A recombinant strain of pichia pastoris for producing lycopene, a construction method and application thereof

Abstract

The application discloses a kind of production lycopene Pichia yeast recombinant strain and its construction method and application.The construction method of the application is: with Pichia yeast as operating platform, lycopene is target product, first realize Pichia yeast heterogenous synthesis lycopene and carry out synthesis pathway optimization reconfiguration, improve lycopene yield.In addition, using new, simple, efficient Pichia yeast CRISPR-Cpf1 system is carried out recombination strain lycopene synthesis branch modification, promotes recombination strain lycopene synthesis.On this basis, to recombination strain lipid synthesis optimization, improve lycopene storage, further improve lycopene yield, obtain lycopene high-yield strain.The application utilizes Pichia yeast combination control strategy and makes lycopene yield reach 7.5g / L, higher than model strain saccharomyces cerevisiae, for the highest level in present Pichia yeast.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis applications, specifically to a recombinant Pichia pastoris strain for producing lycopene, its construction method, and its applications. Background Technology

[0002] Lycopene is a natural carotenoid pigment that can be produced by plants and microorganisms. [1] It is a type of fat-soluble pigment. It contains 11 conjugated double bonds and 2 non-conjugated double bonds, which gives it color and bioprotective activity. [2] Its molecular formula is C 40 H 56 Due to its antioxidant, anticancer, and anti-inflammatory properties. [3-5] It has been widely used in pharmaceutical compounds, nutritional supplements, cosmetics, food additives and other fields.

[0003] Traditional lycopene production processes include natural extraction and chemical synthesis. [6,7] Although lycopene is the most abundant carotenoid in tomatoes, the low lycopene content in raw tomatoes (approximately 30-50 mg / kg) results in a low lycopene yield. [1] The presence of other carotenoids and the instability of lycopene also complicate the lycopene extraction process. [8] Furthermore, tomato growth requires substantial land, a specific growing season, and suitable weather, resulting in a relatively long production cycle. Therefore, extraction methods are insufficient to meet market demand. Microbial synthesis of lycopene is another method for producing lycopene. Compared to natural extraction and chemical synthesis, the growth and metabolism of microorganisms are less affected by seasonal and climatic conditions and do not involve environmental pollution. Therefore, microbial synthesis of lycopene possesses unique advantages that make it a promising alternative for lycopene production.

[0004] Currently, microbial cell factories successfully used for lycopene production mainly include Escherichia coli. [1,9] ), yeasts (Saccharomyces cerevisiae)

[10] , Yarrowia lipolytica

[11] and Pichia pastoris

[12] ), filamentous fungi (baker's yeast)

[13] Streptomyces

[14] In recent years, yeast has been considered a preferred host for isoprene production. In addition, with the rapid development of synthetic biology, the synthesis of lycopene using genetically engineered yeast has also attracted increasing attention.

[0005] Pichia pastoris exhibits a significant advantage in the expression of exogenous proteins, especially in methanol-induced high-level protein expression.

[15] Compared with traditional model yeast strains such as Saccharomyces cerevisiae and other commonly used industrial microorganisms, Pichia pastoris has the following advantages in addition to its good protein production characteristics: (1) It can carry out high-density fermentation production using methanol, glycerol and other carbon sources.

[16] Moreover, it has a mature methanol-regulated fermentation strategy; (2) Pichia pastoris has a well-developed and well-studied peroxisomal mechanism.

[17] (3) It belongs to Crabtree-negative yeast, which can effectively improve substrate utilization.

[18] (4) It has an effective ethanol-acetyl-CoA metabolic pathway. Therefore, Pichia pastoris has become a very promising research platform for advanced metabolic engineering.

[0006] Currently, research on Pichia pastoris as a microbial cell factory, using methanol as a carbon source, is underway to produce various chemicals and high-value-added compounds.

[19] It has broad application prospects, especially in the areas of acetyl-CoA derivatives and fatty acid derivatives. The increasing number of studies and reports on Pichia pastoris metabolic engineering signifies that the genetic manipulation platform for Pichia pastoris metabolic engineering research is being continuously improved and supplemented, laying a solid foundation for in-depth research and industrial application of metabolic engineering based on Pichia pastoris.

[0007] Currently, there is no effective and reliable synthetic biology and industrial microbiology technology in the field for the efficient synthesis of lycopene using Pichia pastoris. Therefore, heterologous synthesis of lycopene using Pichia pastoris holds potential and is expected to enable the inexpensive and efficient production of lycopene.

[0008] References:

[0009] [1]SHI J.Lycopene in Tomatoes:Chemical and Physical PropertiesAffected by Food Processing[J].Critical Reviews in Biotechnology,2008,20(4):293-334.

[0010] [2]BHATAYA A, SCHMIDT-DANNERT C, LEE P C. Metabolic engineering of Pichia pastoris X-33 for lycopene production[J]. Process Biochemistry, 2009, 44(10):1095-102.

[0011] [3]WU T,YE L,ZHAO D,et al.Engineering membrane morphology and manipμlating synthesis for increased lycopene accumμlation in Escherichia coli cellfactories[J].Biotech,2018,8(6):269.

[0012] [4]XUE D,ABDALLAH I I,DE HAAN I E M,et al.Enhanced C30 carotenoidproduction in Bacillus subtilis by systematic overexpression of MEP pathwaygenes[J].Applied Microbiology and Biotechnology,2015,99(14):5907-15.

[0013] [5]VERWAAL R,WANG J,MEIJNEN J-P,et al.High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation withCarotenogenic Genes from Xanthophyllomyces dendrorhous[J].Applied andenvironmental microbiology,2007,73(13):4342-50.

[0014] [6]LADOLE M R,NAIR R R,BHUTADA Y D,et al.Synergistic effect ofμltrasonication and co-immobilized enzymes on tomato peels for lycopeneextraction[J].Μltrasonics Sonochemistry,2018,48(453-62.

