CN115851807A

CN115851807A - Recombinant organism and method for producing multiple cyclosporine-like amino acids

Info

Publication number: CN115851807A
Application number: CN202211236882.2A
Authority: CN
Inventors: 王博祥; 余俊
Original assignee: Shenzhen Lingspider Technology Co ltd
Current assignee: Shenzhen Lingspider Technology Co ltd
Priority date: 2022-10-10
Filing date: 2022-10-10
Publication date: 2023-03-28
Anticipated expiration: 2042-10-10
Also published as: WO2024078367A1; CN115851807B

Abstract

A recombinant organism and a method for producing a plurality of cyclosporine-like amino acids, the method comprises an organism construction step, wherein the organism is provided with a knockout endogenous gene, a xylose metabolism gene is integrated into the genome of the organism, and an organism which can produce S7P by using xylose is obtained; a DDGS-OMT construction step, which comprises integrating at least one of a DDGS gene and an OMT gene into the genome of the organism obtained in the organism construction step to obtain an organism capable of producing 4DG by using xylose. According to the invention, the yield of cyclosporine-like amino acid is obviously improved by knocking out endogenous genes.

Description

Recombinant organism and method for producing multiple cyclosporine-like amino acids

Technical Field

The invention relates to the field of genetic engineering, in particular to a recombinant organism and a method for producing a plurality of cyclosporine-like amino acids.

Background

The ultraviolet rays in the sunlight can cause damage to human skin. Ultraviolet rays irradiated to the ground are classified into UVA (315 to 400 nm) and UVB (280 to 315 nm). UVB has high energy and can directly damage skin cells to cause skin red swelling, inflammation, apoptosis and DNA damage; UVA has stronger penetrating power, can reach deep skin and slowly generate oxidation free radicals, and causes DNA damage, skin aging and darkness. Sun protection is important to everyone, however, existing sun protection means have many problems. Physical sunscreens use zinc oxide or titanium dioxide metal oxide particles to reflect ultraviolet light, but these particles have poor skin feel and also tend to occlude pores. Chemical sunscreens such as benzophenone, ethylhexyl salicylate, etc., which can penetrate into the skin and retain the ability to absorb ultraviolet light, are lighter in skin feel. However, the existing chemical sunscreen substances have low light stability and can slowly release oxidizing free radicals to damage the skin. These sunscreen molecules can also pose a hazard to marine organisms.

Cyclosporine-like amino acids (MAAs or MAA for short, also called Mycosporine-like amino acids) are a family of molecules widely existing in marine life, which can absorb ultraviolet rays and release the ultraviolet rays in a heat energy manner. The natural, mild and non-irritant characteristics make them promising as a new class of sunscreen ingredients. MAAs are produced in nature mainly by algal microorganisms or macroalgae, and their low carbon fixation efficiency and low growth rate result in low MAAs production. In recent two years, academia have successfully used engineering microorganisms such as escherichia coli, saccharomyces cerevisiae and the like to produce MAAs, and the microorganisms grow rapidly and are convenient to genetically modify, so that higher yield can be brought. In the prior art, saccharomyces cerevisiae is used for producing a MAA molecule, shinorine. Saccharomyces cerevisiae has considerable potential for producing a variety of MAA molecules, but the former attempts only to produce shinorine, a molecule, and the yield is low.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided a method of constructing a recombinant organism, comprising:

an organism construction step, which comprises providing an organism after knocking out an endogenous gene, integrating xylose metabolism genes into the genome of the organism, and obtaining an organism capable of producing S7P by using xylose;

a DDGS-OMT construction step, which comprises integrating at least one of a DDGS gene and an OMT gene into the genome of the organism obtained in the organism construction step to obtain an organism capable of producing 4DG by using xylose.

According to a second aspect, in an embodiment, there is provided a recombinant organism obtained by the method of any one of the first aspect.

According to a third aspect, in an embodiment, there is provided a compound produced by a recombinant organism obtained by the method of any one of the first aspect.

According to a fourth aspect, in an embodiment, there is provided a recombinant organism having at least a portion of its endogenous genes knocked out and having integrated in its genome 1) a xylose metabolism gene, and 2) at least one of a DDGS gene, an OMT gene.

According to the recombinant organism and the method for producing the plurality of cyclosporine-like amino acids, the yield of the cyclosporine-like amino acids is obviously improved by knocking out endogenous genes.

In one embodiment, the ultraviolet absorption intensity of the cyclosporine-like amino acid is significantly increased.

In one embodiment, a plurality of cyclosporin amino acids or analogs thereof may also be produced by a recombinant organism.

Drawings

FIG. 1 is a schematic representation of yeast engineering;

FIG. 2 is a schematic of the metabolic pathways for Gadusol and 3 MAA production;

FIG. 3 shows the MAA production pathway and production genes of different organisms;

FIG. 4.1 is a schematic diagram of the knockout of TAL1 gene;

FIG. 4.2 is a diagram showing the results of colony PCR;

FIGS. 4.31 and 4.32 are the sequencing results after TAL1 gene knockout;

FIG. 5.1 is a schematic drawing of the knock-in of the xylose metabolic pathway;

FIG. 5.2 is a graph showing the results of electrophoresis in which the left, right, xyl1, xyl2, and xyl3 genes were amplified by PCR;

FIG. 5.3 is a graph of colony PCR results for the xylose metabolic pathway;

FIG. 5.4 is a graph showing the results of sequencing after insertion of xyl1, xyl2 and xyl3 genes;

FIG. 6.1 is a schematic diagram of the knock-in process of DDGS-OMT;

FIG. 6.2 is a diagram showing the results of PCR after knockin of DDGS-OMT;

FIG. 6.3 is a diagram showing the PCR results of the colonies after the knockin of DDGS-OMT;

FIG. 6.4 is a diagram showing the sequencing results after knocking-in of DDGS-OMT;

FIG. 7 is a growth curve of engineered strains under different carbon sources;

FIG. 8 is a schematic representation of the combinatorial expression of amino acid ligases;

FIGS. 9.1-9.4 are graphs of the results of fermentation and production of Shinorine or Porphyra-334;

FIGS. 10.1-10.4 are graphs of the results of High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) of yeast fermentation products;

FIG. 11.1 is a schematic representation of reinsertion of the OMT-DDGS gene to increase gene copy number, while knocking out the Nqm gene;

FIG. 11.2 is a diagram showing the result of PCR amplification of the Nqm gene knockout with the OMT-DDGS gene reinserted to increase the gene copy number;

FIG. 11.3 is a diagram showing the results of colony PCR in which OMT-DDGS gene was reinserted to increase the gene copy number and the Nqm gene was knocked out;

FIG. 11.4 is the sequencing result of reinserting the OMT-DDGS gene to increase the gene copy number while knocking out the Nqm gene;

FIG. 12.1 is a schematic diagram of the production process of optimized shinorine and porphyra-334;

FIG. 12.2 is the 334nm UV absorption peak of optimized shinorine and porphyra-334;

FIG. 12.3 is the optimized values of OD334 for shinorine and porphyra-334;

FIG. 13.1 is a schematic diagram of the process for producing palythine;

FIG. 13.2 shows the absorption peak of palythine at 320nm in the ultraviolet band;

FIG. 13.3 is the OD320 value of palythine;

FIG. 14.1 is a schematic diagram of the process of engineering the gadusol-producing strain;

FIG. 14.2 is a graph showing the results of PCR amplification of the gadusol-producing strain;

FIG. 14.3 is a graph showing the colony PCR results of the gadusol producing strain;

FIG. 14.4 is a graph showing the sequencing results of the gadusol-producing strain;

FIG. 15.1 is the ultraviolet band absorption peak at 290nm of gadusol;

FIG. 15.2 is a graph of the absorbance of L4 minus the absorbance of the corresponding control;

FIG. 16.1 is an absorption peak from 275nm to 360nm for four molecules gadusol, palythine, shinorine, and porphyra-334;

FIG. 16.2 is a graph showing the UV absorption curves of the four molecules gadusol, palythine, shinorine, and porphyra-334;

FIG. 17.1 is the UV absorbance peak of the combination inserted into the genome;

FIG. 17.2 is the OD334nm absorbance of the combination inserted into the genome.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of clearly describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where a certain sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.

Herein, "a sequence having at least 90% similarity" includes a nucleotide sequence having the same or similar function by substitution and/or deletion and/or addition of one or several nucleotides, or an amino acid sequence having the same or similar function by substitution and/or deletion and/or addition of one or several amino acid residues. Similarities include, but are not limited to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%.

In one embodiment, in the organism constructing step, the knocked-out endogenous gene includes, but is not limited to, at least one of TAL1 gene, nqm gene.

In one embodiment, in the organism constructing step, the xylose metabolism gene includes, but is not limited to, at least one of xyl1 gene, xyl2 gene, and xyl3 gene.

In one embodiment, the xylose metabolism genes in the organism construction step include, but are not limited to, xyl1 gene, xyl2 gene, and xyl3 gene.

In one example, in the organism constructing step, the amino acid encoded by the xyl1 gene comprises a sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO. 6.

