Engineered saccharomyces cerevisiae for producing squalene by endogenous and synthetic pathway and its application

Abstract

The application provides a saccharomyces cerevisiae engineering bacterium for producing squalene through endogenous and artificial synthetic pathways and an application thereof, the saccharomyces cerevisiae engineering bacterium takes ZS00 strain as a starting strain, overexpresses acetyl coenzyme A acetyltransferase gene, hydroxymethylglutaryl coenzyme A synthase gene and N-terminal truncated hydroxymethylglutaryl coenzyme A reductase gene, and effectively improves the yield of squalene. Heterologous expression of mevalonate kinase gene and mevalonate pyrophosphate decarboxylase gene, overexpression of the key gene squalene synthase gene for promoting the conversion of precursors into squalene effectively promotes the synthesis of squalene. The introduction of isopentenol utilization pathway genes and their mutants, through the regulation of IU pathway key substrate, overexpression of isopentenyl pyrophosphate isomerase gene and farnesyl pyrophosphate synthase gene, the final strain can effectively accumulate squalene to 687.93 mg / L. The application realizes the breakthrough of squalene yield and efficiency through complementation and synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, and in particular relates to an engineered Saccharomyces cerevisiae strain that produces squalene through a combination of endogenous and artificial synthesis pathways, and its applications. Background Technology

[0002] Squalene (C 30 H 50 As a triterpenoid compound containing six isoprene double bonds, it has become a key raw material in cosmetics, pharmaceuticals, food processing and vaccine adjuvants due to its excellent antioxidant, anti-inflammatory, moisturizing and immune-enhancing core functions. Its market application scenarios continue to expand, industry demand is showing a steady growth trend, and the industry development potential is significant.

[0003] While squalene is widely distributed in nature, found in plants, fungi, animals, and human tissues, its naturally occurring content is generally low, making large-scale extraction and utilization difficult. Squalene in human sebum is a crucial component of the skin barrier; however, the body's own squalene synthesis capacity gradually declines with age, further increasing the demand for external supply. Currently, traditional methods of obtaining squalene mainly include shark liver extraction, plant extraction, and chemical synthesis, all of which face significant technological and industrial bottlenecks. Traditional shark liver extraction is the primary method for squalene production, but this method is extremely resource-intensive, requiring approximately 3,000 sharks to produce one ton of squalene, severely damaging the biodiversity of marine ecosystems and posing a serious challenge to ecological sustainability. Plant extraction is limited by long plant growth cycles, low squalene content in raw materials, and complex and costly separation and purification processes, resulting in low production efficiency and failing to meet the market's demand for large-scale supply. While chemical synthesis can achieve artificial preparation of squalene, it suffers from cumbersome synthesis steps, harsh reaction conditions, numerous byproducts, and low yields, lacking feasibility for industrial-scale production. In summary, existing methods for obtaining squalene are insufficient to balance ecological sustainability, economic viability, and scalability, highlighting the urgent need for technological innovation in the industry.

[0004] To address the limitations of traditional preparation methods, microbial synthesis technology, with its advantages of being environmentally friendly, having controllable production processes, and providing a wide range of raw materials, has become an important development direction for the large-scale production of squalene. Among these, the biomanufacturing strategy using Saccharomyces cerevisiae as the chassis cell has shown outstanding application potential. Saccharomyces cerevisiae, as a classic microbial engineered strain, possesses a complete organelle system, efficient lipid accumulation capabilities, and good genetic manipulation, making it highly compatible with the synthetic metabolic characteristics of lipid-soluble products like squalene. This makes it an ideal chassis for the microbial synthesis of squalene. Simultaneously, the rapid development of synthetic biology and metabolic engineering technologies provides technical support for the precise regulation of the metabolic network of Saccharomyces cerevisiae and the targeted optimization of the squalene synthesis pathway.

[0005] Currently, squalene microbial synthesis technology based on Saccharomyces cerevisiae has made some progress, but a core technological bottleneck remains: there is significant metabolic competition between Saccharomyces cerevisiae cell growth and squalene synthesis, exhibiting an imbalance between "growth and production," which greatly limits the synthesis efficiency and yield of squalene. To address this issue, existing technologies have developed various metabolic regulation strategies, such as organelle engineering, precursor engineering, and cofactor engineering, attempting to improve squalene yield through optimization of a single pathway. However, due to the inherent metabolic flux limitations of the endogenous synthesis pathway in Saccharomyces cerevisiae and the insufficient adaptability of artificial synthesis pathways, the effectiveness of single-pathway optimization is limited, making it difficult to achieve a breakthrough in squalene yield.