[0015] [7]BARROS H D F Q,GRIMALDI R,CABRAL F A.Lycopene-rich avocado oilobtained by simμltaneous supercritical extraction from avocado pμlp andtomato pomace[J].The Journal of Supercritical Fluids,2017,120(1-6.

[0016] [8]HERN NDEZ-ALMANZA A,MONTA EZ J,MART NEZ G,et al.Lycopene:Progressin microbial production[J].Trends in Food Science&Technology,2016,56(142-8.

[0017] [9]WU T,YE L,ZHAO D,et al.Membrane engineering-A novel strategy toenhance the production and accumμlation ofβ-carotene in Escherichia coli[J].Metabolic Engineering,2017,43(85-91.

[0018]

[10] SHI B,MA T,YE Z,et al.Systematic Metabolic Engineering ofSaccharomyces cerevisiae for Lycopene Overproduction[J].J Agric Food Chem,2019,67(40):11148-57.

[0019]

[11] ZHANG X-K,NIE M-Y,CHEN J,et al.Mμlticopy integrants of crt genesand co-expression of AMP deaminase improve lycopene production in Yarrowialipolytica[J].Journal of Biotechnology,2019,289(46-54.

[0020]

[12] ARAYA-GARAY J M,FEIJOO-SIOTA L,ROSA-DOS-SANTOS F,etal.Construction of new Pichia pastoris X-33strains for production of lycopeneandβ-carotene[J].Applied Microbiology and Biotechnology,2011,93(6):2483-92.

[0021]

[13] SEVGILI A,ERKMEN O.Improved Lycopene Production from DifferentSubstrates by Mated Fermentation of Blakeslea Trispora[J].Foods,2019,8(4):120.

[0022]

[14] BAI C,ZHANG Y,ZHAO X,et al.Exploiting a precise design ofuniversal synthetic modμlar regμlatory elements to unlock the microbialnatural products in Streptomyces[J].Proceedings of the National Academy ofSciences,2015,112(39):12181-6.

[0023]

[15] KARBALAEI M,REZAEE S A,FARSIANI H.Pichia pastoris:A highlysuccessfμl expression system for optimal synthesis of heterologous proteins[J].Journal of Cellμlar Physiology,2020,235(9):5867-81.

[0024]

[16] PE ADA, GASSER B, ZANGHELLINI J, et al. Metabolic engineering of Pichia pastoris[J]. Metabolic Engineering, 2018, 50(2-15.

[0025]

[17] WRIESSNEGGER T, GUBITZ G, LEITNER E, et al. Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources [J]. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 2007, 1771(4): 455-61.

[0026]

[18] SCHWARZHANS JP, LUTTERMANN T, GEIER M, et al. Towards systemsmetabolic engineering in Pichia pastoris[J]. Biotechnology Advances, 2017, 35(6): 681-710.

[0027]

[19] GUO Y, LIAO Y, WANG J, et al. Methylotrophy of Pichia pastoris: Current Advances, Applications, and Future Perspectives for Methanol-BasedBiomanufacturing[J]. ACS Sustainable Chemistry&Engineering, 2022, 10(5):1741-52. Summary of the Invention

[0028] To address the existing deficiencies and shortcomings, this invention combines the advantages of Pichia pastoris to conduct research on its metabolic regulation as a platform for synthesizing terpenoids. Metabolic engineering of Pichia pastoris is performed using genetic engineering techniques to obtain high-lycopene-producing strains, thus promoting the discovery of Pichia pastoris as a new generation of terpenoid cell factories.

[0029] This invention uses *Pichia pastoris* as the starting strain, expressing lycopene synthase from *Phaffia rhodozyma* in *Pichia pastoris*. The lycopene synthesis pathway in *Pichia pastoris* is optimized and reconstructed through strategies such as gene dosage optimization and NADPH supply optimization, gradually increasing lycopene yield. Then, the FPP (farnesyl pyrophosphate) branch of the lycopene synthesis pathway in the recombinant strain is blocked using CRISPR-Cpf1 gene editing, and the squalene synthesis pathway, another essential branch of the lycopene synthesis pathway, is weakened using CRISPR-dCpf1 gene inhibition, further increasing lycopene yield in the recombinant strain. Finally, lipid synthesis is optimized by overexpressing the sterol regulatory element SREBP (Sre), improving lycopene storage and further increasing lycopene yield in the recombinant strain, resulting in a high-yield lycopene strain. During high-density fermentation in a 5L fermenter, the lycopene yield of this strain is 7.5 g / L, higher than the model strain *Saccharomyces cerevisiae*, and is the highest level currently achieved in *Pichia pastoris*.

[0030] According to the construction method provided by the present invention, firstly, optimized lycopene synthase genes Octree, OctreeB, and OctreeI are inserted into the genome of Pichia pastoris; further enhancing the synthesis of NADPH in cells. As one embodiment of the present invention, NADH kinase POS5 and glucose-6-phosphate dehydrogenase ZWF are overexpressed.

[0031] Then, based on the above-mentioned engineered bacteria, the FPP synthesis pathway genes were blocked using the CRISPR-Cpf1 gene editing method, and the squalene synthesis pathway was weakened using the CRISPR-dCpf1 gene inhibition method. As one embodiment of the invention, the DPP1 and LPP1 genes of the recombinant strain were knocked out, and the squalene synthesis gene ERG9 was weakened to promote lycopene synthesis.

[0032] To optimize lipid synthesis and promote lycopene production, this invention optimizes lipid synthesis to promote lycopene storage. As one embodiment of this invention, the stearoyl-CoA-9-desaturase gene OLE2 and the sterol regulatory element Sre were overexpressed, with Sre showing better efficacy than OLE2.

[0033] The primary objective of this invention is to provide a method for constructing engineered bacteria that efficiently produce lycopene.

