CN112980709B - Engineering strain and construction method and application thereof - Google Patents

Abstract

The application discloses an engineering strain, which is LDP1 gene and CALS gene function-inactivated engineering strain delta CALS delta LDP 1. The application provides a method for realizing lipid droplet size regulation through knocking out a lipid droplet protein gene. According to the method, the lipid drop size can be regulated and controlled and the lipid anabolism path can be weakened through gene engineering, so that the metabolic flow can flow to other metabolic paths more for engineering transformation of the natural oil-producing yeast strain, a foundation is laid for synthesis of terpenoids, and the metabolic engineering research of synthesis of terpenoids of rhodosporidium toruloides or other oil-producing yeasts in future can be powerfully promoted.

Description

Engineering strain and construction method and application thereof

Technical Field

The application relates to an engineering strain and a construction method and application thereof, belonging to the technical field of biology.

Background

A group of yeasts exists in nature, which can accumulate more than 20% of oil and fat in cells per se, and are collectively called oleaginous yeasts (Ratledge C, et al. adv Appl Microbiol 2002, 51: 1-51.). Rhodosporidium toruloides is an oleaginous yeast with wide application prospect, which can produce two products with industrial value, namely carotenoid and grease, and the yield can exceed 0.1 percent and 60 percent of the self cell dry weight respectively.

In rhodosporidium toruloides, the synthesis of oil takes acetyl coenzyme A generated by the cracking of citric acid as a precursor, fatty acid is firstly synthesized and then converted into oil, and the oil is mainly stored in lipid drops. At present, it is generally accepted that lipid droplets originate from the endoplasmic reticulum and are generated by the "budding model" (Walther TC, et al. Annu Rev Biochem 2012,81: 687-714.). The endoplasmic reticulum is provided with a plurality of proteins related to oil synthesis, neutral lipids generated by the catalysis of the proteins cannot be mutually dissolved with phospholipids on the endoplasmic reticulum, so that the neutral lipids are continuously accumulated in a nonpolar area in a phospholipid layer, and after the neutral lipids are accumulated to a certain degree, the endoplasmic reticulum wraps the phospholipid layer, and finally a new organelle-lipid droplet with a phospholipid monomolecular layer is formed. Lipid droplets are shed from the endoplasmic reticulum while carrying associated proteins on the endoplasmic reticulum membrane, which we collectively refer to as lipocalins. These lipins are mainly involved in cellular metabolism, protein synthesis and processing, mitochondrial function, membrane vesicle transport, and other functions. Among them, there are two lipid droplet structural proteins (perilipin-like protein Ldp1 and oil body calpain Cals) which play important roles in lipid droplet formation and structure maintenance. Lipid droplets, which are the reservoir of energy, are ubiquitous in the cytoplasm and serve as neutral lipid stores and the center of lipid metabolism.

With the rise of carotenoid extraction from microorganisms, Rhodotorula has attracted attention as a natural carotenoid-producing strain. However, the carotenoid content in the wild-type strain is very low. At present, the improvement of the yield of beta-carotene through genetic engineering is mainly obtained by over-expressing key genes in a carotenoid synthesis way. The three beta-carotene synthetic genes from Blakeslea trispora are simultaneously expressed by connecting 2A sequences in Rhodosporidium toruloides, and the content of the beta-carotene in the obtained engineering strain can reach 1.7mg/g DCW, which is improved by 4.5 times compared with that of a wild strain (Jiano X, et al. Fems Yeast Res 2018,18(7), foy 086). However, there is a limit to the increase in carotenoid production by this operation. Since in the oleaginous yeast Rhodosporidium toruloides, the acetyl-CoA precursor flows mainly to the lipid synthesis pathway even if the carotenoid pathway gene is overexpressed. Then the lipid droplet structural protein Ldp1 and Cals are inactivated by gene editing, so that the purpose of weakening lipid metabolism can be achieved, and more metabolic flow flows to terpene synthesis.

Therefore, there is a need to provide a method for controlling lipid droplet size and weakening lipid metabolism.

Disclosure of Invention

According to the first aspect of the application, an engineering strain is provided, the functions of the genes LDP1 and CALS of the key protein of lipid droplets in the engineering strain are inactivated, and the important function of the lipid droplets in lipid synthesis and storage and the influence on the growth and metabolic pathways of natural oleaginous yeast are further revealed by observing the change of the size of the lipid droplets in the strain with the lipid droplet function defect.

The engineering strain is an LDP1 gene and an engineering strain delta CALS delta LDP1 with CALS gene function inactivated.

Optionally, the preservation number of the engineering strain Δ CALs Δ LDP1 is CGMCC: 18930.

optionally, the engineered strain Δ CALs Δ LDP1 is obtained by regulating the expression of LDP1 gene and CALs gene in the original strain.

Optionally, the starting strain is a natural oleaginous yeast.

Optionally, the starting strain comprises Rhodosporidium toruloides, Rhodosporidium toruloides.

Optionally, the starting strain is an engineered strain expressing Cas9 protein.

According to a second aspect of the present application, there is provided a method for constructing an engineered strain, the method comprising: and knocking out LDP1 gene and CAL s gene in the genome of the starting strain to obtain the engineering strain with LDP1 and CAL s function inactivation.

Optionally, the method comprises: (1) knocking out the genes of CAL in the genome of the original strain to obtain an engineering strain delta CAL; (2) knocking out the LDP1 gene in the delta CALS genome of the engineering strain to obtain the delta CALS delta LDP 1.

Optionally, the step (1) comprises: construction of a peptide having the sequence of SEQ ID NO: 1, a CALs-sgRNA vector having a nucleotide sequence set forth in 1; introducing a CAL-sgRNA vector into an engineering strain expressing Cas9 protein, and screening and culturing to obtain an engineering strain delta CAL;

optionally, the step (2) comprises: construction of a peptide having the sequence of SEQ ID NO: 2, LDP1-sgRNA vector having a nucleotide sequence shown in fig. 2; and introducing the LDP1-sgRNA vector into the engineering strain delta CALS, and screening and culturing to obtain the engineering strain delta CALS delta LDP 1.

Optionally, the starting strain is a natural oleaginous yeast;

optionally, the starting strain comprises Rhodosporidium toruloides, Rhodosporidium toruloides.

According to a third aspect of the present application, there is provided a vector kit, characterized in that the vector kit comprises a vector having the sequence of SEQ ID NO: 1 and a CALs-sgRNA vector having the nucleotide sequence shown in SEQ ID NO: 2, and at least one of LDP1-sgRNA vectors having the nucleotide sequence shown in fig. 2.

According to a fourth aspect of the present application, there is provided a use of the engineered strain provided according to the first aspect of the present application or the engineered strain constructed according to the construction method provided by the second aspect of the present application for regulating lipid droplet size.

Alternatively, the use comprises modulating the size of lipid droplets by modulating the expression level of LDP1 and CALs genes in the engineered strain.

The engineering strain of the invention lacks lipid drop synthesis key protein gene LDP1 or CALS, and the change of lipid drops in the lipid drop function defective strain is observed by a microscope, so that the important function of the lipid drops in lipid synthesis and storage and the influence on the growth and metabolic pathways of rhodosporidium toruloides are further disclosed, and the construction method and the application of the recombinant strain are provided.

In order to achieve the above purpose, the invention provides the following technical scheme:

the detailed sequence and functional information of the knocked-out gene LDP1 in the invention can be described in patent CN 103965307B.

The Rhodosporidium toruloides NP11-SaCas9 expressing Cas9 protein is used as a starting strain.

Selecting target site on LDP1 gene to design vector and construct pZPK-P vector_GPD-NAT-Tnos-U6b-LDP 1-sgRNA; selecting target site on CALS gene for vector design to construct pZPK-P vector_GPDHYG-Tnos-U6 b-CALS-sgRNA. As above, the resistance gene uses GPD promoter and Tnos terminator, and the target gene guide sequence is integrated into the same genetic element using RNA polymerase type III promoter U6b and sgRNA. The sgRNA expression cassettes can be introduced into a target strain by agrobacterium-mediated transformation or electric transformation.