[0006] Therefore, how to overcome the metabolic limitations of the single synthetic pathway of Saccharomyces cerevisiae, integrate the advantages of endogenous and artificial synthetic pathways, construct a synergistic and efficient squalene synthesis metabolic network, solve the core problem of the imbalance between "growth and production", and realize the efficient and large-scale microbial synthesis of squalene has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a *Saccharomyces cerevisiae* engineered strain that synergistically produces squalene through endogenous and artificial synthesis pathways, and its applications. This invention utilizes both endogenous and artificial synthesis pathways of squalene in *Saccharomyces cerevisiae* to construct an engineered *Saccharomyces cerevisiae* strain that synergistically and efficiently produces squalene. This method overcomes the inherent limitations of single-pathway production, achieving breakthroughs in squalene yield and efficiency through complementary advantages and synergistic effects. Compared to extraction from shark liver, plants, and chemical synthesis, it has significant advantages, meets the requirements of green chemistry, and lays the foundation for the industrial production of squalene.

[0008] To address the aforementioned technical problems, this invention provides a brewer's yeast strain that synergistically produces squalene through endogenous and artificial synthesis pathways. The brewer's yeast strain uses ZS00 as the starting strain and overexpresses the acetyl-CoA acetyltransferase gene, the hydroxymethylglutaryl-CoA synthase gene, and the N-terminally truncated hydroxymethylglutaryl-CoA reductase gene.

[0009] The acetyl-CoA acetyltransferase gene is ERG10 (endogenous in Saccharomyces cerevisiae), and the amino acid sequence of ERG10 is shown in SEQ ID NO: 1; specifically: MSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICNGGGGASSIVIEKI*。

[0010] The hydroxymethylglutaryl-CoA synthase gene is ERG13 (endogenous to Saccharomyces cerevisiae), and the amino acid sequence of ERG13 is as shown in SEQ ID NO: 2; specifically: MKLSTKLCWCGIKGRLRPQKQQQLHNTNLQMTELKKQKTAEQKTRPQNVGIKGIQIYIPTQCVNQSELEKFDGVSQGKYTIGLGQTNMSFVNDREDIYSMSLTVLSKLIKSYNIDTNKIGRLEVGTETLIDKSKSVKSVLMQLFGENTDVEGIDTLNACYGGTNALFNSLNWIESNAWDGRDAIVVCGDIAIYDKGAARPTGGAGTVAMWIGPDAPIVFDSVRASYMEHAYDFYKPDFTSEYPYVDGHFSLTCYVKALDQVYKSYSKKAISKGLVSDPAGSDALNVLKYFDYNVFHVPTCKLVTKSYGRLLYNDFRANPQLFPEVDAELATRDYDESLTDKNIEKTFVNVAKPFHKERVAQSLIVPTNTGNMYTASVYAAFASLLNYVGSDDLQGKRVGLFSYGSGLAASLYSCKIVGDVQHIIKELDITNKLAKRITETPKDYEAAIELRENAHLKKNFKPQGSIEHLQSGVYYLTNIDDKFRRSYDVKK*。

[0011] The N-terminally truncated hydroxymethylglutaryl-CoA reductase gene is tHMGR (endogenous in Saccharomyces cerevisiae), and the amino acid sequence of tHMGR is shown in SEQ ID NO: 3, specifically: MVLTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKNKEVAALVIHGKLPLYALEKKLGDTTRAVAVRRKALSILAEAPVLASDRLPYKNYDYDRVF GACCENVIGYMPLPVGVIGPLVIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTVLTKDGMTRGPVVRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTCLAGDLLFMRFRT TTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIEGRGKSVVAEATIPGDVVRKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVE SSNCITLMKEVDGDLRISVSMPSIEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLAGELSLCAALAAGHLVQSHMTHNRKPAEPTKPNNLDATDINRLKDGSVTCIKS*.