[0034] Another object of the present invention is to provide an engineered strain for producing lycopene efficiently obtained by the above-described construction method.

[0035] Another object of the present invention is to provide the application of the above-mentioned engineered bacteria for the efficient production of lycopene.

[0036] The technical solution of this invention is summarized as follows:

[0037] A method for constructing an engineered strain for efficient lycopene production involves using Pichia pastoris as the starting strain, integrating an optimized lycopene synthase encoding gene into the Pichia pastoris genome, and enhancing the NADPH synthesis pathway; wherein:

[0038] The lycopene synthases include Octree, OctreeB, and OctreeI, and the encoding genes for Octree, OctreeB, and OctreeI are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. Each lycopene synthase is integrated into the Pichia pastoris genome in one or more copies. In a preferred embodiment of the present invention, Octree, OctreeB, and OctreeI are each integrated into the Pichia pastoris genome in two copies.

[0039] The enhancement of the synthetic pathway involves overexpressing key genes of the synthetic pathway.

[0040] In a preferred embodiment of the present invention, the key genes of the NADPH synthesis pathway include NADH kinase POS5 and glucose-6-phosphate dehydrogenase ZWF. The coding gene for POS5 is shown in NCBI accession number XM_002490189.1, and the coding gene for ZWF is shown in NCBI accession number XM_002491158.1.

[0041] In a preferred embodiment of the present invention, the above-described construction method further includes blocking the FPP synthesis pathway and / or weakening the squalene synthesis pathway; wherein...

[0042] The blocking of the synthetic pathway involves knocking out key genes in the synthetic pathway.

[0043] The weakening of the synthetic pathway is a key gene for weakening the synthetic pathway.

[0044] More preferably, the key genes in the FPP synthesis pathway include DPP1 and LPP1. The coding gene for DPP1 is shown in NCBI accession number XM_002492361.1, and the coding gene for LPP1 is shown in NCBI accession number XM_002489953.1.

[0045] More preferably, the key gene in the squalene synthesis pathway includes ERG9, and the gene encoding ERG9 is shown in NCBI accession number: XM_002492490.1.

[0046] In a preferred embodiment of the present invention, the above construction method further includes overexpression of the stearoyl-CoA-9-desaturase gene OLE2 and / or the sterol regulatory element Sre. The encoding gene of OLE2 is shown in NCBI accession number XM_002493625.1, and the encoding gene of Sre is shown in NCBI accession number XM_002490953.1.

[0047] In a preferred embodiment of the present invention, the starting strain is Pichia pastoris GS115.

[0048] In a preferred embodiment of the present invention, the integration of the lycopene synthase encoding gene and the overexpression of key genes in the NADPH synthesis pathway are both achieved using the pZACH plasmid.

[0049] In a preferred embodiment of the present invention, the promoters of the expression cassettes for the lycopene synthase encoding gene and the expression cassettes for the key genes in the NADPH synthesis pathway are both P. AOX1 The promoter, with a base sequence as shown in SEQ ID NO.4, and the terminator of the expression cassette are all AOXTT terminators, with a base sequence as shown in SEQ ID NO.5.

[0050] In a preferred embodiment of the present invention, the knockout of key genes in the synthetic pathway is achieved using the CRISPR-Cpf1 gene editing system.

[0051] More preferably, the gene editing system includes a crRNA for knocking out DPP1 with the sequence 5'-AAATGGCGTATATGTGACGTAAT-3', and a crRNA for knocking out LPP1 with the sequence 5'-ATCAGGGCAGTCCAATTAACAGA-3'.

[0052] In a preferred embodiment of the present invention, the squalene synthase ERG9, which weakens the squalene synthesis pathway, is achieved using the CRISPR-dCpf1 gene editing system.

[0053] More preferably, the gene editing system includes a CRRNA for attenuating ERG9, the sequence of which is 5'CTCGACCTGATATTGTTCGTTTA-3'.

[0054] In a preferred embodiment of the present invention, the overexpression of the stearoyl-CoA-9-desaturase gene OLE2 and / or the sterol regulatory element Sre is achieved by using the pZACB plasmid.

[0055] In a preferred embodiment of the present invention, the promoter of the expression cassette for the stearoyl-CoA-9-desaturase gene OLE2 and / or the sterol regulatory element Sre is P. AOX1 The promoter and expression box terminator are both AOXTT terminators.

[0056] Further optimization is needed to obtain engineered bacteria that efficiently produce lycopene. The resistance gene needs to be removed: positive clone transformants are inoculated into BMMY liquid medium containing 1% methanol, and 1% methanol is added every 24 hours for induction, continuing for 3 days to remove the resistance gene. Approximately 10 μl of bacterial culture is streaked onto YPD plates and cultured for 2-3 days. Single colonies are picked and streaked first on YPDZ (YPD with added Zeocin antibiotic) plates, then on YPD plates, and cultured at 30°C for 2-3 days. If a single colony grows only on a YPD plate and not on the corresponding YPDZ plate, it preliminarily indicates that the Zeocin resistance of the strain has been successfully removed.

[0057] An engineered bacterium that efficiently produces lycopene was obtained through the above-described construction method.

[0058] To evaluate the production capacity of the engineered strain of Pichia pastoris for lycopene production, this invention conducted fermentation scale-up tests. As one embodiment of this invention, a high-density fermentation method with dual-stage regulation of the growth and product synthesis stages was achieved during fermentation scale-up using the AOX promoter-induced expression system of a high-yield engineered strain, resulting in high lycopene production.

[0059] The fermentation process is divided into two stages: the first stage uses glycerol as a carbon source to enable rapid cell growth. After the cell biomass reaches a certain concentration and the glycerol is depleted, the second stage of fermentation begins. The second stage is the lycopene production stage, in which methanol is added as a carbon source and also as an inducer to initiate the large-scale synthesis of lycopene, ultimately obtaining a high yield of lycopene.

