Complex microbial inoculant and application thereof in production of 3-hydracrylic acid

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a complex microbial inoculant and application thereof in production of 3-hydracrylic acid. The invention provides a complex microbial inoculant. The complex microbial inoculant comprises a rhodosporidium toruloides engineering strain and bacillus subtilis. The rhodosporidium toruloides engineering strain has the characteristic of being capable of producing 3-HP, and is combined with bacillus subtilis, so that the problem that the utilization capacity of microorganisms with the capacity of producing 3-HP on lignocellulose is limited at present is solved, and the problems of product inhibition and low substrate conversion rate are further solved. The engineering strain provided by the invention can effectively improve the utilization rate of lignocellulose, reduce the production cost, realize the recycling and high-value utilization of agricultural wastes, and provide a new technical approach for the development of biological economy.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound bacterial agent and its application in the production of 3-hydroxypropionic acid. Background Technology

[0002] 3-Hydroxypropionic acid (3-HP) is an important platform compound, serving as a precursor for various chemicals such as acrylic acid, polyesters, and bioplastics, and has broad application prospects in the fields of chemical engineering, materials, and energy. Currently, the biological production of 3-HP using renewable carbon sources (such as lignocellulose) has the advantages of being green and sustainable.

[0003] Lignocellulose is the most abundant renewable resource in nature, mainly derived from agriculture and forestry. It contains abundant cellulose, hemicellulose, and lignin, but its complex structure makes it difficult for microorganisms to utilize directly, thus limiting its efficient biotransformation. Currently, the microorganisms that produce 3-HP are mainly model bacteria such as Escherichia coli and Klebsiella pneumoniae, but these strains have limited ability to utilize lignocellulose and often face problems such as product inhibition and low substrate conversion rates.

[0004] Currently, the production of 3-HP using microbial fermentation suffers from low yield and low substrate utilization efficiency. These microorganisms have certain limitations in utilizing lignocellulose raw materials and are subject to high environmental requirements. Therefore, developing a technical solution for producing 3-HP using lignin as a raw material has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the low efficiency of 3-HP production using lignin as a raw material in existing technologies, this invention provides a compound microbial agent and its application in the production of 3-hydroxypropionic acid, specifically including the following technical solutions: The present invention also provides a compound microbial agent comprising an engineered strain of Rhodotorula buergerianum and Bacillus subtilis; the engineered strain of Rhodotorula buergerianum contains a target gene, and the amino acid encoded by the target gene has the NCBI Reference Sequence WP_012258473.1 in the NCBI database.

[0006] Preferably, the method for constructing the engineered strain of Rhodotorula circotensivea includes: constructing an overexpression vector using a binary vector and a target gene; transforming the overexpression vector into bacteria to construct an overexpressing recombinant microorganism; and screening and purifying the overexpressing recombinant microorganism and Rhodotorula circotensivea to obtain the engineered strain of Rhodotorula circotensivea.

[0007] Preferably, in the compound microbial agent, the ratio of Bacillus subtilis and Rhodotorula glutinis engineered strains is 1-3:1 based on the volume of the seed liquid; the effective viable cell concentration of the Bacillus subtilis seed liquid is 1.0 × 10⁻⁶. 8 CFU / mL ~5.0×10 8 CFU / mL; the effective viable cell concentration of the engineered strain of *Rhodotorula circophylla* is 5.0 × 10⁻⁶ CFU / mL. 7 CFU / mL ~2.0×10 8 CFU / mL; the Bacillus subtilis includes Bacillus subtilis AC83.

[0008] The present invention also provides the application of the compound microbial agent as described above in the production of 3-hydroxypropionic acid.

[0009] The present invention also provides a method for producing 3-hydroxypropionic acid from lignocellulose as raw material, using the compound microbial agent as described above to produce 3-hydroxypropionic acid; the steps of the method are as follows: mixing straw and sludge at a mass ratio of 1:2 to obtain inoculum; inoculating the inoculum with the compound microbial agent and carrying out co-culture aerobic fermentation to obtain 3-hydroxypropionic acid.

[0010] Preferably, the straw includes wheat straw; before mixing the straw and sludge at a mass ratio of 1:2, the process further includes pretreatment of the straw or sludge; the pretreatment of the straw is as follows: air-drying to a moisture content of 5%~10%, and crushing through a 10~30 mesh sieve; the pretreatment of the sludge is as follows: mixing the sludge and water at a mass ratio of 1:1~5, and incubating at a constant temperature of 30~40℃ for 15~30 days.

[0011] Preferably, after obtaining the inoculum, the method further includes adjusting the C / N ratio of the inoculum to 25~30:1 and the water content to 55%~65%.

[0012] Preferably, the temperature of the co-culture aerobic fermentation is 30~35℃; the pH of the co-culture aerobic fermentation is 6.5~7.0.

