Application of lignin monomer transporter

Abstract

The invention belongs to the technical field of biology, and discloses application of a lignin monomer transporter. The amino acid sequence of the lignin monomer transporter is as shown in SEQ ID No: 1. The application is used for constructing a recombinant strain growing in a culture medium of lignin-derived aromatic compounds, or used for constructing a recombinant strain for producing microbial proteins and / or lipids. According to the novel lignin monomer transport protein driven by deep learning, the metabolic bottleneck of lignin utilization is broken through, the key protein transported by the lignin monomer is excavated, the lignin monomer transport capacity can be endowed to a microbial chassis, and a multifunctional microbial platform is provided for sustainable biological refining.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the application of a lignin monomer transporter protein. Background Technology

[0002] Lignin, a major component of plant cell walls, accounts for one-third of biopolymers in the terrestrial biosphere, making it the second most abundant natural polymer after cellulose and the most common aromatic polymer. With the increasing global demand for sustainable and renewable resources, lignin has become a highly attractive raw material due to its wide availability and potential for conversion into various high-value-added products. The effective utilization of lignin or its derived aromatic compounds is crucial for advancing bio-based manufacturing and achieving carbon neutrality goals in materials science, energy, and environmental protection. However, the conversion and utilization of lignin-derived aromatic compounds by microorganisms faces key bottlenecks: the transmembrane transport systems of aromatic compounds are inefficient, and traditional methods rely on sequence homology prediction, making it difficult to discover novel transport proteins. Although existing research has explored lignin-degrading enzyme systems (such as DyP-type peroxidases, laccases, and demethylases), progress in the identification and functional characterization of aromatic transport proteins in fungi has been slow.

[0003] Current machine learning methods remain limited by sequence similarity prediction, resulting in the inability to effectively annotate the functions of a large number of uncharacterized proteins (such as at least one-third of proteins in microorganisms). This limitation severely restricts the efficient biorefining of lignin, highlighting the urgent need for novel computational tools and engineering strategies. Summary of the Invention

[0004] This invention addresses the aforementioned problems by utilizing the deep learning-based protein-ligand affinity prediction model EACR, which overcomes the dependence of traditional methods on three-dimensional structural data. This model enables rapid screening of novel lignin-derived aromatic transporters from multi-omics data. Through heterologous expression of the novel lignin-derived Aromatic Transporter (LAT), this invention successfully transformed non-lignin-depolymerized Pichia pastoris chassis into an engineered strain with highly efficient lignin utilization capabilities, achieving a breakthrough in simultaneous protein and lipid biosynthesis. This achievement not only provides a novel pathway for lignin utilization but also demonstrates the transformative potential of transporter center synthetic biology in expanding the metabolic capabilities of industrial microbial chassis.

[0005] This invention marks the first discovery of a novel lignin monomer transporter driven by deep learning, overcoming the metabolic bottleneck in lignin utilization and identifying a key protein for lignin monomer transport. The encoding gene sequence is the candidate sequence yc_08654 (GenBank: USP81380.1), which endows microbial chassis with lignin monomer transport capabilities, providing a multifunctional microbial platform for sustainable biorefining. Engineered Pichia pastoris can directly utilize lignin-derived aromatic compounds as a carbon source, achieving simultaneous production of high-value products and overcoming the carbon flow conflict problem in traditional engineering pathways. This strategy not only improves the economic feasibility of lignin utilization but also provides a new paradigm for the design of microbial cell factories, with broad industrial application prospects.

[0006] Therefore, the present invention provides an application of a lignin monomer transporter protein, wherein the amino acid sequence of the lignin monomer transporter protein is shown in SEQ ID No: 1; the application is for constructing recombinant strains grown in a culture medium containing lignin-derived aromatic compounds, or for constructing recombinant strains for producing microbial proteins and / or lipids.

[0007] Specifically, it is obtained by introducing a lignin monomer transporter protein with an overexpressed amino acid sequence as shown in SEQ ID No: 1 into the starting bacteria.

[0008] More specifically, the starting strain is Pichia pastoris. Preferably, the starting strain is Pichia pastoris strain HTX-33.