[0060] The application of the aforementioned engineered bacteria for efficient lycopene production in the fermentation production of lycopene.

[0061] In a preferred embodiment of the present invention, the fermentation is carried out according to the following steps: Yeast seed culture is inoculated into a 5L sterile fermenter containing 2L of BSM medium at an inoculation rate of 8%, and glycerol supplementation is initiated for cell growth. The glycerol supplementation is performed by adding a 50% (w / w) glycerol solution containing 1.2% (v / v) PTM1 at a feed rate of 13-23 g / (L·min).

[0062] (1) Cell growth stage: Adjust relevant parameters, control pH at 5.5, maintain dissolved oxygen (DO) concentration at 30%–60%, control temperature at 30℃, control stirring speed at 200–1200 r / min, and air flow rate at 2 L / (L·min); cell density OD 600 Once the dissolved oxygen concentration reaches 440, stop adding glycerol solution; when the dissolved oxygen concentration rebounds to above 80%, start methanol-feeded fermentation.

[0063] (2) Methanol addition stage: Adjust relevant parameters, temperature at 30℃, pH at 5.5, stirring speed at 800 r / min, air flow rate at 4 L / min, pressure at 0.05 MPa, dissolved oxygen concentration at 15% to 25%, and add 50% (w / w) of methanol solution containing 1.2% (v / v) of PTM1 at variable speed: add at 3 g / L / h first, and increase by 1 to 2 g / L / h every 1 to 2 h until the addition rate reaches 8 to 9 g / L / h. If there is no methanol accumulation, this flow rate can be maintained throughout the remaining fermentation process. If necessary, it can be adjusted appropriately according to dissolved oxygen.

[0064] More preferably, the BSM culture medium has the following formulation: 4% glycerol, 0.093% CaSO4·2H2O, 1.147% MgSO4, 1.82% K2SO4, 0.413% KOH, 2.67% H3PO4, and 0.435% trace metal solution PTM1.

[0065] The formula of the trace metal solution PTM1 is as follows: 0.05% CoCl2·6H2O, 0.6% CuSO4·5H2O, 6.5% FeSO4·7H2O, 0.002% H3BO3, 0.008% NaI, 0.5% H2SO4, 0.02% Na2MoO4·2H2O, 2% ZnCl2, 0.3% MnSO4·H2O, and 0.02% biotin.

[0066] The present invention has the following advantages over the prior art:

[0067] The Pichia pastoris strain provided by this invention, which produces high levels of lycopene, achieves a lycopene yield of 7.5 g / L through a two-stage fermentation process. This is the highest production level of Pichia pastoris to date, and it is higher than that of the model strain, Saccharomyces cerevisiae.

[0068] This invention demonstrates the potential for developing high-yield lycopene production from Pichia pastoris and provides guidance for the efficient production of other sesquiterpenes or other terpenes in Pichia pastoris. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to specific preparation and application examples, but the implementation of the present invention is not limited thereto.

[0070] The culture media and their preparation are involved in the following examples:

[0071] YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (YPD solid medium, additional 20 g / L agar powder needs to be added). Zeocin antibiotic can be added to the medium as needed.

[0072] BMGY medium: 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 0.1 mM potassium phosphate buffer pH 6.0, 10 g / L glycerol.

[0073] BMMY medium: 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 0.1 mM potassium phosphate buffer pH 6.0, 10 g / L methanol.

[0074] The basal medium / basal salt medium (BSM) for fermentation in the upper tank consisted of: 4% glycerol, 0.093% CaSO4·2H2O, 1.147% MgSO4, 1.82% K2SO4, 0.413% KOH, 2.67% H3PO4, and 0.435% trace metal solution PTM1. The trace metal solution PTM1 consisted of: 0.05% CoCl2·6H2O, 0.6% CuSO4·5H2O, 6.5% FeSO4·7H2O, 0.002% H3BO3, 0.008% NaI, 0.5% H2SO4, 0.02% Na2MoO4·2H2O, 2% ZnCl2, 0.3% MnSO4·H2O, and 0.02% biotin.

[0075] The lycopene detection method involved in the following examples:

[0076] a. The standard for lycopene was purchased from Sigma Biotech Ltd.

[0077] b. Preparation of lycopene standard solution: Weigh 16 mg of lycopene standard using an analytical balance and dissolve it in 100 mL of n-hexane:acetone (3:2, V:V). Sonicate the solution until it is completely dissolved, and then dilute it to 100 mL using a volumetric flask. The concentration of the stock solution is 160 μg / mL. The stock solution is then subjected to a series of gradient dilutions of 2, 4, 6, 8, and 10 times.

[0078] c. Qualitative and quantitative analysis of lycopene was performed using high-performance liquid chromatography (HPLC). The qualitative and quantitative analysis of lycopene was conducted using a Waters 2695 series HPLC system (Waters Technologies, Milford, MA, USA), employing a Waters C18 column (4.6 mm × 250 mm) and a UV / VIS detector at a detection wavelength of 450 nm. The sample was eluted using an isocratic elution program: acetonitrile-isopropanol-methanol (50:20:30, v / v) at a flow rate of 1 mL / min and a column temperature of 40 °C. The injection volume was set to 10 μL.

[0079] The biomaterials involved in the following embodiments:

[0080] The plasmid PHKA has been published in the literature “Hao Yuechuo, Lin Ying, Liang Shuli. High-efficiency expression of Candida pleuropneumoniae lipase CRL1 in Pichia pastoris and its catalytic synthesis of vitamin E acetate [J]. Modern Food Science and Technology, 2018, 34(10):8”.

[0081] The plasmid pZACH has been published in the paper “Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris”;

[0082] The plasmid pZACB was prepared by replacing the zeocin resistance gene in the commercially available vector pPICZA with the blasidin resistance gene, and then inserting the histidine HIS gene at the MluI restriction site to obtain the pZACB recombinant plasmid.