[0013] The beneficial effects of this invention are as follows: This invention provides a compound microbial agent comprising engineered strains of Bacillus subtilis and Rhodotorula glutinis. The Rhodotorula glutinis engineered strain contains a target gene, and the amino acid encoded by the target gene has the NCBI Reference Sequence WP_012258473.1 in the NCBI database.

[0014] The compound microbial agent described in this invention can produce 3-HP using straw as raw material. Bacillus subtilis is a key substance in the biological pretreatment of straw degradation; its produced cellulase can accelerate the hydrolysis of straw and other biomass into smaller molecules. The engineered strain of *Rhodotorula glutinis* contains the target gene; therefore, this engineered strain has the characteristic of producing 3-HP. It can utilize the small-molecule sugars obtained from the decomposition of Bacillus subtilis for aerobic fermentation to further produce 3-HP, solving the problem that current microorganisms capable of producing 3-HP have limited utilization of lignocellulose, resulting in product inhibition and low substrate conversion rates.

[0015] This invention also provides a method for producing 3-hydroxypropionic acid from lignocellulose. By using straw as a substrate and co-culturing and fermenting with engineered strains of Bacillus subtilis and Rhodotorula glutinis, 3-hydroxypropionic acid can be produced efficiently. This method has advantages such as simple operation, low raw material cost, high product yield, and environmental friendliness. It can effectively improve the utilization rate of lignocellulose, reduce production costs, realize the resource-based and high-value utilization of agricultural waste, and provide a new technological approach for the development of the bioeconomy. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 Schematic diagram of the fermentation of engineered Rhodotorula buergerianum to produce 3-hydroxypropionic acid; Figure 2 Bacillus subtilis AC83 was inoculated into Congo red medium, and clear circles were produced after cellulase decomposed cellulose. Figure 3 The graph shows the dry matter degradation rate of wheat straw by Bacillus subtilis AC83. Figure 4 A schematic diagram showing the pH changes during aerobic composting fermentation using straw as a carbon source after adding engineered Bacillus subtilis AC83 and Rhodotorula buergerianum. Figure 5 A schematic diagram showing the change in electrical conductivity during aerobic composting fermentation using straw as a carbon source after adding engineered strains of Bacillus subtilis AC83 and Rhodotorula buergerianum. Figure 6 A schematic diagram showing the addition of engineered Bacillus subtilis AC83 and Rhodotorula buergerianum to produce 3-hydroxypropionic acid through aerobic composting fermentation using straw as a carbon source; Figure 7 A schematic diagram showing the changes in total volatile solids (TVS) during aerobic composting fermentation using straw as a carbon source, with the addition of engineered Bacillus subtilis AC83 and Rhodotorula buergerianum. Where TS represents total solids and VS represents volatile solids; Figure 8 This diagram illustrates the changes in cellulose, hemicellulose, and lignin content in straw after aerobic composting with straw as the carbon source under different treatments. Detailed Implementation

[0018] This invention provides a compound microbial agent comprising an engineered strain of Rhodotorula buergerianum and Bacillus subtilis; the engineered strain of Rhodotorula buergerianum contains a target gene, and the amino acid encoded by the target gene has the NCBI Reference Sequence WP_012258473.1 in the NCBI database.

[0019] As one implementation method, the preparation method of the engineered strain of Rhodotorula circotensivea includes: constructing an overexpression vector using a binary vector and a target gene; transforming the overexpression vector into bacteria to construct an overexpressing recombinant microorganism; and screening and purifying the overexpressing recombinant microorganism and Rhodotorula circotensivea to obtain the engineered strain of Rhodotorula circotensivea.

[0020] In one embodiment, the *Rhodotorula glutinis* comprises the strain described in the literature “Zheng, X., Liu, Y., Li, Y., Wang, Y., & Yang, X. (2024). De novo biosynthesis of 2-phenylethanol by metabolic engineering the oleaginous yeast”. Rhodotorula toruloide The strain of Rhodotorula circophylla described in the Journal of Agricultural and Food Chemistry, 72(48).

[0021] This invention also provides a method for constructing the engineered strain of *Rhodotorula buergerianum* as described above, comprising the following steps: The target gene was transferred into Rhodotorula buergerianum using a transgenic method to obtain an engineered strain of Rhodotorula buergerianum; the amino acid encoded by the target gene has the NCBI Reference Sequence WP_012258473.1 in the NCBI database.

[0022] In one embodiment, the transgenic method includes constructing an overexpression vector using a binary vector and a target gene, transforming the overexpression vector into bacteria to construct an overexpressing recombinant microorganism, and then screening and purifying the overexpressing recombinant microorganism with *Rhodotorula buergerianum* to obtain an engineered *Rhodotorula buergerianum* strain. In one embodiment, the binary vector includes the pCAMBIA series, pBIN series, or derivatives of the above vectors. In one embodiment, the binary vector includes the following core elements: left and right boundaries, a selection marker, a multiple cloning site, a prokaryotic replicon, and a prokaryotic selection marker. In one embodiment, constructing the overexpression vector using the binary vector and the target gene includes cloning the target gene into the binary vector using a restriction endonuclease. In one embodiment, the bacteria include *Agrobacterium*. In one embodiment, the screening method includes culturing *Agrobacterium* on YEP solid plates containing the corresponding antibiotic. In one embodiment, the purification method includes inoculating the engineered strain in YEP liquid medium containing antibiotics for purification.