[0012] The aforementioned engineered Saccharomyces cerevisiae further incorporates a mevalonate kinase gene derived from Enterococcus faecalis; Enterococcus faecalis The mevalonate pyrophosphate decarboxylase gene was overexpressed, and the squalene synthase gene, a key gene that promotes the conversion of the precursor to squalene, was overexpressed.

[0013] The mevalonate kinase gene is MvaE, and the amino acid sequence of MvaE is shown in SEQ ID NO: 4; specifically: MKTVVIIDALRTPIGKYKGSLSQVSAVDLGTHVTTQLLKRHSTISEEIDQVIFGNVLQAGNGQNPARQIAINSGLSHEIPAMTVNEVCGSGMKAVILAKQLIQLGEAEVLIAGGIENMSQAPKLQRFNYETESYDAPFSSMMYDGLTDAFSGQAMGLTAENVAEKYHVTREEQDQFSVHSQLKAAQAQAEGIFADEIAPLEVSGTLVEKDEGIRPNSSVEKLGTLKTVFKEDGTVTAGNASTINDGASALIIASQEYAEAHGLPYLAIIRDSVEVGIDPAYMGISPIKAIQKLLARNQLTTEEIDLYEINEAFAATSIVVQRELALPEEKVNIYGGGISLGHAIGATGARLLTSLSYQLNQKEKKYGVASLCIGGGLGLAMLLERPQQKKNSRFYQMSPEERLASLLNEGQISADTKKEFENTALSSQIANHMIENQISETEVPMGVGLHLTVDETDYLVPMATEEPSVIAALSNGAKIAQGFKTVNQQRLMRGQIVFYDVADAESLIDELQVRETEIFQQAELSYPSIVKRGGGLRDLQYRAFDESFVSVDFLVDVKDAMGANIVNAMLEGVAELFREWFAEQKILFSILSNYATESVVTMKTAIPVSRLSKGSNGREIAEKIVLASRYASLDPYRAVTHNKGIMNGIEAVVLATGNDTRAVSASCHAFAVKEGRYQGLTSWTLDGEQLIGEISVPLALATVGGATKVLPKSQAAADLLAVTDAKELSRVVAAVGLAQNLAALRALVSEGIQKGHMALQARSLAMTVGATGKEVEAVAQQLKRQKTMNQDRALAILNDLRKQ*。

[0014] The mevalonate pyrophosphate decarboxylase gene is MvaS, and the amino acid sequence of MvaS is as shown in SEQ ID NO: 5, Specifically: MTIGIDKISFFVPPYYIDMTALAEARNVDPGKFHIGIGQDQMAVNPISQDIVTFAANAAEAILTKEDKEAIDMVIVGTESSIDESKAAAVVLHRLMGIQPFARSFEIKEACYGATAGLQLAKNHVALHPDKKVLVVAADIAKYGLNSGGEPTQGAGAVAMLVASEPRILALKEDNVMLTQDIYDFWRPTGHPYPMVDGPLSNETYIQSFAQVWDEHKKRTGLDFADYDALAFHIPYTKMGKKALLAKISDQTEAEQERILARYEESIIYSRRVGNLYTGSLYLGLISLLENATTLTAGNQIGLFSYGSGAVAEFFTGELVAGYQNHLQKETHLALLDNRTELSIAEYEAMFAETLDTDIDQTLEDELKYSISAINNTVRSYRN*。

[0015] The squalene synthase gene is ERG9, and the amino acid sequence of ERG9 is as shown in SEQ ID NO: 6, specifically: MGKLLQLALHPVEMKAALKLKFCRTPLFSIYDQSTSPYLLHCFELLNLTSRSFAAVIRELHPELRNCVTLFYLILRALDTIEDDMSIEHDLKIDLLRHFHEKLLLTKWSFDGNAPDVKDRAVLTDFESILIEFHKLKPEYQEVIKEITEKMGNGMADYILDENYNLNGLQTVHDYDVYCHYVAGLVGDGLTRLIVIAKFANESLYSNEQLYESMGLFLQKTNIIRDYNEDLVDGRSFWPKEIWSQYAPQLKDFMKPENEQLGLDCINHLVLNALSHVIDVLTYLAGIHEQSTFQFCAIPQVMAIATLALVFNNREVLHGNVKIRKGTTCYLILKSRTLRGCVEIFDYYLRDIKSKLAVQDPNFLKLNIQISKIEQFMEEMYQDKLPPNVKPNETPIFLKVKERSRYDDELVPTQQEEEYKFNMVLSIILSVLLGFYYIYTLHRA*。

[0016] Based on a general technical concept, the present invention provides an application of the above-mentioned engineered Saccharomyces cerevisiae in the production of squalene, wherein the method of application includes: placing the engineered Saccharomyces cerevisiae in a culture medium for fermentation to synthesize squalene.