[0009] The present invention also provides a method for preparing microbial protein with high lipid content, which uses recombinant bacteria obtained by introducing a lignin monomer transporter protein with an overexpressed amino acid sequence as shown in SEQ ID No: 1 into a starting strain to produce microbial protein.

[0010] Specifically, the starting strain is Pichia pastoris. Preferably, the starting strain is Pichia pastoris strain HTX-33.

[0011] Specifically, the nucleotide sequence encoding the lignin monomer transporter was codon-optimized according to the expression preferences of Pichia pastoris and fused with an affinity tag (such as a Flag tag) sequence before being introduced into the starting strain.

[0012] In a specific implementation, the plasmid backbone used during import is the pPIC9K plasmid.

[0013] In addition, the fermentation process uses a culture medium with aromatic compounds as the sole carbon source, which are selected from one or more of the following: protocatechuic acid, syringic acid, coumaric acid, gallic acid, p-coumaric acid, catechin, cinnamic acid, ferulic acid, p-hydroxybenzoic acid, vanillin, and coniferaldehyde.

[0014] In a specific implementation, a batch feeding fermentation process is adopted, with a pH of 4.8 and a culture temperature of 30°C during fermentation. After the initial carbon source is exhausted, 0.1% to 1% w / v of aromatic compounds as carbon sources are added, and fermentation continues under the condition of dissolved oxygen maintained at 25%. Attached Figure Description

[0015] Figure 1 The EACR model was evaluated for its predictions of protein-substrate binding patterns and binding sites. (a) Predictions of interactions between the protein sequence 5L4Q from the David dataset and randomly selected compounds from the Davis dataset; (b) Matches 11 out of 22 binding sites predicted by BIOVIA Discovery Studio 2019 Client42 software; (c) Analysis of docking sites between the protein sequence 5L4Q and 68 molecules using BIOVIA Discovery Studio software; (d) Analysis of docking sites between the protein sequence 5L4Q and 68 molecules using the EACR model.

[0016] Figure 2 Growth differences between strains *P. pastoris* HTX-33 and *P. pastoris* OLAT using different substrates as the sole carbon source were analyzed. Glycerol, catechin, syringic acid, coumaric acid, gallic acid, p-coumaric acid, catechin, cinnamic acid, ferulic acid, p-hydroxybenzoic acid, vanillin, and coniferaldehyde were used as substrates.

[0017] Figure 3 Using eugenol at different concentrations (0, 0.1%, 0.25%, 0.5%, 0.75%, 1% w / v) as fed-batch carbon sources, the total lipid and total protein content in Pichia pastoris cells was analyzed.

[0018] Figure 4 Structural and property analysis of lignin transporter LAT. (a) Three-dimensional structural analysis of transmembrane protein LAT; (b) Molecular weight determination of transmembrane protein LAT; (c) Localization of transmembrane protein LAT on the cell membrane; (d) Transmembrane pore structure of the trimer formed by transmembrane protein LAT; (e) Molecular weight analysis of the trimer formed by transmembrane protein LAT. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1: Developing the EACR model to mine the transport protein LAT

[0021] 1.1 Experimental Materials and Data Sources

[0022] (1) Strains: Highly efficient lignin-degrading mutant strain Curvularia clavata J1 (see Chinese patent application document: 202411022370.5, invention title: A lignin-degrading bacterium and its application), deposited on June 20, 2024. Pichia pastoris strain P. pastoris HTX-33 (see Chinese patent application document: 202211146356.7, invention title: A Pichia pastoris strain with high methanol conversion rate and high protein and its preparation method).

[0023] (2) Data source: Transcriptome data of C. clavata J1 cultured to the exponential phase in media with lignin and glucose as carbon sources.

[0024] (3) Small molecule ligands: lignin structural analogs ABTS and guaiacol are used as molecular decoys for screening candidate proteins.