The construction methods of promoters and vectors used by NP11 engineering strain and knockout box for expressing Sacas9 disclosed by the invention can be disclosed in Jiano X, Zhang Y, Liu XJ, Zhang Q, Zhang SF, Zhao ZB, Biotechnol J,2019,1900036 and patent 201910005293.5, and the linear DNA fragment electrotransformation method can be disclosed in Liu HD, et al FEMS Yeast Res,2017,17 and fox 017.

According to the invention, specific yeast endogenous genes and exogenous genes are utilized to construct gene elements and gene modules, and the gene modules are transferred to the genome of the oleaginous yeast expressing SacAS9, so that the reduction of grease synthesis of recombinant strains is realized, and meanwhile, metabolic flow is guided to the generation of terpenoid, and the yield of carotenoid is improved. Wherein, the resistance marker, the promoter and the terminator in the related yeast include but are not limited to NAT, HYG, GPD, Tnos and U6b, the gene elements can be obtained by taking the rhodotorula rubra genome or other vectors carrying the corresponding elements as templates and obtaining or carrying out artificial gene synthesis through PCR amplification; in the invention, the U6b promoter is obtained by PCR amplification in a Rhodosporidium toruloides NP11 genome; NAT and HYG resistance marker gene, GPD promoter and Tnos terminator from vector pZPK-P_GPD-HYG-Tnos or pZPK-P_GPDNAT-Tnos is obtained through PCR amplification, LDP1 gene target site guide sequence and sgRNA are obtained through artificial gene synthesis, and then specific primers are designed for RF cloning to obtain the used vector.

The foreign gene protein comprises SacAS9 derived from Staphylococcus aureus (Staphylococcus aureus) and artificially synthesized and obtained after codon optimization. Codon optimization and whole gene synthesis: there are 64 genetic codes, but organisms have a preference for the use of these codons. Therefore, when the foreign gene is introduced, in order to reduce the influence of the foreign gene on the expression of the translation protein in a heterologous system, the foreign gene needs to be redesigned according to the codon preference of rhodosporidium toruloides, namely, codon optimization. Both codon optimization and whole-gene synthesis were entrusted to Suzhou Hongsn Biotechnology GmbH, and ligated to pUC57 vector, which was stored as dry powder plasmid. Amplifying a SaCas9 fragment by corresponding primers, and then cutting the target fragment and a target vector pZPK-P by EcoRV/SpeI double enzyme_PGK-BLE-Tnos-P_GPD-mcs-Thsp, connected by DNA Ligation Kit to obtain pZPK-P_PGK-BLE-Tnos-P_GPD-SaCas 9-Thsp. The vector obtained by enzyme digestion and connection is directly transformed into the electrotransformation competence of the escherichia coli.

The plasmid constructed above is introduced into Rhodosporidium toruloides by Agrobacterium-mediated transformation, and the specific procedures can be referred to in Lin XP, et al FEMS Yeast Res,2014,14, 547-555.

Preferably, the sequence and functional information of the SaCas9 is detailed in the literature Jiao X, Zhang Y, Liu XJ, Zhang Q, Zhang SF, Zhao zb.

Another objective of the invention is to provide a method for regulating and controlling the lipid droplet size of an engineered strain of Rhodosporidium toruloides, wherein the Rhodosporidium toruloides haploid NP11 is Rhodosporidium toruloides (Rhodosporidium toruloides). In addition, the Rhodosporidium toruloides NP5-2 can be modified by knocking out the gene elements and modules defined in the invention according to the resolved lipocalin gene sequence of the strains.

In the application, the starting strain is an engineering strain expressing SaCas9, and a CRISPR/Cas9 system-guided non-homologous end-linked repair mechanism is adopted.

In the construction method of the engineering strain, a target site is selected from LDP1 gene for vector design, and a vector pZPK-P is constructed_GPD-NAT-Tnos-U6b-LDP 1-sgRNA; selecting target site on CALS gene for vector design to construct pZPK-P vector_GPD-HYG-Tnos-U6 b-CALS-sgRNA. The transformation methods are ATMT and electrotransformation methods. After transformation, screening of the Δ CALs engineered strains was performed on hygromycin-added resistant plates, and screening of Δ LDP1 and Δ CALs Δ LDP1 engineered strains was performed on nourseothricin-added resistant plates. The resistance plate culture medium is YPD, 1.5 percent of agar powder and 50 mu g/mL of cephalosporin are added, and the addition amount of hygromycin or nourseothricin is 10 mu g/mL. The YPD medium consists of 20g/L glucose, 20g/L peptone and 10g/L yeast extract powder, and the pH value is 6.0.

The method comprises the steps of inoculating an engineering strain delta CAL delta LDP1 to a culture medium for culture and activation, then inoculating the culture medium to a fermentation culture medium again for fermentation and culture, collecting somatic cells after fermentation to extract oil, and observing the change of the size of a lipid drop through a fluorescence microscope. Specifically, the Δ CALs Δ LDP1 engineered strain was inoculated into 5mL YPD medium for activation for 24h to prepare a seed solution, the seed solution was transferred to a fresh 50mL nitrogen-limited medium, initial OD was controlled at 2, and fermentation was carried out for 144h, wherein the nitrogen-limited medium was 70g/L glucose, 0.75g/L yeast extract powder, 0.1g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1.5g/L crystalline magnesium sulfate, and 1% (V/V) trace elements (trace element stock solution consisting of 4.0g/L crystalline calcium chloride, 0.55g/L crystalline ferrous sulfate, 0.52g/L citric acid monohydrate, 0.1g/L crystalline zinc sulfate, 0.076g/L crystalline manganese sulfate, and 100 μ L/L18M), and concentrated sulfuric acid pH was 6.0. The culture conditions were all 30 ℃ and 200 rpm. The oil extraction method after the fermentation is an acid-heating method. And (4) taking 1mL of fermentation liquor after fermentation is finished, and adding a nile red dye for fluorescence observation.

According to the construction method, a sgRNA expression vector of an LDP1 or CAL target gene is constructed, a sgRNA expression cassette is linearized through PCR amplification, and the sgRNA expression cassette is electrically converted into rhodosporidium toruloides engineering bacteria expressing Cas9 protein, so that the knockout of the lipin gene is realized, and engineering strains delta CAL and delta LDP1 are obtained; then performing PCR amplification on an LDP1-sgRNA expression cassette and performing electric transformation to an engineering strain delta CALS to obtain an engineering strain delta CALS delta LDP 1; fluorescence observation shows that the engineering strain for knocking out the lipin gene has the characteristic of obviously changing the size of the lipin, and the oil yield is obviously reduced after fermentation. The invention can weaken the oil metabolism path by adjusting the size of the lipid droplets, and lays a foundation for engineering transformation of natural oil-producing yeast strains and more metabolic flows to other metabolic paths, such as synthesis of terpenoids.

The terms of terminology related to the present invention are explained as follows:

LDP1 gene: the gene for coding the lipocalin of the oleaginous yeast has the functions of promoting the fusion and growth of the lipocalin, increasing the accumulation of the lipid in the lipocalin, stabilizing the form and the function of the lipocalin and participating in the regulation and control of the lipid storage and transportation and the metabolism.

The genes of CALS: is a caleosin family protein coding gene, wherein the caleosin is lipid droplet Ca2+ binding protein, is named as CALS, and has important function on the synthesis of lipid droplets of natural oleaginous yeast.

CRISPR/Cas9 technology: the immune system of Regularly Clustered Interspaced Short Palindromic Repeats (Clustered regulated Short Palindromic Repeats/CRISPR-Associated Systems CRISPR/Cas9) is a third class of reported technologies that are capable of specific gene editing. Compared with ZFN and TALEN, the CRISPR system develops fastest in all gene editing technologies by the obvious advantages of convenient operation, high editing efficiency, low cost and the like, and is also the most widely applied technology at present.

sgRNA: guide RNAs (grnas), also known as small guide RNAs (sgrnas). Is used in a post-transcriptional modification process called RNA editing (RNA editing) in the kinetoplast (kinetoplastid). Is also a small non-coding RNA. Can pair with pre-mRNA and insert some uracil (U) therein, resulting in mRNA having a role.

Knocking out: the gene knockout is to modify the target cell genome in a fixed point way by the prior art, thereby modifying a specific gene on a chromosome and losing the function of the specific gene, so that partial functions are shielded, and the influence on organisms can be further caused, thereby further deducing the biological function of the gene.

