Method for enhancing nutritional value of microbial mycoprotein

Abstract

The invention discloses a method for enhancing the nutritional value of microbial mycoprotein. Protein rich in essential amino acid is heterologously expressed in the recombinant kluyveromyces marxianus, and the contents of crude protein, essential amino acid required for feeding human bodies and animals and branched chain amino acid in yeast obtained by fermentation are increased to different extents, so that the nutritional value of mycoprotein is enhanced. The method can be used for producing high-quality microbial protein, and has wide application value in the fields of food and feed.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and biotechnology, and specifically relates to a method for enhancing the nutritional value of microbial cell proteins. Background Technology

[0002] By 2050, the global population will approach 10 billion, and protein demand will increase by more than 50%, resulting in a global protein shortage of 250 million tons. Microbial protein (MP), also known as single-cell protein (SCP), is an important protein source besides plant and animal-derived proteins. Compared with traditional plant and animal-derived proteins, microbial protein has the following advantages: 1) Protein content is as high as 40%–80%, 10%–30% higher than soybeans, containing a wide variety of essential amino acids, and some are also rich in vitamins; 2) Rapid reproduction and high production efficiency, with protein production efficiency per unit area 8,000 times higher than soybeans and 80,000 times higher than cattle; 3) Abundant and inexpensive raw materials, enabling resource utilization of waste; 4) Industrialized production, not limited by region, season, or climate, saving land and water resources, and enabling green and sustainable production. As a new resource for feed and edible protein, microbial protein will play an increasingly important role, with a broad market prospect.

[0003] The content of essential amino acids (EAAs) is one of the important indicators for evaluating the nutritional value of protein. The nine essential amino acids required by the human body are histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), tryptophan (W), and valine (V). The twelve essential amino acids required for animal feed are lysine (K), methionine (M), histidine (H), isoleucine (I), leucine (L), phenylalanine (F), tyrosine (Y), valine (V), cysteine ​​(C), threonine (T), arginine (R), and tryptophan (W). Branched-chain amino acids (BCAAs, including leucine, isoleucine, and valine) have physiological functions such as promoting human protein synthesis, enhancing immunity, and promoting embryonic development. They also play an important role in poultry growth, production performance, immune function, and gut health. A deficiency in certain essential amino acids in protein can lead to the inefficient utilization of other amino acids, thus reducing the overall digestibility of the protein. Ingesting protein containing essential amino acids can stimulate the body to produce essential non-protein substances, such as serotonin, thyroid hormones, and glutathione. Therefore, protein sources that provide all essential amino acids in high amounts are considered "high-quality" proteins.

[0004] Improving the essential amino acid content of proteins through molecular genetic modification is an important way to enhance the nutritional value of protein sources from different species. Currently, this method has been extensively studied in the field of plant-derived protein fortification. Examples include replacing non-essential amino acids in proteins with essential amino acids (CN1638785A); expressing heterologous or modified genes rich in essential amino acids to encode storage proteins (CN101340814B); regulating amino acid metabolic pathways to increase the supply of target free amino acids, such as lysine, methionine, and threonine, while overexpressing proteins rich in target amino acids (CN1195377A); and using protein engineering to artificially design and optimize proteins rich in essential amino acids (WO2023090987A, CA2404050A1), etc. In contrast, the fortification of essential amino acids in microbial proteins mainly relies on methods optimized using traditional fermentation processes (CN101134942A, CN104839445A). There are also a few examples of engineered proteins rich in essential amino acids or branched-chain amino acids being expressed in recombinant microorganisms, such as *Pichia pastoris* (Revista Mexicana De Ingenieria Quimica, 2021.20(3)), *Escherichia coli* (Mol Biol Rep, 2019.46(2):1593-1601, US9605040B2), cyanobacteria (CN104364259A), *Aspergillus niger*, and *Bacillus subtilis* (CN104936466A). However, the focus of these studies is on producing engineered proteins, rather than enhancing the overall nutritional value of microbial proteins. Therefore, further research and development are needed to increase the content of essential amino acids in microbial proteins and thus enhance their nutritional value through molecular genetic modification. Summary of the Invention

