Non-natural short-chain nucleotides and their applications in recombinant organisms

Abstract

The non-natural short-chain nucleotide provided by the application has a nucleotide sequence selected from any one of SEQ ID NO: 1-10, or a non-natural short nucleotide with a sequence identity of at least 92% and still having the same function. The non-natural short-chain nucleotide can change the expression characteristics of gene elements in a recombinant organism, thereby strengthening the growth and development, product accumulation, stress tolerance and other characteristic phenotypes of the recombinant organism, and has wide application value.

Description

Technical Field

[0001] The present invention discloses a method for modifying the genome of an organism using non-natural short-chain nucleotides, thereby changing characteristic traits. The present invention discloses multiple short-chain nucleotide sequences and their use as expression regulatory elements in engineering the genome of an organism. Background Art

[0002] The fundamental basis of life is gene expression and protein translation within cells. By regulating these processes at both the timing and intensity levels, we can ensure the orderly progression of life activities, such as growth and development, and environmental responses. The control of gene expression and protein translation is based on the transcriptional and translational machinery and expression regulatory elements, which primarily include promoters, terminators, 5' untranslated regions, and 3' untranslated regions. By altering the sequence of these expression regulatory elements, the expression characteristics of the target gene can be altered, thereby enhancing or weakening the characteristic phenotype of the recombinant organism.

[0003] The ability to synthesize target products is a key characteristic phenotype of recombinant organisms. The efficient synthesis of bulk chemicals, fine chemicals, and high-value natural products through biofermentation using highly genetically modified recombinant organisms (cell factories) is also a cornerstone of biomanufacturing. Precisely regulating the expression intensity of genes such as synthesis and degradation pathways and transcription factors is a key foundation for enhancing the levels of target products synthesized by recombinant organisms. This can be achieved through modifications of expression regulatory elements. Therefore, the development of expression regulatory elements is particularly necessary to enhance the characteristic phenotypes of recombinant organisms.

[0004] Commonly used expression regulatory elements, such as terminators and 3' untranslated regions, are primarily derived from the organism's own genome. Frequent use of these natural elements increases the number of repetitive sequences in the genome, and these endogenous elements are relatively long, which can easily lead to genomic instability. Developing novel, non-natural short nucleotide sequences is an effective means of enriching the repertoire of expression regulatory elements. Applying these short nucleotides can alter the expression characteristics of single or multiple genes, thereby reshaping the metabolic network of recombinant organisms and enhancing target phenotypes such as product synthesis capacity and stress tolerance. Summary of the Invention

[0005] The present invention trained a machine learning model based on terminator sequences of highly expressed genes. Experimental validation was then conducted on numerous sequences generated by the model, resulting in 10 non-natural short nucleotide chains that altered the expression characteristics of genetic elements in recombinant organisms, thereby enhancing the recombinant organisms' characteristic phenotypes, such as growth and development, product accumulation, and stress tolerance. This led to the present invention.

[0006] The present application provides a non-natural short nucleotide, the nucleotide sequence of which is selected from any one of SEQ ID NO: 1-10; or a non-natural short nucleotide which differs from the sequence by only one, two or three nucleotides, or has at least 92% sequence identity with the sequence, and still has the same function.

[0007]

[0008] The present application further provides the use of the non-natural short nucleotide in regulating the expression characteristics of a gene.

[0009] The present application also provides an expression cassette and an expression vector of the non-natural short nucleotide.

[0010] The present application further provides a recombinant host cell containing the non-natural short nucleotide.

[0011] The present application particularly provides a method for genetically modifying an organism using the non-natural short nucleotide to enhance a characteristic phenotype, wherein the resulting recombinant organism contains at least one copy (e.g. one, two, three or more copies) of the non-natural short nucleotide within 1000 nucleotides upstream and downstream of the gene to be regulated; more preferably, it contains a combination of two, three or more different non-natural short nucleotides.