In one example, in the organism construction step, the amino acid encoded by the xyl2 gene comprises a sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO. 7.

In one example, in the organism construction step, the amino acid encoded by the xyl3 gene comprises a sequence having at least 90% similarity to the amino acid sequence as set forth in SEQ ID NO. 8.

In one embodiment, in the organism construction step, the genome of the organism comprises a sequence that has at least 90% similarity to the nucleotide sequence set forth in SEQ ID NO. 9.

In one example, in the organism construction step, expression of xyl1 gene is promoted using the pTDH3 promoter.

In one example, the expression of xyl2 gene is driven using the pPGK1 promoter in the organism construction step.

In one example, the expression of xyl3 gene is driven using the pTEF2 promoter in the organism construction step.

In one example, in the organism constructing step, the insertion site of at least one of xyl1 gene, xyl2 gene and xyl3 gene is the his3 site.

In one example, the insertion sites of xyl1 gene, xyl2 gene and xyl3 gene in the organism constructing step are his3 sites.

In one embodiment, the DNA sequence for cleavage comprises: TAL1, nqm, 308, 106, his3 site.

In one example, the insertion sites of xyl1 gene, xyl2 gene and xyl3 gene are his3 site, the insertion sites of DDGS gene and OMT gene are 308 site, the insertion sites of EEVS gene and MTOx gene are 308 site, and the insertion sites of AGL gene and ALAL gene are 106 site. The AGL gene is a gene for expressing AGL enzyme, and the ALAL gene is a gene for expressing ALAL enzyme.

In one embodiment, in the organism constructing step, the DNA for cleavage comprises at least one of the nucleotide sequences shown in SEQ ID NOS: 1 to 5.

In one embodiment, in the organism constructing step, the DNA for cleavage comprises at least one of the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 5.

In one embodiment, in the step of constructing the DDGS-OMT, the amino acid encoded by the DDGS gene comprises a sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO. 10.

In one embodiment, in the step of constructing the DDGS-OMT, the amino acid encoded by the OMT gene comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 11.

In one example, in the step of constructing DDGS-OMT, the expression of the DDGS gene is promoted using pTDH3 promoter.

In one example, in the step of constructing DDGS-OMT, the pPGK1 promoter is used to drive the expression of the OMT gene.

In one example, the DDGS-OMT is constructed by using pPGK1 promoter to drive the expression of DDGS gene.

In one embodiment, in the step of constructing the DDGS-OMT, the copy number of the OMT gene in the genome of the organism is 1 or more.

In one embodiment, in the step of constructing the DDGS-OMT, the copy number of the DDGS gene in the genome of the organism is more than or equal to 1.

In one embodiment, in the step of constructing the DDGS-OMT, the genome of the organism has a copy number of the OMT gene of 2 or more.

In one embodiment, in the step of constructing the DDGS-OMT, the copy number of the DDGS gene in the genome of the organism is more than or equal to 2.

In one embodiment, in the step of constructing the DDGS-OMT, the insertion site of the DDGS gene and/or the OMT gene is 308 th site.

In one embodiment, the DDGS-OMT is constructed such that the genome of the organism comprises a sequence that is at least 90% similar to the nucleotide sequence set forth in SEQ ID NO. 12.

In one embodiment, the method further comprises a ligase gene construction step, wherein a gene capable of expressing amino acid ligase is integrated into the genome of the organism obtained in the DDGS-OMT construction step, and the organism capable of producing the cyclosporine-like amino acid is obtained.

In one embodiment, the amino acid ligase comprises at least one of AGL and ala enzymes.

In one embodiment, the AGL enzyme comprises at least one of an Np5598 protein, an nlysc protein, an Am4257 protein and the ala enzyme comprises at least one of an Am4256 protein, an Np5597 protein, an nlysd protein.

In one embodiment, the Np5598 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID No. 16.

In one embodiment, the NlmysC protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 23.

In one embodiment, the Am4257 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 24.

In one embodiment, the Am4256 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 18.

In one embodiment, the Np5597 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID No. 25.

In one embodiment, the NlmysD protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO 17.

In one embodiment, in the ligase gene construction step, the amino acid ligase comprises Np5598 protein and nlysd protein.

In one embodiment, in the step of constructing the ligase gene, the amino acid ligase comprises Np5598 protein and Am4256 protein.

In one embodiment, in the ligase gene construction step, the amino acid ligase comprises Np5598 protein and nlysc protein.

In one embodiment, in the step of constructing the ligase gene, the amino acid ligase comprises Np5598 protein and Np5597 protein.

In one embodiment, in the ligase gene constructing step, the promoter of the AGL gene includes pTDH3 promoter.

In one embodiment, in the ligase gene construction step, the promoter of the ALAL gene comprises the pPGK1 promoter.

In one embodiment, the insertion site of the AGL gene and/or the ala gene in the ligase gene constructing step is 106 sites.

In one embodiment, the ligase gene construction step comprises a sequence in the genome of the organism that has at least 90% similarity to the nucleotide sequence set forth in SEQ ID NO. 19.

In one embodiment, the ligase gene construction step comprises a sequence in the genome of the organism that has at least 90% similarity to the nucleotide sequence set forth in SEQ ID NO. 22. The sequence can improve the yield of MAA.

In one embodiment, the ligase gene constructing step further comprises recovering the product from the organism producing the cyclosporine-like amino acid.

In one embodiment, the product comprises a cyclosporin-like amino acid.

In one embodiment, the cyclosporin-like amino acid used in the ligase gene constructing step includes Shinorine (CAS number: 73112-73-9,N- [3- [ (carboxymethyl) amino group]-5-hydroxy-5- (hydroxymethyl) -2-methoxy-2-cyclohexene-1-methylene]-L-serine, formula: c ₁₃ H ₂₀ N ₂ O ₈ Molecular weight: 332.31 Pora-334 (CAS No.: 70579-26-9).

In one embodiment, a mysH construction step is further included, comprising integrating a mysH gene into the genome of the organism resulting from the ligase gene construction step, resulting in an organism that produces a cyclosporin-like amino acid. The mysH gene is the gene for expression of the mysH protein.

In one embodiment, the mysH construction step uses the pTEF2 promoter to drive expression of the mysH gene.

In one embodiment, the mysH protein comprises a sequence that is at least 90% similar to the amino acid sequence set forth in SEQ ID NO. 20.

In one embodiment, the mysH construction step comprises a sequence in the genome of the organism that has at least 90% similarity to the nucleotide sequence set forth in SEQ ID NO. 21.

In one embodiment, the mysH construction step further comprises recovering a cyclosporine-like amino acid from the cyclosporine-like amino acid-producing organism.

In one embodiment, the mysH construction step, the cyclosporine-like amino acid comprises palythine (CAS number: 67731-19-5).

In one embodiment, the method further comprises a step of constructing EEVS-MTOx, which comprises integrating at least one of EEVS gene and MTOx gene into the genome of the organism obtained from the step of constructing the organism to obtain gadusol (CAS No.: 76663-30-4, molecular formula: C) ₈ H ₁₂ O ₆ ) The organism of (1).

In one example, in the construction step of EEVS-MTOx, the expression of EEVS gene is initiated using pTDH3 promoter.

In one example, in the step of constructing EEVS-MTOx, pPGK1 promoter is used to drive the expression of MTOx gene.

In one embodiment, in the step of constructing the EEVS-MTOx, the insertion site of the EEVS gene and/or the MTOx gene is at position 308.

In one embodiment, in the step of constructing EEVS-MTOx, the amino acid encoded by the EEVS gene comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 13.

In one embodiment, in the step of constructing EEVS-MTOx, the amino acid encoded by the MTOx gene comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 14.

In one embodiment, the EEVS-MTOx construct step comprises a sequence having at least 90% similarity to the nucleotide sequence set forth in SEQ ID NO. 15 in the genome of the organism.

In one embodiment, the step of constructing EEVS-MTOx further comprises recovering gadusol from the organism producing gadusol.

In one embodiment, in the organism constructing step, the organism includes, but is not limited to, a fungus.

In one embodiment, in the organism construction step, the fungus includes, but is not limited to, yeast.

In one embodiment, in the organism construction step, the yeast includes, but is not limited to, saccharomyces cerevisiae.

In one embodiment, the compound comprises a cyclosporin-like amino acid or an analog thereof.

In one embodiment, the cyclosporin-like amino acids include, but are not limited to, 3 MAA molecules such as shinorine (absorption peak 334 nm), porphyra-334 (absorption peak 334 nm), palythine (absorption peak 320 nm), and the like.

In one embodiment, the cyclosporine-like amino acid analogs (MAA analogs) include, but are not limited to, gadusol (absorption peak 300 nm).

In one embodiment, the endogenous gene that is knocked out includes, but is not limited to, at least one of TAL1 gene, nqm gene.

In one embodiment, the xylose metabolism genes include, but are not limited to, at least one of xyl1 gene, xyl2 gene, and xyl3 gene.