[0023] In one embodiment, the ratio of *Bacillus subtilis* and *Rhodotorula glutinis* engineered strains in the compound microbial agent, based on the volume of the seed liquid, is 1-3:1. In another embodiment, the effective viable cell concentration of the *Bacillus subtilis* seed liquid is 1.0-5.0 × 10⁻⁶. 8 CFU / mL. As one embodiment, the effective viable cell concentration of the engineered *Rhodotorula buergerianum* strain is 5.0 × 10⁻⁶ CFU / mL. 7 ~2.0×10 8 CFU / mL; In one embodiment, the Bacillus subtilis includes Bacillus subtilis AC83. In another embodiment, the Bacillus subtilis AC83 can be conventionally replaced with other microbial strains capable of cellulolysis. This invention utilizes the characteristic of Bacillus subtilis to decompose lignocellulose substrates. The cellulase secreted by Bacillus subtilis itself performs "in-situ" and mild biological pretreatment of the lignocellulose substrate, providing fermentable sugars for subsequent fermentation by engineered Rhodotorula glutinis. This invention provides a low-cost pretreatment strategy for the continuous and efficient production of 3-HP.

[0024] In one embodiment, the effective viable cell concentration of the Bacillus subtilis seed solution can be 1.0 × 10⁻⁶. 8 CFU / mL, 2.0×10 8 CFU / mL, 3.0×10 8 CFU / mL, 4.0×10 8 CFU / mL or 5.0×10 8CFU / mL. As one embodiment, the effective viable cell concentration of the engineered *Rhodotorula buergerianum* strain can be 5.0 × 10⁻⁶ CFU / mL. 7 CFU / mL, 6.0×10 7 CFU / mL, 7.0×10 7 CFU / mL, 8.0×10 7 CFU / mL, 9.0×10 7 CFU / mL, 1.0×10 8 CFU / mL or 2.0×10 8 CFU / mL.

[0025] The present invention also provides the application of the engineered strain of Rhodotorula circophylla, the engineered strain of Rhodotorula circophylla constructed by the above-described method, or the composite bacterial agent in the production of 3-hydroxypropionic acid.

[0026] In one embodiment, the compound microbial agent is capable of producing 3-hydroxypropionic acid using lignin as a substrate. In another embodiment, the lignin-containing substrate includes straw.

[0027] This invention also provides a method for producing 3-hydroxypropionic acid from lignocellulose as raw material, using the compound microbial agent shown above to produce 3-hydroxypropionic acid; the steps of the method are as follows: straw and sludge are mixed at a mass ratio of 1:2 to obtain inoculum; the compound microbial agent is inoculated into the inoculum, and co-cultured aerobic fermentation is carried out to obtain 3-hydroxypropionic acid.

[0028] In one embodiment, the straw includes wheat straw. In another embodiment, before mixing the straw and sludge at a 1:2 mass ratio, pretreatment of the straw or sludge is included. In one embodiment, the straw pretreatment method is: natural air drying to a moisture content of 5%~10%, followed by crushing through a 10~30 mesh sieve. In another embodiment, the moisture content of the straw can be 5%, 6%, 7%, 8%, 9%, or 10%. In another embodiment, the particle size of the crushed straw can be 10 mesh, 15 mesh, 20 mesh, 25 mesh, or 30 mesh. In another embodiment, the sludge pretreatment method is: mixing the sludge and water at a mass ratio of 1:1~5, and incubating at a constant temperature of 30~40℃ for 15~30 days. In another embodiment, the mass ratio of sludge to water can be 1:1, 1:2, 1:3, 1:4, or 1:5. In one embodiment, the culture temperature can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. In another embodiment, the culture time can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days.

[0029] In one embodiment, after obtaining the inoculum, the C / N ratio of the inoculum is adjusted to 25-30:1, and the water content is adjusted to 55%-65%. In another embodiment, the C / N ratio of the inoculum can be 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In yet another embodiment, the water content of the inoculum can be 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%.

[0030] In one embodiment, the temperature for the co-culture aerobic fermentation is 30-35°C. In another embodiment, the pH for the co-culture aerobic fermentation is 6.0-7.0.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a compound microbial agent provided by the present invention and its application in the production of 3-hydroxypropionic acid, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] The culture medium used in this invention and its composition are described below: YEP liquid medium components: 10 g yeast extract, 20 g peptone, 5 g NaCl, 1000 mL distilled water, pH value approximately 6.5; YEP solid medium is YEP liquid medium with an additional 20 g / L agar powder.