[0005] In view of the above-mentioned problems of the prior art, the object of the present invention is to provide recombinant microorganisms expressing proteins rich in essential amino acids, thereby enhancing the nutritional value of microbial cell proteins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention first provides a recombinant microorganism expressing a protein rich in essential amino acids, wherein the crude protein content of the recombinant microorganism is increased relative to the original microorganism, and the characteristics of the expressed protein rich in essential amino acids satisfy any one or more of (a) to (c):

[0008] (a) The mass percentage content of the nine essential amino acid residues for the human body is within the range of 28.0%-47.0% or 51.0%-77.0%;

[0009] (b) The mass percentage content of the 12 essential amino acid residues for feeding animals is in the range of 41.0%-58.0% or 61.0%-79.0%; (c) The mass percentage content of branched-chain amino acid residues is in the range of 10.0%-21.0% or 23.0%-35.0%.

[0010] Preferably, the recombinant microorganism is yeast; more preferably, the yeast is Kluyveromyces martensii; preferably, the amino acid sequence of the protein rich in essential amino acids is as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, SEQ ID NO: 4.

[0011] The present invention also provides a recombinant microorganism expressing a protein rich in essential amino acids. The content of the nine essential amino acids in the cells of the recombinant microorganism is increased compared with the original strain, and the characteristics of the protein rich in essential amino acids expressed meet the following conditions: the mass percentage content of the nine essential amino acid residues is within the range of 28.0%-39.0% or 48.0%-77.0%.

[0012] Preferably, the recombinant microorganism is yeast; more preferably, the yeast is Kluyveromyces martensii; preferably, the amino acid sequence of the protein rich in essential amino acids is as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_001371279.1, SEQ ID NO: 1, XP_024848751.1, SEQ ID NO: 3, SEQ ID NO: 4.

[0013] The present invention also provides a recombinant microorganism expressing a protein rich in essential amino acids, wherein the content of the 12 essential amino acids for feeding animals in the cells of the recombinant microorganism is increased compared with the original strain, and the characteristics of the expressed protein rich in essential amino acids meet the following conditions:

[0014] The recombinant microorganism contains 12 essential amino acid residues in the protein, which are essential for feeding animals (pigs, chickens, ducks, grass carp, conger eels, loaches, crucian carp, silver carp, common carp, shrimp, etc.) at a mass percentage range of 41.0%-61.0% or 62.0%-79.0%. Preferably, the recombinant microorganism is yeast. More preferably, the yeast is Kluyveromyces martensii. Preferably, the amino acid sequence of the protein rich in essential amino acids is as shown in NP_776803.1, SEQ ID NO: 1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, and SEQ ID NO: 4.

[0015] The present invention also provides a recombinant microorganism expressing a protein rich in essential amino acids, wherein the content of branched-chain amino acids in the cells of the recombinant microorganism is increased compared with that of the originating microorganism, wherein the branched-chain amino acids refer to leucine, isoleucine and valine; the expressed protein rich in essential amino acids has the following characteristics: the mass percentage content of branched-chain amino acid residues is in the range of 10.0%-35.0%.

[0016] Preferably, the recombinant microorganism is yeast; more preferably, the yeast is Kluyveromyces martensii; preferably, the amino acid sequence of the protein rich in essential amino acids is as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_001371279.1, SEQ ID NO: 1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, SEQ ID NO: 4.

[0017] The present invention further provides a method for constructing the recombinant Kluyveromyces martensii, comprising the following steps: inserting a codon-optimized nucleotide sequence encoding a protein rich in essential amino acids into an expression plasmid, using a PDC1 promoter and terminator, and fusing a green fluorescent protein tag at the C-terminus of the protein to obtain a yeast exogenous protein expression plasmid; introducing the plasmid into a Kluyveromyces martensii host to obtain the recombinant Kluyveromyces martensii.

[0018] Preferably, the nucleotide sequence of the PDC1 promoter. Preferably, the nucleotide sequence encoding the open reading frame of the HIS and eGFP tag. Preferably, the nucleotide sequence of the PDC1 terminator.