In one embodiment, the xylose metabolism genes include xyl1 gene, xyl2 gene and xyl3 gene.

In one embodiment, the promoter of xyl1 gene comprises the pTDH3 promoter;

in one embodiment, the promoter of xyl2 gene comprises the pPGK1 promoter.

In one embodiment, the promoter of xyl3 gene comprises the pTEF2 promoter.

In one embodiment, the insertion site of xyl1 gene, xyl2 gene, and xyl3 gene is a his3 site.

In one embodiment, the copy number of the OMT gene in the genome of the organism is 1 or more.

In one embodiment, the copy number of the DDGS gene in the genome of the organism is greater than or equal to 1.

In one embodiment, the copy number of the OMT gene in the genome of the organism is 2 or more.

In one embodiment, the copy number of the DDGS gene in the genome of the organism is greater than or equal to 2.

In one embodiment, the insertion site of the DDGS gene and/or OMT gene comprises site 308.

In one embodiment, the organism further has integrated into its genome a gene capable of expressing an amino acid ligase.

In one embodiment, the amino acid ligase includes, but is not limited to, at least one of an AGL enzyme, an ala enzyme.

The Np5598 protein comprises a sequence which has at least 90 percent of similarity with the amino acid sequence shown in SEQ ID NO. 16.

The NlmysD protein comprises a sequence which has at least 90% similarity to the amino acid sequence shown in SEQ ID NO. 17.

The Am4256 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 18.

In one embodiment, the amino acid ligase comprises Np5598 protein and nlysd protein.

In one embodiment, the amino acid ligase comprises Np5598 protein and Am4256 protein.

In one embodiment, the amino acid ligase comprises Np5598 protein and nlysc protein.

In one embodiment, the amino acid ligase comprises an Np5598 protein and an Np5597 protein.

In one embodiment, the promoter of the AGL gene comprises pTDH3 promoter.

In one embodiment, the promoter of the ala gene comprises the pgk1 promoter.

In one embodiment, the insertion site of the AGL gene and/or the ala gene comprises 106 sites.

The promoter of the mysH gene comprises a pTEF2 promoter, and a gene capable of expressing mysH protein, namely the mysH gene, is also integrated in the genome of the organism.

In one embodiment, the promoter of the mysH gene comprises the pTEF2 promoter.

In one embodiment, the organism further has integrated into its genome at least one of an EEVS gene, a MTOx gene.

In one embodiment, the promoter of the EEVS gene comprises pTDH3 promoter.

In one embodiment, the promoter of the MTOx gene comprises the pPGK1 promoter.

In one embodiment, the insertion site of the EEVS gene and/or the MTOx gene is position 308.

In one embodiment, the organism includes, but is not limited to, a fungus.

In one embodiment, the fungus includes, but is not limited to, yeast.

In one embodiment, the yeast includes, but is not limited to, saccharomyces cerevisiae.

In one embodiment, the present invention enables the use of Saccharomyces cerevisiae to obtain more MAA molecules.

In one embodiment, the invention provides a method for systematically transforming saccharomyces cerevisiae, which can enable the saccharomyces cerevisiae to produce 3 MAA molecules such as shinorine (absorption peak 334 nm), porphyra-334 (absorption peak 334 nm), palythine (absorption peak 320 nm) and the like, and an MAA analogue gadosol (absorption peak 300 nm). The molecules can absorb UVB and UVA with high energy level to reach full coverage of UVB-UVA-1 wavelength, thereby being used as a series of novel cosmetic raw materials.

In one embodiment, the invention constructs various MAA production approaches in Saccharomyces cerevisiae, and the specific modification strategy is as follows: MAA production starts with S7P, which is produced from a carbohydrate molecule such as glucose via the pentose phosphate pathway. In microorganisms with high efficiency of sugar utilization, S7P molecules have higher accumulation, which is also an advantage of Saccharomyces cerevisiae.

In one embodiment, the present invention provides a method comprising the steps of:

1. S7P is first subjected to DDGS and OMT enzymes to produce 4DG, which forms the basic backbone of the MAA molecule, the cyclosporin-like structure. And adding amino acid into the skeleton by using different amino acid ligases to produce various different MAAs. For example, L-glycine can be linked to 4DG using ATP-grapp ligand to form M-glycine molecules, and L-Serine can be linked to M-glycine through D-ALA-D-ALA ligand to form shinorine molecules. And when L-Alanine is used as a substrate, porphyra-334 is formed. On the basis of these two molecules, palythine can be produced by adding MysH enzyme to remove amino acid residues.

2. Different algae and microorganisms have different enzyme combinations. By CRISPR technology, we combined the expression and fermentation of different amino acid ligases from nosonic punctiforme, nosonic linckia and Actinosynnema mirum in yeast, finding the optimal combination to reach the highest yield.

3. Increasing the concentration of the common precursor S7P of the MAA molecule is key to increasing yield. S7P is produced by conversion of glucose through a multi-step reaction, but intracellular glucose mainly proceeds through glycolytic pathway reactions to ensure normal growth of microorganisms. To increase the yield of S7P, we introduced the xylose metabolic system (xyl 1, xyl2, xyl 3) from Schiffersomyces stipitis, so that the yeast rapidly produces the precursor X5P of S7P using xylose as a substrate, thereby increasing the total yield of S7P. At the same time, we knocked out the enzyme Tal1 downstream of S7P to reduce the possibility of S7P producing other substances.

The molecule gadusol with similar properties to MAA is also produced by conversion with EEVS and MTOx enzymes using S7P as precursor, without the need for addition of other amino acids. The same strategy was used in the present invention for the production of gadusol using EEVS and MTOx from zebrafish.

Example 1

FIG. 1 is a schematic representation of yeast engineering. This example introduced xyl1, xyl2 and xyl3 systems in yeast for the mass production of S7P. Meanwhile, the downstream genes TAL1 and Nqm were knocked out in this example to reduce the loss of S7P. To produce various MAAs, this example introduced two copies of DDGS and OMT, combining various AG-L and ALA-L amino acid ligases, and finally produced shinorine, porphyra-334, palythine. Meanwhile, this example introduced EEVS and MT-OX in another strain to produce gadusol.

FIG. 2 is a schematic of the metabolic pathways for Gadusol and 3 MAA production. S7P as a co-substrate for the reaction, through EEVS and MT-OX, produces gadusol, a UVB-absorbing (. Lamda.) (Lambda.) _max =290 nm). S7P can produce 4-deoxygadusol (4-DG), which is a cyclosporine-like framework, through DDGS and OMT. Mycrosporine-glycine (MG) can be produced by ATP-grap sugar (AG-L), and Shinorine (lambda) can be produced by using serine as substrate by different D-Ala-D-Ala sugar _max =334 nm), porphyra-334 (lambda) can be produced by using Alanine as a substrate _max =334 nm). Shinorine can be transformed by MysH to produce Palythine (lambda) _max ＝320nm)。

FIG. 3 shows the MAA production pathway and production genes of different organisms. Both Nostoc Puncidiform, a genus of Nostoc, and Actinosynnema mirum, a genus of Actinosynnema, produce shinorine and porphyra-334, while Nostoc Linckia, a genus of Nostoc, produces palythine.

The operation steps of this embodiment are as follows:

1. knockout of saccharomyces cerevisiae endogenous DNA and insertion of exogenous DNA:

cleavage and knockout of endogenous DNA was performed using the pCRCT gene. Specifically, a commercial pCRCT plasmid was used, and DNA sequences for cleavage were mixed and assembled using the Golden gate kit supplied by NEB corporation. After transformation of escherichia coli DH5a and confirmation of successful assembly of the DNA sequence by sequencing, CRISPR plasmids were extracted and transformation of saccharomyces cerevisiae (cen. Pk2 strain) was performed by lithium acetate transformation. If the foreign DNA is inserted at the same time as the DNA is cleaved, 1000ng of the foreign DNA is transformed together with the plasmid. Culturing the transformed yeast on SC-URA solid culture medium, and identifying whether the gene is successfully knocked out or whether foreign DNA is successfully inserted or not by colony PCR and DNA sequencing technology. If the monoclonal detection is successful, the monoclonal is picked and diluted in a conventional YPD medium for spread culture until the monoclonal is grown out, and then the monoclonal is subjected to negative screening. And selecting a monoclonal strain only growing in the YPD medium to obtain the modified yeast strain.

In this example, the DNA sequence for cleavage contained: TAL1, nqm1, 308, 106, his3 site (see sequence below). Wherein the endogenous genes knocked out are TAL1 and Nqm genes, which can convert S7P endogenous to Saccharomyces cerevisiae into other substances so as to reduce the yield of MAA. Thus, knocking out both genes contributes to the accumulation of MAA product in yeast. In this way, we first knock out the endogenous TAL1 gene to obtain the L1 strain.

There are various ways to knock out endogenous DNA, and the CRISPR system, ZFN system, TALEN system can be used to cut the same site of the gene, or any other site on the gene.