[0033] YPD (Yeast Extract Peptone Glucose Medium) liquid medium components: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L; YPD solid medium consists of YPD liquid medium components with the addition of 20 g / L agar powder.

[0034] IM medium: K2HPO4 2.05 g, KH2PO4 1.45 g, NaCl 0.15 g, MgSO4·7H2O 0.5 g, CaCl2·2H2O 67 mg, FeSO4·7H2O 2.5 mg, (NH4)2SO4 0.5 g, glucose 2.0 g, distilled water 1000 mL, pH 7.0.

[0035] Beef extract peptone medium: 2.5 g peptone, 0.75 g beef extract, 1.25 g sodium chloride and 1000 mL water, pH adjusted to 7.4-7.6.

[0036] Carboxymethyl cellulose sodium (CMC-Na) medium: Carboxymethyl cellulose sodium (CMC-Na) 20.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, disodium hydrogen phosphate 2.5 g / L, peptone 2.0 g / L, yeast extract 0.5 g / L, pH 7.2±0.2.

[0037] NL medium: glucose 20 g / L, yeast extract 0.5 g / L, ammonium chloride 0.33 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium dihydrogen phosphate 1 g / L, sodium dihydrogen phosphate 1 g / L.

[0038] Congo red medium (1 L): sodium carboxymethyl cellulose 10.0 g, agar 15.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, NaNO3 1.0 g, KCl 0.5 g, Congo red 0.1 g; among which, Congo red needs to be prepared in advance as a 1% (w / v) filtered sterile aqueous solution.

[0039] Example 1: Construction and testing of engineered strains of Rhodotorula buergerianum The steps are as follows: The Rhodotorula buergerianum used in this invention ( Rhodotorula toruloides) is the strain described in “Zheng, X., Liu,Y., Li, Y., Wang, Y.,&Yang,

[0040] 1.1 Selection of Skeleton Carrier Use binary vectors suitable for Agrobacterium-mediated transformation (such as pCAMBIA series, pBIN series, or derivatives of the above vectors) as the basic framework. The vector should contain the following core elements: left and right boundaries, selection markers, multiple cloning sites, prokaryotic replicons, and prokaryotic selection markers.

[0041] The core elements function as follows: the left and right boundaries define the T-DNA region; the selection marker provides antibiotic resistance in yeast; the multiple cloning site is used to insert the target gene; and the prokaryotic replicon and prokaryotic selection marker are used to amplify and screen plasmids in E. coli, including genes such as kanamycin resistance genes.

[0042] In this embodiment, the pZPK vector was used for the experiment. The pZPK vector backbone carries a key enzyme gene driven by a strong promoter (Ppgk) and carrying a mitochondrial localization signal (MTS4). MCR It is equipped with a highly efficient terminator (Thsp) and is screened in yeast by resistance markers (Ntc).

[0043] 1.2 Construction of overexpression vectors The target gene used in this embodiment is the MCR gene. The amino acid encoded by the MCR gene is listed in the NCBI Reference Sequence of the NCBI database as WP_012258473.1. The malonyl-CoA reductase encoded by the MCR gene is responsible for directly converting the metabolic intermediate malonyl-CoA into the target product 3-HP.

[0044] This embodiment uses PCR technology to amplify the desired target gene coding sequence from template DNA and clone the target gene. The desired gene fragment is amplified using polymerase chain reaction (PCR). PCR amplification employs a high-fidelity DNA polymerase premixed reagent; specifically, the kit used is Takara's PrimeSTAR® Max DNA Polymerase. The reaction system and cycling program were prepared according to the kit's standard instructions.

[0045] Using restriction endonucleases XbaI and SacI and DNA ligase, the purified target gene fragment was cloned into the multiple cloning site of the pZPK vector to construct a recombinant overexpression vector.

[0046] Specifically, the vector linearization steps are as follows: the pZPK vector plasmid is double-digested with XbaI and SacI restriction endonucleases, and the linearized vector fragment is then recovered by agarose gel electrophoresis. Similarly, the PCR product or intermediate plasmid containing MTS4-MCR is double-digested with XbaI and SacI to recover the target gene fragment.

[0047] The successfully constructed recombinant overexpression vector was transformed into competent E. coli cells, and then screened on LB agar plates containing 50 µg / mL kanamycin. Single clones were picked, amplified by shaking, and plasmids were extracted for restriction enzyme digestion identification and DNA sequencing to verify that the target gene was correctly inserted and the reading frame was accurate.

[0048] 1.3 Construction of Agrobacterium engineered strain The verified recombinant plasmid was introduced into the *Agrobacterium* competent cells using either a freeze-thaw method or an electroporation method. The engineered *Agrobacterium* strain was then screened. Transformed *Agrobacterium* was plated on YEP solid plates containing 50 µg / mL spectinomycin and incubated upside down at 28°C for 2–3 days. Positive clones were screened. Single colonies growing on the plates were selected for verification using colony PCR and plasmid digestion. Rapid verification was performed using the colony PCR method described in the reference “Ullah A, Bashir A, Rehman B, et al. Optimization of colony polymerase chain reaction for the 16S rRNA of different strains of *Escherichia coli*[J]. Innovare Journal of Life Sciences, 2023: 32-35.”. Amplification was performed using the colonies as templates. Single *Agrobacterium* clones containing the correct recombinant plasmid were screened. These engineered strains were inoculated into YEP liquid medium containing antibiotics and cultured at 28°C with shaking at 200 rpm until the late logarithmic growth phase to obtain engineered *Agrobacterium* strains for subsequent co-culture.