[0025] The inventors designed a model to analyze substrate-protein interactions and decipher protein-ligand binding patterns to accurately predict protein-ligand binding sites. For example, the predicted interactions between the protein sequence 5L4Q and randomly selected compound molecules from the Davis dataset (e.g.) Figure 1 (a) matches 11 of the 22 binding sites predicted by BIOVIA Discovery Studio 2019 Client42 (a software based on molecular docking, scoring functions, energy minimization optimization, and molecular dynamics simulations). Figure 1 (b). The designed model exhibits high consistency, with an average compatibility rate of 74.89% across 68 molecules. Figure 1 (cd). Based on the matrix relationship between proteins and substrates in the designed model, the binding sites between proteins and substrates can be easily located.

[0026] 1.3 Candidate Gene Mining Process

[0027] 1.3.1 Transcriptome Data Analysis

[0028] (1) Culture medium: Lignin medium (using lignin as the sole carbon source, with a suitable amount of inorganic salts in the basic components to maintain the growth of the strain); glucose medium (20 g / L glucose, with the other basic components the same as lignin medium).

[0029] (2) Culture conditions: C. clavata J1 strain was inoculated into the two culture media mentioned above and cultured at 30℃ and 200 rpm until the exponential phase.

[0030] (3) Experimental procedure: Total RNA was extracted using TRIzol reagent and TruSeq was used. TM The Stranded Total RNA Library Prep Kit for Illumina was used to construct sequencing libraries. Differentially expressed genes were screened using RSEM and DEGseq software, with |log2FC|≥1 and P < 0.05 as the criteria. 992 upregulated genes were obtained, and 250 uncharacterized proteins were then selected from their encoded proteins as a candidate pool.

[0031] 1.3.2 EACR model for screening candidate proteins

[0032] Using a model designed by the inventors, novel lignin monomer transport proteins were discovered, and *Curvularia lanceolata* J1, a fungus known for its high efficiency in degrading lignin, was screened. Comparative transcriptomics data revealed 992 differentially expressed genes responding to lignin upregulation and 1961 differentially expressed genes responding to lignin downregulation. The designed model simulated the upregulated gene pool of 992 genes, using lignin structural analogs ABTS and guaiacol as molecular baits. The model predicted the binding affinity of 250 uncharacterized proteins to the bait molecules. 65 proteins with binding affinity higher than their homolog laccase (yc_07441) were screened. Combined with transmembrane property analysis, a candidate protein possessing both high binding affinity and transmembrane properties was finally selected. Its encoding gene sequence is the candidate sequence yc_08654 (GenBank: USP81380.1), with the amino acid sequence: MTRPVYAAGLVLTLACTVMTIASISMPRW. VSYSPNGERQYSYGLHTRCSAVTGTCVSFPKSSDCTKDPSFCNMWRTVGFLTSFGVVVELCAIVSFIVIISGGVQRRAAGWQVAVGVLSLSAIVQCGGMAIVAFLFDHDERF WDGWHLDLSWSLCTASWTILILTSIGMAASAFYLPAEGDYELIPEDHYGPPDDRLLSRISAWDNGFKGPGSGMQYSYQREQDSMSDVVSIAASSIAASHRRESDVRK (SEQID No:1).

[0033] The protein encoded by this sequence has a significantly higher affinity for ABTS and guaiacol than its homologous laccase and also possesses transmembrane properties. Phylogenetic analysis shows that it is mainly found in the Pleosporales species and is not annotated, suggesting that it is a novel transporter protein, which is named Lignin-derived Aromatic Transporter (LAT).

[0034] Example 2: Analysis of the function and mechanism of action of the lignin transporter LAT

[0035] 2.1 Culture medium

[0036] Culture media: YPD medium was used for strain culture, specifically YPD medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, natural pH) supplemented with 100 mg / L genimycin (G418). BMGY medium (10 g / L yeast extract, 20 g / L peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% amino acid-free yeast nitrogen source, 4 × 10⁻⁶ ppm) was used for transformant screening. -5 % Biotin, 10 g / L glycerol) were used for seed culture; BMMY medium (yeast extract 10 g / L, peptone 20 g / L, 100 mM potassium phosphate buffer (pH 6.0), 1.34% amino acid-free yeast nitrogen source, 4×10 -5 Biotin (5 g / L) and methanol (5 g / L) were used to induce expression; Delft medium supplemented with different lignin-derived aromatic compounds (KH2PO4 3.0 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.1 g / L, (NH4)2SO4 2.0 g / L, FeSO4·7H2O 0.01 g / L, ZnSO4·7H2O 0.002 g / L, MnSO4·H2O 0.002 g / L, CoCl2·6H2O 0.0005 g / L) was used to verify transport function.