Fig. 4.1 is a schematic diagram of the knockout of TAL1 gene. CRISPR-cas9 plasmid pCRCT-TAL1 and the upstream and downstream homologous arms of TAL1 gene are jointly transformed into yeast, and the sequence of the TAL1 gene can be knocked out. Fig. 4.2 is a colony PCR result diagram, wherein 11 yeast clones are selected for colony PCR, fig. 4.31 and fig. 4.32 are sequencing result diagrams after TAL1 gene knockout, and the strain knockout is confirmed through the sequencing result to obtain the L1 strain.

2. Construction of xylose metabolism System:

this example introduces the xylose metabolic system (xyl 1, xyl2, xyl 3) from Schiffersomyces stipitis, specifically, synthesizes the sequences of xyl1, xyl2, xyl3, and simultaneously performs codon optimization of s.cerevisiae. Then, the pTDH3, pPGK1 and pTEF2 promoters of Saccharomyces cerevisiae are used to start the three genes respectively, and the complete DNA sequence constructed is shown as SEQ ID NO. 9.

The genome was inserted by the method in step 1. Specifically, we used pCRCT-his3 site to cleave the plasmid and simultaneously transformed the L1 strain with 1000ng of the constructed xyl1, xyl2, xyl3 DNA sequences, and equivalent amounts of the 1000bp homology arm sequences upstream and downstream of the cds sequence of the his3 gene by lithium acetate transformation. The strain L2 producing S7P using xylose was obtained by screening the strain according to the method in step 1.

FIG. 5.1 is a knock-in schematic of the xylose metabolic pathway. The his3 gene site of strain L1 was cleaved using plasmid pCRCT-his3, and then transformed into yeast together with the upstream and downstream homology arms, xyl1, xyl2, and xyl3 genes, and the entire DNA was combined in vivo by recombinant enzymes endogenous to the yeast.

FIG. 5.2 shows the results of electrophoresis in which the left, right, xyl1, xyl2, and xyl3 genes were amplified by PCR, and after transformation, yeast clones were selected and colony PCR was performed (FIG. 5.3), and the completion of the insertion of the xyl1, xyl2, and xyl3 genes was confirmed by sequencing (FIG. 5.4), thereby obtaining L2 strain.

By comparing the growth curves of the strains under different carbon sources, we found that the growth was significantly reduced in pure xylose after introduction of the xylose gene, while there was almost no change in pure glucose culture. This is because xylose uses a large amount of the production precursor substance S7P and thus cannot supply the energy required for growth to the microorganisms.

3. Construction of DDGS-OMT:

this example introduced DDGS and OMT genes from the Nostoc punctiform. Specifically, the sequences of DDGS and OMT were synthesized and codon-optimized in Saccharomyces cerevisiae. Then, the pTDH3 promoter of Saccharomyces cerevisiae was used to start OMT gene, the pPGK1 promoter was used to start DDGS gene, and the complete DNA sequence was shown in SEQ ID NO. 12.

The genome was inserted by the method in step 1. Specifically, we used the modified yeast L2, using pCRCT-308 site to cut plasmid, and transformed 1000ng constructed DDGS-OMT DNA sequence and equivalent 308 gene upstream and downstream 1000bp homologous arm sequence simultaneously by lithium acetate transformation. The strain L3 producing 4DG using xylose was obtained by screening the strain according to the method described in step 1.

By comparing the growth curves of the strains under different carbon sources, we found that the ability of the strains to utilize xylose as a carbon source was further reduced in pure xylose after the introduction of the DDGS-OMT gene, because the metabolic flow rate of xylose was accelerated by the production of 4DG, further affecting growth.

FIG. 6.1 is a schematic diagram of the knockin process of DDGS-OMT. The plasmid pCRCT-308 is used to cut the 308 gene site of the L2 strain, and then the gene is transformed into yeast together with the upstream and downstream homology arms, DDGS and OMT genes, and the complete DNA (A) can be combined in vivo by recombinant enzymes endogenous to the yeast. We amplified the left and right homology arms, DDGS, and OMT genes by PCR (FIG. 6.2), and after transformation, we picked yeast clones and performed colony PCR (FIG. 6.3), and confirmed the completion of insertion of DDGS-OMT gene by sequencing results (FIG. 6.4), and obtained strain L3.

4. Construction of EEVS-MTOx:

this example introduced the EEVS and MTOx genes from brachycelanio rerio. Specifically, the sequences of EEVS, MTOx were synthesized and simultaneously codon-optimized for Saccharomyces cerevisiae. Then, we used the pTDH3 promoter of Saccharomyces cerevisiae to start the expression of EEVS gene and pPGK1 promoter to start the expression of MTOx gene, and the complete DNA sequence constructed is shown in SEQ ID NO. 15.

The genome was inserted by the method in step 1. Specifically, the yeast strain L2 modified in step 2 is used for cutting plasmids by using pCRCT-308 site, and 1000ng of constructed DDGS-OMT DNA sequence and equivalent homologous arm sequences of 1000bp respectively at the upstream and downstream of 308 site are simultaneously transformed by a lithium acetate transformation method. The strains were screened according to the procedure in step 1, to obtain a strain producing gadusol using xylose.

This production strain was fermented using 1% xylose and glucose as carbon sources. After the cells and the organic phase were disrupted and extracted with chloroform, they were centrifuged with a centrifuge to remove the cells and the organic phase, and only the aqueous phase was taken. Then, 150 μ L of the product is taken to carry out absorption peak scanning in a microplate reader, and the absorption peak under 270-400 nm is tested.

FIG. 7 is a growth curve of engineered strains under different carbon sources. The top left panel of FIG. 7 is the growth curve of 4 strains in 2% glucose. The upper right graph of FIG. 7 shows the growth curves of 4 strains in 1% glucose and 1% xylose. The lower left panel of FIG. 7 shows the growth curves of 4 strains in 0.4% glucose and 1.6% xylose. The bottom right panel of FIG. 7 is the growth curve for 4 strains in 2% xylose.

FIG. 14.1 is a schematic diagram of the engineering process of the gadusol producing strain. We used plasmid pCRCT-308 to cut 308 gene site on the basis of strain L2, then transformed into yeast together with upstream and downstream homology arms, EEVS, MTOX genes, and the complete DNA can be combined in vivo by recombinant enzymes endogenous to the yeast. We amplified the left homology arm, right homology arm, EEVS, MTOX genes by PCR (FIG. 14.2), we picked yeast clones after transformation for colony PCR (FIG. 14.3), confirmed by sequencing results that the insertion of EEVS-MTOX gene was completed (FIG. 14.4), and obtained strain L4.

And (3) producing gadusol. Fermentation experiments were performed on strain L4 and L2 strain was used as a negative control. After fermentation for 72h, supernatant is taken to detect an absorption curve, and L4 can be seen to have an obvious ultraviolet band absorption peak at 290nm (figure 15.1), and the light absorption value of the corresponding control group is subtracted from the light absorption value of the L4 to obtain a more obvious gadusol absorption peak graph (figure 15.2).

5. Selection of amino acid ligase produced by Shinorine and Porphyra-334:

shinorine and porphyra-334 are produced by the same enzyme (AGL and ALAL for short), but the amino acids connected with the two molecules are different: shinorine requires L-Serine (L-Serine) for its production, while porphyra-334 requires L-Alanine (L-aminopropionic acid). Since different amino acid ligases exist in different organisms, we selected amino acid ligases of three organisms, nostoc punctiform, nostoc linckia and Actinosynnema mirum, and confirmed the optimal enzyme combination by permutation and combination. Specifically, AGL (glycogen debranching enzyme, ATP-gram ligand) of 3 organisms was expressed using a pTDH3 promoter, ALAL (D-Ala-D-Ala ligand) was expressed using pPGK1 gene, and these two types of DNA were combined with pYT vector by enzymatic ligation to obtain 9 combinations (Np 5598-Np5597, np5598-NlmysD, np5598-Am4256, nlmysC-Np5597, nlmysC-NlmysD, nlmysC-Am4256, am4257-Np5597, am4257-NlmysD, and Am4257-Am4256, respectively). These 9 combined plasmid DNAs were transformed into yeast L3, and the yeast was cultured in SC-Ura medium. Clones were obtained in all combinations except the combination Am4257-Am4256, which failed to grow. And (4) carrying out fermentation culture, extraction and analysis on the 8 groups according to the method in the step (4) under the condition of 1% of xylose and glucose. The results showed that each yeast had a distinct absorption peak at 334 nm. In the combination, the combination of Np5598-NlmysD achieves the optimal absorption effect, which is obviously improved by 32.8 percent compared with the combination of Np5598-Np5599 reported in the literature, and meanwhile, the combination of Np5598-Am4256 is also improved by 11.4 percent compared with the combination of the literature. The DNA sequence of the most preferred combination of AGL-ALAL is shown in SEQ ID NO 19.

MAA standards are not commercially available, so we used laver extract (mainly comprising shinorine and porphyra-334) as a standard, and compared it with the combination product Np5598-Np 5597. High Performance Liquid Chromatography (HPLC) tests and Mass Spectrometry (MS) tests show that the combination of Np5598-NlmysC produces almost only porphyra-334, and that the combination of Np5598-Np5597 produces almost only shinorine, which shows that mysC enzyme has high preference for Alanine, and Np5597 has high preference for serine. Am4256 produces two substances simultaneously.