[0049] 1.4 Co-culture and transformation of Agrobacterium-mediated transformation with yeast: First, prepare the yeast recipient by inoculating the Rhodotorula buergerianum strain into YPD liquid medium and shaking it at 30°C and 200 rpm until the mid-log growth phase.

[0050] Then, an antibiotic-free induction medium is prepared, which is either YEP medium containing 200 μM acetylsylgenone or IM medium containing 200 μM acetylsylgenone.

[0051] The Agrobacterium engineered bacteria obtained in step 1.3 were collected by centrifugation at 3000-5000 g for 5-10 min, resuspended in induction medium, and the bacterial concentration was adjusted to OD. 600 The concentration was between 0.5 and 1.0 to obtain an Agrobacterium engineered bacterial suspension.

[0052] Yeast cells cultured in YEP liquid medium to mid-logarithmic growth phase were collected by centrifugation at 3000~5000 g for 5~10 min, and washed with sterile water or induction medium to obtain yeast cells.

[0053] Agrobacterium suspension was mixed with yeast cells at a 2:1 ratio, and the mixture was evenly spread onto YPD solid medium plates lined with microporous membranes. The plates were then incubated in the dark at 20–25°C for 2–3 days to complete T-DNA transfer and integration. After incubation, the co-cultured yeast cells were washed off the membrane and spread onto YPD selective medium plates to screen for yeast transformants resistant to antibiotics such as hygromycin. The resistant transformants were then incubated at 30°C for 3–5 days. After the transformants emerged, they were continuously streaked 2–3 times for purification to obtain homozygous transformants.

[0054] Select a single colony that has been verified above and inoculate it into 3-5 mL of YPD liquid medium. Incubate at 30°C and 200 rpm for 24 h with shaking to obtain a primary activated culture. Take 1 mL of the primary activated culture and, based on the selected vector and strain resistance, transfer the primary activated culture product to 50 mL of fresh YPD medium containing the same antibiotic. Continue incubation at 30°C and 200 rpm with shaking until mid-logarithmic growth (OD2). 600 (≈4.0, specific time approximately 16~18 h), to obtain highly active engineered Rhodotorula buergerianum seed culture.

[0055] 1.5 Detection method for purified resistant yeast transformants Genomic DNA was extracted from the resistant yeast clone and verified by PCR. PCR amplification was performed using primers specific to the target gene to confirm that the T-DNA had been integrated into the yeast genome.

[0056] The specific primer sequences for the target gene are shown below: Upstream primer F (SEQ ID NO:1): 5'-CGGGTTGCATTTGGGGAAAG-3'; Downstream primer R (SEQ ID NO:2): 5'-GCAATGTGGACGCGATCTTC-3'.

[0057] The verified overexpression engineered strains were subjected to shake-flask fermentation or fermenter culture. The fermentation process was as follows: *Rhodotorula glutinis* engineered strains were streaked onto xylose-free YPD solid medium and incubated at 28°C for 36 h. The culture was then stored at 4°C for later use. Single colonies were picked and inoculated into YPD liquid culture tubes and incubated at 28°C and 180 rpm for 24 h to activate the cells. Then, 2% of the activated cells were inoculated into YPD liquid culture vials and incubated at 28°C and 180 rpm for 24 h to obtain the fermentation seed culture. The glucose carbon source in the YPD liquid medium was then replaced with xylose, and the seed culture was added at a 1% (v / v) inoculation ratio for continuous aerobic fermentation. Fermentation products were collected every 24 h, and the 3-HP yield was measured.

[0058] The yield of 3-HP was determined by high performance liquid chromatography (HPLC). The detection conditions were as follows: HPX-87H column (300 mm × 7.8 mm, 5 μm); mobile phase 5 mmol / L H2SO4, without organic phase; mobile phase flow rate 0.6 mL / min; column temperature 65 ℃; injection volume 20 μL; and UV detector (210 nm).

[0059] 1.6 Experimental Results: The 3-HP yield of the engineered Rhodotorula glutinis during fermentation in a xylose-only carbon source medium was as follows: Figure 1 As shown, the yield of 3-HP exhibits a continuous increasing trend throughout the fermentation experiment. The growth rate of 3-HP yield is relatively rapid within 0-96 h, but slows down significantly after 96 h. These experimental results demonstrate that the engineered *Rhodotorula glutinis* strain described in this invention can produce 3-HP and exhibits good yield performance.