[0017] Furthermore, the aforementioned engineered Saccharomyces cerevisiae strain is further modified by introducing choline kinase gene or its mutant, and isopentenyl phosphokinase gene or its mutant into the engineered Saccharomyces cerevisiae strain.

[0018] The choline kinase gene is CK (derived from Saccharomyces cerevisiae), and the amino acid sequence of CK is shown in SEQ ID NO: 7; specifically: MPMDLRDNKQSQKKWKNRTLTSSLEFALTGIFTAFKEERNMKKHAVSALLAVIAGLVFKVSVIEWLFLLLSIFLVITFEIVNSAIENVVDLASDYHFSMLAKNAKDMAAGAVLVISGFAALTGLIIFLLKIWFLLFH*.

[0019] The isopentenyl phosphokinase gene is IPK (derived from Arabidopsis thaliana), and the amino acid sequence of IPK is shown in SEQ ID NO: 8; specifically: MELNISESRSRSIRCIVKLGGAAITCKNELEKIHDENLEVVACQLRQAMLEGSAPSKVIGMDWSKRPGSSEISCDVDDIGDQKSSEFSKFVVVHGAGSFGHFQASRSGVHKGGLEKPIVKAGFVATRISVTNLNLEIVRALAREGIPTIGMSPFSCGWSTSKRDVAS ADLATVAKTIDSGFVPVLHGDAVLDNILGCTILSGDVIIRHLADHLKPEYVVFLTDVLGVYDRPPSPSEPDAVLLKEIAVGEDGSWKVVNPLLEHTDKKVDYSVAAHDTTGGMETKISEAAMIAKLGVDVYIVKAATTHSQRALNGDLRDSVPEDWLGTIIRFSK*.

[0020] The mutant of CK is CK mut (Derived from brewer's yeast), the CK mentioned mut The amino acid sequence is shown in SEQ ID NO: 9; specifically: MPMDLRDNKQSQKKWKNRTLTSSLEFALTGIFTAFKEERNMKKHAVAALLAVIAGLVFKVSVIEWLFLLLSIFLVITFEIVNSAIENVVDLASDYHFSMLAKNAKDMAAGAVLVISGFAALTGAIIFLLKIWFLLFH*.

[0021] The mutant of IPK is IPK. mut (Derived from Arabidopsis thaliana), the IPK mut The amino acid sequence is shown in SEQ ID NO: 10, specifically: MELNISESRSRSIRCIVKLGGAAITCKNELEKIHDENLEVVACQLRQAMLEGSAPSKVIGMDWSKRPGSSEISCDVDDIGDQKSSEFSKFVVVHGAGSFGHFQASRSGVHKGGLEKPIVKAGFVATRISVTNLNLEIVRALAREGIPTIGMSPFSCGWSTSKRDVAS ADLATVAKTIDSGFVPVLHGDAVLDNILGCTILSGDVIIRHLADHLKPEYVVFLTDVLGVYDRPPSPSEPDAVLLKEIAVGEDGSWKVVNPLLEHTDKKVDYPVRAHDTTGGMETKISEAAMIAKLGVDVYIVKAATTHSQRALNGDLRDSVPEDWLGTIIRFSK*.

[0022] Furthermore, in the aforementioned engineered Saccharomyces cerevisiae strain, the expression cassettes of the isopentenyl pyrophosphate isomerase gene IDI1 and the farnesyl pyrophosphate synthase gene ERG20 are integrated into the XII4 site of the strain genome.

