Patent Application
20250092161
This patent application claims the benefit and priority of Chinese Patent Application No. 2023112177340, filed with the China National Intellectual Property Administration on Sep. 20, 2023, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
A computer readable XML file entitled “GWP20220801010_seglist”, that was created on Nov. 10, 2023, with a file size of about 17,834 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.
The present disclosure belongs to the technical fields of cell metabolic engineering and synthetic biology, and specifically relates to a promotion system for cellular protein synthesis and use thereof.
Over the past 30 years, metabolic engineering has emerged as a powerful tool to improve cell performance and enable sustainable biomanufacturing production of compounds. However, changes in metabolic pathways and the introduction of exogenous proteins increase the demand for cellular resources and disrupt the normal physiological balance of cells. Overexpression of pathway proteins can hinder cell growth and thus reduce productivity; simply reducing the expression level of pathway enzymes may form a bottleneck in the reaction pathway, thereby limiting yield and titer. Therefore, more refined regulation of pathways is needed to achieve a balance between cell growth and product synthesis.
Metabolic engineering improves cellular synthetic capabilities by modifying the metabolic network of chassis cells, while synthetic biology aims to create new cellular functions and/or reprogram existing cellular functions based on mechanical engineering principles. Artificially designed and synthesized regulatory elements theoretically have less interaction with the endogenous transcriptional regulatory network of chassis cells and are orthogonal. Accordingly, in the future, it is necessary to construct predictable, orthogonal, and quantitatively-controllable gene circuits based on standardized characterization elements, hoping to achieve “ground-up” balanced expression of genes in cells. In addition, the development of synthetic biology allows researchers to control cell phenotypes at the temporal or spatial level through intelligent methods, but there are still poor level of control and flexibility yet. As a result, the transformation technology of cell regulation should also develop in the direction of programmability, multi-target, and automatic feedback control in the future.
Currently, synthetic biology can achieve multi-level coordinated regulation of metabolic networks, significantly improving biosynthetic capabilities. However, it needs to be emphasized that achieving precise and effective balance of bacterial physiology and efficient synthesis of target compounds is the ultimate goal pursued by industrial microbial technology. Metabolic engineering has stringent standards for the economics of production performance. Compared with general synthetic biology research, the regulatory tools used in actual metabolic engineering show more stringent restrictions on the loading and toxicity of components. Although the library of synthetic biology regulation tools has been enriched in recent years, the currently available switches or regulation tools are still extremely limited compared to the diverse application scenarios and evolving technical needs of metabolic engineering. It is necessary to further optimize and develop new intelligent dynamic control systems oriented to metabolic engineering applications. With the development of synthetic biology and artificial intelligence technology, biomanufacturing may have a major impact on many fields, while there may be more urgent demand for new expression components and control methods. In addition, the current RNA regulatory elements regulating metabolic pathways are all in negative regulatory modes at the transcription or translation level. At present, metabolic engineering or synthetic biology has an urgent need for regulatory elements in positive regulatory modes and has diverse application requirements.
In order to solve the current urgent need and practical application of regulatory elements or regulatory systems in positive regulatory modes in cell metabolic engineering or synthetic biology, a purpose of the present disclosure is to provide a promotion system for cellular protein synthesis and use thereof.
The present disclosure provides a RNA-binding protein (RBP) AbrP, where the RBP AbrP has an amino acid sequence shown in SEQ ID NO: 1.
The present disclosure further provides use of RBP AbrP in promoting recombinant protein translation.
Preferably, the recombinant protein translation includes expression of an exogenous protein in a host cell.
Preferably, the host cell includes a prokaryotic cell and/or a eukaryotic cell.
The present disclosure further provides an mRNA translation-promoting complex based on sRNA targeting, where the mRNA translation-promoting complex includes a AbrP combined with a translation-promoting factor, and the RBP AbrP has an amino acid sequence shown in SEQ ID NO: 1.
Preferably, the translation-promoting factor includes FusA, LepA, and Era.
