An engineered strain of *Acetoin-producing* *Morphozoa* and its application

Abstract

This invention relates to the field of biotechnology, and more particularly to an engineered strain of *Fermentomonas motilityis* that produces high levels of acetoin and its applications. The engineered strain is derived from *Fermentomonas motilityis* ZM4Δpdc-BDO. The strain was obtained by replacing the ZMO0038 gene with the NADH oxidase gene noxE, knocking out the 2,3-butanediol dehydrogenase gene bdh, and further knocking out the potential reductase genes ZMO0318 and ZMO1576, which are outside the conventional knowledge system. This resulted in a high-acetoin-producing engineered strain. When fermenting typical complex non-grain biomass feedstocks, this strain can maintain approximately 80% of its production capacity, demonstrating good potential for industrial production using renewable lignocellulosic biomass.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an engineered strain of *Acetoin-producing* *Morphozoa* and its applications. Background Technology

[0002] Acetoni (3-hydroxy-2-butanone) is an important platform chemical, listed by the U.S. Department of Energy as one of the 30 priority platform compounds for development. Due to its unique buttery and fatty aroma, acetoin is widely used in the food, flavoring, cosmetics, and chemical industries, serving as a food flavor enhancer and as an intermediate in the synthesis of chiral drugs and a monomer for polymers. Currently, the industrial production of acetoin mainly relies on chemical synthesis, using petroleum derivatives (such as 2,3-butanediol) as raw materials through incomplete oxidation reactions. This process suffers from high energy consumption, cumbersome steps, the need for toxic catalysts, and severe environmental pollution, which not only contradicts green and sustainable development strategies but also poses safety risks when used as a food additive. Therefore, developing microbial fermentation methods based on renewable resources has become an important research direction in the industry.

[0003] While microbial fermentation offers advantages such as environmental friendliness and relatively simple processes, it still faces significant bottlenecks. Firstly, most reported high-yield acetoin strains are opportunistic pathogens like *Klebsiella pneumoniae* or *Serratia marcescens*, whose application is strictly limited by biosafety concerns, making large-scale industrial production difficult, especially in sensitive industries like food and pharmaceuticals. Secondly, even attempts to construct synthetic pathways using safe model microorganisms (such as *Escherichia coli*) often encounter problems like intense competition for central metabolic pathways, insufficient precursor supply, and imbalanced cofactor regeneration, resulting in unsatisfactory final yields and conversion rates of acetoin. *Fermentomonas motilityis*, a recognized safe strain, possesses advantages such as simple nutritional requirements, rapid growth, high sugar utilization, and strong stress resistance, making it an excellent chassis cell for developing high-value-added chemicals. However, a metabolic pathway capable of overcoming intrinsic metabolic limitations and efficiently and stably synthesizing acetoin has not yet been constructed in this host. How to utilize this safe host and match it with low-cost non-grain raw materials to achieve efficient acetoin production remains a pressing technical challenge. Summary of the Invention

[0004] In view of this, the present invention proposes an engineered strain of *Acetoin-producing* motile fermentation monoclonal bacteria and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides an engineered strain that produces high levels of acetoin, wherein the ZMO0038 gene of *Z. motile fermentum* ZM4Δpdc-BDO is replaced with the NADH oxidase gene noxE, and in the engineered strain, the 2,3-butanediol dehydrogenase genes bdh, ZMO0318, and ZMO1576 are all knocked out.

[0007] Secondly, the present invention provides a method for preparing an engineered strain that produces high levels of acetoin. Using *Zygomorpha motileis* ZM4Δpdc-BDO as the starting strain, the ZMO0038 gene of the starting strain is replaced with the NADH oxidase gene noxE. Then, the 2,3-butanediol dehydrogenase gene bdh, the ZMO0318 gene, and the ZMO1576 gene are knocked out to obtain the engineered strain.

[0008] Furthermore, the preparation method includes the following steps:

[0009] First, second, third, and fourth edit plasmids targeting ZMO0038, bdh gene, ZMO0318, and ZMO1576, respectively, were constructed.

[0010] The first edited plasmid was transferred into the ZM4Δpdc-BDO strain to obtain a strain with the ZMO0038 gene edited.