[0023] The dimethylallyl diphosphate isomerase gene is IDI1 (derived from Saccharomyces cerevisiae), and the amino acid sequence of IDI1 is shown in SEQ ID NO: 11, specifically: MTADNNSMPHGAVSSYAKLVQNQTPEDILEEFPEIIPLQQRPNTRSSETSNDESGETCFSGHDEEQIKLMNENCIVLDWDDNAIGAGTKKVCHLMENIEKGLLHRAFSVFIFNEQGELLLQQRATEKITFPDLWTNTCCSHPLCI DDELGLKGKLDDKIKGAITAAVRKLDHELGIPEDETKTRGKFHFLNRIHYMAPSNEPWGEHEIDYILFYKINAKENLTVNPNVNEVRDFKWVSPNDLKTMFADPSYKFTPWFKIICENYLFNWWEQLDDLSEVENDRQIHRML*.

[0024] The farnesyl pyrophosphate synthase gene is ERG20 (derived from Saccharomyces cerevisiae), and the amino acid sequence of ERG20 is shown in SEQ ID NO: 12, specifically: MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPE DKVDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKKIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK*.

[0025] Based on a general technical concept, the present invention provides an application of engineered Saccharomyces cerevisiae in the production of squalene. The method of application includes: placing engineered Saccharomyces cerevisiae in a culture medium for fermentation, adding a substrate during the fermentation process, and synthesizing squalene; wherein the substrate is 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol.

[0026] In the above application, further, the molar ratio of 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol is 6:4 to 4:6; and / or, the substrate is added at 24 h to 48 h after fermentation.

[0027] In the above application, the molar ratio of 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol is 4:6.

[0028] Furthermore, in the above-described application, the culture medium contains yeast extract, peptone, and glucose.

[0029] In the above application, the fermentation conditions are further specified as follows: fermentation at 30°C and 220 rpm in a shaker for 72 h to 120 h.

[0030] Compared with the prior art, the advantages of the present invention are as follows: This invention provides a *Saccharomyces cerevisiae* engineered strain that synergistically produces squalene through endogenous and synthetic pathways. Using metabolic engineering and synthetic biology techniques, the invention first identified key rate-limiting genes in the *Saccharomyces cerevisiae* squalene synthesis pathway. Overexpression of the rate-limiting module significantly increased squalene yield. Subsequently, MvaE and MvaS genes from *Enterococcus faecalis* were introduced, and overexpression of the pathway gene ERG9 significantly promoted squalene yield. Finally, key genes of the synthetic IUP pathway were introduced to enhance squalene synthesis. By regulating the addition time and molar ratio of 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol, and overexpressing the key genes IDI1 and ERG20 to improve precursor transformation, squalene yield was further increased. This engineered yeast strain utilizes both endogenous and synthetic squalene pathways in *Saccharomyces cerevisiae* to construct a *Saccharomyces cerevisiae* strain that synergistically and efficiently produces squalene. This method breaks through the inherent limitations of a single pathway in the synthesis of squalene in Saccharin yeast. By combining the strengths of different approaches and achieving synergistic effects, it achieves breakthroughs in squalene yield and efficiency, providing important reference and ideas for the industrial production of squalene and showing broad application prospects. Attached Figure Description

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0032] Figure 1 This invention describes the principle of squalene production by engineered brewer's yeast.

[0033] Figure 2 This is for the identification of the MVA path speed limiting module in Experiment 1 of this invention.

[0034] Figure 3 The effect of introducing heterologous pathway genes MvaE and MvaS on squalene production in Experiment 2 of this invention.

[0035] Figure 4 This refers to the chromosomal integration of the squalene synthesis pathway gene in Experiment 2 of this invention.

[0036] Figure 5 This is the effect of ERG9 on squalene yield in Experiment 3 of this invention.

[0037] Figure 6 This study investigates the effect of introducing the IU pathway gene on squalene production in Experiment 4 of this invention.

[0038] Figure 7 This study investigates the effect of substrate addition time via the IU pathway on squalene yield in Experiment 4 of this invention.

[0039] Figure 8 This study investigates the effect of the substrate molar ratio in the IU pathway on squalene yield in Experiment 4 of this invention.

[0040] Figure 9 This study examines the effect of precursor optimization in Experiment 5 of this invention on squalene yield. Detailed Implementation

[0041] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0043] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Unless otherwise specified, the methods in the following examples are conventional methods in the art.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0046] In the following examples, squalene, 3-methyl-3-buten-1-ol, and 3-methyl-2-buten-1-ol were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., acetone was purchased from Hunan Huihong Reagent Co., Ltd., and chromatographic grade acetonitrile was purchased from Thermo Fisher Scientific.