[0060] Example 2 Straw Degradation Experiment This embodiment uses cellulase activity detection experiments of Bacillus subtilis AC83 as evaluation indicators, with the results of Congo red transparent zone and the results of cellulose, hemicellulose and lignin content detection after 30 days of aerobic fermentation of wheat straw as substrate. The Bacillus subtilis AC83 has cellulose decomposition function. The Bacillus subtilis strain AC83 is disclosed in the literature "Chen Huan, et al. Screening, mutagenesis and breeding of high-yield cellulose-degrading bacteria and their straw degradation effect[J]. Journal of Animal Nutrition, 2024, 36(12):8126-8136.DOI:10.12418 / CJAN2024.693."; Rhodotorula rubrum was donated by Professor Yang Xiaobing's laboratory of Northwest A&F University. The laboratory number of this strain is: YH-2024-01.

[0061] Congo red can form a red complex with cellulose. When cellulose is broken down by cellulase produced by microorganisms, this complex cannot form, and a clear zone centered on the cellulose-decomposing bacteria appears in the culture medium. Therefore, the presence and size of the clear zone can be used to screen for cellulose-decomposing bacteria. In this example, Bacillus subtilis AC83 was inoculated into Congo red medium for screening cellulose-decomposing bacteria. In Congo red medium, cellulase breaks down cellulose and produces a clear zone. Enzyme activity was assessed by the ratio of the clear zone diameter to the colony diameter. 2 µL of the seed culture described in Example 1 was inoculated into the center of a Congo red medium plate using the spot inoculation method. Each strain was replicated in triplicate. The inoculated plates were placed in a 37°C incubator and inverted for 48–72 h. The culture results are shown below. Figure 2 As shown. By Figure 2As can be seen, Bacillus subtilis AC83 formed clear, transparent hydrolysis zones after cultivation. Measurements showed that the average diameter of the hydrolysis zones (D_h) was 3 ± 0.5 mm, and the average colony diameter (D_c) was 1.2 ± 0.2 mm. These results confirm that Bacillus subtilis AC83 possesses significant cellulose-degrading capabilities and can serve as an effective biodegrading strain for lignocellulose raw materials.

[0062] Wheat straw was dried at 65℃ to constant weight, chopped to 0.5 cm, and used as the sole carbon source to prepare a culture medium. Bacillus subtilis AC83 bacterial suspension was then inoculated onto the straw medium at a 2% inoculum. An equal volume of sterile water was used as a control. Fermentation was carried out at 35℃ and 180 r / min for 30 days. After fermentation, the straw was filtered through gauze, thoroughly washed, and dried at 65℃ to constant weight. The dry matter degradation rate was calculated using the weight loss method. The results are shown below. Figure 2 As shown, samples from the control group after 30 days and the experimental groups after 15 days and 30 days were measured. The calculation formula is: Straw dry matter degradation rate (%) = [(W0-Wt) / W0]×100.

[0063] In the formula: W0 is the initial dry weight of straw; Wt is the dry weight of straw after t days of cultivation.

[0064] The test results of dry matter degradation rate in wheat straw are as follows: Figure 3 As shown.

[0065] Depend on Figure 3 It is evident that the Bacillus subtilis AC83 described in this invention has a strong ability to degrade straw dry matter, and can significantly improve the dry matter degradation rate in straw within 15 to 30 days.

[0066] Example 3: Preparation method of engineered compound bacterial agent of Bacillus subtilis and Rhodotorula glutinis The steps are as follows: Bacillus subtilis AC83 was inoculated into beef extract peptone medium for activation culture at 35°C overnight to obtain activated Bacillus subtilis inoculum. The activated Bacillus subtilis was then inoculated into sodium carboxymethyl cellulose medium at a volume ratio of 2% for fermentation culture at 35°C, 180 rpm overnight to obtain Bacillus subtilis seed culture.

[0067] The engineered strain of *Rhodotorula circotensivea* was first inoculated into YPD medium for large-scale culture at 30℃ and 180 rpm for 24-48 hours to obtain activated *Rhodotorula circotensivea* inoculum. The activated *Rhodotorula circotensivea* inoculum was then inoculated into NL medium for fermentation culture at 30℃ and 180 rpm for 24-48 hours to obtain *Rhodotorula circotensivea* seed culture.