[0011] The second editing plasmid was transferred into the strain in which the ZMO0038 gene was edited, resulting in a strain in which the ZMO0038 and bdh genes were edited.

[0012] The third editing plasmid was transferred into the strain in which the ZMO0038 and bdh genes were edited, resulting in a strain in which the ZMO0038, bdh, and ZMO0318 genes were all edited.

[0013] The fourth editing plasmid was transferred into a strain in which the ZMO0038, bdh, and ZMO0318 genes were all edited, resulting in a strain in which the ZMO0038, bdh, ZMO0318, and ZMO1576 genes were all edited, which is the engineered strain that produces high levels of acetoin.

[0014] Furthermore, the first edit plasmid carries a first donor sequence and a first target element. The first donor sequence includes an upstream sequence of ZMO0038, the NADH oxidase gene noxE, and a downstream sequence of ZMO0038. The first target element includes two repeating sequences as shown in SEQ ID NO:1 and a first guide sequence as shown in SEQ ID NO:2 located between the two repeating sequences.

[0015] The second editing plasmid carries a second donor sequence and a second targeting element. The second donor sequence includes an upstream sequence of the bdh gene and a downstream sequence of the bdh gene. The second targeting element includes two repeat sequences as shown in SEQ ID NO:1 and a second guide sequence as shown in SEQ ID NO:19 located between the two repeat sequences.

[0016] The third editing plasmid carries a third donor sequence and a third targeting element. The third donor sequence includes an upstream sequence of ZMO0318 and a downstream sequence of ZMO0318. The third targeting element includes two repeating sequences as shown in SEQ ID NO:1 and a third guide sequence as shown in SEQ ID NO:29 located between the two repeating sequences.

[0017] The fourth edit plasmid carries a fourth donor sequence and a fourth target element. The fourth donor sequence includes an upstream sequence of ZMO1576 and a downstream sequence of ZMO1576. The fourth target element includes two repeating sequences as shown in SEQ ID NO:1 and a fourth guide sequence as shown in SEQ ID NO:30 located between the two repeating sequences.

[0018] Thirdly, the present invention provides a method for preparing acetoin, comprising: obtaining the engineered strain or the engineered strain obtained by the preparation method, inoculating the engineered strain into a culture medium containing non-grain biomass for fermentation; and harvesting the acetoin from the fermentation product, wherein the non-grain biomass is a plant raw material hydrolysate.

[0019] Fourthly, the present invention provides a method for preparing acetoin, comprising: obtaining the engineered strain or the engineered strain obtained by the preparation method, inoculating the engineered strain into a glucose-containing culture medium for fermentation; and harvesting the acetoin from the fermentation product.

[0020] Fifthly, the present invention provides the application of the engineered strain or the engineered strain obtained by the preparation method in the preparation of acetoin.

[0021] The beneficial effects of the present invention include at least the following:

[0022] This invention uses *Zygomorpha motileis* ZM4Δpdc-BDO as the starting strain. First, it optimizes intracellular cofactor balance by replacing the ZMO0038 gene with the NADH oxidase gene noxE, and knocks out the 2,3-butanediol dehydrogenase gene bdh to block major-effect pathways and guide carbon flow to the target product. Further, through transcriptomic analysis of the metabolic response network of the bdh knockout strain under high glucose stress and other conditions, this invention successfully identified two potential reductase genes, ZMO0318 and ZMO1576, which are outside the conventional knowledge system and may divert carbon sources or affect redox balance. Knocking out these two genes resulted in a synergistic yield increase of approximately 18%. Through the above multi-target synergistic intervention, this invention effectively removes the main obstacles to carbon flow to the target product acetoin, obtaining a high-acetoin-producing engineered strain. This engineered strain achieved an acetoin yield of approximately 73 g / L in fed-batch fermentation.