FIG. 8 is a schematic representation of the combinatorial expression of amino acid ligases. The AG-L genes from three organisms are respectively expressed by using a yeast strong promoter pTDH3, the ALA-L genes from the three organisms are expressed by using a yeast secondary strong promoter pPGK1, and then the genes are randomly combined and inserted into a 2micro vector to be transformed into the transformed saccharomyces cerevisiae.

FIGS. 9.1-9.4 are graphs of the results of fermentation and production of Shinorine or Porphyra-334. FIG. 9.1 shows that the fermentation broth obtained from 9 groups of yeast after culturing for 72h is scanned by UV absorption, and shows a distinct absorption peak at 334 nm. FIG. 9.2 compares the OD334 values of the above broths and found that the combination of the Np5598 series had the most significant absorbance. FIG. 9.3 is a graph showing the results of the examination of yeast lysates after disruption of 6 groups of yeast cells with the highest yield, showing that the cell lysates have a more pronounced absorption peak, and the OD334 of the lysates are shown in FIG. 9.4.

FIGS. 10.1-10.4 are High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) detection results of yeast fermentation products. Fig. 10.1 is a HPLC result chart, fig. 10.2 is a mass spectrum of the fermentation liquid in a chart of fig. 10.1, fig. 10.3 is a mass spectrum of the fermentation liquid in B chart of fig. 10.2, and fig. 10.4 is a mass spectrum of the fermentation liquid in C chart of fig. 10.2. In FIG. 10.1, the peaks of the graph A at porphyra-334 are 4.917 and 4.975, but there is actually only one peak, but the software of the instrument determines two values.

Shinorine and Porphyra-334 lack the standards on the market, so we chose to purify MAA from laver (Porphyra spp.). The HPLC result (FIG. 10.1, panel A) and the MS result (FIG. 10.2) show that the peaks of shinorine and porphyra-334 are clearly present and the extract has good purity. The fermentation broth of Np5598-NlmysD was examined, and HPLC results (panel B of FIG. 10.1) and MS results (FIG. 10.3) showed the predominant presence of porphyra-334, whereas in the fermentation broth of Np5598-Np5597, HPLC results (panel C of FIG. 10.1) and MS results (FIG. 10.4) showed the predominant presence of shinorine.

Production of palythine:

palythine is a molecule obtained by decarboxylation on the basis of shinorine or porphyra-334 molecules. Therefore, we used the Np5598-Np5597 combined plasmid of step 5, and then added mysH from Nostoc linckia (using pTEF2 promoter to drive gene expression) by means of enzymatic ligation, the sequence of which is shown in SEQ ID NO: 21. Plasmid DNA was transformed into yeast L3 and L6, and the yeast was cultured with SC-Ura medium. The samples were subjected to fermentation culture, extraction, and analysis as in step 4 under conditions of 1% xylose and glucose. The result shows that the L6 strain has a remarkable absorption peak at 320nm compared with the L3 and the control strain, and the absorption value of the L6 strain reaches 2.62 times of that of the control group, which indicates that the palythine is successfully produced.

7. And (3) optimizing yield:

to increase the production of MAA, we increased the production of the precursor 4DG by increasing the copy number of the DDGS and OMT genes. We used the pTDH3 promoter to drive expression of OMT and the pTDH3 promoter to drive expression of DDGS, after which the two DNA fragments were combined to form an insert (see SEQ ID NO: 22). Then, we transformed the insertion sequence, pCRCT-Nqm plasmid and Nqm gene upstream and downstream 1000bp homology arm sequence into L3 strain. After the culture, screening and negative screening of the processes, 2 DDGS-OMT copy yeast strains L6 are obtained.

The plasmid for producing shinorine and palythine is transformed into a strain L6, and after fermentation, a more obvious absorption peak is generated, and compared with the numerical value of OD334 of a supernatant of fermentation liquid, the ultraviolet absorption peak of Np5598-NlmysD is increased by 91% compared with that of L3, and the ultraviolet absorption peak of Np5598-Np5597 is increased by 70% compared with that of L3. The two combinations were inserted into the genome at position 106 of L6, which gave 3.72 times the plasmid.

FIG. 11.1 is a schematic representation of reinsertion of the OMT-DDGS gene to increase gene copy number, while knocking out the Nqm gene. The plasmid pCRCT-Nqm1 is used for cutting Nqm gene locus, then the gene is transformed into yeast together with upstream and downstream homologous arms, OMT and DDGS genes, and complete DNA can be combined in vivo through recombinase endogenous to the yeast. We amplified the left and right homology arms, OMT and DDGS genes by PCR (FIG. 11.2), and after transformation we picked yeast clones for colony PCR (FIG. 11.3), and confirmed the completion of the insertion of OMT-DDGS gene by sequencing (FIG. 11.4), to obtain L6 strain.

FIG. 12.1 is a schematic diagram of the production process of optimized shinorine and porphyra-334. We transformed the plasmid for porphyra-334 production (Np 5598-NlmysD) and the plasmid for shinorine production (Np 5598-Np 5597) into L3 and L6 strains, and compared the yields after fermentation. The ultraviolet band absorption peak at 334nm in the supernatant of the fermentation broth after 72h culture is significantly improved (fig. 12.2), and compared with the combination of L3, the yield of Porphyra-334 in L6 yeast is improved by 91.8% and the yield of shinorine is improved by 70.9% according to the judgment of the OD334 value (fig. 12.3).

FIG. 13.1 is a schematic diagram of the production process of palythine. We transformed the plasmid Np5598-Np5597-NlmysH producing palythine into the L3 and L6 strains and compared the yields after fermentation. In the supernatant of the fermentation broth after 72h culture, an ultraviolet band absorption peak of 320nm (fig. 13.2) can be seen, and compared with the combination of the control plasmid and the L3, the absorbance in the L6 yeast is significantly improved by judging with an OD320 value, and the improvement effect is about 2.62 times that of the control group (fig. 13.3).

Production comparison of MAA. From the literature, we found four molecules, i.e., gadosol, palythine, shinorine and porphyra-334, which can absorb the ultraviolet band from 275nm to 360nm, so as to absorb UVB and most of high-energy UVA (FIG. 16.1), while from the ultraviolet absorption curve of the detection culture solution after fermentation of the yeast strain L6 of the embodiment, a remarkable absorption peak from 280nm to 360nm can be seen, and the design goal is perfectly met (FIG. 16.2).

Results of the insertion into the genome. FIG. 17.1 shows that the combination of the inserted genome produces a very high UV absorbance peak at OD334 nm. FIG. 17.2 shows that the OD334nm absorbance data of the inserted genome was increased to 3.72 times that of the control group compared to the uninserted plasmid DNA.

The sequences are illustrated below:

1. DNA sequence for cleavage:

TAL1：CTAGAACAATTGAAAGCCTCGTTTT(SEQ ID NO:1)

Nqm1：ACGATCAAGATAGCTTCTACGTGTTTT(SEQ ID NO:2)

308：ACCACTTGTCAAACAGAATATAGTTTT(SEQ ID NO:3)

106：ACATACGGTCAGGGTAGCGCCCGTTTT(SEQ ID NO:4)

His3：ACTGCCTCGCAGACAATCAACGGTTTT(SEQ ID NO:5)

2. amino acid sequence of xylose metabolic system enzyme:

xyl1：

MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLDINLRFNDPWDWDKIPIFV(SEQ ID NO:6)

xyl2：

MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAHGRIGNFVLTKPMVLGHESAGTVVQVGKGVTSLKVGDNVAIEPGIPSRFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHVSLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGAKGVIVVDIFDNKLKMAKDIGAATHTFNSKTGGSEELIKAFGGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPITVFAMKELTLFGSFRYGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRAGKGAVKCLIDGPE(SEQ ID NO:7)

xyl3：

MTTTPFDAPDKLFLGFDLSTQQLKIIVTDENLAALKTYNVEFDSINSSVQKGVIAINDEISKGAIISPVYMWLDALDHVFEDMKKDGFPFNKVVGISGSCQQHGSVYWSRTAEKVLSELDAESSLSSQMRSAFTFKHAPNWQDHSTGKELEEFERVIGADALADISGSRAHYRFTGLQIRKLSTRFKPEKYNRTARISLVSSFVASVLLGRITSIEEADACGMNLYDIEKREFNEELLAIAAGVHPELDGVEQDGEIYRAGINELKRKLGPVKPITYESEGDIASYFVTRYGFNPDCKIYSFTGDNLATIISLPLAPNDALISLGTSTTVLIITKNYAPSSQYHLFKHPTMPDHYMGMICYCNGSLAREKVRDEVNEKFNVEDKKSWDKFNEILDKSTDFNNKLGIYFPLGEIVPNAAAQIKRSVLNSKNEIVDVELGDKNWQPEDDVSSIVESQTLSCRLRTGPMLSKSGDSSASSSASPQPEGDGTDLHKVYQDLVKKFGDLYTDGKKQTFESLTARPNRCYYVGGASNNGSIIRKMGSILAPVNGNYKVDIPNACALGGAYKASWSYECEAKKEWIGYDQYINRLFEVSDEMNLFEVKDKWLEYANGVGMLAKMESELKH(SEQ ID NO:8)

3. DNA sequence of xylose metabolism system:

the sequence marked by single underlining is pTDH3, the sequence marked by double underlining is xyl1, the sequence marked by bold underlining is tTGH3, the sequence marked by dotted underlining is pPGK1, the sequence marked by dashed underlining is xyl2, the sequence marked by single underlining is tPGK1, the sequence marked by dot-dash short-line is pTEF2, the sequence marked by double underlining is xyl3, and the sequence marked by dot-dash short-line is tSSA1.