[0019] Preferably, the codon-optimized nucleotide sequence encoding a protein rich in essential amino acids is shown in SEQ ID No: 5-14.

[0020] The present invention also provides the application of the recombinant Kluyveromyces martensii in the fermentation production of high-quality microbial protein, characterized in that the high-quality microbial protein refers to the content of crude protein in the bacterial cells being higher than that of the original strain, or the high-quality microbial protein refers to the content of the nine essential amino acids for humans in the bacterial cells being higher than that of the original strain, or the application is to increase the content of the twelve essential amino acids for feeding animals in the bacterial cells, or the application is to increase the content of branched-chain amino acids in the bacterial cells.

[0021] Preferably, the recombinant bacteria are fermented to produce microbial cell protein; preferably, expression is achieved by purifying proteins rich in essential amino acids through fusion tags.

[0022] This invention enhances the nutritional value of microbial protein by expressing proteins rich in essential amino acids, thereby increasing the content of crude protein, essential amino acids required for human and animal feeding, and branched-chain amino acids in recombinant microbial cells. It has broad application value in the food and feed industries. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the expression and detection process of the target protein in Kluyveromyces martensii.

[0024] Figure 2 This is a schematic diagram of the construction of the yeast expression plasmid for the target protein.

[0025] Figure 3 It utilizes laser scanning confocal microscopy to detect the expression of target proteins indicated by green fluorescent protein in yeast expression cell factories.

[0026] Figure 4 These are the SDS-PAGE and Western blot results for expressing and purifying the target protein.

[0027] Figure 5 This is a coverage map of the target protein identified by mass spectrometry. The green text indicates the peptides detected by mass spectrometry.

[0028] Figure 6 This is a trend graph showing the effects of different target proteins on the accumulation of yeast cells.

[0029] Figure 7 This is a trend graph showing the influence of different target proteins on the crude protein content of yeast cells.

[0030] Figure 8 This is a trend chart showing the influence of different target proteins on the essential amino acid composition of yeast cells. Detailed Implementation

[0031] The process is as follows Figure 1As shown in the diagram, firstly, proteins rich in essential amino acids and functional elements such as promoters and terminators required for protein expression were identified, and yeast exogenous protein expression plasmids were constructed. The nucleotide sequences of the proteins were codon-optimized, and fluorescent protein tags were fused to the exogenous proteins to facilitate subsequent evaluation and screening of exogenous protein expression levels. Then, the plasmids were transformed into yeast cells, and transformants were obtained. Flow cytometry was used for fluorescence screening and plate culture. Transformants with high protein expression were selected, and growth curves, fluorescence values, and laser confocal microscopy were used to analyze the growth capacity and protein expression status of the transformants. Further protein purification and expression identification were then performed. Finally, fermentation experiments were conducted to analyze the crude protein and amino acid content in the cells, thereby assessing the degree to which the expression of proteins rich in essential amino acids enhances the nutritional value of the bacterial protein.

[0032] Example 1: Selection of proteins rich in essential amino acids

[0033] According to statistics from the China Feed Database, conventional protein sources, including soybeans, soybean meal, fishmeal, and brewer's yeast, contain crude protein, the nine essential amino acids for humans, the twelve essential amino acids for animal feed, and branched-chain amino acids at mass percentages ranging from 35.5% to 67.0%, 12.1% to 25.6%, 16.0% to 32.1%, and 5.5% to 11.0%, respectively (Table 1). Through literature review, patent searches, and NCBI database searches, we selected 10 yeast exogenous proteins with different amino acid compositions that have higher levels and wider ranges of the above-mentioned amino acid content compared to conventional protein sources, as the target proteins of this invention (Table 2). Table 3 shows the amino acid composition of these target proteins. The mass percentages of the nine essential amino acids for humans, the twelve essential amino acids for animal feed, and branched-chain amino acids in these target proteins range from 28.8% to 76.6%, 41.7% to 78.2%, and 10.4% to 34.9%, respectively.

