was selected as the vector and linearized, and the vector was ligated with the gRNA using a DNA ligation reaction to construct the CRISPRa-VP64 vector; amplify the vector and extract the CRISPRa-VP64 plasmid; use electroporation to introduce the CRISPRa-VP64 vector into the target cell strain *Moorelh themoaceca* + thlΔptaΔptb, and after electroporation, transfer the bacterial culture to YPD medium for culture, and culture on a medium containing selective antibiotics to screen for positive strains to obtain *Moorelh themoaceca* + thlΔptaΔptb+H2 strain with activated hydrogenase gene; this is the *Moorelh themoaceca* strain *Moorelh themoaceca* GTLB-iB2403 used for the synthesis of n-butanol.

6. The preparation method according to claim 5, characterized in that, In the steps of activating the th1 gene, the forward sequence for activating the th1 gene is: 5'-GGCCGGCGAGGCTGCCGGTG-3'; the reverse sequence is: 5'-CACCGGCAGCCTCGCCGGCC-3'. And / or, in the step of activating the hydrogenase gene, the gRNA sequence for activating the hydrogenase gene is: positive strand gRNA: 5'-AGGCTGGAGTGGTGTCCTGC-3'; negative strand gRNA: 5'-GCAGACACCCTCCAGCCT-3'; And / or, in the step of activating the hydrogenase gene, the electroconversion conditions are: voltage: 1.8kV, capacitance: 25uF, resistance: 200Ω, and time constant: 8.5ms; And / or, in the steps of silencing the pta and ptb genes, the gRNA sequence for silencing the pta gene is as follows: Positive chain forward sequence: 5'-GGCCGGCGAGGCTGCCGGTG-3'; Reverse sequence: 5'-CACCGGCAGCCTCGCCGGCC-3'; gRNA sequence for silencing the ptb gene: Forward sequence: 5'-GGGCTGCCGGCGGCGCCGGC-3' Reverse sequence: 5'-GCCGGCGCCGCCGGCAGCCC-3'.

7. A method for producing n-butanol by syngas fermentation, characterized in that, n-Butanol is prepared by fermentation using the *Heat-Acetobacter murine* strain for synthesizing n-butanol as described in any one of claims 1 to 4.

8. The method according to claim 7, characterized in that, Syngas includes hydrogen, and also includes at least one of carbon dioxide and carbon monoxide.

9. The method according to claim 7, characterized in that, The culture pH is controlled at 6.8; and / or the culture temperature is controlled at 58℃±0.5℃.

10. A method for producing n-butanol by syngas fermentation, characterized in that, Syngas includes at least two of hydrogen, carbon dioxide, and carbon monoxide. The fermentation strain includes at least one of Moorella thermoacetica, Clostridium tyrobutyricum, Clostridium acetobutylicum, or Butyribacterium methylotrophicum. The strain is obtained by overexpressing the thl gene and hydrogenase gene using CRISPR-Cas9 technology and knocking out the pta and ptb genes using CRISPRi technology.

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