[0023] This invention significantly enhances the industrial robustness of the engineered strain. When fermenting complex and typical non-grain biomass feedstocks, the strain maintains approximately 80% of its production capacity, demonstrating its strong adaptability to various inhibitors in actual hydrolysates. This characteristic, combined with the use of non-grain feedstocks, effectively reduces reliance on grain-based raw materials and showcases the enormous potential for production using renewable lignocellulosic biomass. Therefore, the entire technical solution exhibits significant advantages in terms of economic feasibility for industrial production, environmental friendliness, and compliance with green manufacturing strategies. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows the construction of the engineered strains ZMA1, ZMA2, ZMA4 and ZMA7 of the present invention and their corresponding fermentation performance test results.

[0026] Figure 2 This is a schematic diagram comparing the key physiological metabolism and molecular response mechanisms in *Mammotrophic motility-fermenting* bacteria under high glucose stress and after knockout of the 2,3-butanediol dehydrogenase (bdh) gene.

[0027] Figure 3 The results of the fermentation performance test in the hydrolysate of corn cob residue from the ZMA7 strain of this invention;

[0028] Figure 4The results are the fed-batch fermentation performance test results of the engineered strain ZMA7 of this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] Example 1

[0031] This application uses the engineered strain ZM4Δpdc-BDO (see patent CN117778291A) of *Lactococcus lactis* with key genes for ethanol synthesis knocked out as the starting strain. Using the endogenous CRISPR-Cas system, the NADH oxidase gene (noxE from *Lactococcus lactis*) is first introduced to construct strain ZMA1 (i.e., ZM4Δpdc-BDO-noxE). Then, the major gene bdh encoding butanediol dehydrogenase is knocked out to construct strain ZMA2 (i.e., ZM4Δpdc-BDO-noxE-Δbdh).

[0032] Knocking out bdh directly disrupts the primary pathway for the conversion of acetoin to 2,3-butanediol, removing the catalytic enzyme required for this reduction reaction and forcing the metabolic flux to remain at the acetoin node. The acetoin synthesis pathway involves redox reactions. Knocking out reductases such as bdh may lead to NADH accumulation, resulting in metabolic imbalance. Introducing and expressing exogenous noxE continuously oxidizes excess NADH to NAD⁺, which alleviates reduction stress and prevents impaired cell growth. Furthermore, sufficient NAD⁺ also facilitates the oxidation step in the acetoin synthesis pathway. Simultaneously, the lack of excess reducing power further inhibits any remaining reduction reactions, stably pushing the carbon flux towards acetoin. The construction process of strains ZMA1 and ZMA2 is as follows:

[0033] 1. Integration of the noxE gene into ZM4Δpdc-BDO

[0034] The gene editing method based on the endogenous IF CRISPR-Cas system established in the literature (reference "Yanli, Zheng, Jiamei, Han, Baiyang, Wang et al. Characterization and repurposing of the endogenous Type IF CRISPR-Cas system of Zymomonas mobilis for genome engineering.[J]. Nucleic Acids Res, 2019, 47: 11461-11475.") is as follows:

[0035] (1) gRNA target design

[0036] PAM sequences were selected from the ZMO0038 locus, and the downstream 32 bp was extracted as a spacer (i.e., gRNA-0038). Targeting primer sequences (0038gr-F / 0038gr-R) were designed to edit the gRNA in the plasmid.

[0037] (2) Construction of endogenous CRISPR-Cas editing plasmids

[0038] The gRNA-0038 sequence was obtained by PCR amplification and cloned into the pL2R plasmid (containing a CRISPR expression unit) between two repetitive sequences as shown in SEQ ID NO:1 using enzyme digestion / ligation to obtain the target plasmid backbone. The upstream (US), downstream (DS), and noxE genes of ZMO0038 were amplified using primers (0038US-F / 0038US-R, 0038DS-F / 0038DS-R, and NoxE-F / NoxE-R), respectively. The US, noxE, and DS fragments were mixed and assembled into a complete "0038US-noxE-0038DS" donor fragment using overlap PCR with the outer primer (0038US-F / 0038DS-R).

[0039] The target plasmid backbone was linearized by reverse PCR using primers pl2r-fk-F / pl2r-fk-R. The linearized plasmid backbone and donor fragment were mixed at a 1:3 molar ratio and assembled using Gibson chromatography. The assembly product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing spectinomycin (100 μg / mL). Positive clones were picked and colony PCR was performed using primers (pEZ15A-F / pEZ15A-R). All constructed editing plasmids were verified by sequencing.