The beneficial effect that this application can produce includes:

1) the application provides an engineering strain by regulating and controlling the function inactivation of LDP1 gene and CAL gene in an original strain.

2) The functions of gene LDP1 and CALS which are key proteins for synthesizing lipid droplets by the engineering strain are inactivated, and microscopic examination shows that the lipid droplets in the strain with the lipid droplet function defect become small, so that the important function of the lipid droplets in lipid synthesis and storage and the influence on the growth and metabolic pathways of natural oil-producing yeast are further disclosed.

3) The construction method of the engineering strain is provided by firstly constructing an LDP1-sgRNA vector and a CALS-sgRNA vector, and then introducing an LDP1-sgRNA expression cassette and a CALS-sgRNA expression cassette into an original strain.

4) The application also provides a vector kit for constructing the engineering strain.

5) The application provides a method for realizing the regulation and control of lipid droplet size through the knockout of lipid droplet gene. According to the method, the lipid drop size can be regulated and controlled and the lipid anabolism path can be weakened through gene engineering, so that the metabolic flow can flow to other metabolic paths more for engineering transformation of the natural oil-producing yeast strain, a foundation is laid for synthesis of terpenoids, and the metabolic engineering research of synthesis of terpenoids of rhodosporidium toruloides or other oil-producing yeasts in future can be powerfully promoted.

Drawings

FIG. 1 is a schematic diagram of the composition of LDP1-sgRNA vector and CAL-sgRNA vector constructed according to examples 1 and 2 of the present application.

FIG. 2 is a schematic diagram showing the design site and sequence deletion information after knockout of Rhodosporidium toruloides NP11 target gene according to example 3 of the present application.

FIG. 3 shows the comparison of oil content and biomass after fermentation of the starting strain NP11-SaCas9 and the engineered strains Δ CAL, Δ LDP1 and Δ CAL Δ LDP1 according to example 3 of the present application.

FIG. 4 is a lipid drop staining pattern of the starting strain NP11-SaCas9 and the engineered strains Δ CALS, Δ LDP1 and Δ CALS Δ LDP1 of the invention after fermentation according to the example 3 of the present application.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.

The invention discloses a construction method of an engineering strain for regulating and controlling the size of an oil-producing yeast lipid droplet and application thereof, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the regulation and control. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The present invention will be further described with reference to the accompanying drawings and examples, which will assist those skilled in the art in understanding the present invention, but are not intended to limit the invention in any way. All primer synthesis and sequencing work in the following examples was performed by Shanghai Biotech, Inc., unless otherwise specified. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

Toruloides (r. toruloides) CGMCC 2.1389: purchased from China general microbiological culture Collection center (CGMCC, with the collection number of CGMCC 2.1389).

PrimeSTAR DNA polymerase: purchased from Takara, Inc.

Agrobacterium tumefaciens AGL1 (available from American Standard Collection of Biotechnology (ATCC) under the accession number ATCC BAA-100).

Fluorescence observation was performed by EVOS fluorescence microscope observation.

Example 1: LDP1-sgRNA vector construction

Specifically, the method for obtaining the rhodosporidium toruloides NP11 comprises the following steps: toruloides NP11 haploid strain, which is obtained by taking R.toruloides Y4 (from CGMCC 2.1389 ═ CBS 6016 ═ IFO 0559x IFO 0880) domesticated by lignocellulose hydrolysate as a parent strain by Zhang Fangji chemist institute of chemical and physical institute of Chinese academy of sciences, and carrying out operations such as sporulation, germination of winter spores, separation of basidiospores and the like, wherein the specific operation method is shown in Nat. Commun.2012 and 3,1112, and the R.toruloides NP11 haploid is stored in Guangdong province microorganism strain collection center, the collection number is GDMCC 2.224 and can be purchased by the public.

Taking Rhodosporidium toruloides NP11 as an example, primers are designed for amplification and cloning to obtain an LPD1-sgRNA vector. All plasmids used were constructed using the RF cloning method. With plasmid pZPK-P_GPDUsing NAT-Tnos-U6b-sgRNA as a template, and amplifying by using primers Tnos-U6b-sgRNA-F/LPD 1-sgRNA-R (see table 1) to obtain a U6b-LPD1-sgRNA fragment. Then integrating the fragment into a target vector by an RF cloning method to obtain a plasmid pZPK-P_GPD-NAT-Tnos-U6b-LDP1- sgRNA。

pZPK-P_GPDThe preparation method of the NAT-Tnos-U6b-sgRNA comprises the following steps: amplifying a U6b promoter by using a Rhodosporidium toruloides NP11 genome as a template and Tnos-U6b-F (5'-GCAGCATGCAAGCTTGGAGCTTGAGCTTGGGGCGGGATGACCCAGC GCTTTCAC-3') and U6-sgRNA-R (5'-TAACTTGCTATTTCTAGCTCTAAAACGTATGGGGCTCAGTAGGCAGTG TGGCTCGACGAACAAGTT-3'); the sgRNA base sequence was synthesized by Suzhou Hongsn Biotechnology GmbH. Then, the amplified U6b sequence is connected with sgRNA by fusion PCR, and then is integrated into a vector pZPK-P according to an RF cloning method_GPDNAT-Tnos (FEMS Yeast Research 2014,14:547-555) to obtain the vector pZPK-P_GPD- NAT-Tnos-U6b-sgRNA。

Plasmid pZPK-P_GPD-HYG-Tnos-U6b-sgRNA as pZPK-P_GPD-HYG-Tnos is used as a template and is obtained by the same construction method and the primer.

pZPK-P_GPDNAT-Tnos andpZPK-P_GPDpreparation of HYG-Tnos reference is XinPing Lin, et al FEMS Yeast Research 2014,14: 547-555.

RF cloning: 10.0. mu.L of 5 XPCR buffer, 1.0. mu.L of dNTPs (10mM), 300ng of the above amplified fragment as a large primer (mega-primer), 20ng of plasmid pZPK-P_GPDPrimeSTAR DNA polymerase (Takara macrocephala) 0.5. mu.L, ddH using NAT-Tnos-U6b-sgRNA as template₂Adding O to 50 mu L, keeping the temperature at 94 ℃ for 3min, then keeping the temperature at 98 ℃ for 10s, at 62 ℃ for 10s, at 72 ℃ for 10min, performing 15 cycles, at 72 ℃ for 15min, and finishing the reaction at 4 ℃.

DpnI digestion and shock transformation: adding 1 μ L DpnI (from TaKaRa) and 1 μ L DpnI buffer into 8 μ L RF reaction product, mixing, and treating at 37 deg.C for 120min to remove pZPK-P_GPDAfter NAT-Tnos-U6b-sgRNA plasmid, 10 μ L of electric shock was taken and transformed into 100 μ L of DH10B competent cells prepared according to standard methods (third edition of molecular cloning instructions, translation of sambrook, huang petang et al, published by scientific press), parameters of electric shock transformation: 2200 ℃ and 2500V, 400 omega, 25 muF, 0 ℃ and 4-8 ms. Immediately after the electric shock, 900. mu.L of LB medium was added and rejuvenated at 37 ℃ and 200rpm for 45 min. And then coating the bacterial liquid on a kanamycin-resistant plate, selecting transformants for enrichment culture and plasmid extraction after 10 hours, carrying out colony PCR identification by using primers HSPT-R (5'-AGAGGAAAAGCGGACGACTG-3') and PZPK-F (5'-GCTGGTGGCAGGATATATTG-3'), and sending the identified positive recombinant vector to Takara for sequencing to obtain the correct vector. Meanwhile, the recombinant vector is named as LDP 1-sgRNA.

LB culture medium: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract powder and the balance of water, wherein the pH value is 7.0.

Kanamycin-resistant plates: the LB medium was supplemented with 50. mu.g/mL kanamycin.

The constructed LDP1-sgRNA vector has the sequence shown in SEQ ID NO: 2.

The LDP1-sgRNA vector composition is shown in fig. 1.