[0083] Template P GAP -Cpf1-pSER-SUP4TT has been disclosed in the paper "A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a / Cpf1 for Pichia pastoris";

[0084] P AOX1 The base sequence of the promoter is shown in SEQ ID NO.4;

[0085] The base sequence of the terminator AOXTT is shown in SEQ ID NO.5.

[0086] Example 1: Construction of recombinant bacteria 1

[0087] Obtaining the target genes: Based on the lycopene synthase encoding genes provided by NCBI, and after specific codon optimization by the inventors, Shanghai Jierui Biotechnology Co., Ltd. synthesized the optimized lycopene synthase encoding genes OcrtE, OcrtB, and OcrtI, and inserted them into plasmid PHKA, obtaining plasmids PHKA-OcrtE, PHKA-OcrtB, and PHKA-OcrtI. The expression cassettes of the three genes were tandemly linked, and then the tandem expression cassettes were inserted into plasmid PHKA, obtaining the recombinant plasmid PHKA-OcrtEBI. The optimized nucleotide sequences of OcrtE, OcrtB, and OcrtI are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0088] Using the Pichia pastoris GS115 genome as a template, POS5 and ZWF were amplified. The nucleotide sequence of POS5 is shown in NCBI accession number: XM_002490189.1, and the nucleotide sequence of ZWF is shown in NCBI accession number: XM_002491158.1.

[0089] The vector used in this invention is the pZACH resistance cycling vector, with PAOX1 as the promoter and AOXTT as the terminator. Octree was amplified by PCR using PHKA-OcrtE as a template. Octree was then inserted into pZACH using the restriction enzymes PmlI and NotI to obtain the recombinant plasmid pZACH-OcrtE. Similarly, recombinant plasmids pZACH-OcrtB, pZACH-OcrtI, pZACH-POS5, and pZACH-ZWF were obtained.

[0090] Different expression cassettes containing the target gene were tandemly cascaded using appropriate restriction sites BamHI or MluI to obtain recombinant plasmids pZACH-OcrtE-OcrtB, pZACH-OcrtB-OcrtB, pZACH-OcrtB-OcrtI, and pZACH-OcrtE-OcrtB-OcrtI, as shown in Table 1.

[0091] The circular ligation system was transformed into E. coli Top10F' competent cells, and the positive recombinant plasmid was successfully constructed by screening with Zeocin-resistant plates and verifying by colony PCR and sequencing.

[0092] The recombinant plasmid was linearized using the restriction endonuclease Kpn2I. It was then transformed into Pichia pastoris GS115 competent cells via electroporation. Recombinant bacteria containing different target genes were obtained by selection using Zeocin-resistant YPD plates, as shown in Table 2. Taking the recombinant strain EOZPP as an example, a recombinant strain integrating the Octree+OcrtE+OcrtB tandem expression cassette was first obtained using PHKA-OcrtEBI, and then the Octree expression cassette was further integrated using pZACH-OcrtE.

[0093] The vector used in this invention is the pZACH resistance cycling vector, which requires zeocin resistance excision before fermentation. Recombinant Pichia pastoris transformants carrying zeocin resistance are inoculated into BMMY liquid medium containing 1% methanol. 1% methanol is added every 24 hours for induction, continuing for 3 days. Approximately 10 μl of the bacterial culture is streaked onto YPD plates, aiming to obtain single colonies. The culture is incubated for 2-3 days. Single colonies are picked up with a toothpick and streaked first on a YPDZ plate, then on a YPD plate. The culture is incubated at 30°C for 2-3 days. If single colonies grow only on YPD plates and not on the corresponding YPDZ plates, this preliminarily indicates that the zeocin resistance of the strain has been successfully excised.

[0094] Fermentation for lycopene production: Select a single yeast colony with good growth from a solid plate and inoculate it into a 50mL Erlenmeyer flask containing 5mL of BMGY medium. Incubate at 30℃ and 250rpm for about 24h. Measure the OD600 value of the seed culture, calculate and transfer it to 25mL of BMMY medium with its actual OD600 value set to 1. Centrifuge at 6000rpm for 2min at room temperature, discard the supernatant and collect the cells. Wash the cells 2-3 times with 800μL of BMMY medium. Transfer all the washed cells to a 250mL Erlenmeyer flask containing 25mL of BMMY medium and incubate at 30℃ and 250rpm. Add 1% methanol every 24h.

[0095] Lycopene isolation and purification: Lycopene was extracted from Pichia pastoris using organic solvent extraction, as detailed below:

[0096] (1) Take 1 mL of fermentation broth into a 2 mL centrifuge tube, centrifuge at room temperature for 2 min and then discard the supernatant;

[0097] (2) Add 1 mL of distilled water to a 2 mL centrifuge tube, centrifuge at 10,000 rpm for 2 min at room temperature, and then discard the supernatant; repeat the above steps 2-3 times.

[0098] (3) Transfer the washed cells to a 2 mL lysis tube, weigh 700 mg of 0.5 mm silica beads (BioSpec Products, Bartlesville, OK, (USA)) and mix them with 1 mL of pre-cooled hexane and acetone (3:2 v / v) organic solvent and add them to the lysis tube containing the yeast cells; use a cell disruption device to disrupt the sample for 30 s.

[0099] (4) Place the broken suspension in a pre-cooled cryogenic centrifuge and centrifuge at 10,000 rpm for 10 min at 4 °C. Then transfer the supernatant to a 2 mL Eppendorf tube. Evaporate the supernatant at 40 °C under constant airflow conditions in a fume hood.

[0100] (5) Repeat steps (3) and (4) above three to four times until the broken bacterial sample is colorless; evaporate the extract to dryness, redissolve the sample with 1 mL of pre-cooled n-hexane and acetone (3:2 v / v), filter the sample with a 0.22 μm organic filter membrane, and transfer the supernatant of the filtered sample to a brown 2 mL screw cap glass sample bottle. Store at -20℃ for no more than two days under light protection or perform HPLC detection and analysis directly.