[0040] (3) Transformation and screening of edited plasmids

[0041] 1 μg of the edited plasmid was added to 50 μL of ZM4Δpdc-BOD competent cells of *Syntrophus amoebae*, mixed well, and then transferred to a 0.1 cm electroporation cuvette. Electroporation was performed according to the program of 1800 V, 25 μF, and 200 Ω. After electroporation, the plasmid was transferred to 1 mL of RM medium and incubated statically at 30°C for 4–6 h. Then, 200 μL of the plasmid was evenly spread onto 100 μg / mL spectinomycin-resistant plates and incubated upside down at 30°C for 2–3 days. After colony growth, colony PCR was performed using primers (0038check-F / 0038check-R) for verification. Colonies with PCR products matching the expected size were sequenced, confirming that the noxE gene had been accurately integrated into the target site in the genome. The correct strain was named ZMA1. The relevant sequence information is as follows:

[0042]

[0043] 2. Knockout of the bdh gene in ZMA1

[0044] The 32 bp downstream sequence of PAM within the bdh gene was selected as gRNA-bdh. During plasmid construction, the upstream and downstream homologous arms of the bdh gene were amplified, and these two homologous arms were directly spliced ​​into the "US-DS" donor fragment using Overlap PCR. Final colony PCR was performed using primers bdh-check-F / bdh-check-R for verification; other procedures were the same as above. The strain that correctly knocked out the bdh gene, verified by colony PCR and Sanger sequencing, was named ZMA2. Relevant sequence information is as follows:

[0045]

[0046] Growth and fermentation performance tests of recombinant strains:

[0047] To evaluate the performance of the recombinant strains, each strain was first streaked onto RMG5 agar plates and incubated at 30 °C for 48 h to obtain single colonies. Subsequently, three independent and well-isolated single colonies were randomly selected from each strain's plates. Each selected colony was aseptically inoculated into 1 mL of RMG5 liquid medium and incubated at 30 °C with shaking at 180 rpm for 24 h to obtain the primary seed culture for each strain. Then, the primary seed culture for each strain was transferred to 5 mL of RMG5 medium, and the initial OD was controlled. 600 nmThe initial OD was set at 0.1, and the culture was carried out at 30 °C with shaking at 180 rpm for 24 h to obtain secondary seed cultures for each strain. Finally, the secondary seed cultures of each strain were transferred to RMG medium (with different concentrations of glucose added according to the experimental design requirements) to control the initial OD. 600 nm The concentration was 0.1, and the liquid volume was 20% (V / V). The cultures were incubated at 30 °C with shaking at 180 rpm. Three independent biological replicates were set up for each strain.

[0048] In this application RMG medium The names correspond to the glucose concentrations, such as RMG5 indicating 50 g / L of glucose, and RMG8 indicating 80 g / L of glucose, and so on; their basic components are all fixed at 10 g / L yeast extract and 2 g / L potassium dihydrogen phosphate.

[0049] Detection of glucose, acetoin, and 2,3-BDO concentrations:

[0050] During fermentation, samples at different time points were centrifuged and the supernatant was collected (12000 rpm, 2 min). The supernatant was filtered through a 0.22 μm filter and then analyzed using an HPLC (LC-20 AD, Shimadzu, Japan) system equipped with an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad) and a RID-20A differential refractive index detector. The concentrations of glucose, acetoin, and 2,3-BDO in the fermentation broth were determined. The detection conditions were as follows: mobile phase: 0.005 mol / L H2SO4; flow rate: 0.5 mL / min; detector temperature: 40 ℃; column temperature: 60 ℃; injection volume: 20 μL.

[0051] Data Statistical Analysis

[0052] All experiments were performed in triplicate (n=3). Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used to compare the significance of differences between groups. When ANOVA results showed significant differences, Tukey's honest significance test (HSD) was used for post-hoc comparisons. A p-value < 0.05 was considered statistically significant.