[0068] A co-culture inoculum was prepared by mixing Bacillus subtilis AC83 seed culture, cultured to the logarithmic growth phase, with engineered Rhodotorula buergerianum seed culture at a volume ratio of 2:1. The effective viable cell concentration of the Bacillus subtilis seed culture should be adjusted to 1.0 × 10⁻⁶. 8 CFU / mL ~5.0×10 8 CFU / mL. The effective viable cell concentration of the engineered Rhodotorula rubra seed culture should be adjusted to 5.0 × 10⁻⁶ CFU / mL. 7 CFU / mL ~2.0×10 8 CFU / mL. After mixing at this ratio and concentration, the viable cell ratio of the two bacteria (Bacillus subtilis: Rhodotorula glutinis) in the initial co-culture system is approximately 1:1 to 5:1. The specific ratio is determined based on the total sugar and reducing sugar content in the fermentation system. This strain ratio ensures that the free sugars produced by Bacillus subtilis can supply the engineered Rhodotorula glutinis, guaranteeing its survival and transformation. In the mixed inoculum, the addition of Bacillus subtilis serves as a key step in the biological pretreatment of straw degradation; its produced cellulase accelerates the hydrolysis of straw and other biomass into smaller molecules. Rhodotorula glutinis utilizes the small-molecule sugars decomposed by Bacillus subtilis for aerobic fermentation to further produce 3-HP. Mixing Bacillus subtilis and Rhodotorula glutinis in the correct ratio ensures that Bacillus subtilis initially dominates in terms of quantity and metabolism, rapidly initiating the straw degradation process.

[0069] Example 4: Fermentation Method and Effect Verification Using Compound Microbial Agents This embodiment uses the mixed bacterial agent described in Example 3 as a sample to investigate the yield of 3-HP during aerobic fermentation.

[0070] 1. Raw material collection and processing: Wheat straw and sludge from near the sewage treatment plant in Yangling Demonstration Zone, Shaanxi Province were collected as experimental raw materials.

[0071] Air dry the wheat straw naturally until the moisture content is about 7%, then crush it into particles that can pass through a 20-mesh sieve. Store in an airtight container for later use.

[0072] Sludge from a wastewater treatment plant near the Yangling Demonstration Zone in Shaanxi Province was manually collected using sampling bottles as inoculum material. The sludge was mixed with tap water at a mass ratio of 1:2 and then incubated at a constant temperature of 35℃ for 15-30 days to obtain the desired acclimatized sludge inoculum.

[0073] 2. Co-culture fermentation experiment A 250 mL feed bottle was used as the co-culture fermentation reactor, and the fermentation temperature was 35℃. Wheat straw and sludge inoculum were mixed evenly at a mass ratio of 1:2, with the initial C / N ratio controlled at 30:1, and the moisture content adjusted to 55%–65%. The mixed material and prepared culture medium were placed into a 250 mL feed bottle, and 20 mL of Bacillus subtilis and 10 mL of Rhodotorula glutinis engineered strain seed culture were inoculated as the experimental group. The Bacillus subtilis and Rhodotorula glutinis engineered strain seed culture were replaced with an equal volume of sterile water as the control group. The Bacillus subtilis AC83 group was formed by replacing the Bacillus subtilis and Rhodotorula glutinis engineered strain seed culture with an equal volume of Bacillus subtilis culture.

[0074] The experimental and control groups were co-cultured and fermented simultaneously at a temperature of 35℃ and a pH of 6.5. Changes in pH and conductivity during fermentation were measured, and the results are as follows: Figure 4 , Figure 5 As shown in the figure. During the co-fermentation process, the fermentation reactor was thoroughly stirred once every 24 hours, and the fermentation broth was sampled and measured every 24 hours. The yield of 3-HP, TSVS, and other indicators in the fermentation broth were statistically analyzed. The results are shown in the figure. Figures 6-7 As shown in the figure. The contents of cellulose, hemicellulose, and lignin in straw after fermentation in the experimental group, control group, and Bacillus subtilis AC83 group were detected. The results are shown in the figure. Figure 8 As shown.

[0075] 3. Experimental Results: Depend on Figure 4 As can be seen, during the aerobic composting process of co-culturing Bacillus subtilis and Rhodotorula glutinis engineered strain seed liquid, the pH value of the culture medium continuously decreased from the initial 7.57 (0 h) to a minimum of 5.58 (72 h). The pH change mainly stemmed from the acidity of 3-HP itself and the accumulation of a small amount of organic acid byproducts. Furthermore, based on the product yield, cell growth was not significantly inhibited in the first 72 h, indicating that the engineered strain in this embodiment inherited the excellent low pH tolerance of wild-type Rhodotorula glutinis. The Rhodotorula glutinis engineered strain provided by this invention provides a foundation for the high yield of acidic products (3-HP).

[0076] Depend on Figure 5It is evident that changes in conductivity can serve as an indicator of changes in ionic strength within the fermentation broth. During the aerobic composting process involving the co-cultivation of Bacillus subtilis and Rhodotorula glutinis engineered strains in seed culture, changes in conductivity indirectly reflected the strains' metabolism of inorganic salt ions and inhibitors in the hydrolysate. The conductivity value gradually increased from 5.8 mS / cm to 7.2 mS / cm, then decreased and stabilized at around 3.2 mS / cm, suggesting that the strains may have metabolized certain organic acid ions, leading to the decrease in conductivity.

[0077] Depend on Figure 6 It is evident that the highest 3-HP yield, reaching 1.25 g / L, was observed during the 72-hour co-fermentation period. After 72 hours, the 3-HP content decreased continuously as fermentation progressed.