[0034] Table 1. Crude protein content and amino acid composition of common protein sources

[0035]

[0036] Table 2 Yeast exogenous proteins rich in essential amino acids

[0037]

[0038] Note: Document 1, Sanchez-Crisostomo, MI, et al., Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes. Mol Biol Rep, 2019.46(2):1593-1601; Document 2, Bartolo-Aguilar, Y., et al., Expression of a synthetic protein with a high proportion of essential aminoacids by Pichia pastoris. Revista Mexicana De Ingenieria Quimica, 2021.20(3).

[0039] Table 3. Amino acid composition of proteins rich in essential amino acids

[0040]

[0041]

[0042] Example 2: Construction of yeast exogenous protein expression plasmid

[0043] The plasmid construction process is illustrated below. Figure 2As shown in Table 4, the primers used for constructing the expression vector are shown in Table 4. First, the expression cassette plasmid for the foreign protein was constructed. On one hand, the backbone plasmid pMTU-DO-G418-C4 was purchased from Addgene (catalog number: 160342). This plasmid contains the high-copy autonomous replication sequence ARS2 from *Kluyveromyces martensii* and the G418 selection marker gene KanMX, enabling autonomous replication in this yeast and allowing transformations to be selected using YPD plates containing G418. On the other hand, the expression cassette used the PDC1 promoter (1000bp) and terminator (250bp) from *Kluyveromyces martensii*. The 6×His and green fluorescent protein (eGFP) tag were located at the C-terminus of the foreign protein, and the codons were optimized. The above expression cassette was synthesized by Genewiz Biotechnology Co., Ltd., and the superfolderGFP coding sequence in pMTU-DO-G418-C4 was replaced. The resulting plasmid was named pMTU-HIS-eGFPopt, which also served as a blank plasmid control for expressing the foreign protein. Then, using the empty plasmid as a backbone, codon-optimized target protein coding sequences (SEQ ID NO: 5 to 14) were inserted downstream of the start codon ATG in the expression cassette. These sequences were synthesized by Tianjin Zhonghe Gene Technology Co., Ltd., and the cloning method followed the homologous recombination rapid cloning kit. Follow the instructions (Brand: TransGen, Catalog No.: CU201-03). Using first-generation sequencing (sequencing primers in Table 4), select correctly sequenced plasmids and extract them, naming them pMTU-1btαLB, pMTU-2Bra, pMTU-3chαLB, pMTU-4hαLB, pMTU-5hαLBm, pMTU-6hLF, pMTU-7hβC, pMTU-8btOPN, pMTU-9HEAAP, and pMTU-10SPA2 (the nucleotide sequences of these genes have undergone codon optimization, and their sequences correspond to SEQ ID NO: 5-14, respectively). These plasmids will be used for subsequent transformation with Kluyveromyces martensii.

[0044] Table 4 Primers for expression vector construction

[0045]

[0046]

[0047] Example 3: Obtaining and screening yeast transformants

[0048] Ten target protein expression plasmids and one blank control plasmid obtained in Example 2 were transformed into Kluyveromyces martensii strain GQ7 (purchased from Beijing Beina Chuanglian Biotechnology Research Institute, BNCC191517) using a chemical transformation method (kit: Frozen-EZ Yeast Transformation II Kit, catalog number: T2001, brand: Zymo). The transformation products were plated on YPD plates (G418 selection plates) containing 200 μg / mLG418 for screening. After incubation at 30°C for 2-4 days, the single colonies that grew were the yeast transformants carrying the expression plasmids, and were named KM-1btαLB, KM-2Bra, KM-3chαLB, KM-4hαLB, KM-5hαLBm, KM-6hLF, KM-7hβC, KM-8btOPN, KM-9HEAAP, KM-10SPA2, and KM-CK, respectively.