The present disclosure further provides an AbrP protein translation-promoting system, including the mRNA translation-promoting complex.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering regulation of a prokaryotic chassis cell.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering regulation of a eukaryotic chassis cell.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering and/or biosynthesis of animal and plant cells.
Beneficial effects: the present disclosure provides a RBP AbrP, an mRNA translation-promoting complex based on sRNA targeting, and a corresponding AbrP protein translation-promoting system. It is confirmed by the examples that the AbrP protein binds to translation-promoting factors FusA, LepA, and Era separately and then promotes translation of a target mRNA. The translation-promoting process is mainly characterized by promoting a stability of bacterial ribosomes, promoting an initiation process of translation, and promoting an elongation process of translation.
In the present disclosure, a bacterial translation-promoting molecular system for a target mRNA based on sRNA targeting is discovered and verified for the first time. A core protein of this system is a novel RBP AbrP On one hand, the AbrP binds to translation-promoting factors FusA, LepA, and Era separately; on the other hand, the AbrP binds to a characteristic sequence of an sRNA. A complex resulting from the combination of the AbrP and the promoting factor reaches a translation system of a target mRNA under a guidance of the sRNA, thus releasing the translation-promoting factors FusA, LepA, and Era to stimulate and promote the translation.
In the examples of the present disclosure, representative strains of hydrocarbon-degrading bacteria (Alcanivorax) widely distributed in the ocean are used as research materials. It is discovered and confirmed that the novel bacterial RBP AbrP combines with multiple sRNAs to promote the translation of multiple target genes involved in hydrocarbon metabolism in a sensing and signal transduction system, an uptake transport system, a chemotaxis and movement system, a regulatory and degradation system, and a secretion system of bacteria. Sequence analysis has confirmed that multiple sRNAs that bind to the RBP AbrP have a conserved sequence region of 20 nt to 25 nt, and it is also verified that this conserved sequence region binds to an S1 domain of the AbrP (
The present disclosure provides a RNA-binding protein (RBP) AbrP, where the RBP AbrP has an amino acid sequence shown in SEQ ID NO: 1. The RBP AbrP can be applied to prokaryotic and eukaryotic cells. However, when applied in prokaryotic cells, an encoding gene of the RBP AbrP is preferably shown in SEQ ID NO: 2; when applied to eukaryotic cells, the encoding gene is preferably shown in SEQ ID NO: 3.
In the present disclosure, the biological species distribution of the AbrP protein shown in SEQ ID NO: 1 is analyzed. The results show that AbrP protein is widely distributed in bacterial taxa (
In the present disclosure, the RBP AbrP is preferably derived from a representative strain of hydrocarbon-degrading bacteria (Alcanivorax). It is discovered and confirmed that the novel bacterial RBP AbrP combines with multiple sRNAs to promote the translation of multiple target genes involved in hydrocarbon metabolism in a sensing and signal transduction system, an uptake transport system, a chemotaxis and movement system, a regulatory and degradation system, and a secretion system of bacteria. Sequence analysis has confirmed that multiple sRNAs that bind to the RBP AbrP have a conserved sequence region of 20 nt to 25 nt, and it is also verified that the conserved sequence region binds to an S1 domain of the AbrP (
The present disclosure further provides use of the RBP AbrP in promoting recombinant protein translation.
In the present disclosure, the recombinant protein translation includes preferably expression of an exogenous protein in a host cell, and the host cell preferably includes a prokaryotic cell and/or a eukaryotic cell. The prokaryotic cell preferably includes E. coli, Bacillus subtilis, and Corynebacterium glutamicum. The eukaryotic cell preferably includes fungi, human tumor cells HeLa cells, animal cells, and plant cells, and the fungi preferably include Saccharomyces cerevisiae and filamentous fungi.
The present disclosure further provides an mRNA translation-promoting complex based on sRNA targeting, where the mRNA translation-promoting complex includes a RBP AbrP combined with a translation-promoting factor, and the RBP AbrP has an amino acid sequence shown in SEQ ID NO: 1.