TABLE 1 sequence Listing of primers used to construct vectors

Example 2: construction of CALS-sgRNA vector

Taking Rhodosporidium toruloides NP11 as an example, primers are designed for amplification and cloning to obtain a CALS-sgRNA vector. With plasmid pZPK-P_GPDusing-HYG-Tnos-U6 b-sgRNA as a template, and amplifying by using a primer Tnos-U6b-sgRNA-F/CALS-sgRNA-R (shown in table 1) to obtain a U6b-CALS-sgRNA fragment. Then integrating the fragment into a target vector by an RF cloning method to obtain a plasmid pZPK-P_GPD-HYG-Tnos-U6b-CALS-sgRNA as shown in FIG. 1.

Plasmid pZPK-P_GPD-HYG-Tnos-U6b-sgRNA as pZPK-P_GPDHYG-Tnos (XinPing Lin, et al. FEMS Yeast Research 2014,14:547-555) was used as a template and obtained by using the same construction method and primers as described in example 1.

Vector pZPK-P_GPDThe construction of HYG-Tnos was as described in example 1.

The constructed CALS-sgRNA vector has the sequence shown in SEQ ID NO: 1.

The CALs-sgRNA vector composition is shown in fig. 1.

Example 3: construction of engineering strain with functional defect of rhodosporidium toruloides NP11 lipid droplet

YPD medium (seed medium): 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder, and the balance water, wherein the pH value is 6.0.

TMLSD medium: 10mM Tris-HCl, 1mM MgCl₂100mM LiOAc, 270mM sucrose, 5mM DTT (filter sterilized dispensed, -20 ℃ storage), 0.22 μ M filter sterilized, pH 7.5.

TMS culture medium: 10mM Tris-HCl, 1mM MgCl₂270mM sucrose, 0.22 μ M filter sterilized, pH 7.5.

TES buffer solution: 300mM NaCl, 50mM Tris-HCl, 25mM EDTA, 0.2% (V/V) SDS, 2mg/ml proteinase K (added at the time), pH 8.0.

TE buffer solution: 10mM Tris-HCl, 1mM EDTA, pH 8.0.

Hygromycin-resistant plates: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder, 1.5% agar powder, 20 μ g/mL hygromycin, 300 μ g/mL cephalosporin, and the balance water, wherein the pH value is 6.0.

Nourseothricin resistant plates: 20g/L glucose, 20g/L peptone, 10g/L yeast extract powder, 1.5% agar powder, 10 ug/mL nourseothricin, 300ug/mL cephalosporin, and the balance water, and the pH is 6.0.

The amino acid sequence of LDP1 is shown as SEQ ID NO: 3, the gDNA sequence of the LDP1 gene is shown as SEQ ID NO: 4, the amino acid sequence of CALs is shown as SEQ ID NO: 5, the gene gDNA sequence of the CALs is shown as SEQ ID NO: and 6.

Taking rhodosporidium toruloides NP11 as an example, taking NP11-SaCas9 engineering strains as initiating strains, and constructing LDP1 and CAL (RHTO _05627 and RHTO _03414) gene knockout strains delta LDP1 and delta CAL; and further taking the engineering strain delta CALS as a starting strain to construct a double-gene knockout strain delta CALS delta LDP 1. The specific implementation process is as follows:

construction of NP11-SaCas9 engineering Strain:

the SaCas9 fragment is amplified through primers EcoRV-Cas9-F (5 ' -CCGGATATCATGCACCACCATCACCAT CACGATAAGAAG) and Cas9-SpeI-R (5'-CGGACTAGTCTAGTGATGGT GATGGTGGTGGACCTTCCGCTTCTTCTTCGGATC-3'), and then the target fragment and a target vector pZPK-P are subjected to double enzyme digestion through EcoRV/SpeI_PGK-BLE- Tnos -P_GPD-mcs-Thsp, connected by DNA Ligation Kit to obtain pZPK-P_PGK-BLE- Tnos-P_GPD-SaCas 9-Thsp. The vector obtained by enzyme digestion and connection is directly transformed into the electrotransformation competence of the escherichia coli.

The plasmid constructed above is introduced into Rhodosporidium toruloides through Agrobacterium-mediated transformation to obtain NP11-SaCas9 engineering strain. The specific step reference method can be found in Lin XP, et al FEMS Yeast Res,2014,14, 547-555.

Firstly, introducing a constructed CALS-sgRNA vector into NP11-SaCas9 through an electric conversion method, and coating thalli on a hygromycin resistant plate; 5 generations of transformants obtained by screening are subjected to stability verification; then extracting genome DNA of the stably-passaged engineering strain; using gene upstream and downstream primers: CALS-F (5'-ATGTCACCCTCTTACGCACAAGC-3') and CALS-R (5'-CTTCACGGAAGGTCAAGCCGCCT-3') amplify genome DNA, and sequencing the obtained PCR product to obtain a gene deletion type delta CALS engineering strain, wherein the gene deletion type delta CALS engineering strain is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) No.1 Hospital 3 of North West Lu No. 3 of the sunward quarter of Beijing, the preservation time is 11 months and 8 days in 2019, the preservation number is CGMCC No.18928, and the classification and the designation of the preserved strain are as follows: rhodosporidium toruloides, Rhodosporidium toruloides.

The LDP1-sgRNA vector is led into NP11-SaCas9 by the same method to obtain a gene deletion type delta LDP1 engineering strain, the gene deletion type delta LDP1 engineering strain is preserved in China general microbiological culture Collection center (CGMCC) of No.1 Hospital 3 of Xilu north Chen located in the sunward area of Beijing, the preservation time is 11 months and 8 days in 2019, the preservation number is CGMCC No.18929, and the classification and the name of the preserved strain are as follows: rhodosporidium toruloides, Rhodosporidium toruloides; the screening plate is a nourseothricin resistant plate, and the amplification primers are LDP1-F (5'-CAAACAACGAGCACAGCGACAC-3') and LDP1-R (5'-AAACCGAGAAGAAACCCGAAC-3').

The LDP1-sgRNA vector is introduced into an engineering strain delta CAL by the same method to obtain a double-gene deletion type delta CAL delta LDP1 engineering strain, the gene deletion type delta CAL delta LDP1 engineering strain is preserved in the China general microbiological culture Collection center (CGMCC) of China microbiological culture Collection management Committee No. 3 of Beijing area of facing Yang, the preservation time is 2019, 11 and 8 days, the preservation number is CGMCC No.18930, and the classification and the naming of the preserved strain are as follows: rhodosporidium toruloides, Rhodosporidium toruloides; the screening plate is a nourseothricin resistant plate, and the amplification primers are LDP1-F (5'-CAAACAACGAGCACAGCGACAC-3') and LDP1-R (5'-AAACCGAGAAGAAACCCGAAC-3').

The target gene sequence and the sequencing comparison result are shown in FIG. 2, and the target gene with base deletion represents the realization of the gene and the functional knockout thereof.

Electrotransformation method reference (Liu HD, et al. fems Yeast Res,2017,17, fox 017): exogenous DNA is electrically shocked and transformed into rhodosporidium toruloides, and firstly, the electrically transformed competent cells of the rhodosporidium toruloides are prepared. The Rhodosporidium toruloides strain is selected from the activated plate and inoculated into 10 mL YPD medium, cultured at 30 ℃ and 200rpm for 24h to prepare seed liquid. Inoculating the seed solution into 200mL YPD medium at a ratio of 1:100, culturing at 30 deg.C and 200rpm for 7-8h to OD₆₀₀About 1.0. Centrifuge at 4000g for 5min at 4 ℃ and discard the supernatant. 200mL of freshly prepared TMLSD was resuspended in cells, centrifuged at 4000g for 5min at 4 ℃ and the supernatant discarded. 100mL of freshly prepared TMLSD was resuspended in cells, incubated at 25 ℃ for 60min at 50rpm, centrifuged at 4 ℃ at 1300g for 5min, and the supernatant discarded. The pellet was washed with 100mL of precooled TMS, centrifuged at 1300g for 5min at 4 ℃ and the supernatant discarded. The washing was repeated once. Finally the mycelia were resuspended in 0.5mL of precooled TMS. Each 100. mu.L of the electroporation competent cells was dispensed into 2mL centrifuge tubes. The linearized vector with the concentration of more than 800 ng/. mu.L is added into the electrotransformation competence of 100. mu.L and ice-cooled for 5-10 min. The mixture was then transferred to a 0.2cm pre-chilled electric rotor for shock conversion at 700V. Immediately after electric shock, 1mL of TMS was added, and the mixture was subjected to static culture at 30 ℃ for 2 hours for rejuvenation. 600g, centrifuging for 2min, removing the supernatant, adding 1mL YPD medium, and culturing at 30 ℃ and 200rpm for 2h for rejuvenation again. 600g, centrifuging for 2min, taking a proper amount of thalli, coating the thalli on a corresponding antibiotic plate, and culturing at 30 ℃ until transformants grow out.