[0053] Test results

[0054] ZMA1 and engineered strain ZM4Δpdc-BDO (as a control) were inoculated into RMG8 medium and cultured at 30 ℃ with shaking at 180 rpm for 24 hours. The test results are as follows:

[0055]

[0056] Note: Data are expressed as mean ± standard deviation (n=3). Different letters on the right side of the data in the same column indicate significant differences at the p < 0.05 level (based on one-way ANOVA and Tukey HSD post-hoc test).

[0057] The results showed that the introduction of the noxE gene significantly increased acetoin production (16%).

[0058] 3. Screening for potential acetoin reductase targets using a combination of BLAST homology analysis and transcriptomics ( Figure 2 )

[0059] (1) The amino acid sequence of 2,3-butanediol dehydrogenase BDH was used as the query sequence. The NCBI BLASTP tool was used to compare against the reference protein database of Zymomonas mobilis ZM4. The expected threshold (E-value) was set to be less than 1e-5, and proteins with high sequence identity and similarity were given priority.

[0060] (2) The strain ZMA1 and the engineered strain ZM4Δpdc-BDO (as a control) were inoculated into fermentation medium containing 80 g / L glucose and cultured at 30 ℃ with shaking at 180 rpm. Samples were taken at mid-log intervals for RNA-seq transcriptome sequencing. Differential expression analysis was performed using DESeq2. The significance criteria were set as follows: adjusted p-value (padj) < 0.05 and fold change in expression (log2FoldChange) > 1.

[0061] The analysis results showed that the ZMO0318 and ZMO1576 genes simultaneously met the following two conditions: they had significant homology with BDH at the protein level; and at the transcriptional level, their expression was significantly upregulated due to the knockout of BDH.

[0062] Based on this, it can be further inferred that after losing the main active enzyme bdh, the strain upregulated these functionally redundant bypass enzymes (such as ZMO0318 and ZMO1576) to compensate for the reduction reaction of acetoin in order to maintain metabolic homeostasis. Therefore, ZMO0318 and ZMO1576 were identified as priority targets for genetic modification.

[0063] 4. Fermentation performance tests were conducted on strains ZMA4, ZMA5, and ZMA7, respectively.

[0064] The construction pathways for strains ZMA4, ZMA5, and ZMA7 are shown in the table below:

[0065]

[0066] The preparation method follows the aforementioned "bdh gene knockout" strategy. First, a 32 bp region downstream of PAM within the target gene is selected as the gRNA target sequence. Based on this, targeting primers for the guide RNA in the editing plasmid are designed, and the corresponding oligonucleotide chains are synthesized and cloned into the editing plasmid backbone. Subsequently, the upstream and downstream sequences of each target gene are amplified using primers, and then spliced ​​into a complete donor fragment using overlap PCR. Simultaneously, the editing plasmid is amplified by inverse PCR, and the resulting linearized vector and donor fragment are assembled using Gibson sequencing to obtain the complete editing plasmid. This plasmid is transformed into competent host cells, and after screening for positive clones using spectinomycin-resistant plates, colony PCR and Sanger sequencing confirm the successful knockout of the target gene. The relevant sequence information is as follows:

[0067]

[0068] Fermentation performance test

[0069] Fermentation of strains ZMA2, ZMA4, ZMA5, and ZMA7 was carried out in RMG8 medium. 20 mL of RMG8 medium was added to 100 mL shake flasks, and the initial OD was controlled. 600 nm The concentration was 0.1, and the mixture was incubated at 30 °C with shaking at 180 rpm for 24 hours. The detection methods for glucose, acetoin, and 2,3-BDO concentrations were the same as described above.

[0070] The test results are as follows:

[0071]

[0072] The results showed that the acetoin yield of the double-knockout strain ZMA7 (34.22 g / L) was higher than that of any single-knockout strain (ZMA4: 31.03 g / L, ZMA5: 33.64 g / L), indicating a positive synergistic effect of double knockout. That is, the effect of simultaneously knocking out two genes (ZMO0318 and ZMO1576) is superior to the simple summation of individual knockouts. Meanwhile, the 2,3-BDO yield was the lowest in ZMA7 (4.93 g / L), and lower than that of the single-knockout strains (ZMA4: 6.37 g / L, ZMA5: 6.59 g / L), indicating that double knockout more thoroughly blocked the 2,3-BDO synthesis pathway.