[0101] HPLC analysis of lycopene: Qualitative and quantitative analysis of lycopene was performed using a Waters 2695 series HPLC system (Waters Technologies, Milford, MA, USA). A Waters C18 column (4.6 mm × 250 mm) and a UV / VIS detector were used, with a detection wavelength of 450 nm. An isocratic elution program of acetonitrile-isopropanol-methanol (50:20:30, v / v) was used to elute the sample at a flow rate of 1 mL / min and a column temperature of 40 °C. The injection volume was set to 10 μL.

[0102] Cell dry weight measurement:

[0103] (1) Take 10 mL of bacterial fermentation broth into a 15 mL centrifuge tube, centrifuge at room temperature, 10000 rpm for 2 min, and then discard the supernatant.

[0104] (2) Add 5 mL of distilled water to a 15 mL centrifuge tube containing the bacterial cells, vortex to suspend the cells, centrifuge at room temperature, 10,000 rpm for 2 min, and then discard the supernatant; repeat this operation 2-3 times.

[0105] (3) Place the washed bacterial cells in an 80℃ oven and dry them to constant weight, and determine their dry weight;

[0106] As shown in Table 2, increasing the lycopene gene dose can promote lycopene synthesis. The recombinant strain 2OZPP showed the greatest increase in lycopene production, while further increasing the gene dose resulted in a lower lycopene production in strain 3OZPP compared to 2OZPP. Therefore, optimizing the lycopene gene dose can effectively promote lycopene synthesis, but overexpression may lead to protein accumulation and metabolic burden, negatively impacting the synthesis of the target product by the chassis cells. Overexpression of POS5 and ZWF can optimize the synthesis of the Pichia pastoris cofactor NADPH and promote lycopene synthesis in recombinant yeast strains. The recombinant yeast strain P2OZPP showed better results than Z2OZPP. Therefore, recombinant strain P2OZPP (i.e., recombinant strain 1) was used for subsequent research.

[0107] Table 1. Plasmids used

[0108] PHKA Original carrier PHKA-OcrtE Amplification of OcrtE PHKA-OcrtB Amplification of OcrtB PHKA-OcrtI Amplification of OcrtI PHKA-OcrtEBI OcrtEBI Expression Box Serial pZACH Resistance selection marker cyclic vector pZACH-OcrtE OcrtE gene expression plasmid pZACH-OcrtB OcrtB gene expression plasmid pZACH-OcrtI OcrtI gene expression plasmid pZACH-OcrtE-OcrtB OcrtE,OcrtB tandem expression plasmids pZACH-OcrtB-OcrtB OcrtB, OcrtB tandem expression plasmid pZACH-OcrtB-OcrtI OcrtB,OcrtI tandem expression plasmid pZACH-OcrtE-OcrtB-OcrtI OcrtE, OcrtB, and OcrtI tandem expression plasmids pZACH-POS5 POS5 gene expression plasmid pZACH-ZWF ZWF gene expression plasmid

[0109] Table 2. Production Statistics

[0110]

[0111]

[0112] Example 2: Construction of recombinant bacteria 2

[0113] The FPP pathway and the squalene synthesis pathway are competing pathways in lycopene synthesis. To further increase lycopene production, the FPP pathway of lycopene synthesis in the recombinant engineered strain P2OZPP was blocked using the CRISPR-Cpf1 gene editing system, and the squalene synthesis pathway, another pathway in the recombinant strain's synthesis pathway, was weakened using the CRISPR-dCpf1 gene inhibition system to further improve lycopene synthesis in Pichia pastoris.

[0114] First, the FPP pathway-related genes DPP1 and LPP1 were knocked out, both as single and double genes. Then, the Pichia pastoris gene Erg9 was weakened.

[0115] Construction of plasmids related to the CRISPR-Cpf1 system:

[0116] 1. Taking the CRISPR-Cpf1 gene editing system for the recombinant Pichia pastoris target gene DPP1 as an example, the DPP1 gene editing plasmid P was constructed using circular PCR. GAP -Cpf1-△DPP1-CrRNA1 plasmid, template is P GAP -Cpf1-pSER-SUP4TT.

[0117] 2. Donor DNA of the target gene DPP1 was prepared by fusion PCR. Using the genome of Pichia pastoris GS115 as a template, approximately 250 bp of each homologous arm of the DPP1 gene was amplified. Then, the two homologous arms were fused by PCR to obtain ΔDPP1-Donor DNA.

[0118] 3. Construct the ΔLPP1 gene editing plasmid P using the same method. GAP -Cpf1-△LPP1-CrRNA2 plasmid, template is P GAP -Cpf1-pSER-SUP4TT; △LPP1-Donor DNA acquisition method is the same as above.

[0119] 4. Obtain the △DPP1△LPP1 double gene editing plasmid P using enzyme digestion and ligation methods. GAP -Cpf1-△DPP1-CrRNA1-△LPP1-CrRNA2: with P GAP Using -Cpf1-△LPP1-CrRNA2 as a template, the △LPP1-CrRNA2 expression cassette fragment was obtained, and plasmid P... GAP The recombinant plasmid P was obtained by using -Cpf1-△DPP1-CrRNA1 as a vector, with the restriction enzyme site BamHI, and ligation at T4. GAP -Cpf1-ΔDPP1-CrRNA1-ΔLPP1-CrRNA2.

[0120] Construction of plasmids related to the CRISPR-dCpf1 system:

[0121] 1. Construction of PGAP-dCpf1 recombinant plasmid

[0122] The RuvC domain of Cpf1 was mutated with D917A and E1006A to obtain the mutant dCpf1. (The last part, "using P," appears to be an unrelated fragment and is omitted from the translation.) GAP Based on -Cpf1-pSER-SUP4TT, the recombinant plasmid PGAP-dCpf1 was obtained by homologous recombination.