[0047] In the following examples, the fermentation and detection methods for squalene are as follows: A single colony was picked from the plate and inoculated into 5 mL of YPD medium. The culture was incubated at 30°C and 220 rpm with shaking for 16 hours to obtain the seed culture. The inoculation amount was 1% ( v / v The culture medium was inoculated into a 250 mL shake flask containing 25 mL of YPD medium and fermented at 30 °C and 220 rpm for 96 h. After fermentation, an appropriate amount of zirconia beads (0.5 mm in diameter) was added to a 2 mL centrifuge tube, and 500 μL of fermentation broth was mixed with 1 mL of acetone. The mixture was then ground at 60 Hz for 35 min. The resulting mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm membrane and analyzed by HPLC using a reversed-phase C18 column (4.6 × 250 mm, 5 μm, Shimadzu). Isocratic elution was performed with acetonitrile at a flow rate of 1.2 mL / min, the column temperature was maintained at 40 °C, and the detection wavelength was 195 nm.

[0048] This invention provides a brewer's yeast strain that produces squalene through a combination of endogenous and artificial synthesis pathways, and its applications.

[0049] Figure 1 This invention relates to the principle of squalene production by engineered Saccharomyces cerevisiae strains. The invention utilizes genetic engineering, metabolic engineering, and synthetic biology techniques to construct a Saccharomyces cerevisiae strain that synergistically produces squalene through artificial and endogenous synthetic pathways. Starting with Saccharomyces cerevisiae ZS00, the key rate-limiting module of the squalene synthesis pathway was first identified. This was achieved by overexpressing the endogenous pathway gene acetyl-CoA acetyltransferase (ACE). ERG10 ), hydroxymethylglutaryl-CoA synthase gene ( ERG13 ), N-terminally truncated hydroxymethylglutaryl-CoA reductase gene ( tHMGR This effectively increases squalene production. Subsequently, heterologous expression of the mevalonate kinase gene ( MvaE ), mevalonate pyrophosphate decarboxylase gene ( MvaS Overexpression of squalene synthase gene, a key gene that promotes the conversion of precursors to squalene. ERG9 This effectively promoted the synthesis of squalene. Furthermore, the isopentenol utilization pathway (IUP) gene and its mutants were introduced, and the isopentenol pyrophosphate isomerase gene was overexpressed by regulating the key substrate addition time and molar ratio of the IU pathway. IDI1 ) and farnesyl pyrophosphate synthase gene ( ERG20This method enabled the final strain to effectively accumulate squalene up to 687.93 mg / L. By utilizing endogenous squalene in *Saccharomyces cerevisiae* and introducing an artificial synthesis pathway, a *Saccharomyces cerevisiae* engineered strain capable of synergistically and efficiently producing squalene was constructed. This method overcomes the inherent limitations of single-pathway synthesis, achieving breakthroughs in squalene yield and efficiency through complementary advantages and synergistic effects.

[0050] Example 1 A series of engineered Saccharomyces cerevisiae strains, ZS01–ZS03, were developed. Starting with Saccharomyces cerevisiae ZS00, which can produce trace amounts of squalene, the mevalonate pathway (MVA pathway) was divided into upper, middle, and lower modules to identify key rate-limiting modules in the squalene synthesis pathway. The genes involved include the upper module (ERG10, ERG13, and tHMGR), the middle module (ERG12, ERG8, ERG9, and IDI1), and the lower module (ERG20 and ERG9). By constructing free expression plasmids, the genes of the three modules were expressed in Saccharomyces cerevisiae.

[0051] The method for constructing the engineered Saccharomyces cerevisiae ZS01 is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, three fragments, PGK1p-ERG10-ADH1t, SED1p-ERG13-TJID1t, and INO1p-tHMGR-CYC1t, were amplified respectively. The three fragments were ligated into the plasmid backbone in the order of PGK1p-ERG10-ADH1t-SED1p-ERG13-TJID1t-INO1p-tHMGR-CYC1t to obtain the recombinant plasmid. The recombinant plasmid was then transformed into competent Saccharomyces cerevisiae cells to obtain ZS01.