[0037] 2.2 Construction and Expression Validation of Engineered Strains

[0038] 2.2.1 Plasmid Construction and Transformation

[0039] The C-terminus of the codon-optimized LAT gene sequence was fused with a Flag tag sequence to construct the "LAT-Flag" fusion gene fragment. ECORI and EAGI restriction enzyme sites were introduced at both ends of the gene using polymerase chain reaction (PCR). The fusion gene fragment and the Pichia pastoris expression vector pPIC9K were then double-digested using these two enzymes. After purification by gel electrophoresis, the target fragment was directionally cloned into the vector using T4 DNA ligase to construct the recombinant expression plasmid pPIC9K-LAT-Flag. After confirmation by sequencing, the recombinant plasmid was linearized using the restriction endonuclease SalI and transformed into competent Pichia pastoris strain HTX-33 cells via electroporation. The transformed cells were plated on MD plates containing G418 for antibiotic selection. Finally, yeast genomic PCR was used to verify the integration of the target gene. After methanol induction, the expression of the fusion protein was detected by Western blotting, thus completing the construction and validation of the recombinant Pichia pastoris expression system. The engineered strain P. pastoris OLAT was constructed by plating the transformants onto the screening medium. After the transformants grew, single colonies were picked and cultured at 30°C for 48-72 h before PCR verification. Positive transformants were inoculated into BMGY medium and cultured at 30°C and 200 rpm for 24 h. Then, they were transferred to BMMY medium and cultured at 30°C and 200 rpm for 4-5 days. 1% methanol was added every 24 h to maintain AOX1 promoter expression.

[0040] 2.3 Testing of the transport capacity of lignin-derived aromatic compounds

[0041] 2.3.1 Strains Culture and Treatment

[0042] The engineered strain of P. pastoris OLAT and the parent strain P. pastoris HTX-33 were inoculated into YPD medium and cultured to mid-log (OD600≈0.5). The cells were collected by centrifugation and washed twice with PBS buffer (pH7.4) for later use.

[0043] 2.3.2 Solid-state flat panel growth verification

[0044] The treated bacterial cells were prepared into bacterial suspensions, and 2 μL of each suspension was spotted onto Delft solid medium containing different lignin-derived aromatic compounds. The suspensions were then incubated at 30°C for 5-7 days, and the colony growth was observed.

[0045] like Figure 2As shown, the validation results indicated that the parental strain *P. pastoris* HTX-33 could not utilize lignin-derived aromatic compounds for growth, while *P. pastoris* OLAT could grow on media with 11 tested aromatic compounds as the sole carbon source. These 11 substrates included protocatechuic acid, syringic acid, coumaric acid, gallic acid, p-coumaric acid, catechol, cinnamic acid, ferulic acid, p-hydroxybenzoic acid, vanillin, and coniferaldehyde. The utilization rate of syringic acid was close to that of glycerol, with an OD600 of 6 within 25 h, and the growth rate within 30 h was more than 10 times higher than that of syringaldehyde.

[0046] 2.3.4 Analysis of Fermentation Products

[0047] A fed-batch fermentation process was employed, with culture at pH 4.8 and 30℃. After the initial carbon source was depleted, different concentrations (0, 0.1%, 0.25%, 0.5%, 0.75%, 1% w / v) of syringic acid were added as a carbon source. Fermentation continued under dissolved oxygen conditions maintained at 25%. Samples were taken every 24 hours to determine the protein and lipid content in the biomass. Figure 3 As shown, the analysis of fermentation products indicates that this strain can achieve adjustable lipid accumulation of 5.46% to 16.54% DCW while maintaining a relatively stable protein content. The highest lipid content is 3.03 times higher than that of the glycerol culture control group.

[0048] Table 1. Protein and lipid content in biomass when different concentrations of syringic acid are added as a carbon source.