Amino acid sequence of DDGS-OMT:

DDGS：

MSNVQASFEATEAEFRVEGYEKIEFSLVYVNGAFDISNREIADSYEKFGRCLTVIDANVNRLYGKQIKSYFRHYGIDLTVVPIVITEPTKTLATFEKIVDAFSDFGLIRKEPVLVVGGGLTTDVAGFACAAYRRKSNYIRVPTTLIGLIDAGVAIKVAVNHRKLKNRLGAYHAPLKVILDFSFLQTLPTAQVRNGMAELVKIAVVANSEVFELLYEYGEELLSTHFGYVNGTKELKAIAHKLNYEAIKTMLELETPNLHELDLDRVIAYGHTWSPTLELAPMIPLFHGHAVNIDMALSATIAARRGYITSGERDRILSLMSRIGLSIDHPLLDGDLLWYATQSISLTRDGKQRAAMPKPIGECFFVNDFTREELDAALAEHKRLCATYPRGGDGIDAYIETQEESKLLGV(SEQ ID NO:10)

OMT：

MTSILGRDTARPITPHSILVAQLQKTLRMAEESNIPSEILTSLRQGLQLAAGLDPYLDDCTTPESTALTALAQKTSIEDWSKRFSDGETVRQLEQEMLSGHLEGQTLKMFVHITKAKSILEVGMFTGYSALAMAEALPDDGRLIACEVDSYVAEFAQTCFQESPHGRKIVVEVAPALETLHKLVAKKESFDLIFIDADKKEYIEYFQIILDSHLLAPDGLICVDNTLLQGQVYLPSEQRTANGEAIAQFNRIVAADPRVEQVLLPIRDGITLIRRLV(SEQ ID NO:11)

DNA sequence of DDGS-OMT:

the sequence marked by single straight line is pTDH3, the sequence marked by double straight line is DDGS, the sequence marked by thick straight line is tTGH3, the sequence marked by dot underline is pPGK1, the sequence marked by single wavy line is OMT, and the sequence marked by dotted underline is tPGK1.

Amino acid sequence of EEVS-MTOx:

EEVS：

MERPGETFTVSSPEEVRLPSVHRDNSTMENHNKQETVFSLVQVKGTWKRKAGQNAKQGMKGRVSPAKIYESSSSSGTTWTVVTPITFTYTVTQTKNLLDPSNDTLLLGHIIDTQQLEAVRSNTKPLKRFIVMDEVVYNIYGSQVTEYLEARNVLYRILPLPTTEENKSMDMALKILEEVHQFGIDRRTEPIIAIGGGVCLDIVGLAASLYRRRTPYIRVPTTLLSYIDASVGAKTGVNFANCKNKLGTYIAPVAAFLDRSFIQSIPRRHIANGLAEMLKMALMKHRGLFELLEVHGQFLLDSKFQSASVLENDRIDPASVSTRVAIETMLEELAPNLWEDDLDRLVDFGHLISPQLEMKVLPALLHGEAVNIDMAYMVYVSCEIGLLTEEEKFRIICCMMGLELPVWHQDFTFALVQKSLCDRLQHSGGLVRMPLPTGLGRAEIFNDTDEGSLFRAYEKWCDELSTGSPQ(SEQ ID NO:13)

MTOx：

MQTAKVSDTPVEFIVEHLLKAKEIAENHASIPVELRDNLQKALDIASGLDEYLEQMSSKESEPLTELYRKSVSHDWNKVHADGKTLFRLPVTCITGQVEGQVLKMLVHMSKAKRVLEIGMFTGYGALSMAEALPENGQLIACELEPYLKDFAQPIFDKSPHGKKITVKTGPAMDTLKELAATGEQFDMVFIDADKQNYINYYKFLLDHNLLRIDGVICVDNTLFKGRVYLKDSVDEMGKALRDFNQFVTADPRVEQVIIPLRDGLTIIRRVPYTPQPNSQSGTVTYDEVFRGVQGKPVLDRLRLDGKVAYVTGAGQGIGRAFAHALGEAGAKVAIIDMDRGKAEDVAHELTLKGISSMAVVADISKPDDVQKMIDDIVTKWGTLHIACNNAGINKNSASEETSLEEWDQTFNVNLRGTFMCCQAAGRVMLKQGYGKIINTASMASLIVPHPQKQLSYNTSKAGVVKLTQTLGTEWIDRGVRVNCISPGIVDTPLIHSESLEPLVQRWLSDIPAGRLAQVTDLQAAVVYLASDASDYMTGHNLVIEGGQSLW (SEQ ID NO: 14) 7. DNA sequence of EEVS-MTOx:

the sequence marked by single-straight line is pTDH3, the sequence marked by double-straight line is EEVS, the sequence marked by thick straight line is tTGH3, the sequence marked by dot underline is pPGK1, the sequence marked by single-wavy line is MTOx, and the sequence marked by dotted underline is tPGK1.

8. Amino acid sequences of AGL and ala in optimal combination:

Np5598：

MAQSISLSLPQSTTPSKGVRLKIAALLKTIGTLILLLIALPLNALIVLISLMCRPFTKKPAVATHPQNILVSGGKMTKALQLARSFHAAGHRVILIEGHKYWLSGHRFSNSVSRFYTVPAPQDDPEGYTQALLEIVKREKIDVYVPVCSPVASYYDSLAKSALSEYCEVFHFDADITKMLDDKFAFTDRARSLGLSAPKSFKITDPEQVINFDFSKETRKYILKSISYDSVRRLNLTKLPCDTPEETAAFVKSLPISPEKPWIMQEFIPGKELCTHSTVRDGELRLHCCSNSSAFQINYENVENPQIQEWVQHFVKSLRLTGQISLDFIQAEDGTAYAIECNPRTHSAITMFYNHPGVAEAYLGKTPLAAPLEPLADSKPTYWIYHEIWRLTGIRSGQQLQTWFGRLVRGTDAIYRLDDPIPFLTLHHWQITLLLLQNLQRLKGWVKIDFNIGKLVELGGD(SEQ ID NO:16)

NlmysD：

MPVLRILHLVGSAQDDFYCDLSRLYAQDCLAAMAELPYDSAIAYITPDGQWRFPRSLSREDIAQAKPMPVSEAIEFIAAQNIDIVLPQMFCIPGMTYYRALFDLLEIPYIGNTPDLMAITAHKARTKAIVEAAGVKVPRGEVLRRGDVPTITPPVVIKPVSSDNSLGVTLVKDAAEYEAALEKAFEHGDEAIVETFIEGREVRCGIIVKDGELIGLPLEEYLIDSQEKPIRTYADKLKKTDDGSLGFAAKGNNKSWILDPNDPITQKVQEVAKKCHQALGCRHYSLFDFRIDSQGQPWFLEAGLYCSFAPKSVISSMAKAVGIPLNELLTIAIAETLGSNKYSDRISVVEINEPSKTPRKERELSQMI(SEQ ID NO:17)

Am4256：

MLRVLHLTGSPVSPFFAELSTVYGRGCLGAAADPARYEFLVAHVTPDGRWRFPADLTPEALAAAPRLGLPEALGLIESRSVDVAVPQLFCPPGMTTYRALLDALGVPYPGNPPDVMALGADKAMTRAVVAAAGVPVPEGRVVTSADPCPLPPPFVVKPVDADNSDGLTLVHDRADYHAALDAAFACSPRRRALVERYVPPGREVRCGVLVRSGVPTPLPLEEYPLPSGVRPRADKLADDGGGSLSLVAKADGRSWIVDHDDPVTAAVQEQALRCHEALGCRDYSLFDFRIDPEGRPWFLEAGLYCSFAPTSVITTMAGAAGIGLAELFAEAVTTAARRG(SEQ ID NO:18)

9. DNA sequence of AGL-ALAL in optimal combination:

the sequence marked by single line and double lines is pTDH3, the sequence marked by double lines and double lines is Np5598, the sequence marked by thick lines and double lines is tTGH3, the sequence marked by dot underline is pPGK1, the sequence marked by wavy lines is NlmysD, and the sequence marked by dotted underline is tPGK1.