[0052] The method for constructing the engineered Saccharomyces cerevisiae strain ZS02 is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, four fragments, PGK1p-ERG12-TTYS1t, SED1p-ERG8-TJID1t, INO1p-ERG19-CYC1t, and BGL2p-IDI1-TPS1t, were amplified respectively. The four fragments were ligated into the plasmid backbone in the order of PGK1p-ERG12-TTYS1t-SED1p-ERG8-TJID1t-INO1p-ERG19-CYC1t-BGL2p-IDI1-TPS1t to obtain the recombinant plasmid. The recombinant plasmid was then transformed into competent Saccharomyces cerevisiae cells to obtain ZS02.

[0053] The method for constructing the engineered Saccharomyces cerevisiae ZS03 is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, two fragments, PGK1p-ERG20-ADH1t and SED1p-ERG9-TJID1t, were amplified respectively. The two fragments were ligated into the plasmid backbone in the order of PGK1p-ERG20-ADH1t-SED1p-ERG9-TJID1t to obtain the recombinant plasmid. The recombinant plasmid was transformed into competent Saccharomyces cerevisiae cells to obtain ZS03.

[0054] Experiment 1: To investigate the squalene content produced by engineered Saccharomyces cerevisiae ZS00~ZS03.

[0055] The engineered strain ZS00-ZS03 was subjected to shake-flask fermentation, and the fermentation products were detected by HPLC after 96 h.

[0056] Figure 2 The figure shows the effect of the rate-limiting module of the MVA pathway on squalene content. As shown in the figure, the starting strain ZS00 could only synthesize 3.11 mg / L of squalene. After expressing the genes of the three modules of the MVA pathway, it was found that the overexpression of the upper module gene had the most significant effect on squalene yield, reaching 17.76 mg / L, an increase of 5.71 times. This indicates that the upper module is the key rate-limiting module for squalene synthesis.

[0057] Example 2 A series of engineered Saccharomyces cerevisiae strains, ZS04–ZS05 and SS01–SS02, were developed by introducing MvaE and MvaS genes from Enterococcus faecalis into background strains ZS01 and ZS00, respectively. After verification using defective plate selection, engineered strains ZS04–ZS05 were obtained. To improve the genetic stability of the strains, module genes (ERG10, ERG13, and tHMGR) along with the upper module genes and exogenous genes (ERG10, ERG13, tHMGR, MVAE, and MVAS) were integrated into the X3 and X4 loci of the starting strain ZS00 genome. After verification using defective plate selection, engineered strains SS01–SS02 were obtained.

[0058] The method for constructing the ZS04 engineered strain is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, four fragments, PGK1p-MVAE, MVAE-ter22t, ter22-MVAS, and MVAS-FBA1p, are amplified respectively and ligated into the plasmid backbone in the order of PGK1p-MVAE-ter22t-MVAS-FBA1p to obtain the recombinant plasmid; the recombinant plasmid is transformed into competent Saccharomyces cerevisiae cells to obtain ZS04.

[0059] The method for constructing the ZS05 engineered strain is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS01 as a template, four fragments, PGK1p-MVAE, MVAE-ter22t, ter22-MVAS, and MVAS-FBA1p, are amplified respectively and ligated into the plasmid backbone in the order of PGK1p-MVAE-ter22t-MVAS-FBA1p to obtain the recombinant plasmid; the recombinant plasmid is transformed into competent Saccharomyces cerevisiae cells to obtain ZS05.

[0060] The method for constructing the SS01 engineered strain is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, the module genes (ERG10, ERG13 and tHMGR) on the MVA pathway are integrated into the X3 site of the starting strain ZS00 genome according to the method in Example 1. After screening and verification by defective plates, the engineered strain SS01 is obtained (that is, the genes ERG10, ERG13 and tHMGR are expressed on a free plasmid).

[0061] The method for constructing the SS02 engineered strain is as follows: using the genomic DNA of Saccharomyces cerevisiae ZS00 as a template, the module genes (ERG10, ERG13 and tHMGR) of the MVA pathway, as well as the upper module genes and exogenous genes (ERG10, ERG13, tHMGR, MVAE, MVAS) are integrated into the X3 and X4 sites of the starting strain ZS00 genome, respectively. After screening and verification by defective plates, the engineered strain SS02 is obtained (that is, the genes are expressed on free plasmids).