[0012] Specifically, the organism is an animal, a plant or a microorganism; further, the organism includes but is not limited to a eukaryote, which includes but is not limited to a yeast, which includes but is not limited to Yarrowia lipolytica.

[0013] Preferably, the characteristic phenotype includes but is not limited to the growth and development of the recombinant organism, product accumulation, stress tolerance; the gene to be regulated includes but is not limited to a natural product synthesis pathway gene; the natural product synthesis pathway gene includes but is not limited to an aromatic compound synthesis gene; the aromatic compound synthesis gene includes but is not limited to a synthesis pathway gene of p-coumaric acid and resveratrol; the synthesis pathway gene of p-coumaric acid and resveratrol includes but is not limited to one of the genes shown in SEQ ID NO: 11-13.

[0014] Further preferably, the position of the non-natural short nucleotide is within 1000 nucleotides downstream of the 3' end of the gene stop codon.

[0015] More preferably, the position of the non-natural short nucleotide is between the 3' end of the gene stop codon and the terminator.

[0016] In addition, preferably, the products include but are not limited to biological macromolecules, cellular metabolites, bulk chemicals, fine chemicals, nutritional chemicals, and natural products; preferably, the accumulation of the products is achieved through microbial fermentation; the biological macromolecules include but are not limited to proteins, polysaccharides, and oil molecules; the natural products include but are not limited to terpenes, aromatics, alkaloids, and fatty acid compounds; the aromatic compounds include but are not limited to p-coumaric acid and resveratrol.

[0017] The non-natural short-chain nucleotides provided by the present invention can change the expression characteristics of gene elements in recombinant organisms, thereby enhancing the growth and development, product accumulation, adversity tolerance and other characteristic phenotypes of the recombinant organisms, and have wide application value. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0019] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0020] In the following examples, p-coumaric acid and resveratrol content analysis were performed using p-coumaric acid (Beijing Solaibao Technology Co., Ltd., product catalog number 501-98-4) and resveratrol (Tanmo Quality Inspection Standard Material Center, product catalog number 501-36-0) as standards, respectively. HPLC was used to draw standard curves, and then quantitatively analyze the p-coumaric acid and resveratrol contents in the test samples.

[0021] Example 1: Using non-natural short-chain nucleotides to regulate single gene expression characteristics and change the level of yeast synthesis of p-coumaric acid

[0022] 1) Using the Gibson assembly method, ten non-natural short-chain nucleotides (sequences are shown in SEQ ID NO: 1-10) were respectively attached to the promoter PrTefintron and the tyrosine ammonia lyase encoding gene TAL (sequence shown in SEQ ID NO: 11), terminator Tlip2 and other modules were used to construct 10 expression plasmids targeting the same genomic integration site. The short nucleotide is located between the stop codon of the tyrosine ammonia lyase gene and the terminator Tlip2 (structure is PrTefintron- TAL -short nucleotide-Tlip2), and the control plasmid does not contain the short nucleotide module.

[0023] 2) Double enzyme digestion of the above 10 plasmids and control plasmid, 11 integration type DNA fragments were obtained.

[0024] 3) By transformation, the above integration type DNA fragments were integrated into the same neutral site of Yarrowia lipolytica chromosome by the principle of homologous recombination, and confirmed by genome PCR, thus 11 recombinant Yarrowia lipolytica strains were obtained.

[0025] 4) The above recombinant Yarrowia lipolytica strains were inoculated into basic mineral salt culture solution, pre-cultured for 16-18 h, and then inoculated into 0.05 of basic mineral salt culture solution, cultured for 72 h, 100 μL culture was taken, 100 μL anhydrous ethanol was added, vortexed, filtered, and 100 μL was taken into liquid phase injection bottle for HPLC detection of p-coumaric acid. The results are shown in Table 1, which shows that the application of new short chain nucleotides affects the expression characteristics of cells, and further changes the p-coumaric acid synthesis ability of Yarrowia lipolytica. OD 600 for 72 h, 100 μL culture was taken, 100 μL anhydrous ethanol was added, vortexed, filtered, and 100 μL was taken into liquid phase injection bottle for HPLC detection of p-coumaric acid. The results are shown in Table 1, which shows that the application of new short chain nucleotides affects the expression characteristics of cells, and further changes the p-coumaric acid synthesis ability of Yarrowia lipolytica. TAL