In the present disclosure, the translation-promoting factor includes preferably FusA, LepA, and Era.
The present disclosure further provides an AbrP protein translation-promoting system, including the mRNA translation-promoting complex.
In the examples of the present disclosure, through a series of biochemical and molecular biology experiments such as CoIP-MS experiments, bacterial two-hybrid verification, dual-plasmid protein translation verification experiments, quantitative mass spectrometry technology, bacterial translation group (Ribo-seq) technology, surface plasmon resonance (SPR) technology, and bacterial ribosome in vitro translation experiments, it is confirmed that the AbrP binds to three types of non-classical GTP hydrolase translation-promoting factors (FusA, LepA, and Era) in bacteria and promotes the translation of target mRNAs (
In the present disclosure, the AbrP protein translation-promoting system developed in the present disclosure becomes a metabolic network regulatory element in chassis cells of synthetic biology, and also becomes an important tool for studying gene expression regulation in molecular biology and cell biology.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering regulation of a prokaryotic chassis cell.
In the present disclosure, the use is preferably the same as the above, and is not repeated here.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering regulation of a eukaryotic chassis cell. In the present disclosure, the use is preferably the same as the above, and is not repeated here.
The present disclosure further provides use of the AbrP protein translation-promoting system in metabolic engineering and/or biosynthesis of animal and plant cells. In the present disclosure, the use is preferably the same as the above, and is not repeated here.
In order to further illustrate the present disclosure, the promotion system for cellular protein synthesis and the use thereof provided by the present disclosure are described in detail below with reference to the accompanying drawings and examples, but the accompanying drawings and the examples should not be construed as limiting the protection scope of the present disclosure.
The E. coli had a key gene for succinic acid synthesis, namely a phosphoenolpyruvate carboxylase gene (ppc, NC_000913.3); a targeting gRNA was designed at the +465 position of the CDS interval of the ppc gene, and named as ppc-gRNA, with a sequence shown in SEQ ID NO: 5.
A cell factory of metabolic engineering of 1,3-propanediol-producing E. coli was derived from a glycerol-3-phosphate dehydrogenase gene (gpd, NC_001139.9) and a glycerol-3-phospholipase gene (gpp, NC 001146.8) of Saccharomyces cerevisiae; a targeting gRNA was designed at the +311 position of the CDS interval of the gpd gene, and named as gpd-gRNA, with a sequence shown in SEQ ID NO: 6; a targeting gRNA was designed at the +141 position of the CDS interval of the gpp gene, and named as gpp-gRNA, with a sequence shown in SEQ ID NO: 7.
There were 3 squalene synthase (SQS) genes from different sources in a cell factory of squalene-producing Bacillus subtilis: BmSQS (CP001982.1), PgSQS (KJ939264.1), and ScSQS (U00030.1); a targeting gRNA was designed at the +61 position of the CDS interval of the BmSQS gene, and named as bmsq-gRNA, with a sequence shown in SEQ ID NO: 8; a targeting gRNA was designed at the +121 position of the CDS interval of the PgSQS gene, and named as pgsq-gRNA, with a sequence shown in SEQ ID NO: 9; a targeting gRNA was designed at the +201 position of the CDS interval of the ScSQS gene, and named as scsq-gRNA, with a sequence shown in SEQ ID NO: 10.
There was a glutamine synthetase gene (glnA, AF005635) in a cell factory of L-glutamic acid-producing Corynebacterium glutamicum; a targeting gRNA was designed at the +421 position of the CDS interval of the GlnA gene, and named as glnA-gRNA, with a sequence shown in SEQ ID NO: 11.
The synthesized gRNA was ligated into the vector pgRNA according to a method in “Molecular Biology Experimental Technology” to form gRNA expression vectors, which were named as ppc-pgRNA and gpd-pgRNA in sequence.