[0049] Since the target protein is fused with the green fluorescent protein eGFP, the expression intensity of the target protein is directly proportional to the green fluorescence intensity of the transformant cells. To further isolate transformant cells with high target protein expression intensity, we used flow cytometry (BDFACSAriaFusionSORP) to screen and isolate transformants with high fluorescence intensity. After sorting, the plates were incubated at 30℃ for 1-2 days until single colonies grew. For each yeast transformant expressing the target protein, 8 single colonies were randomly selected from its flow cytometry sorting plate. These single colonies were then transferred to 48-well plates containing 1 ml of LG418 liquid selection medium and incubated at 30℃ and 800 rpm for 20 hours. The fluorescence value and cell growth rate (OD) were measured using a BioTek multi-plate reader. 600 The ratio of cell fluorescence value to biomass is calculated, which is the cell fluorescence intensity value. Transformants with the highest fluorescence intensity are then screened.

[0050] Figure 3 This image shows the expression of target proteins indicated by green fluorescent protein (eGFP) in a yeast cell factory for target protein expression, detected using laser scanning confocal microscopy. The presence of clear eGFP green fluorescence in the target protein expression cells indicates successful expression of all 10 target proteins. The fluorescence signals of target protein expression bacteria KM-2Bra, KM-7hβC, KM-8btOPN, and KM-CK are distributed throughout the cell, with strong fluorescence signals, indicating high expression levels of the corresponding target proteins. The fluorescence signals of target protein expression bacteria KM-1btαLB, KM-3chαLB, KM-4hαLB, KM-5hαLBm, KM-6hLF, KM-9HEAAP, and KM-10SPA2 show some regional clustering, possibly due to increased expression levels during the expression process.

[0051] Example 4: Expression, purification, and identification of proteins rich in essential amino acids

[0052] In this embodiment, the target protein cell factories KM-2Bra, KM-8btOPN, KM-10SPA2, and the control strain KM-CK were randomly selected for protein expression, purification, and identification. Since the target protein has a 6×His tag fused to its C-terminus, protein purification can be performed using a Ni affinity chromatography column.

[0053] The yeast cells were inoculated into 5 mL of YD40 liquid medium containing G418 (20 g / L yeast extract, 40 g / L glucose, 200 μg / mL G418) and cultured overnight at 30°C and 220 rpm. The overnight cells were then diluted and inoculated into 50 mL of YD40 liquid medium containing G418, with the initial OD... 600 The cells were cultured at 0.2°C, 30°C, and 220 rpm for 40 hours, and an equal amount of biomass was collected. The cells were resuspended in 50 mL of PBS buffer (phosphate buffer containing 1.06 mM KH₂PO₄, 155.17 mM NaCl, 2.97 mM Na₂HPO₄, 1 mmol / L PMSF protease inhibitor, pH 7.4), and the cell walls were disrupted by high-pressure homogenization. The obtained supernatant was added to a Ni affinity chromatography column (1 mL), and protein purification was performed using an AKTAavant 150 automated protein purification process optimization system. The purified samples were collected for SDS-PAGE analysis, Western blot identification, and mass spectrometry detection of the target protein. Mass spectrometry sample processing and detection were performed by the Center for Systems Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

[0054] Figure 4 This presents the SDS-PAGE and Western blot results for the expression and purification of the target protein. During protein purification, gradient elution was performed using elution buffer B with different imidazole concentrations. Samples were collected before and after the elution peaks for SDS-PAGE and Western blot analysis. Compared to the purification results of 6×His-eGFP expressed in the control strain KM-CK, the target protein purified from KM-2Bra, KM-8btOPN, and KM-10SPA2 cells may have undergone varying degrees of protein degradation during cell disruption and purification. Therefore, we selected multiple bands for mass spectrometry detection.

[0055] Based on the Western blot results, the corresponding bands from SDS-PAGE were selected for gel cutting and mass spectrometry analysis. The gel-cut bands are shown below. Figure 4 As indicated by the arrow in section A. Figure 5This is a mass spectrometry (MS) coverage map of the target proteins identified, with sequences highlighted in green indicating those detectable by MS. The target protein detection coverage is shown in Table 5. The 6×His-eGFP MS detection coverage of the control strain KM-CK was 60%. For the exogenous protein 2Bra, three bands were selected from the gel image for MS detection, with one band showing detectable target protein, resulting in a coverage of 17%. For the exogenous protein 8btOPN, four bands were selected from the gel image for MS detection, all showing detectable target protein, with coverage ranging from 25% to 55%, indicating significant degradation during cell disruption and purification. For the exogenous protein 10SPA2, three bands were selected from the gel image for MS detection, with one band showing detectable target protein, resulting in a coverage of 8%. The results indicate that all target proteins were successfully expressed.