[0078] Depend on Figure 7 It can be seen that the TS degradation rate of co-culture fermentation reached 10.87%, which was greater than that of the control group. The VS degradation rate of co-culture fermentation reached 0.09%, which was greater than that of the control group (0.035%).

[0079] Depend on Figure 8 As can be seen, the changes in straw composition in different groups during the aerobic composting process of co-culturing Bacillus subtilis and Rhodotorula glutinis engineered strains of straw seed liquid are as follows: The control group (naturally degraded) had the highest proportion of cellulose residue (approximately 62%), indicating that the natural degradation process of straw is slow and incomplete without the intervention of exogenous highly efficient functional bacteria.

[0080] The proportion of cellulose residue in the group treated with Bacillus subtilis AC83 alone was significantly reduced to about 36%, and the proportion of hemicellulose residue was also reduced to about 29%. This fully demonstrates that Bacillus subtilis AC83 itself has a strong ability to decompose cellulose and hemicellulose and can be used as an excellent strain for biological pretreatment.

[0081] The treatment group using a mixed microbial agent of Bacillus subtilis and engineered Rhodotorula glutinis achieved the best degradation effect, with the residual cellulose content significantly reduced to approximately 39%. Overall, the mixed agent showed better overall performance; it not only degraded lignin more efficiently (at least 5% in all groups), but also increased the relative accumulation of hemicellulose to 41%, while reducing the formation of non-target products, demonstrating a more comprehensive lignocellulose degradation capability. The degradation efficiency of the mixed agent on cellulose was significantly better than that of Bacillus subtilis alone. This is not a simple superposition of the functions of the two, but a direct manifestation of the "decomposition-synthesis" synergistic mechanism designed in this invention.

[0082] In summary, this invention provides a compound microbial agent and its application in the production of 3-hydroxypropionic acid. The compound microbial agent of this invention can solve the problem that current 3-HP-producing microorganisms have limited utilization capacity of lignocellulose, resulting in product inhibition and low substrate conversion rates. It can effectively improve the utilization rate of lignocellulose, reduce production costs, realize the resource-based and high-value utilization of agricultural waste, and provide a new technical approach for the development of the bioeconomy.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent includes engineered strains of Rhodotorula buergerianum and Bacillus subtilis; The engineered strain of *Rhodotorula buergerianum* contains the target gene, and the amino acid encoded by the target gene has the NCBI Reference Sequence WP_012258473.1 in the NCBI database.

2. The compound microbial agent as described in claim 1, characterized in that, The method for constructing the engineered strain of Rhodotorula circotensivea includes: constructing an overexpression vector using a binary vector and a target gene; transforming the overexpression vector into bacteria to construct an overexpressing recombinant microorganism; and screening and purifying the overexpressing recombinant microorganism and Rhodotorula circotensivea to obtain the engineered strain of Rhodotorula circotensivea.

3. The compound microbial agent as described in claim 1, characterized in that, In the compound microbial agent, the ratio of Bacillus subtilis and Rhodotorula glutinis engineered strains is 1~3:1 based on the volume of the seed liquid; The effective viable cell concentration of the Bacillus subtilis seed solution is 1.0 × 10⁻⁶. 8 CFU / mL ~5.0×10 8 CFU / mL; the effective viable cell concentration of the engineered strain of *Rhodotorula circophylla* is 5.0 × 10⁻⁶ CFU / mL. 7 CFU / mL ~2.0×10 8 CFU / mL; The Bacillus subtilis includes Bacillus subtilis AC83.

4. The use of the compound microbial agent according to any one of claims 1 to 3 in the production of 3-hydroxypropionic acid.

5. A method for producing 3-hydroxypropionic acid from lignocellulose, characterized in that, Production of 3-hydroxypropionic acid using the compound microbial agent according to any one of claims 1 to 3; The steps of the method include: Straw and sludge were mixed at a mass ratio of 1:2 to obtain the inoculum; The compound bacterial agent was inoculated into the inoculum, and co-cultured aerobic fermentation was carried out to obtain 3-hydroxypropionic acid.

6. The method as described in claim 5, characterized in that, The straw includes wheat straw; Before mixing straw and sludge at a mass ratio of 1:2, the process also includes pretreatment of the straw or sludge. The pretreatment of the straw is as follows: air-drying to a moisture content of 5% to 10%, and crushing it through a 10 to 30 mesh sieve; The sludge pretreatment involves mixing the sludge with water at a mass ratio of 1:1 to 5 and incubating at a constant temperature of 30 to 40°C for 15 to 30 days.

7. The method as described in claim 5, characterized in that, After obtaining the inoculum, the process further includes adjusting the C / N ratio of the inoculum to 25~30:1 and the water content to 55%~65%.

8. The method as described in claim 5, characterized in that, The temperature for the co-culture aerobic fermentation is 30~35℃; the pH for the co-culture aerobic fermentation is 6.5~7.0.

Images