[0026] Table 1 Yield of recombinant Yarrowia lipolytica strains carrying non-natural short chain nucleotides for fermentation synthesis of p-coumaric acid

[0027] unnatural short-chain nucleotides p-coumaric acid production (mg / L) none 56.20±0.02 SEQ ID NO. 1 68.43±0.13 SEQ ID NO. 2 67.40±0.25 SEQ ID NO. 3 61.50±0.17 SEQ ID NO. 4 68.45±0.24 SEQ ID NO. 5 50.20±0.15 SEQ ID NO. 6 45.37±0.17 SEQ ID NO. 7 46.28±0.08 SEQ ID NO. 8 61.25±0.19 SEQ ID NO. 9 50.18±0.28 SEQ ID NO. 10 61.37±0.98

[0028] The nucleotide sequence of SEQ ID NO: 11 is as follows:

[0029] ​

[0030] Example 2: Modulating the expression characteristics of multiple genes using a combination of non-natural short-chain nucleotides to alter the level of resveratrol synthesis in yeast

[0031] 1) Two non-natural short-chain nucleotides (sequences shown in SEQ ID NO: 1 and 2) were combined with three sets of expression cassettes respectively by Gibson assembly to form an integration plasmid containing three expression cassettes; the control plasmid did not contain non-natural short-chain nucleotides. The three sets of expression cassettes were (1) promoter PrTefintron, tyrosine ammonia lyase encoding gene (sequence shown in SEQ ID NO: 11), and terminator Tlip2; (2) promoter PrPGD, 4-coumarinoyl-CoA ligase encoding gene (sequence shown in SEQ ID NO: 12), and terminator Tlip2; (3) promoter PrTefintron, resveratrol synthase encoding gene (sequence shown in SEQ ID NO: 13), and terminator TPex20. One sequence represented by SEQ ID NO: 1 is located between the stop codon of the gene encoding tyrosine ammonia lyase and the terminator Tlip2, another sequence represented by SEQ ID NO: 1 is located between the stop codon of the gene encoding resveratrol synthase and the terminator TPex20, and the sequence represented by SEQ ID NO: 2 is located between the stop codon of the gene encoding 4-coumarinoyl-CoA ligase and the terminator Tcyc.

[0032] 2) The two integration plasmids are double-digested to obtain two integration DNA fragments targeting the same genomic site.

[0033] 3) Through transformation, the aforementioned integrative DNA fragments were integrated into the same neutral site of the Yarrowia lipolytica chromosome by homologous recombination. After confirmation by genomic PCT, two recombinant Yarrowia lipolytica strains were obtained: ST-1NL, which incorporated the non-natural short-chain nucleotides, and ST-1N, a control recombinant strain that did not contain the two non-natural short-chain nucleotides.

[0034] 4) The two recombinant Yarrowia lipolytica strains were inoculated into the basal mineral salt culture medium and pre-cultured for 16-18 hours. OD 600 The basal mineral salt culture medium was inoculated with 0.05% aliquots of the culture medium and cultured for 72 h. 100 μL of the culture was taken and 100 μL of anhydrous ethanol was added. The mixture was vortexed and filtered. 100 μL of the culture medium was placed in a liquid injection vial for HPLC detection of resveratrol.

[0035] 5) The results are shown in Table 2, which demonstrate that the combined application of non-natural short-chain nucleotides in Yarrowia lipolytica alters the expression characteristics of multiple gene elements in the recombinant organism, thereby enhancing the resveratrol synthesis capacity of Yarrowia lipolytica.