The carrier linearization method comprises the following steps: designing universal primers PZPK-F (about 100bp upstream of a promoter PGD, 5'-CCGGACTGATGGGCTGCCTG-3') and PZPK-R (about 100bp downstream of sgRNA, 5'-ATCCCGAGGGGAACCCTGTG-3'), carrying out PCR amplification on the constructed vector and recovering an amplification product.

Genomic DNA extraction method (well documented molecular biology protocols): the strain with the verified stability was cultured in YPD medium at 30 ℃ for 24 hours. 2-5mL of the bacterial solution is taken, centrifuged and washed once with ultrapure water. Adding 400 μ L TES buffer solution and appropriate amount of glass beads, and using

-24 cell disruptor shaking for 1min, ice-cooling for 1min, repeating for 2-3 times. Then, 400. mu.L of a Tris-saturated phenol mixture (Tris-saturated phenol: chloroform: isoamyl alcohol: 25:24:1) was added thereto, and the mixture was mixed by repeated inversion and then allowed to stand at room temperature for 5 minutes. Centrifuging at 15000g for 10min at 4 deg.C, transferring 200 μ L to new oneA1.5 mL centrifuge tube was charged with 1/10 volumes of sodium acetate (3M pH 5.2), 2 volumes of ice cold absolute ethanol and precipitated at-20 ℃ for 30-60 min. Centrifuge at 15000g for 10min at 4 ℃ and discard the supernatant. The precipitate was washed with ice-cold 75% ethanol and then centrifuged to air-dry. Adding a proper amount of TE buffer solution for dissolving, and measuring the concentration of the NanoDrop for later use.

Example 4: shake flask fermentation of engineering strain with lipid drop function defect of rhodosporidium toruloides NP11

Strain: NP11 (original strain, same as example 3), NP11-SaCas9 (original strain, same as example 3), engineered strains delta CALS (preservation number: CGMCC 18928) prepared in example 3, delta LDP1 (preservation number: CGMCC 18929), and delta CALS delta LDP1 (preservation number: CGMCC 18930).

The test method comprises the following steps:

seed culture medium: as described in example 3.

Nitrogen-limited culture medium: glucose 70g/L, yeast extract powder 0.75g/L, ammonium sulfate 0.1g/L, potassium dihydrogen phosphate 1g/L, crystalline magnesium sulfate 1.5g/L, and water in balance, wherein the pH value is 6.0.

Inoculating the strain into 5mL of seed culture medium, activating for 24h to prepare seed liquid, transferring the seed liquid into fresh 50mL of nitrogen-limited culture medium, and setting three biological parallels; culturing at 30 deg.C and 200rpm for 144h with initial OD of 2, collecting bacterial cells after fermentation culture, extracting oil, and observing fluorescence of lipid drop staining.

The oil extraction method comprises the following steps: taking 30mL of fermentation liquor, and calculating biomass after centrifugal drying. Then extracting oil by an acid heating method. 5mL of 4M hydrochloric acid was added to the dried cells, and the mixture was treated in a 78 ℃ water bath for 1.5 hours. After cooling, adding chloroform/methanol solvent with the same volume, carrying out vortex oscillation for 5min, centrifuging for 5min at 8000g, and transferring the lower organic phase to a new centrifuge tube. The upper layer was extracted with an equal volume of chloroform and repeated twice. Adding equal volume of 0.1% sodium chloride solution into the collected organic phase, vortex and shake for 1min, centrifuging for 5min at 8000g, transferring the lower organic phase into a glass funnel containing anhydrous sodium sulfate, drying, and continuously adding chloroform until the red color on the anhydrous sodium sulfate disappears. And removing the solvent from the oil bottle for collecting the oil through reduced pressure distillation, weighing and calculating the oil content.

The fluorescence observation method comprises the following steps: 1mL of fermentation broth is taken, 8000g of the fermentation broth is centrifuged for 5min, and the supernatant is discarded. The cells were resuspended in 1mL of ultrapure water, 8000g centrifuged for 5min, and the supernatant discarded. Add 50. mu.L of 20 XNile Red dye and stain for 5-10 min. An appropriate amount of the cells were placed on a slide glass and observed by an EVOS fluorescence microscope (excitation wavelength of 475nm, emission wavelength of 580 nm).

And (3) test results: as can be seen from FIG. 3, after the fermentation is finished, the biomass or oil content of NP11-SaCas9 is almost the same as that of wild strain NP11, which indicates that the expression of SaCas9 protein in NP11 does not affect the growth and oil metabolism of the strain; compared with NP11 or NP11-SaCas9, the engineering strains delta CALS, delta LDP1 and delta CALS delta LDP1 are obviously reduced in biomass and grease content; wherein the average oil content of the engineering strains delta LDP1 and delta CALS delta LDP1 is respectively 0.42g/g and 0.33g/g, which are respectively reduced by 29 percent and 44 percent compared with the 0.59g/g of the original strain NP11-SaCas9, and the oil anabolism is obviously weakened; the corresponding biomass decreases by 25% and 36%, respectively. FIG. 4 shows the lipid drop staining patterns of the starting strain NP11-SaCas9 and the engineering strains of the invention delta CALS, delta LDP1 and delta CALS delta LDP1 after the fermentation is finished; wherein, the left side is shot by white light, the middle is shot by Nile red dyeing, and the rear side is a composite image of the two; the green fluorescence area represents the lipid droplet range. Fluorescence staining imaging results show that the lipid droplet volumes of the engineering strains delta CAL, delta LDP1 and delta CAL delta LDP1 are reduced compared with that of the wild strain NP11 or the starting strain NP11-SaCas9, and the lipid droplet reduction of the engineering strains delta LDP1 and delta CAL delta LDP1 is obvious (figure 4), and is consistent with the change trend of the oil content, so that the construction method of the engineering strain for regulating the lipid droplet size of the oil-producing yeast is feasible.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Sequence listing

<110> institute of chemistry and physics, large connection of Chinese academy of sciences