Amino acid sequence of mysh:

NlmysH：

MLKVDTQKISPQQVEAFERDGVICVKNAVDDIWVERMRTAVDKNISIPGPLEDKNVPKPQGSAEHASSIWLIDADFRALAFESPLPTLAAQVLKSKKLNFLADGFFVKKPESNGRIGWHNDLPYWPVQGWQCCKIWLALDTVKQENGRLEYIKGSHQWGKELRERSNPSWFIEPEPHEILSWDMEAGDCLIHHFLTIHHSVTNISSTQRRAIVTNWTGDDVTYYQRPKAWPFKPLEEIDLPEFNSLKTKKSGEPIDCDIFPRVQVHR(SEQ ID NO:20)

DNA sequence of AGL-ALAL-mysH:

the sequence marked by single straight line is pTDH3, the sequence marked by double straight line is Np5598, the sequence marked by thick straight line is tTGH3, the sequence marked by dot underline is pPGK1, the sequence marked by single wavy line is Np5597, the sequence marked by virtual underline is tPGK1, the sequence marked by double wavy line is pTEF2, the sequence marked by dot-short underline is NlmysH, and the sequence marked by thick wavy line is tSSA1.

OMT-DDGS sequence:

the sequence marked by single line drawn downwards is pTDH3, the sequence marked by double lines drawn downwards is DDGS, the sequence marked by thick line drawn downwards is tTGH3, the sequence marked by dot underline is pPGK1, the sequence marked by wavy line drawn downwards is OMT, and the sequence marked by dotted underline is tPGK1.

Nlysc amino acid sequence:

MAQSISVSSSPAIPSFPSETKIAVIIQNLLTLALLLLALPINAAIVLVTLLWHTISRPFQQPATKAANPKNILISGGKMTKALQLARSCAAAGHRVILIETHKYWLSGHRFSQAVDKFYTVPAPQENPERYTQALIDIIKQENIDVYIPVTSPLGSYYDSLAKPLLSEYCEVFHFDIDITEKLDDKFAFAETARSLGLSVPKSFKITSAEQVLNFDFSQESRKYILKSIPYDSVRRLDLTKLPCATPEETAAFVRSLPISPDKPWIMQEFIPGKEFCTHSTVRDGELRLHCCCESSAFQVNYENVENSQIREWVRHFVKELKLTGQVSFDFIQAEDGRVYAIECNPRTHSAITTFYDHPQVAQAYLDNEPMAETLQPLPSSKPTYWTYHEVWRLTGIRSFTQLKKWIANIWRGTDAIYKPDDPLPFLMVHHWQIPLLLLKNLRQIKGWTRIDFNIGKLVELGGD(SEQ ID NO:23)

am4257 amino acid sequence:

MSDAVAPQRVPGRVPGRSGSGRVSRTLGALALLLAALPFSAALTAVAALRAAVRPSPARATPRRPRTVLLTGGKMTKALHLARAFHRAGHRVVLVETARYRLTAHRFSRAVDAFHVVPDSADPRYPQALLAIVEREGVDVFVPVCSPASSVHDAAAAPLLATRCEVLHAGLEVVELLDDKHRFAELSAELGLPVPRSHRITAPEQVLDLGLDGPHVLKSIPYDPVNRLDLTPLPRPTPEATLEFLRGKDVRDGHPWVLQEFVAGKEYCTHSTVRNGRVVVYGCCESSAFQVNYEMVDKPEIERWVRAFAEATGVTGQVSFDFIESADGRALAIECNPRTHSAITMFHDHPDLARAYLDPDAPQIRPLPSSRPTYWLFHELWRALSEPGTARERLRVVARGKEAVFDWSDPLPFLLLHHVHVPLLLLRALVRGQDWVRVDFNIGKLVAPSGD(SEQ ID NO:24)

np5597 amino acid sequence:

MPVLNILHLVGSAHDKFYCDLSRLYAQDCLAATADPSLYNFQIAYITPDRQWRFPDSLSREDIALTKPIPVFDAIQFLTGQNIDMMLPQMFCIPGMTQYRALFDLLKIPYIGNTPDIMAIAAHKARAKAIVEAAGVKVPRGELLRQGDIPTITPPAVVKPVSSDNSLGVVLVKDVTEYDAALKKAFEYASEVIVEAFIELGREVRCGIIVKDGELIGLPLEEYLVDPHDKPIRNYADKLQQTDDGDLHLTAKDNIKAWILDPNDPITQKVQQVAKRCHQALGCRHYSLFDFRIDPKGQPWFLEAGLYCSFAPKSVISSMAKAAGIPLNDLLITAINETLGSNKKVLQN(SEQ ID NO:25)

np5597 nucleic acid sequence:

Atgccagtacttaatatccttcatttagttgggtctgcacacgataagttttactgtgatttatcacgtctttatgcccaagactgtttagctgcaacagcagatccatcgctttataactttcaaattgcatatatcacacccgatcggcagtggcgatttcctgactctctcagtcgagaagatattgctcttaccaaaccgattcctgtgtttgatgccatacaatttctaacaggccaaaacattgacatgatgttaccacaaatgttttgtattcctggaatgactcagtaccgtgccctattcgatctgctcaagatcccttatataggaaataccccagatattatggcgatcgcggcccacaaagccagagccaaagcaattgtcgaagcagcaggggtaaaagtgcctcgtggagaattgcttcgccaaggagatattccaacaattacacctccagcagtcgtcaaacctgtaagttctgacaactctttaggagtagtcttagttaaagatgtgactgaatatgatgctgccttaaagaaagcatttgaatatgcttcggaggtcatcgtagaagcattcatcgaacttggtcgagaagtcagatgcggcatcattgtaaaagacggtgagctaataggtttaccccttgaagagtatctggtagacccacacgataaacctatccgtaactatgctgataaactccaacaaactgacgatggcgacttgcatttgactgctaaagataatatcaaggcttggattttagaccctaacgacccaatcacccaaaaggttcagcaagtggctaaaaggtgtcatcaggctttgggttgtcgccactacagtttatttgacttccgaatcgatccaaagggacaaccttggttcttagaagctggattatattgttcttttgcccccaaaagtgtgatttcttctatggcgaaagcagccggaatccctctaaatgatttattaataaccgctattaatgaaacattgggtagtaataaaaaggtgttacaaaattga(SEQ ID NO:26)

Am4256:

MLRVLHLTGSPVSPFFAELSTVYGRGCLGAAADPARYEFLVAHVTPDGRWRFPADLTPEALAAAPRLGLPEALGLIESRSVDVAVPQLFCPPGMTTYRALLDALGVPYPGNPPDVMALGADKAMTRAVVAAAGVPVPEGRVVTSADPCPLPPPFVVKPVDADNSDGLTLVHDRADYHAALDAAFACSPRRRALVERYVPPGREVRCGVLVRSGVPTPLPLEEYPLPSGVRPRADKLADDGGGSLSLVAKADGRSWIVDHDDPVTAAVQEQALRCHEALGCRDYSLFDFRIDPEGRPWFLEAGLYCSFAPTSVITTMAGAAGIGLAELFAEAVTTAARRG(SEQ ID NO:27)

in one embodiment, the present invention provides for the first time a method for the systematic production of various MAAs using yeast.

In one example, for shinorine, we found an optimal set of expressed gene combinations, which could improve the yield by 32.8% over the sequences reported in the literature. We claim to protect this group of amino acid sequences and DNA sequences preferentially.

In one embodiment, the present invention first produces palythine using Saccharomyces cerevisiae, and we claim to protect this set of amino acid and DNA sequences.

In one embodiment, the present invention uses reverse insertion with two copies of DDGS-OMT, increasing the yield of various MAAs. We claim to protect this set of amino acid and DNA sequences.

In one example, the strains of the invention have the ability to produce gadusol, shinorine, porphyra-334, palythine, which we claim to protect.

In one example, for enzyme selection, based on codon degeneracy, and the amino acid sequence modifications before and after the enzyme may not affect the activity of the enzyme. Thus, we claim sequences having at least 90% similarity to the amino acid sequence of each gene.

In one embodiment, the promoter is selected such that a constitutive promoter is used at the same or similar transcription level to achieve the corresponding effect, or an inducible promoter is used to achieve the corresponding transcription strength to achieve the corresponding effect. The present invention preferentially protects promoter selection and claims that any promoter sequence is used as long as the combination of enzymes disclosed in the present invention is used. The terminator is chosen substantially independently of the expression of the gene, so in principle any terminator sequence can be used, and it is within the scope of the invention to replace any terminator sequence in the sequence.

In one example, for the Chassis organism, the Saccharomyces cerevisiae CEN. PK2 was chosen for the present invention, but this set of gene combinations could work in any yeast strain. We therefore claim that the selection of similar Chassis organisms is protected BY the present invention, i.e. it is within the scope of the present invention to use the above MAA-producing enzymes and DNA sequences in saccharomyces cerevisiae (S288C, cen. Pk2, BY4741, BY4742, etc.) and other yeasts such as pichia, kluyveromyces, yarrowia lipolytica, etc.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. Numerous simple deductions, modifications or substitutions may also be made by those skilled in the art in light of the present teachings.