[0049]

[0050] Example 3: Characterization of the structure and transport mechanism of the yc_08654 protein

[0051] 3.1 LAT protein sequence and structural characterization

[0052] 3.1.1 Protein Model Construction

[0053] Templates were obtained from the PDB database, and protein homology modeling was performed using SwissModel. Figure 4 (a) The number of membrane transport protein subunits was predicted using the DeepSub online tool, and the transmembrane structure of LAT protein trimers was modeled using AlphaFold. A Pichia pastoris phospholipid bilayer membrane environment was constructed using CHARMM-GUI, and the LAT trimer model was embedded into the membrane. Water molecules and ions were added to form a protein-membrane complex system. Figure 4 (d).

[0054] 3.1.2 Structural Quality Assessment

[0055] The dihedral distribution of residues was analyzed using Ramachandran plots, and the overall quality factor of the protein structure was calculated using ERRAT 2.0. The secondary structure composition, transmembrane regions, and signal peptide characteristics of the LAT protein were analyzed. The LAT monomer contains an SP-type signal peptide composed of 23 hydrophobic amino acids. Its secondary structure comprises 50.61% α-helices, 15.26% β-sheets, and 34.13% random coils, with three transmembrane regions (amino acids 78-100, 113-135, and 150-172). The trimer structure was validated using Ramachandran plots and ERRAT 2.0, with 94.4% of residues located in preferred regions. The overall quality factor reached 94.268, meeting the standards for a high-quality model.

[0056] 3.1.3 Molecular weight analysis of LAT protein

[0057] Bacterial culture of *P. pastoris* OLAT engineered strain after induction and expression was collected. The cells were collected by centrifugation, and proteins were extracted. Protein molecular weight and expression levels were analyzed by SDS-PAGE or Native-PAGE and Western blotting using Flag antibody as the primary antibody. Figure 2 The results showed that yc_08654 was expressed normally, with a monomer molecular weight of 27 kDa, and formed an 85 kDa trimer in its native state. Figure 4 (b, e). Thus, the engineered strain of P. pastoris OLAT was obtained through experimental overexpression of LAT.

[0058] 3.1.4 Subcellular localization analysis

[0059] The LAT gene was fused with a GFP tag at its C-terminus to construct the pPIC9K-LAT-GFP recombinant plasmid, which was then transformed into *P. pastoris* HTX-33 cells. Positive transformants were cultured overnight at 30°C in Delft medium containing 0.5% methanol. After washing, the distribution of GFP fluorescence was observed using the ImageXpress Confocal HT.AI high-quality imaging analysis system. Subcellular localization showed that the LAT protein was located on the *Pichia pastoris* cell membrane. Figure 4 (c)

[0060] 3.2 Characterization of LAT protein transport mechanism

[0061] 3.2.1 Molecular docking and identification of key sites

[0062] Using syringic acid as a substrate, 20 potential binding sites were obtained through molecular docking calculations, and the complex model with the highest binding fraction was screened. Hydrogen bonding and hydrophobic interactions in the complex were analyzed to identify key binding sites. Single-point mutants of Arg29Ala, Ser50Ala, and Ser58Ala were constructed, and their transport efficiency for syringic acid was determined through heterologous expression in Pichia pastoris. Molecular docking and mutation validation showed that Arg29, Ser50 (A chain), and Ser58 (B chain) are key substrate binding sites, and mutations in these sites significantly reduce syringic acid transport efficiency.

[0063] 3.2.2 Molecular Dynamics (MD) Simulation

[0064] Based on the complex model obtained from molecular docking, a protein-POPC membrane complex system was constructed, and a neutralization system with balanced ions was added. Molecular dynamics simulations were performed in Amber22 software using the GPU-accelerated pmemd.cuda module and the NPT ensemble at 300K to analyze the protein backbone RMSD, residue RMSF, hydrogen bond dynamics, and transport trajectory. MD simulations revealed that syringic acid binds to LAT protein through hydrogen bonds and hydrophobic interactions (binding energy -5.94 kcal / mol), undergoing a complete transport process from extracellular binding to intracellular channel shuttle. The protein remained generally stable in the simulation (RMSD fluctuated within 0.8 nm).