Preparation of LB medium: 1% (W/V) tryptone, 0.5% (W/V) yeast extract, and 1% (W/V) sodium chloride were sterilized at 121° C. for 15 min.
Preparation of GM medium (growth medium): tryptone 1% (W/V), yeast extract 0.5% (W/V), NaCl 0.5% (W/V), hydrolyzed casein 0.2% (W/V) V), 500 mM sorbitol, 500 mM glucose, 50 mM K2HPO4, and 50 mM KH2PO4 were sterilized at 115° C. for 15 min.
Preparation of cell wall weakening agent (WA solution Ox): 5% (W/V) glycine, 10% (W/V) serine, and 5 mmol DTT were used. The glycine and serine were filtered and sterilized using a 0.22 μm filter membrane, and the DTT was sterilized at 121° C. for 15 min.
Preparation of WB buffer (electric shock buffer): 500 mM trehalose, 500 mM sorbitol, 500 mM mannitol, 15 mM MgCl2, 0.5 mM K2HPO4, 0.5 mM KH2PO4, and 10% glycerol were mixed, a resulting mixture was adjusted to a pH value of 7.2, and sterilized at 121° C. for 15 min. 0.5% (W/V) 500 mM sorbitol and 350 mM mannitol were added, and sterilized at 121° C. for 15 min.
Preparation of RM medium (resuscitation medium): tryptone 1% (W/V), yeast extract 0.5% (W/V), NaCl 0.5% (W/V), sorbitol 500 mM, and mannitol 350 mM were sterilized at 121° C. for 15 min.
Preparation of Bacillus subtilis Competent Cells
The BHI medium included: brain heart infusion 37 g/L, (NH4)2SO4 10 g/L, K2HPO4 0.2 g/L, NaH2PO4 0.3 g/L, and MgSO4 7H2O 0.5 g/L, pH=7.2.
The LBHIS medium included: peptone 5 g/L, NaCl 10 g/L, yeast powder 2.5 g/L, brain heart infusion 18.5 g/L, and sorbitol 91.1 g/L.
The synthesized gRNA was ligated into the vector pgRNA according to a method in “Molecular Biology Experimental Technology” to form gRNA expression vectors, which were named as ppc-pgRNA, gpd-pgRNA, gpp-gRNA, bmsq-gRNA, pgsq-gRNA, scsq-gRNA, and glnA-gRNA in sequence.
A construction method was as follows (taking ppc-pgRNA as an example): a DNA fragment at position +465 of the CDS interval of artificially-synthesized phosphoenolpyruvate carboxylase gene with EcoRI and NheI enzyme cleavage sites (ppc, NC_000913.3) was digested with restriction endonucleases EcoRI and NheI and then gel recovered, while the pgRNA vector was also digested and gel recovered using restriction endonucleases EcoRI and NheI. The DNA fragment at the +465 position of the CDS interval of the phosphoenolpyruvate carboxylase gene (ppc, NC_000913.3) after digestion and the pgRNA vector were ligated using T4 ligase. An obtained ligation product was mixed with the competent cells of a receptor strain E. coli Top10, placed on ice for 30 min, and heat shocked at 42° C. for 90 s. 400 μL LB liquid medium was added, resuscitated at 37° C. and 160 rpm for 45 min, spread on LB solid medium containing 50 μg/mL kanamycin, and cultured at 37° C. overnight. A recombinant plasmid was selected and subjected to double enzyme digestion and sequencing verification to obtain a ppc-pgRNA recombinant plasmid. In a same way, the recombinant vectors of gpd-pgRNA, gpp-gRNA, bmsq-gRNA, pgsq-gRNA, scsq-gRNA, glnA-gRNA, and ArbP protein vector pCarbP were constructed.
Determination of glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase, glycerol dehydratase, and alanine dehydrogenase in E. coli was conducted by a PRM-based targeted mass spectrometry (MS) method; the Bacillus subtilis included 4 squalene synthases (SQS): BaSQS, BmSQS, PgSQS and ScSQS genes; the Corynebacterium glutamicum included protein expression of AAR (acyl ACP reductase) and ADO (aldehyde-deformylating oxygenase).