[0073] This suggests that ZMO0318 and ZMO1576 may be involved in different steps of 2,3-BDO synthesis or possess isomerase activity. Double knockout simultaneously inhibits multiple bypass or redundant enzymes, avoiding metabolic compensation and thus more efficiently directing the precursor to acetoin. Further optimization of acetoin yield in ZMA7 indicates functional redundancy or interaction between ZMO0318 and ZMO1576, requiring double knockout to fully unleash their potential. This strategy is suitable for constructing high-yield industrial acetoin strains while reducing byproduct purification costs.

[0074] This application screened two potential reductase genes, ZMO0318 and ZMO1576, that were specifically induced to be upregulated in the context of bdh knockout across the entire genome. The results of the examples also showed that knocking out bdh alone had limited effect, and further knockout of ZMO0318 and ZMO1576 was necessary to achieve a synergistic increase in acetoin production. This demonstrates that combining bioinformatics homology analysis with transcriptome differential expression analysis is an efficient and systematic strategy for discovering recessive metabolic bottleneck genes. This method overcomes the limitations of traditional metabolic engineering that relies solely on known biochemical knowledge, and can proactively discover and guide the knockout of functionally redundant competing pathways that are activated by cells after the main pathway is blocked, thus providing an effective way to maximize the yield of target products.

[0075] Example 2: Application of strain ZMA7 in fermentation of corn residue hydrolysate

[0076] 1. Method

[0077] Batch fermentation evaluation of engineered strain ZMA7 was conducted using corn cob residue hydrolysate as the basic fermentation medium. The main components of this hydrolysate were glucose (148 g / L) and xylose (18 g / L), along with fermentation inhibitors such as acetic acid (3.2 g / L) and furfural (2 g / L). Undiluted hydrolysate without nutrient supplementation was used as the fermentation substrate; no additional yeast extract or inorganic salts were added. The specific fermentation process is as follows:

[0078] Add 40 mL of corn cob residue hydrolysate to a 100 mL Erlenmeyer flask to control the initial cell concentration (OD). 600nm The concentration of glucose was 0.1. The culture was carried out at 30°C and 180 rpm in a shaker. The control group used RMG15 synthetic medium with well-defined components, while all other culture conditions remained the same. The methods for detecting the concentrations of glucose, acetoin, and 2,3-BDO in the fermentation broth were the same as in Example 1.

[0079] 2. Results

[0080] The fermentation test results are shown in the table below. Figure 3 As shown:

[0081]

[0082] The results showed that the engineered strain ZMA7 achieved a final acetoin concentration of 42.04 g / L in the fermentation of corn cob residue hydrolysate, equivalent to 80.68% of its yield in a synthetic medium (RMG15) with a comparable carbon source. This result highlights the excellent adaptability and robustness of this engineered strain in complex industrial raw materials. Although inhibitors such as acetic acid and furfural in the hydrolysate typically have a significant inhibitory effect on microbial growth and metabolism, strain ZMA7 still maintained relatively high production performance, indicating that its metabolic system has good tolerance to the complex components and stress environment of industrial raw materials. This provides strong technical support for utilizing inexpensive agricultural and forestry waste such as corn cobs for biomanufacturing and reducing production costs.

[0083] Example 3: Fed-batch fermentation test

[0084] 1. Method

[0085] In this embodiment, the engineered strain ZMA7 was subjected to fed-batch fermentation in a 1-L bioreactor. The initial fermentation medium was RMG10, with an initial volume of 0.5 L. Throughout the process, the culture temperature was controlled at 30°C, the stirring rate at 500 rpm, and the aeration rate at 100 CCM to maintain dissolved oxygen and mixing homogeneity. Samples were taken periodically during fermentation, and the concentrations of key metabolites such as glucose and acetoin were monitored using HPLC. When the residual glucose concentration decreased to approximately 10 g / L, 120 mL of a 500 g / L glucose solution was manually added via fed-batch fermentation.