[0056] Table 5 Target protein detection coverage

[0057]

[0058] *Note: SDS-PAGE strip numbering, see details. Figure 4 The annotation in A.

[0059] Example 5: The effect of expressing proteins rich in essential amino acids on the nutritional value of yeast cell protein.

[0060] In this embodiment, the single colony of the cell factory expressing the target protein with the highest fluorescence intensity selected in Example 4 was subjected to cell culture, crude protein and amino acid composition detection and analysis.

[0061] (1) Culture of cell factories expressing target proteins

[0062] Ten yeast transformants carrying the target protein expression plasmid and one carrying the blank control plasmid were inoculated into 10 mL of YD40 liquid medium containing G418 (20 g / L yeast extract, 40 g / L glucose, 200 μg / mL G418) and cultured overnight at 30°C and 220 rpm. The overnight culture was diluted and inoculated into 2 L Erlenmeyer flasks containing 500 mL of YD40 liquid medium containing G418, and the initial OD was determined. 600 Cells were cultured at 0.2°C, 30°C, and 220 rpm for 40 hours, and cell accumulation (OD) was recorded. 600Centrifuge at 6000 rpm for 30 min, collect the bacterial cells, wash the cells 2-3 times with ultrapure water, and freeze at -80℃. Then freeze-dry for 2 days, grind into powder, and determine the crude protein content (Kjeldahl method, Suzhou Mengxi Biomedical Technology Co., Ltd.) and amino acid content (hydrochloric acid hydrolysis method, high performance liquid chromatography-tandem mass spectrometry, Beijing Zhongyuan Yongxin Technology Co., Ltd.). The shake-flask fermentation experiment was repeated twice, and the average value was taken.

[0063] (2) Methods for determining crude protein and amino acid content

[0064] Crude protein content determination: Refer to GB / T6432-1994 "Determination of Crude Protein in Feed". The crude protein content was determined by sulfuric acid-hydrogen peroxide digestion and micro-Kjeldahl nitrogen determination method. The crude protein content of the fermentation product was calculated according to "crude protein = total nitrogen content in solids × 6.25".

[0065] Amino acid content determination: Sample pretreatment was performed using hydrochloric acid hydrolysis, and amino acid quantification in the submitted samples was conducted using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). LC: Dionex Ultimate 3000. MS: AB Laboratories, API 3200Q-TRAP.

[0066] (3) Results

[0067] As shown in Table 6, except for the target proteins 3chαLB and 4hαLB, compared with the control strain KM-CK, the expression of exogenous proteins rich in essential amino acids increased the crude protein and essential amino acid percentages of yeast cells, while decreasing cell accumulation. This indicates that the effect of the target proteins on yeast growth and protein synthesis may be inversely proportional. The highest increases were observed in the following cases: the crude protein content of the control strain KM-CK was 37.76%, which was increased to over 40% by the expression of the target proteins 1btαLB, 6hLF, and 9HEAAP, representing increases of 1.11, 1.16, and 1.13 times, respectively; the content of the nine essential amino acids for humans in the control strain KM-CK was 13.87%, which was increased to 16.28% by the expression of the target protein 9HEAAP, representing an increase of 1.18 times; and the content of the twelve essential amino acids for animals fed with the control strain KM-CK was 17.38%, which was increased to 20.30% by the expression of the target protein 9HEAAP, representing an increase of 1.17 times.

[0068] Table 6. Crude protein and essential amino acid composition of the target protein yeast expression cell factory.