Each sample was ground to a powder in liquid nitrogen and resuspended in a lysis buffer containing 50 mM Tris-HCl, 7 M urea, 2 M thiourea, and 1× protease inhibitor (pH=8), along with 10 mM ice-cold acetic acid and 1,4-dimercaptopropanol (DTT). The suspension was incubated at −20° C. for 2 h and centrifuged at 13,000 g and 4° C. for 20 min. The precipitated pellet was resuspended with 10 mM ice-cold acetic acid and DTT and centrifuged again at 13,000 g and 4° C. for 20 min. The precipitated particles were collected and lysed using lysis buffer. The protein was quantified using a Bradford method. The protein was diluted 5-fold by adding 100 mM triethylammonium bicarbonate and digested using trypsin (Promega, USA) at a substrate-to-enzyme ratio of 50:1 (w/w). Peptides were desalted using a Strata-X C18 column (Phenomenex Inc, California, USA) and then dried using a vacuum centrifuge and frozen at −80° C. until further analysis.
PRM detection was conducted using a Triple TOF 5600+LC-MS/MS system (SCIEX, Framingham, MA, USA). It was worth noting that the resolution of MS1 scans and associated precursor ions was approximately 35,000, while the resolution of MS/MS scans and associated fragment ions was approximately 15,000 (PRM transitions). The MS/MS spectra of the 30 most abundant precursor ions were obtained using data-dependent acquisition. In each cycle (250 ms), a 50 ms/MS/MS spectrum was obtained after each measurement of MS1 scan, with a total cycle time of 1.8 s. The acquired PRM included MS1 scan (250 ms) and target MS/MS scan (cycle time 1.3 s to 3.3 s).
All observed peptides were entered into Skyline software, and peptides for protein quantification were selected based on ion signals in the spectral library. The relevant peptides with retention times and m/z values were exported using Skyline software, and then input into the MS control software Analyst (version 1.7, SCIEX, USA) to construct a PRM acquisition method. A final PRM acquisition method used a quadrupole tandem time-of-flight mass spectrometer (TripleTOF 5600+ system, SCIEX, USA) to collect data from all samples. Typically, precursor ions were selected with a quadrupole and then fragmented, and the fragment ions were quantitatively determined by a TOF mass spectrometer. To eliminate protein cross-contamination, “blanks” were run between adjacent samples to flush the column. The data were then processed using Skyline, followed by manual inspection of the quantification results for the peptides in the target protein.
A. dieselolei B5 ArbP-related sRNAs were evaluated for regulation by target mRNAs using the E. coli system proposed by Urban and Vogel according to previously published protocols. Considering plasmids expressing diverse ArbP-related sRNAs in the PL promoter, genes encoding ArbP-related sRNAs were amplified by PCR and the arbP gene was continuously expressed following a previously published protocol. In this study, translated GFP fusions with ArbP-related potential sRNA target genes were established using gDNA-amplified PCR products as previously described. The fusions were inserted using NheI/BfrBI restriction enzyme digestion and then ligated into a pXG10 plasmid backbone. The DNA sequence was analyzed to verify the authenticity of the construction. The target gfp fusion plasmid was transfected into E. coli TOP10 cells (Invitrogen), and then transfected alone or in combination with sRNA expression plasmid.
Fluorescence assay was conducted to measure GFP levels. An LB medium (2 mL) containing chloramphenicol and ampicillin was added to a single bacterial colony incubated at 37° C. and 220 rpm for 12 h. A volume of culture medium equivalent to OD600/mL was mixed with phosphate-buffered saline (PBS) at pH=7.4 containing 4% formaldehyde (1 mL), and centrifuged at 7,500 g for 2 min. 1 mL of 1×PBS was added to resuspend the cell pellet. For flow cytometry analysis, a 1/1000 dilution of 1×PBS was prepared to conduct measurements on a BD FACS Calibur instrument. Three replicate experiments were set up for different samples. Results were analyzed using Cyflogic software.