[0086] 2. Results

[0087] Experimental results show that ZMA7 exhibits excellent production performance in fed-batch fermentation mode. For example... Figure 4 As shown, the final concentration of acetoin reached 73 g / L during the 72-hour fermentation cycle. This result is significantly higher than the levels reported in most batch fermentations or conventional fed-batch fermentations, indicating that this strain has the ability to efficiently direct carbon to the acetoin synthesis pathway under controlled fermentation conditions.

[0088] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered bacterial strain for high production of acetoin, characterized in that, The engineered strain is *ZM4Δpdc-BDO*, in which the ZMO0038 gene is replaced with the NADH oxidase gene noxE. Furthermore, the 2,3-butanediol dehydrogenase genes bdh, ZMO0318, and ZMO1576 are all knocked out in this engineered strain. The *ZM4Δpdc-BDO* strain originates from patent CN117778291A. The nucleotide sequence of the ZMO0038 gene is shown in NCBI database CP023715.1:39623-40225, the nucleotide sequence of the NADH oxidase gene noxE is shown in SEQ ID NO:5, the nucleotide sequence of the 2,3-butanediol dehydrogenase gene bdh is shown in SEQ ID NO:18, the nucleotide sequence of the ZMO0318 gene is shown in NCBI database CP023715.1:318604-319359, and the nucleotide sequence of the ZMO1576 gene is shown in NCBI database CP023715.1:1610108-1610854.

2. A method for preparing an engineered strain for high production of acetoin, characterized by, Using *Zygomorpha motileis* ZM4Δpdc-BDO as the starting strain, the ZMO0038 gene of the starting strain was replaced with the NADH oxidase gene noxE, and the 2,3-butanediol dehydrogenase genes bdh, ZMO0318, and ZMO1576 were knocked out to obtain the engineered strain; the *Zygomorpha motileis* ZM4Δpdc-BDO is derived from patent CN117778291A. The nucleotide sequence of the ZMO0038 gene is shown in NCBI database CP023715.1:39623-40225, the nucleotide sequence of the NADH oxidase gene noxE is shown in SEQ ID NO:5, the nucleotide sequence of the 2,3-butanediol dehydrogenase gene bdh is shown in SEQ ID NO:18, the nucleotide sequence of the ZMO0318 gene is shown in NCBI database CP023715.1:318604-319359, and the nucleotide sequence of the ZMO1576 gene is shown in NCBI database CP023715.1:1610108-1610854.

3. The production method according to claim 2, characterized by, The preparation method includes the following steps: First, second, third, and fourth edit plasmids targeting ZMO0038, bdh gene, ZMO0318, and ZMO1576 were constructed, respectively. The first edited plasmid was transformed into the ZM4Δpdc-BDO strain to obtain a strain with the ZMO0038 gene edited. The second editing plasmid was transferred into the strain in which the ZMO0038 gene was edited, resulting in a strain in which the ZMO0038 and bdh genes were edited. The third editing plasmid was transferred into the strain in which the ZMO0038 and bdh genes were edited, resulting in a strain in which the ZMO0038, bdh, and ZMO0318 genes were all edited. The fourth editing plasmid was transferred into the strain whose ZMO0038, bdh, and ZMO0318 genes were edited, resulting in a strain whose ZMO0038, bdh, ZMO0318, and ZMO1576 genes were all edited, which is the engineered strain that produces high levels of acetoin.

4. A process for the preparation of acetoin, characterized in that, include: Obtain the engineered strain according to claim 1 or the engineered strain prepared by any of the preparation methods of claims 2-3, inoculate the engineered strain into a culture medium containing non-grain biomass for fermentation; and harvest the acetoin from the fermentation product, wherein the non-grain biomass is corn residue hydrolysate.

5. A process for the preparation of acetoin, characterized in that, include: Obtain the engineered strain according to claim 1 or the engineered strain prepared by any of the preparation methods of claims 2-3, inoculate the engineered strain into a glucose-containing culture medium for fermentation; and harvest the acetoin from the fermentation product.

6. The use of the engineered strain according to claim 1 or the engineered strain obtained by any of the preparation methods according to claims 2-3 in the preparation of acetoin.

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