[0069]

[0070]

[0071] We further analyzed the content of branched-chain amino acids (leucine, isoleucine, and valine) among the essential amino acids in the target protein expression bacteria, and the results are shown in Table 7. Compared with the control bacteria KM-CK, the percentage content of these amino acids in yeast cells was increased to varying degrees after expressing exogenous proteins rich in essential amino acids. The improvements were as follows: Compared with the control strain KM-CK, the expression of target protein 1 btαLB increased the content of these amino acids by 1.11 to 1.20 times; the expression of target protein 2 Bra increased it by 1.09 to 1.21 times; the expression of target protein 3 chαLB increased it by up to 1.07 times; the expression of target protein 4 hαLB increased it by 1.06 to 1.12 times; the expression of target protein 5 hαLBm increased it by 1.08 to 1.13 times; the expression of target protein 6 hLF increased it by 1.03 to 1.09 times; the expression of target protein 7 hβC increased it by 1.02 to 1.10 times; the expression of target protein 8 btOPN increased it by 1.01 to 1.03 times; the expression of target protein 9 HEAAP increased it by 1.11 to 1.21 times; and the expression of target protein 10 SPA2 increased it by 1.07 to 1.09 times.

[0072] Table 7. Branched-chain amino acid composition of the yeast expression cell factory for the target protein.

[0073]

[0074] Figures 6 to 8 The effects of the target protein on yeast cell growth and protein synthesis are shown more clearly. Expression of the target protein inhibits yeast cell growth. Figure 6 Especially when the target protein has the following characteristics: the mass percentage of the nine EAAs in the target protein is within the range of 22.0%-35.0% or 48.0%-77.0%, or the mass percentage of the twelve EAAs is within the range of 52.0%-79.0%, or the mass percentage of BCAAs is within the range of 8.0%-12.0% or 17.0%-35.0%. Target protein characteristics that promote crude protein accumulation ( Figure 7 This includes: the target protein containing 9 EAAs at a mass percentage within the range of 28.0%-47.0% or 51.0%-77.0%; or 12 EAAs at a mass percentage within the range of 41.0%-58.0% or 61.0%-79.0%; or BCAAs at a mass percentage within the range of 10.0%-21.0% or 23.0%-35.0%. Regarding essential amino acids... Figure 8The target protein that promotes the accumulation of 9 types of EAAs in bacterial cells is characterized by a mass percentage of the 9 EAAs in the target protein within the range of 28.0%-39.0% or 48.0%-77.0%. The target protein that promotes the accumulation of 12 types of EAAs in bacterial cells is characterized by a mass percentage of the 12 EAAs in the target protein within the range of 41.0%-61.0% or 62.0%-79.0%. When the mass percentage of the target protein BCAA is within the range of 10.0%-35.0%, it can promote the accumulation of BCAAs in bacterial cells, and the degree of influence is inversely proportional to the accumulation in cells. Figure 6 ).

[0075] In summary, this invention improves the crude protein and essential amino acid content of yeast cells by expressing proteins rich in essential amino acids in Kluyveromyces martensii. The results show that the expression of exogenous proteins increases the crude protein and essential amino acid content of yeast cells to a certain extent, indicating that this strategy is effective in improving the nutritional value of yeast cell protein.

Claims

1. A recombinant microorganism expressing a protein rich in essential amino acids, characterized in that, The crude protein content of the recombinant microorganism is increased relative to the original strain, and the characteristics of the expressed protein rich in essential amino acids satisfy any one or more of (a) to (c): (a) The mass percentage content of the nine essential amino acid residues for the human body is within the range of 28.0%-47.0% or 51.0%-77.0%; (b) The mass percentage content of the 12 essential amino acid residues for feeding animals is within the range of 41.0%-58.0% or 61.0%-79.0%; (c) The mass percentage content of branched-chain amino acid residues is within the range of 10.0%-21.0% or 23.0%-35.0%; Preferably, the recombinant microorganism is yeast; More preferably, the yeast is Kluyveromyces martensii; Preferably, the amino acid sequences of proteins rich in essential amino acids are as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, and SEQ ID NO:

4.