In order to study the regulatory mechanism of Arbp-related gene expression mediated by sRNAs, a modified E. coli two-plasmid system was used. The two-plasmid system could continuously express B5 ArbP protein to regulate the expression of ArbP-related sRNA and target translation fusion. The target transcript containing the ArbP-associated sRNA binding site was selected and fused to superfolded green fluorescent protein (sfgfp). The fusion activity of the reporter gene was assessed by monitoring GFP fluorescence over time. The potential sRNA target GFP gene reporter plasmid and the sRNA overexpression vector were co-transfected into E. coli; and the ArbP-related sRNA targets were activated post-transcriptionally (
To further evaluate the impact of ArbP-related sRNAs on translational regulation, PRM was conducted to determine the protein expression levels of target genes. Compared with the wild-type strain, the protein expression levels of all target genes in the sRNA mutant strain were down-regulated (fold 0.24 to 0.03). Therefore, sRNA related to ArbP had an impact on the translational regulation of sRNA target genes, but showed a low regulatory activity on target mRNA.
The expression levels of the target proteins in the source cells of E. coli and the editing cells obtained by co-transformation with the AbrP protein translation-promoting system were compared, thereby studying the regulatory role of the AbrP protein translation-promoting system in cell factory for metabolic engineering of E. coli. The protein expression levels of the target genes in each cell were detected through the PRM-based targeted mass spectrometry method (Method 1.5). The results showed that compared with the source cells, the protein expression levels of the target genes (glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase, glycerol dehydratase, and alanine dehydrogenase) in edited E. coli cells (by PRM) were significantly increased. The glycerol-3-phosphate dehydrogenase had a 6.65-fold increase in protein level, the glycerol-3-phosphatase had a 2.68-fold increase in protein level, the glycerol dehydratase had a 3.21-fold increase in protein level, the alanine dehydrogenase had a 3.84-fold increase in protein level (
The expression levels of the target proteins in the source cells of Bacillus subtilis and the editing cells obtained by co-transformation with the AbrP protein translation-promoting system were compared, thereby studying the regulatory role of the AbrP protein translation-promoting system in cell factory for metabolic engineering of Bacillus subtilis. The protein expression levels of the target genes in each cell were detected through the PRM-based targeted mass spectrometry method (Method 1.5). The results showed that compared with the source cells, the protein expression levels (by RPM) of the target genes (4 squalene synthases: BaSQS, BmSQS, PgSQS, and ScSQS) in the edited Bacillus subtilis cells were significantly increased. The BaSQS had a 4.12-fold increase in protein level, the BmSQS had a 2.54-fold increase in protein level, the PgSQS had a 3.14-fold increase in protein level, the ScSQS had a 3.49-fold increase in protein level (
The expression levels of the target proteins in the source cells of Corynebacterium glutamicum and the editing cells obtained by co-transformation with the AbrP protein translation-promoting system were compared, thereby studying the regulatory role of the AbrP protein translation-promoting system in cell factory for metabolic engineering of Corynebacterium glutamicum. The protein expression levels of the target genes in each cell were detected through the PRM-based targeted mass spectrometry method (Method 1.5). The results showed that compared with the source cells, the protein expression levels (by RPM) of the target genes (AAR: acyl ACP reductase, ADO: aldehyde-deformylating oxygenase) in the edited Corynebacterium glutamicum cells were significantly increased. The AAR had a 3.88-fold increase in protein level, the ADO had a 5.20-fold increase in protein level (
Although the above example has described the present disclosure in detail, it is only apart of, not all of, the examples of the present disclosure. Other examples may also be obtained by persons based on the example without creative efforts, and all of these examples shall fall within the protection scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2023112177340 | Sep 2023 | CN | national |