<120> engineering strain and construction method and application thereof

<130> DD190634I

<141> 2019-12-13

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8746

<212> DNA

<213> synthetic

<400> 1

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60

ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120

cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180

tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240

cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300

tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360

aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420

gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480

ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540

accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600

gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660

gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720

gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780

gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840

gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900

acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960

ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020

gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080

gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140

cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200

tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260

cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320

cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380

gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440

ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500

gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560

cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620

cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680

gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740

tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800

atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860

agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920

acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980

atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040

ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100

cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160

aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220

aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280

gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340

aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400

gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460

gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520

gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580

tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640

ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700

accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760

acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820

cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880

gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940

gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000

tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060

ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120

gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180

gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240

aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300

catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360

aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420

ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480

tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540

ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600

aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660

gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720

cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780

tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840

gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900

ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960

actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020

aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agccatattc 4920

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4980

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 5040

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 5100

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5160

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5220

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5280

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5340

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5400

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5460

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5520

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5580

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5640

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5700

tgatgctcga tgagtttttc taatcacagg cagcaacgct ctgtcatcgt tacaatcaac 5760

atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 5820

gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 5880

gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 5940

gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca 6000

acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat tgaattcgag 6060

ctcggtacct ggagttcgac gttctcctcg ctccgcaagc attggaatga accttgctct 6120

ctagttccct cctccgtgac ctcgtttcgt cctttagacg gcacgatgga aggaagaaat 6180

ctctgcggac aagcaaatct gctggctcgc cttgtaggtc gcctaccgga gcaagccttg 6240

tgccgccggg atgccaacgt cgttttttga cgtttgcaag acgtagagga cgcttcggac 6300

gacgaaacaa gctgtgagga catggaagtc gtgggaggaa cggcgcagag cggcgccgcg 6360

ggagcataag gcaagcgaga tagtccagaa atcgcggcgc caagtacagt aatttattgg 6420

agcaggcacc agaagcgggc agcagtatgc gcaggcttgg ggtcgacgag agacgactcc 6480

ctcatactcg gttacctcga gcaatacaat caatcgaagc tgcgcgaatc tcggcttgta 6540

agggtcggaa aggaacctcg gagatggcca cgtcacatca ccaacttatc gatctcagcc 6600

gacgtcgcag agagggcgag cgaagcggtg aaggagggaa acaatccctc gagagcatga 6660

tccgtctgaa tctgcagcgc aggaagccgt cacacgcccg cctcgagcgc aggtcgggtc 6720

cagccggggg acgaaacgcg cgagggctga tttcgtgagc gaaggaagcc gcatcgacaa 6780

gttcgcgtcc ctttgccctc tttcccatca cccgctctcg ctctacccgc tcagaacaac 6840

accagatcag tcacaatgcc ggagctcacg gcgacgtcgg tcgagaagtt cctcatcgaa 6900

aagttcgact cggtctcgga cctcatgcaa ctctcggagg gagaggaatc gcgcgcgttc 6960

tcgttcgacg tcggaggccg cggatacgtc ctccgcgtca actcgtgtgc ggacggattc 7020

tacaaggatc ggtacgtcta ccgccatttt gcgtcggcgg cgctcccgat ccccgaagtc 7080

ctcgacatcg gagagttctc ggaatccctc acgtactgta tctcgcgccg ggcgcaagga 7140

gtcacgctcc aagatctccc ggagacggaa ctcccggcgg tcctccaacc ggtcgcggaa 7200

gcgatggacg cgatcgcggc cgcggacctc tcgcaaacgt cgggattcgg accgtttgga 7260

ccgcaaggaa tcggacaata cacgacgtgg cgcgacttca tctgtgcgat cgcggatccc 7320

catgtctacc actggcaaac ggtcatggat gacacggtct cggcgtcggt cgcgcaagcg 7380

ctcgacgagc tcatgctctg ggcggaggac tgtccggagg tccgccacct cgtccacgcg 7440

gactttggat cgaacaacgt cctcacggac aacggacgca tcacggcggt catcgactgg 7500

tcggaggcga tgtttggaga ctcgcaatac gaggtcgcga acatcttctt ctggcgcccg 7560

tggctcgcgt gcatggagca acagacgcgc tacttcgagc gccgccaccc ggagctcgcg 7620

ggatcgccgc gcctccgcgc gtacatgctc cgcatcggcc tcgaccaact ctaccagtcg 7680

ctcgtcgacg gaaacttcga cgatgccgcg tgggcccaag gacgctgcga cgcgattgtc 7740

cgctcgggag cgggaaccgt gggacgcacg caaattgcgc ggcgctcggc ggccgtctgg 7800

acggatggat gtgtcgaagt cctcgcggat tcgggaaacc ggcgcccgtc gacgcgcccg 7860

cgggcgaaag aacaccacca tcaccatcac taggatcgtt caaacatttg gcaataaagt 7920

ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat 7980

tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt 8040

atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca 8100

aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgggc ctggatcctc 8160

tagagtcgac ctgcagcatg caagcttgga gcttgagctt ggggcgggat gacccagcgc 8220

tttcaccctc gcctgaccgg gcgctctcac cgtcgtcggc tgcgctcgct cgagacgacg 8280

ccgccgactc gccgcgctac cactcgtcgt cgtcaacccc ccaaaaagcc aggcctttat 8340

aagcagattc gctgcgtcga cctcagcacc cctacggggg ttgcatacaa taaacttgtt 8400

cgtcgagcca catgcacgga tatcttccag cgacttcggt cttggaggag cacgatgtcc 8460

cgggcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 8520

aaagtggcac cgagtcggtg gtgctttttt tgttttttat gtctatcaga ttgtcgtttc 8580

ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 8640

aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt tatccgttcg 8700

tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaa 8746

<210> 2

<211> 8746

<212> DNA

<213> synthetic

<400> 2

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60

ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120

cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180

tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240

cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300

tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360

aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420

gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480

ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540

accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600

gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660

gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720

gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780

gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840

gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900

acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960

ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020

gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080

gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140

cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200

tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260

cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320

cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380

gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440

ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500

gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560

cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620

cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680

gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740

tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800

atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860

agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920

acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980

atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040

ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100

cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160

aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220

aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280

gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340

aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400

gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460

gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520

gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580

tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640

ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700

accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760

acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820

cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880

gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940

gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000

tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060

ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120

gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180

gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240

aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300

catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360

aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420

ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480

tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540

ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600

aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660

gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720

cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780

tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840

gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900

ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960

actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020

aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agccatattc 4920

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4980

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 5040

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 5100

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5160

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5220

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5280

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5340

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5400

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5460

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5520

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5580

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5640

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5700

tgatgctcga tgagtttttc taatcacagg cagcaacgct ctgtcatcgt tacaatcaac 5760

atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 5820

gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 5880

gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 5940

gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca 6000

acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat tgaattcgag 6060

ctcggtacct ggagttcgac gttctcctcg ctccgcaagc attggaatga accttgctct 6120

ctagttccct cctccgtgac ctcgtttcgt cctttagacg gcacgatgga aggaagaaat 6180

ctctgcggac aagcaaatct gctggctcgc cttgtaggtc gcctaccgga gcaagccttg 6240

tgccgccggg atgccaacgt cgttttttga cgtttgcaag acgtagagga cgcttcggac 6300

gacgaaacaa gctgtgagga catggaagtc gtgggaggaa cggcgcagag cggcgccgcg 6360

ggagcataag gcaagcgaga tagtccagaa atcgcggcgc caagtacagt aatttattgg 6420

agcaggcacc agaagcgggc agcagtatgc gcaggcttgg ggtcgacgag agacgactcc 6480

ctcatactcg gttacctcga gcaatacaat caatcgaagc tgcgcgaatc tcggcttgta 6540

agggtcggaa aggaacctcg gagatggcca cgtcacatca ccaacttatc gatctcagcc 6600

gacgtcgcag agagggcgag cgaagcggtg aaggagggaa acaatccctc gagagcatga 6660

tccgtctgaa tctgcagcgc aggaagccgt cacacgcccg cctcgagcgc aggtcgggtc 6720

cagccggggg acgaaacgcg cgagggctga tttcgtgagc gaaggaagcc gcatcgacaa 6780

gttcgcgtcc ctttgccctc tttcccatca cccgctctcg ctctacccgc tcagaacaac 6840

accagatcag tcacaatgcc ggagctcacg gcgacgtcgg tcgagaagtt cctcatcgaa 6900

aagttcgact cggtctcgga cctcatgcaa ctctcggagg gagaggaatc gcgcgcgttc 6960

tcgttcgacg tcggaggccg cggatacgtc ctccgcgtca actcgtgtgc ggacggattc 7020

tacaaggatc ggtacgtcta ccgccatttt gcgtcggcgg cgctcccgat ccccgaagtc 7080

ctcgacatcg gagagttctc ggaatccctc acgtactgta tctcgcgccg ggcgcaagga 7140

gtcacgctcc aagatctccc ggagacggaa ctcccggcgg tcctccaacc ggtcgcggaa 7200

gcgatggacg cgatcgcggc cgcggacctc tcgcaaacgt cgggattcgg accgtttgga 7260

ccgcaaggaa tcggacaata cacgacgtgg cgcgacttca tctgtgcgat cgcggatccc 7320

catgtctacc actggcaaac ggtcatggat gacacggtct cggcgtcggt cgcgcaagcg 7380

ctcgacgagc tcatgctctg ggcggaggac tgtccggagg tccgccacct cgtccacgcg 7440

gactttggat cgaacaacgt cctcacggac aacggacgca tcacggcggt catcgactgg 7500

tcggaggcga tgtttggaga ctcgcaatac gaggtcgcga acatcttctt ctggcgcccg 7560

tggctcgcgt gcatggagca acagacgcgc tacttcgagc gccgccaccc ggagctcgcg 7620

ggatcgccgc gcctccgcgc gtacatgctc cgcatcggcc tcgaccaact ctaccagtcg 7680

ctcgtcgacg gaaacttcga cgatgccgcg tgggcccaag gacgctgcga cgcgattgtc 7740

cgctcgggag cgggaaccgt gggacgcacg caaattgcgc ggcgctcggc ggccgtctgg 7800

acggatggat gtgtcgaagt cctcgcggat tcgggaaacc ggcgcccgtc gacgcgcccg 7860

cgggcgaaag aacaccacca tcaccatcac taggatcgtt caaacatttg gcaataaagt 7920

ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat 7980

tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt 8040

atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca 8100

aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgggc ctggatcctc 8160

tagagtcgac ctgcagcatg caagcttgga gcttgagctt ggggcgggat gacccagcgc 8220

tttcaccctc gcctgaccgg gcgctctcac cgtcgtcggc tgcgctcgct cgagacgacg 8280

ccgccgactc gccgcgctac cactcgtcgt cgtcaacccc ccaaaaagcc aggcctttat 8340

aagcagattc gctgcgtcga cctcagcacc cctacggggg ttgcatacaa taaacttgtt 8400

cgtcgagcca catgcacgga tatcttccag cgacttcggt cttgtcgaga gggtgtcttt 8460

gatcagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 8520

aaagtggcac cgagtcggtg gtgctttttt tgttttttat gtctatcaga ttgtcgtttc 8580

ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 8640

aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt tatccgttcg 8700

tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaa 8746

<210> 3

<211> 265

<212> PRT

<213> Rhodosporidium toruloides

<400> 3

Met Ala Thr Val Asn Glu Lys Gln Pro Ala Thr Asp Ala Pro Leu Ala

1 5 10 15

His Glu Thr Ala Ile His Arg Ala Leu Asp Tyr Pro Val Ile Lys Asp

20 25 30

Thr Leu Ser Thr Phe Asp His Tyr Ala His Ser His Pro Tyr Ile Ser

35 40 45

Ser Leu Tyr Ser Arg Thr Leu Ser Leu Ser Arg Gln Ile Leu Ala His

50 55 60

Val Gln Pro Val Leu Pro Leu Glu Leu Ala Asp Gln Tyr Ala Asn Lys

65 70 75 80

Thr Leu Asp Val Val Glu Lys Tyr Val Pro Gln Val Lys Met Glu Thr

85 90 95

Gly Glu Leu Ile Gly Lys Ala Arg Gly Pro Ala Asp Ala Ala Phe Gln

100 105 110

Thr Ala Gln Glu Tyr Arg Gln Gly Ile Gln Ser Arg Ile Ser Pro Val

115 120 125

Thr Asp Gln Leu Tyr Gln Arg Ile Thr Thr Ser Gln Ala His Leu Ser

130 135 140

Ser Leu Gln Asp Arg Leu Gln Lys Thr Ile Lys Gln Leu Pro His Asp

145 150 155 160

Thr Glu Ser Leu Gln Ser Thr Leu His Ser Ile Leu Asn Glu Val Asp

165 170 175

Gly Leu Val Lys Ser Ala Gln Ser Ile Pro Ala Asn Ala Gln Ala Thr

180 185 190

Ala Lys Pro Val Phe Asp Gly Val Val Glu Ala Ala Asp His Ile Arg

195 200 205

Lys Glu Val Thr Arg Thr Asp Ile Pro Met Gly Ala Arg Ala Gln Asn

210 215 220

Val Leu Thr Tyr Thr Gln Asp Arg Leu Gln Pro Val Val Glu Gln Ile

225 230 235 240

Lys Ser Phe Val Leu Lys Lys Lys Asp Glu Val Ala Glu Thr Val Glu

245 250 255

Glu Lys Glu Gly Glu Gly Glu Lys Gln

260 265

<210> 4

<211> 1240

<212> DNA

<213> Rhodosporidium toruloides

<400> 4

atggccaccg tcaacgagaa gcagcccgcc accgacgcgc ccctcgcgca cgagaccgcc 60

atccaccgcg tgcgtcccca tctcttcctc gtgaaaaccc gctcaacaac tcacacacgt 120

gcaacacagg cgctcgacta ccccgtgatc aaagacaccc tctcgacctt tgaccactac 180

gcccactcgc acccctacat ctcgtccctc tactcgcgca ccctcagcct ctcgcgccag 240