2. A recombinant microorganism expressing a protein rich in essential amino acids, characterized in that, The recombinant microorganisms have increased levels of the nine essential amino acids for humans compared to the original strain, and the expressed proteins rich in essential amino acids meet the following conditions: The mass percentage content of the nine essential amino acid residues for the human body is within the range of 28.0%-39.0% or 48.0%-77.0%. Preferably, the recombinant microorganism is yeast; More preferably, the yeast is Kluyveromyces martensii; Preferably, the amino acid sequences of proteins rich in essential amino acids are as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_001371279.1, SEQ ID NO: 1, XP_024848751.1, SEQ ID NO: 3, and SEQ ID NO:

4.

3. A recombinant microorganism expressing a protein rich in essential amino acids, characterized in that, The recombinant microorganisms contain increased levels of the 12 essential amino acids required for animal feeding compared to the original strain, and the expressed proteins rich in essential amino acids meet the following conditions: The mass percentage content of the 12 essential amino acid residues for feeding animals is within the range of 41.0%-61.0% or 62.0%-79.0%. Preferably, the recombinant microorganism is yeast; More preferably, the yeast is Kluyveromyces martensii; Preferably, the amino acid sequences of proteins rich in essential amino acids are as shown in NP_776803.1, SEQ ID NO: 1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, and SEQ ID NO:

4.

4. A recombinant microorganism expressing a protein rich in essential amino acids, characterized in that, The recombinant microorganism has an increased content of branched-chain amino acids compared to the original strain, wherein the branched-chain amino acids are leucine, isoleucine, and valine; and the expressed protein rich in essential amino acids meets the following conditions: The mass percentage content of branched-chain amino acid residues is in the range of 10.0%-35.0%; Preferably, the recombinant microorganism is yeast; More preferably, the yeast is Kluyveromyces martensii; Preferably, the amino acid sequences of proteins rich in essential amino acids are as shown in NP_776803.1, AGM38242, NP_001272564.1, NP_001371279.1, SEQ ID NO: 1, NP_002334.2, SEQ ID NO: 2, XP_024848751.1, SEQ ID NO: 3, and SEQ ID NO:

4.

5. The method for constructing recombinant Kluyveromyces martensii as described in any one of claims 1 to 4, comprising the following steps: inserting a codon-optimized nucleotide sequence encoding a protein rich in essential amino acids into an expression plasmid, using a PDC1 promoter and terminator, and fusing a green fluorescent protein tag at the C-terminus of the protein to obtain a yeast exogenous protein expression plasmid; introducing the plasmid into a Kluyveromyces martensii host to obtain the recombinant Kluyveromyces martensii; Preferably, the nucleotide sequence of the PDC1 promoter; Preferably, the nucleotide sequence encoding the open reading frame of the HIS and eGFP tags; Preferably, the nucleotide sequence of the PDC1 terminator; Preferably, the codon-optimized nucleotide sequence encoding an essential amino acid-rich protein is shown in SEQ ID No: 5-14.

6. The application of recombinant Kluyveromyces martensii as described in claim 1 in the fermentation production of high-quality microbial protein, characterized in that, The term "high-quality microbial protein" refers to a higher content of crude protein in the bacterial cell than that in the original strain.

7. The application of recombinant Kluyveromyces martensii as described in claim 2 in the fermentation production of high-quality microbial protein, characterized in that, The high-quality microbial protein refers to the bacterial cells containing a higher content of the nine essential amino acids for humans than the original strain.

8. The application of recombinant Kluyveromyces martensii as described in claim 3 in the fermentation production of high-quality microbial protein, characterized in that, The application is to increase the content of the 12 essential amino acids in the bacteria that are necessary for feeding animals.

9. The application of recombinant Kluyveromyces martensii as described in claim 4 in the fermentation production of high-quality microbial protein, characterized in that, The application is to increase the content of branched-chain amino acids in the bacterial cells.

10. The application as described in any one of claims 6 to 9, characterized in that, The recombinant bacteria are fermented to produce microbial cell protein; preferably, expression is achieved by purifying proteins rich in essential amino acids through fusion tags.

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