[0036] Table 2 Yield of resveratrol synthesized by recombinant Yarrowia lipolytica

[0037] unnatural short-chain nucleotides resveratrol production (mg / L) Recombinant Yarrowia lipolytica strain ST-1N without SEQ ID NO. 1 and 2 (control) 42.57±0.45 Recombinant Yarrowia lipolytica strain ST-1NL with SEQ ID NO. 1 and 2 81.68±0.26

[0038] The nucleotide sequence of SEQ ID NO: 12 is as follows:

[0039]

[0040] The nucleotide sequence of SEQ ID NO: 13 is as follows:

[0041]

[0042] Example 3: Composite application of non-natural short-chain nucleotides to regulate the expression characteristics of multiple copies of multiple genes and increase the level of resveratrol synthesis in Yarrowia lipolytica

[0043] The two integrative DNA fragments from Example 2 (one containing three non-natural short nucleotide sequences, two of which are shown in SEQ ID NO: 1 and the other in SEQ ID NO: 2; the other fragment lacked any non-natural short nucleotide sequences) were cloned into integration vectors targeting six different neutral sites on the genome, yielding two sets of integration plasmids: six in each set, for a total of 12. Double enzyme digestion yielded two sets of integration plasmids (six in each set). These fragments were then transformed and, using homologous recombination, sequentially integrated into the Yarrowia lipolytica chromosome at six different neutral sites. PCR verification confirmed the generation of two recombinant Yarrowia lipolytica strains harboring six copies of genes involved in the resveratrol biosynthesis pathway (one containing the non-natural short nucleotide sequence ST-6NL and one without the non-natural short nucleotide sequence ST-6N).

[0044] 2) The recombinant Yarrowia lipolytica strain was inoculated into YPD liquid medium and pre-cultured for 16-18 h. OD 600 The YPD salt culture medium was inoculated with 0.05% aliquots of the culture medium and cultured for 72 h. 100 μL of the culture was taken and 100 μL of anhydrous ethanol was added. The mixture was vortexed and filtered. 100 μL of the culture medium was placed in a liquid phase injection vial for HPLC detection of resveratrol.

[0045] 3) The results, shown in Table 3, demonstrate that the use of non-natural short-chain nucleotides in Yarrowia lipolytica containing multiple copies of the resveratrol biosynthesis gene altered the expression characteristics of the gene elements within the recombinant organism, thereby enhancing the resveratrol biosynthesis capacity of the yeast. This further demonstrates that the application of these non-natural short nucleotides can alter the expression characteristics of single or multiple genes, thereby reshaping the metabolic network of the recombinant organism and enhancing the biosynthesis capacity of the product.

[0046] Table 3 Yield of resveratrol synthesized by recombinant Yarrowia lipolytica

[0047] unnatural short-chain nucleotides resveratrol production (mg / L) Recombinant Yarrowia lipolytica strain ST-6N without SEQ ID NO. 1 and 2 (control) 372.71±0.85 Recombinant Yarrowia lipolytica strain ST-6NL with SEQ ID NO. 1 and 2 812.29±0.93

[0048] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A non-natural short nucleotide, characterized in that: The nucleotide sequence is selected from SEQ ID NO: 1 or SEQ ID NO:

2.

2. An expression cassette comprising the non-natural short nucleotide sequence according to claim 1.

3. An expression vector comprising the non-natural short nucleotide according to claim 1.

4. A method for genetically modifying an organism using the non-natural short nucleotide of claim 1 to enhance a characteristic phenotype, characterized in that: The resulting recombinant organism contains at least one copy of a non-natural short nucleotide, wherein the non-natural short nucleotide is located within 1000 nucleotides downstream of the 3' end of the stop codon of the gene to be regulated; The organism is a yeast; The characteristic phenotype is product accumulation of the recombinant organism; the gene to be regulated is a natural product synthesis pathway gene, and the natural product synthesis pathway gene is one of the genes shown in SEQ ID NO: 11-13.

5. The method according to claim 4, characterized in that The resulting recombinant organisms contain one, two, or three copies of the unnatural short nucleotide; It contains two different combinations of the non-natural short nucleotides.

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