[0123] 2. Construction of PGAP-dCpf1-Erg9-CrRNA series plasmids

[0124] The PGAP-dCpf1-Erg9-CrRNA plasmid was obtained by circular PCR using the PGAP-dCpf1 plasmid as a template.

[0125] The crRNA for knocking out DPP1 has the sequence 5'-AAATGGCGTATATGTGACGTAAT-3', the crRNA for knocking out LPP1 has the sequence 5'-ATCAGGGCAGTCCAATTAACAGA-3', and the crRNA for attenuating Erg9 has the sequence 5'-CTCGACCTGATATTGTTCGTTTA-3'.

[0126] The circular ligation system was transformed into E. coli Top10F' competent cells, and the positive recombinant plasmid was successfully constructed by screening with Zeocin-resistant plates and verifying by colony PCR and sequencing.

[0127] Recombinant plasmids and Donor DNA were transformed into competent Pichia pastoris cells by electroporation, and recombinant bacteria containing different target genes were obtained by screening with Zeocin-resistant YPD plates.

[0128] As shown in Table 4, the lycopene yield of recombinant bacteria 2 obtained by knocking out and weakening the branch pathway in this invention reached 38.99 mg / g DCW.

[0129] Table 3. Plasmids used

[0130]

[0131] Table 4. Production Statistics

[0132]

[0133]

[0134] Example 3: Construction of recombinant bacteria 3

[0135] Lycopene is a hydrophobic and lipophilic compound, and improving lipid synthesis in cells helps with lycopene synthesis and storage. Although the above strategies significantly improved lycopene production in engineered strain 2, this invention investigated the effect of optimizing lipid synthesis on lycopene production to further increase lycopene yield. To further improve lycopene production, this invention optimized lipid synthesis in the recombinant strain 2.

[0136] Obtaining the target genes: Using the Pichia pastoris GS115 genome as a template, Sre and OLE2 were amplified. The nucleotide sequence of Sre is shown in NCBI accession number XM_002490953.1, and the nucleotide sequence of OLE2 is shown in NCBI accession number XM_002493625.1. Expression cassettes containing Sre and OLE2 were tandemly constructed using appropriate restriction enzyme sites.

[0137] This invention uses pZACB as a vector, with pAOX1 as the promoter and AOXTT as the terminator. Recombinant plasmids were constructed using the restriction enzyme sites PmlI and NotI via T4 ligation. The successfully constructed recombinant plasmids were pZACB-Sre and pZACB-OLE2. Then, expression cassettes containing the Sre and OLE2 genes were tandemly concatenated to obtain pZACB-Sre-OLE2, as shown in Table 5.

[0138] The circular ligation system was transformed into E. coli Top10F' competent cells, and the positive recombinant plasmid was successfully constructed by screening with Zeocin-resistant plates and verifying by colony PCR and sequencing.

[0139] The recombinant plasmid was linearized using the restriction endonuclease Kpn2I. It was then transformed into competent Pichia pastoris cells via electroporation, and recombinant bacteria containing different target genes were obtained by selection using Zeocin-resistant YPD plates.

[0140] As shown in Table 6, the recombinant strain 3 obtained by overexpressing Sre in this invention has the highest lycopene yield. After 120 h of shake-flask fermentation, the lycopene yield reached 469.43 mg / L (43.23 mg / g DCW) as detected by high performance liquid chromatography.

[0141] Table 5. Plasmids used

[0142]

[0143] Table 6. Production Statistics

[0144]

[0145] Example 4: Assessment of the production capacity of lycopene from recombinant strain 3

[0146] To evaluate the production capacity of recombinant strain 3, fed-batch fermentation was conducted in a 5L tank fermenter. Single colonies were initially inoculated into 250mL Erlenmeyer flasks containing 50mL YPD medium and incubated at 30°C and 250rpm for approximately 20 hours. Then, 4% of the seed culture was transferred to 500mL Erlenmeyer flasks containing 100mL YPD medium and incubated under the same conditions and time. Finally, 8% of the seed culture was inoculated into BSM medium in the 5L fermenter for fed-batch fermentation. Following the reference protocol in the *Pichia pastoris Fermentation Process Guide* (Invitrogen), a fed-batch strategy was employed using a 50% (w / v) glycerol solution containing 1.2% (v / v) PTM1 and a methanol solution containing 1.2% (v / v) PTM1.

[0147] The fermentation process of this invention is divided into two stages: the cultured yeast seed liquid is inoculated into a 5L fully automatic mechanically stirred and ventilated fermenter (sterilized) at an inoculation rate of 8%, and glycerol is added to start cell growth (initial culture medium volume 2L, sterilized at 121℃ for 30min). (1) Cell growth stage: 25% concentrated ammonia water is used to control the pH at 5.5, dissolved oxygen is maintained at (30-60)%, temperature is controlled at 30℃, stirring speed is set to an upper limit of 200r / min and a lower limit of 1200r / min, and aeration rate is set to 2vvm. Initial cell density OD 600 After reaching 440, stop feeding glycerol. When DO rebounds to above 80%, start methanol feeding to induce fermentation. (2) Methanol feeding stage: The temperature is controlled at 30℃, the pH is controlled at 5.5 using 25% concentrated ammonia, the rotation speed is controlled at 800r / min, the aeration rate is controlled at 4L / min, the pressure is controlled at 0.05Mpa, and methanol is fed in a variable speed feeding mode, while maintaining dissolved oxygen at 15-25%.

[0148] The present invention achieves a lycopene yield of 7.5 g / L by using the above-mentioned 5L tank for batch fermentation of recombinant bacteria 3.