[0065] 3.2.3 Free Energy and Permeability Analysis

[0066] Three substrates—syringic acid, p-hydroxybenzoic acid, and coniferaldehyde—were selected. The mean potential (PMF) for transmembrane transport was determined using an umbrella sampling technique. Weighted histogram analysis (WHAM) was employed to process the sampling data, and free energy curves were plotted to calculate the transport energy barriers. Based on the sampling data, the Z-dependent diffusion coefficient, resistivity, and effective permeability coefficient (Peff) were calculated to analyze the interaction mechanisms of the substrates in different regions of the channel. Free energy and permeability analysis showed that the transport energy barriers for the three substrates were 9.30 kcal / mol for syringic acid, 13.05 kcal / mol for p-hydroxybenzoic acid, and 2.96 kcal / mol for coniferaldehyde. However, coniferaldehyde exhibited the lowest transport efficiency due to its high dissociation energy barrier (4.12 kcal / mol) and potential covalent interaction with the C56 residue, confirming that the transport efficiency is determined by the substrate structure and functional group characteristics.

[0067] The above candidate protein discovery, functional analysis, and structural characterization results, progressing step by step from gene screening, cell phenotype to molecular mechanism, confirm that the LAT protein, precisely discovered through the EACR model, achieves efficient transport of lignin-derived aromatic compounds through a transport mechanism mediated by the hydrophobic interaction of the trimeric transmembrane structure, key binding sites of Arg29 / Ser50 / Ser58, and hydrogen bonds. This is consistent with the macroscopic high utilization rate of substrates such as syringic acid, stable protein synthesis, and modulated lipid accumulation phenotype of the engineered strain P. pastorris OLAT.

Claims

1. Use of a lignin monomer transporter protein, characterized in that, The amino acid sequence of the lignin monomer transporter is shown as SEQ ID No: 1; the application is for constructing a recombinant strain grown in a culture medium of lignin-derived aromatic compounds, or for constructing a recombinant strain for producing microbial proteins and / or lipids.

2. Use according to claim 1, wherein It is obtained by overexpressing the lignin monomer transporter with the amino acid sequence shown as SEQ ID No: 1 in a starting strain by introduction.

3. Use according to claim 2, wherein the compound is ###0002### The starting strain is Pichia pastoris.

4. Use according to claim 3, wherein the compound is ###0002### The starting strain is Pichia pastoris strain (Pichiapastoris) HTX-33.

5. A method of preparing a high-lipid content microbial protein, characterized in that, It employs a recombinant strain obtained by overexpressing the lignin monomer transporter with the amino acid sequence shown as SEQ ID No: 1 in a starting strain by introduction for fermentation to produce microbial proteins.

6. The method of claim 5, wherein, The starting strain is Pichia pastoris.

7. The method of claim 6, wherein, The starting strain is Pichia pastoris strain (Pichiapastoris) HTX-33.

8. The method of claim 6, wherein, The coding nucleotide sequence of the lignin monomer transporter is codon-optimized according to the expression preference of Pichia pastoris, and is fused with an affinity tag sequence before being introduced into the starting strain.

9. The method of claim 8, wherein, The plasmid backbone used in the introduction is pPIC9K plasmid.

10. The method of claim 8, wherein, An aromatic compound is used as the sole carbon source in the fermentation medium, and the aromatic compound is selected from one or more of protocatechuic acid, syringic acid, coumaric acid, gallic acid, p-coumaric acid, catechol, cinnamic acid, ferulic acid, p-hydroxybenzoic acid, vanillin, and coniferyl aldehyde.

11. The method of claim 10, wherein, A fed-batch fermentation process is used, and the fermentation is carried out at pH 4.8 and a culture temperature of 30℃, and after the initial carbon source is depleted, 0.1% to 1% w / v of the aromatic compound as the carbon source is added, and the fermentation is continued under the condition that the dissolved oxygen is maintained at 25%. A fed-batch fermentation process is used, and the fermentation is carried out at pH 4.8 and a culture temperature of 30℃, and after the initial carbon source is depleted, 0.1% to 1% w / v of the aromatic compound as the carbon source is added, and the fermentation is continued under the condition that the dissolved oxygen is maintained at 25%.

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