atcctcgctc acgtccagcc cgtcctcccc ctcgagttgg cggaccagta cgcgaacaag 300

acgctcgatg tcgtcgagaa gtatgtcccg caggtcaaga tggagacggg agagttgatt 360

ggaaaggcga gggggcccgc cgatgccgcg ttccagacgg cccaggagta caggcaggga 420

atccagtctg tgagtcgcgc gccggcgttt tctctccttc tttgtcgctc gttctcgccg 480

agcgcgaggg cggtcgactg cttcagggag agcgctgtcg ctgacatttc tcccttctcc 540

cttgtgcgcg tcccgcagcg catctcgccc gtgacggacc agctctacca gcgcatcacg 600

acctcgcaag cgcacctctc gtccctccaa gaccgtctcc aaaagacgat caagcagcgt 660

gcgtccctct ccctcccttt cccttcctcc cttctcccgc tgacaggctt gagtggatgc 720

gatgcagtcc cccacgacac ggagagcctc cagtctacgc tccactcgat cctcaacgag 780

gtcgacgggt tggtcaagtc tgctcagtcg atcgtgcgtc cttccctccc ctccctccct 840

tcttccgacc gacgagctga cgataggatt tgtggggatg tgataaagcc cgccaacgcc 900

caagcgaccg ccaagcccgt ctttgacgga gtcgtcgagg ctgcggacca cattcgcaag 960

gaggtcactc gcaccggtgc gtcctcccgc gctctctcct ctcttctcgt cgactttgac 1020

gagataccga gaaagttcag caagacgacg ctgactctct gccctcgctg cgctcgcgca 1080

aacagacatc ccgatgggcg cccgcgcgca aaacgtcctc acctacaccc aggaccgcct 1140

ccagcccgtc gtcgagcaga tcaagagttt cgtgctcaag aagaaggacg aggttgccga 1200

gactgtcgag gagaaggagg gcgaggggga gaagcagtga 1240

<210> 5

<211> 372

<212> PRT

<213> Rhodosporidium toruloides

<400> 5

Met Ser Pro Ser Tyr Ala Gln Ala Thr Ser Ala Trp Leu Pro Ser Pro

1 5 10 15

Ser Glu Met Pro Gln Pro Asp Lys Ala Leu Leu Ala Thr Thr Glu Asp

20 25 30

Val Thr Ala Gly Lys His Ser Pro Ser Thr Ile Ala His Gly Asn Glu

35 40 45

Asn Ala Lys Val Gln Val Val Gly Glu Leu Thr Pro Pro Leu Thr Pro

50 55 60

Pro Gly Glu Lys Glu Asp Ser Ile Ala Ala Gly Glu Gly Val Arg Arg

65 70 75 80

Arg Lys Val Gly Lys Ser Ser Glu Asp Glu Glu His Asp Val Pro Gly

85 90 95

Lys Gly Phe Val Gln Ser Ile Glu Gly Thr Val Ser Ala Glu Arg Tyr

100 105 110

Val Pro Glu Asp Leu Asp Lys Arg Ile Tyr Lys Pro Trp Val Pro Arg

115 120 125

Ala Asn Ile Ala Ala Thr Pro Glu His Pro Tyr Gly Thr Thr Ala Gly

130 135 140

Gly Tyr Ala Glu Lys His Lys Asp Glu Pro Val Leu Ala Gln His Val

145 150 155 160

Ala Phe Phe Asp Lys Asp Arg Asp Asn Ile Leu Trp Pro Leu Asp Thr

165 170 175

Trp Arg Gly Phe Arg Glu Met Gly Tyr Ser Phe Phe Trp Cys Thr Phe

180 185 190

Ala Met Cys Val Ile His Phe Phe Phe Ser Trp Phe Thr Thr Pro Asn

195 200 205

Arg Ile Leu Pro Asp Pro Phe Phe Arg Val Tyr Ile Ser Asn Gly His

210 215 220

Arg Ser Lys His Gly Ser Asp Thr Ala Val Phe Asp Ser Glu Gly Arg

225 230 235 240

Phe Ile Pro Ala Lys Phe Glu Glu Ile Phe Thr Lys Phe Asp Lys Gly

245 250 255

Asn Lys Gly Gly Leu Thr Phe Arg Glu Gly Val Gln Leu Ile His Ala

260 265 270

Gln Arg Gln Ala Val Asp Pro Ile Gly Val Ala Ala Glu Cys Phe Glu

275 280 285

Trp Ala Ser Thr Tyr Leu Leu Ile Trp Pro Lys Asp Gly Ile Cys Asp

290 295 300

Lys Glu Ser Ile Arg Thr Val Tyr Asp Gly Ser Leu Phe Tyr Leu Val

305 310 315 320

Ala Asp Ala Glu Arg Gln Arg Ser Leu Ala Arg Leu Glu Ala Arg Lys

325 330 335

His Met Ser Trp Pro Ala Trp Val Trp Asp Ser Val Pro Gly Pro Trp

340 345 350

Arg Gly Trp Gly Lys Glu Asn Lys Ala Ser Gly Phe Glu Trp Thr Glu

355 360 365

Glu Lys Phe Leu

370

<210> 6

<211> 1800

<212> DNA

<213> Rhodosporidium toruloides

<400> 6

atgtcaccct cttacgcaca agctacttca gcgtggttac cctccccctc cgaaatgccg 60

caacccgaca aggctctcct cgcgacgaca gaggacgtga cggcggggaa acactctcca 120

tcgacgatcg cgcacggaaa cgagaacgca aaggtgcagg tcgttgggga gttgacgccc 180

cctcttacgc cgccgggtga gaaggaggat tcgattgcgg cgggagaggg cgtgaggagg 240

aggaaagtgg ggaagtcttc cgaggacgag gagcacgatg tcccgggcaa gggtttcgtg 300

cagagtattg aggggactgt gagtgcggaa cgctacgtcc cggaggacct cgacaagcgg 360

atctacaagc cttgtgcgtc ctccctctcc tctcttcctc gcgagttcct tggagagctg 420

atggtgggca cgcaggggtt ccgcgcgcga acatcgctgc gacgccggag catccgtacg 480

gcacgaccgc gggcgggtac gcggagaagc acaaggacga gcccgttctc gctcagcacg 540

tcgctttctt cgacaaggtg cgatcccttt cacccccctt aaaccctgcg agtgagactg 600

atggtggcga atgataggac cgcgacaaca tcctgtggcc gctcgatacc tggcgcgggt 660

tccgcgagat ggggtacagt ttcttctggt gcaccttcgc catgtggtgc gtccctgcgc 720

tcttgtttcc cttcccttct ctcccgtcgt ccgccgctcc gtctctcgcc catgccactc 780

ccacgtatcg ctccgctcgg tcgcagtcgc taacgctcct gcacgcagtg tgatccactt 840

cttcttctcc tggttcacca cccccaaccg catcctcccc gaccccttct tccgcgtcta 900

catctcgaac ggacaccgct cgaaacacgg ttccgacacg gccgtgtttg attcagaagg 960

caggttcatc ccggctaagt ttgaggagat ctttaccaag tgcgtggagc gtgcgatgtg 1020

aaagagggag gagggctgat gaggttttgt ttgtttggtg gcgggaacag gttcgataag 1080

ggcaacaaag gcggcttgac cttccgtgaa ggcgtccagg tgcgttcctt ctcccttccc 1140

tcctccgtat gaagcgactg actgacgccc gctcgcggca cacagctcat ccacgcccaa 1200

cgccaggccg tcgacccgat cggcgtagct gccgagtgct tcgagtgggc tagcacgtac 1260

ttgctgagta cgtctctccc tctctctctc caaccttcct tagggtgagt atgaggcgct 1320

gacgagattt gcggtggtcg tggcggtggt gcgcgcagtt tggccgaaag acggggtatg 1380

tttccgcctc tctcatccct ctcttcttcc tcttccctct ctccctagct ctcgaccgtc 1440

gcagagcttc gtactgactc tccgcactcg tctcgtctcg tctcgtctcg cttcgcccgg 1500

aactagattt gcgacaaaga gtccatccgt acagtctacg acgtgcgtct cccctctcct 1560

ccctcgtccc tcccacaagc cctgaccctc tgccttccct tgcttctccg gaattcgcag 1620

ggttcgctct tctacctcgt cgccgacgca gaacgccaac gctccctcgc gcgcctcgaa 1680

gctcgcaaac acatgtcctg gcccgcttgg gtttgggatt cggtgcccgg tccgtggagg 1740

gggtggggga aggagaacaa ggcgagtggg tttgagtgga cggaggagaa gttcctgtaa 1800

Claims (11)

1. An engineering strain is characterized in that the engineering strain is LDP1 gene and CAL gene, namely RHTO _05627 and RHTO _03414, and a double-gene function-inactivated engineering strain delta CAL delta LDP1, and the starting strain of the engineering strain is Rhodosporidium toruloidesRhodosporidium toruloides。

2. The engineered strain of claim 1, wherein the engineered strain Δ CALS Δ LDP1 is deposited at the China general microbiological culture Collection center (CGMCC) at 11/08 in 2019 with the deposit number of CGMCC: 18930.

3. the engineered strain of claim 1, wherein the engineered strain Δ CALS Δ LDP1 is a regulated starting strain Rhodosporidium toruloidesRhodosporidium toruloidesThe expression of LDP1 gene and CALS gene.

4. The engineering strain of claim 1, wherein the starting strain is Rhodosporidium toruloides expressing Cas9 proteinRhodosporidium toruloides。

5. The method for constructing the engineering strain according to any one of claims 1 to 4, wherein the method comprises the following steps:

starting strain rhodosporidium toruloides knockout yeastRhodosporidium toruloidesLDP1 gene and CAL s gene in genome, namely RHTO _05627 and RHTO _03414, obtain double-gene deletion type engineering strain delta CALS delta LDP 1.

6. The building method according to claim 5, characterized in that the building method comprises:

(1) knocking out the genes of CAL in the genome of the original strain to obtain an engineering strain delta CAL;

(2) knocking out the LDP1 gene in the delta CALS genome of the engineering strain to obtain a double-gene deletion type engineering strain delta CALS delta LDP 1.

7. The method of constructing according to claim 6,

the step (1) comprises the following steps:

construction of a peptide having the sequence of SEQ ID NO: 1, a CALs-sgRNA vector having a nucleotide sequence set forth in 1;

Cals-sgRNA vector is introduced into engineering strain rhodosporidium toruloides for expressing Cas9 proteinRhodosporidium toruloidesScreening and culturing to obtain the engineering strain delta CALS;

the step (2) comprises the following steps:

construction of a polypeptide having the sequence of SEQ ID NO: 2, and a nucleotide sequence shown in the specification, wherein the nucleotide sequence is LDP1-sgRNA vector;

and (3) introducing the LDP1-sgRNA vector into the engineering strain delta CALS, and screening and culturing to obtain the double-gene deletion type engineering strain delta CALS delta LDP 1.

8. The construction method according to claim 6,

the engineering strain delta CALS obtained in the step (1) is preserved in China general microbiological culture Collection center on 11 th and 08 th in 2019, and the preservation number is as follows: CGMCC 18928;

the double-gene deletion type engineering strain delta CAL delta LDP1 engineering strain obtained in the step (2) is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 08 months in 2019, and the preservation numbers are as follows: CGMCC 18930.

9. Use of a vector kit in a method of constructing an engineered strain with reduced lipid droplet size, wherein the vector kit comprises a nucleic acid molecule having the sequence of SEQ ID NO: 1 and a CALs-sgRNA vector having the nucleotide sequence shown in SEQ ID NO: 2, LDP1-sgRNA vector having a nucleotide sequence shown in fig. 2; the starting strain of the constructed engineering strain with the reduced lipid droplet size is rhodosporidium toruloidesRhodosporidiumtoruloides。

10. The use according to claim 9, wherein the engineered strain with reduced lipid droplet size is the engineered strain Δ CALS Δ LDP1 with inactivated function of two genes as claimed in claims 1-4 or the engineered strain Δ CALS Δ LDP1 with deletion of two genes constructed according to the construction method as claimed in any one of claims 5-8.

11. The use of claim 10, wherein said use comprises reducing lipid droplet size by knocking out LDP1 and CALs genes, RHTO _05627 and RHTO _03414, in the starting strain.

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