[0149] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing engineered bacteria for producing lycopene, characterized in that: Using Pichia pastoris as the starting strain, a lycopene synthase-encoding gene was integrated into the Pichia pastoris genome, and the NADPH synthesis pathway was enhanced; among which: The lycopene synthase mentioned is OcrtE, OcrtB and OcrtI , OcrtE, OcrtB, OcrtI The encoding genes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; each lycopene synthase is integrated into the Pichia pastoris genome in 2 copies; The enhanced NADPH synthesis pathway is achieved by overexpressing a key gene in the pathway; the key gene in the NADPH synthesis pathway is NADH kinase. POS5 or glucose-6-phosphate dehydrogenase ZWF, POS5 The encoding gene is shown in the NCBI accession number: XM_002490189.

1. ZWF The encoding gene is shown in the NCBI accession number: XM_002491158.1; The integration of the lycopene synthase encoding gene and the overexpression of key genes in the NADPH synthesis pathway were both achieved using the pZACH plasmid.

2. The method for constructing engineered bacteria for producing lycopene according to claim 1, characterized in that: The construction method further includes blocking the FPP synthesis pathway and weakening the squalene synthesis pathway; wherein: The blocking of the synthetic pathway involves knocking out key genes in the synthetic branch pathway. The weakening of the synthetic pathway refers to the weakening of key genes in the synthetic branch pathway. Key genes in the FPP synthesis pathway DPP1 and / or LPP1, DPP1 The encoding gene is shown in the NCBI accession number: XM_002492361.

1. LPP1 The encoding gene is shown in the NCBI accession number: XM_002489953.1; The key genes in the squalene synthesis pathway are ERG9, ERG9 The encoding gene is shown in the NCBI accession number: XM_002492490.

1.

3. The method for constructing engineered bacteria for producing lycopene according to claim 2, characterized in that: The construction method further includes overexpression of the stearoyl-CoA-9-desaturase gene. OLE2 and / or sterol regulating elements Sre , OLE2 The encoding gene is shown in the NCBI accession number: XM_002493625.

1. Sre The encoding gene is shown in the NCBI accession number: XM_002490953.

1.

4. The method for constructing engineered bacteria for producing lycopene according to claim 3, characterized in that: The starting strain was Pichia pastoris GS115; The gene expressing stearoyl-CoA-9-desaturase. OLE2 and / or sterol regulating elements Sre Overexpression was achieved using the pZACB plasmid; The expression cassettes of the lycopene synthase encoding gene, the expression cassettes of key genes in the NADPH synthesis pathway, and the stearoyl-CoA-9-desaturase gene are described. OLE2 Sterol regulating element Sre The promoters of the expression boxes are all P AOX1 The promoter and base sequence are shown in SEQ ID NO.4, and the terminator is AOX. TT The terminator has the base sequence shown in SEQ ID NO.

5.

5. The method for constructing engineered bacteria for producing lycopene according to claim 3, characterized in that: Knockout of key genes in the synthetic pathway was achieved using the CRISPR-Cpf1 gene editing system; The gene editing system includes features for knocking out DPP1 The crRNA has the sequence 5'-AAATGGCGTATATGTGACGTAAT-3' ， Used for knocking LPP1 The crRNA has the sequence 5'-ATCAGGGCAGTCCAATTAACAGA-3'; Squalene synthases that weaken the squalene synthesis pathway ERG9 This was achieved using the CRISPR-dCpf1 gene editing system; The gene editing system includes features for attenuation ERG9 The crRNA has the sequence 5'CTCGACCTGATATTGTTCGTTTA-3' 。 6. An engineered strain for producing lycopene, characterized in that: Obtained by the construction method described in any one of claims 1 to 5.

7. The application of the engineered bacteria for producing lycopene as described in claim 6 in the fermentation production of lycopene.

8. The application of the engineered bacteria for producing lycopene according to claim 7 in the fermentation production of lycopene, characterized in that: The fermentation is carried out as follows: Yeast seed culture is inoculated at an 8% inoculation rate into a 5 L sterile fermenter containing 2 L of BSM medium. Glycerol supplementation is then initiated to promote cell growth. The glycerol supplementation is performed by adding a 50% w / w glycerol solution containing 1.2% v / v PTM1 at a feed rate of 13–23 g / (L·min). (1) Cell growth stage: Adjust relevant parameters, control pH at 5.5, maintain dissolved oxygen concentration at 30%–60%, control temperature at 30℃, control stirring speed at 200–1200 r / min, and air flow rate at 2 L / (L·min); cell density OD 600 Once the dissolved oxygen concentration reaches 440, stop adding glycerol solution; when the dissolved oxygen concentration rebounds to above 80%, start methanol-feeded fermentation. (2) Methanol addition stage: Adjust relevant parameters, temperature at 30℃, pH at 5.5, stirring speed at 800r / min, air flow rate at 4 L / min, pressure at 0.05 MPa, dissolved oxygen concentration at 15%~25%, and add 50%w / w methanol solution containing 1.2%v / v PTM1 at variable speed: first add at a rate of 3g / L / h, and increase by 1~2 g / L / h every 1~2 h until the addition rate reaches 8~9 g / L / h.

9. The application of the engineered bacteria for producing lycopene according to claim 8 in the fermentation production of lycopene, characterized in that: The BSM medium has the following formulation: 4% glycerol, 0.093% CaSO4·2H2O, 1.147% MgSO4, 1.82% K2SO4, 0.413% KOH, 2.67% H3PO4, and 0.435% trace metal solution PTM1. The formula of the trace metal solution PTM1 is as follows: 0.05% CoCl2·6H2O, 0.6% CuSO4·5H2O, 6.5% FeSO4·7H2O, 0.002% H3BO3, 0.008% NaI, 0.5% H2SO4, 0.02% Na2MoO4·2H2O, 2% ZnCl2, 0.3% MnSO4·H2O, and 0.02% biotin.

Images