Claims

1.A method of constructing a recombinant organism, comprising:

2. The method of claim 1, wherein in the organism constructing step, the endogenous gene to be knocked out comprises at least one of TAL1 gene and Nqm gene.

3. The method of claim 1, wherein in the organism constructing step, the xylose metabolism gene comprises at least one of xyl1 gene, xyl2 gene, and xyl3 gene;

preferably, in the organism constructing step, expression of xyl1 gene is promoted using pTDH3 promoter;

preferably, in the organism construction step, expression of xyl2 gene is promoted using the pPGK1 promoter;

preferably, in the organism construction step, expression of xyl3 gene is promoted using the pTEF2 promoter;

preferably, in the organism constructing step, at least one of the xyl1 gene, xyl2 gene and xyl3 gene is inserted at the his3 site.

4. The method of claim 1, wherein in the step of constructing the DDGS-OMT, expression of the OMT gene is promoted using pTDH3 promoter;

preferably, in the step of constructing the DDGS-OMT, the expression of the DDGS gene is promoted by using a pPGK1 promoter;

preferably, in the step of constructing DDGS-OMT, the copy number of OMT gene in genome of the organism is more than or equal to 1;

preferably, in the step of constructing the DDGS-OMT, the copy number of the DDGS gene in the genome of the organism is more than or equal to 1;

preferably, in the step of constructing the DDGS-OMT, the copy number of the OMT gene in the genome of the organism is more than or equal to 2;

preferably, in the step of constructing the DDGS-OMT, the copy number of the DDGS gene in the genome of the organism is more than or equal to 2;

preferably, in the step of constructing the DDGS-OMT, the insertion site of the DDGS gene and/or the OMT gene is 308 th site.

5. The method of claim 1, further comprising a ligase gene construction step comprising integrating an expressible amino acid ligase gene into the genome of the organism resulting from the DDGS-OMT construction step to obtain a cyclosporin-like amino acid producing organism.

6. The method of claim 5, wherein in the ligase gene constructing step, the amino acid ligase comprises at least one of an AGL enzyme and an ALAL enzyme;

the AGL enzyme comprises at least one of Np5598 protein, nlmysC protein and Am4257 protein, and the ALAL enzyme comprises at least one of Am4256 protein, np5597 protein and NlmysD protein;

the Np5598 protein comprises a sequence which has at least 90 percent of similarity with an amino acid sequence shown as SEQ ID NO. 16;

the NlmysC protein comprises a sequence which has at least 90 percent of similarity with an amino acid sequence shown as SEQ ID NO. 23;

the Am4257 protein comprises a sequence which has at least 90 percent of similarity with an amino acid sequence shown as SEQ ID NO. 24;

the Am4256 protein comprises a sequence having at least 90% similarity to the amino acid sequence set forth in SEQ ID NO. 18;

the Np5597 protein comprises a sequence which has at least 90% similarity with an amino acid sequence shown as SEQ ID NO. 25;

the NlmysD protein comprises a sequence which has at least 90% similarity with the amino acid sequence shown in SEQ ID NO. 17.

7. The method of claim 6, wherein in the ligase gene construction step, the amino acid ligase comprises an Np5598 protein and an nlysd protein;

or, in the ligase gene constructing step, the amino acid ligase comprises Np5598 protein and NlmysD protein;

or in the step of constructing the ligase gene, the amino acid ligase comprises Np5598 protein and Am4256 protein;

or in the step of constructing the ligase gene, the amino acid ligase comprises Np5598 protein and NlmysC protein;

or in the step of constructing the ligase gene, the amino acid ligase comprises Np5598 protein and Np5597 protein.

8. The method according to claim 6, wherein in the ligase gene constructing step, the promoter of the AGL gene comprises pTDH3 promoter;

preferably, in the ligase gene constructing step, the promoter of the ALAL gene comprises pPGK1 promoter;

preferably, in the ligase gene constructing step, the insertion site of the AGL gene and/or the ALAL gene is 106 sites;

preferably, the step of constructing the ligase gene further comprises recovering the product from the organism capable of producing the cyclosporin-like amino acid;

preferably, the product comprises a cyclosporin-like amino acid.

9. The method according to any one of claims 5 to 8, further comprising a mysH construction step comprising integrating a mysH gene into the genome of the organism obtained in the ligase gene construction step to obtain an organism that produces a cyclosporin-like amino acid.

10. The method of claim 9, wherein the expression of the mysH gene is driven using the pTEF2 promoter;

preferably, the mysH construction step further comprises recovering the product from said cyclosporin-like amino acid-producing organism;

preferably, in the mysH construction step, the product comprises a cyclosporin-like amino acid;

preferably, in the mysH construction step, the cyclosporine-like amino acid comprises palythine.

11. The method of any one of claims 1 to 4, further comprising a step of constructing EEVS-MTOx, comprising integrating at least one of EEVS gene and MTOx gene into the genome of the organism obtained from the step of constructing the organism, to obtain an organism capable of producing gadusol using xylose.

12. The method of claim 11, wherein in the step of constructing EEVS-MTOx, the expression of EEVS gene is initiated using pTDH3 promoter;

preferably, in the step of constructing EEVS-MTOx, pPGK1 promoter is used to promote the expression of MTOx gene;

preferably, in the step of constructing the EEVS-MTOx, the insertion site of the EEVS gene and/or the MTOx gene is 308 site;

preferably, the step of constructing EEVS-MTOx further comprises recovering gadusol from the organism that produces gadusol.

13. The method of any one of claims 1 to 12, wherein in the organism constructing step, the organism comprises a fungus;

preferably, the fungus comprises a yeast;

preferably, the yeast comprises saccharomyces cerevisiae.

14. A compound produced by a recombinant organism obtained by the method of any one of claims 1 to 13.

15. The compound of claim 14, wherein the compound comprises a cyclosporine amino acid or analog thereof;

preferably, the cyclosporine-like amino acid comprises at least one of shinorine, porphyra-334 and palythine;

preferably, the cyclosporine-like amino acid analog comprises gadusol.

16. A recombinant organism having at least a portion of its endogenous genes knocked out and having integrated into its genome 1) a xylose metabolism gene and 2) at least one of a DDGS gene and an OMT gene.

17. The recombinant organism of claim 16, wherein the knocked-out endogenous gene comprises at least one of a TAL1 gene, a Nqm gene;

preferably, the xylose metabolism gene comprises at least one of xyl1 gene, xyl2 gene, and xyl3 gene;

preferably, the promoter of xyl1 gene includes the pTDH3 promoter;

preferably, the promoter of xyl2 gene comprises the pPGK1 promoter;

preferably, the promoter of xyl3 gene comprises the pTEF2 promoter;

preferably, the insertion site of xyl1 gene, xyl2 gene or xyl3 gene is the his3 site;

preferably, the copy number of the OMT gene in the genome of the organism is more than or equal to 1;

preferably, the copy number of the DDGS gene in the genome of the organism is more than or equal to 1;

preferably, the copy number of the OMT gene in the genome of the organism is more than or equal to 2;

preferably, the copy number of the DDGS gene in the genome of the organism is more than or equal to 2;

preferably, the insertion site of the DDGS gene and/or OMT gene comprises site 308.

18. The recombinant organism according to any one of claims 16 to 17, wherein the genome of the organism further comprises a gene capable of expressing an amino acid ligase;

preferably, the amino acid ligase comprises at least one of an AGL enzyme, an ala enzyme;

preferably, the AGL enzyme comprises at least one of an Np5598 protein, an nlysc protein, an Am4257 protein, the ala enzyme comprises at least one of an Am4256 protein, an Np5597 protein, an nlysd protein;

the NlmysD protein comprises a sequence having at least 90% similarity to the amino acid sequence shown in SEQ ID NO. 17;

preferably, the amino acid ligase comprises Np5598 protein and nlysd protein;

preferably, the amino acid ligase comprises an Np5598 protein and an Am4256 protein;

preferably, the amino acid ligase comprises Np5598 protein and nlysc protein;

preferably, the amino acid ligase comprises an Np5598 protein and an Np5597 protein;

preferably, the promoter of the AGL gene comprises pTDH3 promoter;

preferably, the promoter of the ala gene comprises the pgk1 promoter;

preferably, the insertion site of the AGL gene and/or the ala gene comprises 106 sites.

19. The recombinant organism of claim 18, wherein the organism further has integrated into its genome a mysH gene;

preferably, the promoter of the mysH gene comprises the pTEF2 promoter.

20. The recombinant organism of claim 16, wherein the organism further has integrated into its genome at least one of an EEVS gene, a MTOx gene;

preferably, the promoter of the EEVS gene comprises pTDH3 promoter;

preferably, the promoter of the MTOx gene includes pPGK1 promoter;

preferably, the insertion site of the EEVS gene and/or MTOx gene is at position 308.

21. The recombinant organism according to any one of claims 16-20, wherein the organism comprises a fungus;

preferably, the fungus comprises a yeast;

preferably, the yeast comprises saccharomyces cerevisiae.