[0062] Experiment 2: To investigate the squalene content produced by engineered brewer's yeast strains ZS04, ZS05, SS01, and SS01.

[0063] Engineered bacteria ZS04 and ZS05 were subjected to shake-flask fermentation, and the fermentation products were analyzed by HPLC after 96 h.

[0064] Figure 3 The effect of introducing heterologous pathway genes MvaE and MvaS on squalene production. The figure shows the effect of introducing genes carrying the heterologous pathway. MvaE and MvaS The plasmid discovery significantly increased squalene production, with strain ZS04 increasing it by 43.58 times compared to the starting strain ZS00, and strain ZS05 increasing it by 33.48 times.

[0065] Engineered bacteria ZS04 and ZS05 were subjected to shake-flask fermentation, and the fermentation products were analyzed by HPLC after 96 h.

[0066] Figure 4The figure shows the effect of chromosomal integration of genes involved in the squalene synthesis pathway on squalene production. The figure indicates that the squalene production of strains SS01 and SS02, obtained through genomic integration, was further enhanced, with SS01 reaching 276.20 mg / L and SS02 reaching 241.35 mg / L. This suggests that the synergistic expression of multiple pathways promotes squalene synthesis.

[0067] Example 3 Engineered Saccharomyces cerevisiae strains SS03 and SS04: ERG9 is a key enzyme gene in the MVA pathway that catalyzes the conversion of farnesyl pyrophosphate to squalene. Low ERG9 activity may lead to the accumulation of large amounts of intermediates, hindering the efficient synthesis of squalene precursors and thus limiting the high-efficiency synthesis of squalene. Using SS01 and SS02 as starter strains, the constructed ERG9 expression cassette was integrated into the XII3 locus of the corresponding strain genome. After verification using defective plates, engineered strains SS03-SS04 were obtained.

[0068] The method for constructing the engineered Saccharomyces cerevisiae strain SS03 is as follows: using SS01 as the starting strain, an ERG9 expression cassette is constructed according to the ERG9p-ERG9-ERG9t sequence. The constructed ERG9 expression cassette is integrated into the corresponding strain's genome at the XII3 site. After verification by defective plate screening, the engineered strain SS03 is obtained.

[0069] The method for constructing the engineered Saccharomyces cerevisiae strain SS04 is as follows: using SS02 as the starting strain, an ERG9 expression cassette is constructed according to the ERG9p-ERG9-ERG9t sequence. The constructed ERG9 expression cassette is integrated into the corresponding strain's genome at the XII3 site. After verification by defective plate screening, the engineered strain SS04 is obtained.

[0070] Experiment 3: To investigate the squalene content produced by engineered Saccharomyces cerevisiae SS03 and SS04.

[0071] Engineered bacteria SS03 and SS04 were subjected to shake-flask fermentation, and the fermentation products were analyzed by HPLC after 96 h.

[0072] Figure 5 The effect of ERG9 on squalene production is shown in the figure. The figure shows that overexpression of ERG9 can increase squalene production, with SS03 showing the largest increase, rising by 30.94% compared to SS01, reaching 399.95 mg / L.

[0073] Example 4 Engineered Saccharomyces cerevisiae strains SS10 and SS11 were developed. The IU pathway, through a two-step phosphorylation reaction, directly converts isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from isopentenyl alcohol isomers (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol), thereby enhancing squalene synthesis. Mutating genes related to the IU pathway can improve its metabolic efficiency. Therefore, the wild-type artificially synthesized IU pathway gene and the mutated pathway gene were integrated into the XI2 site of the starting strain SS03, and engineered strains SS010-SS11 were obtained after screening and verification using defective plates.

[0074] Construction of SS10 engineered strain: Saccharomyces cerevisiae SS03 was used as the starting strain for the construction of engineered strain. The wild-type artificial synthetic pathway IU pathway gene PGK1p-AtIPK-TER22t-ScCK-FBA1p was integrated into the XI2 site of the starting strain SS03. After verification by defective plate screening, the engineered strain SS010 was obtained.

[0075] Construction of SS12 engineered strain: Saccharomyces cerevisiae SS03 was used as the starting strain for the construction of the engineered strain. The mutated pathway gene PGK1p-AtIPK was used. S270P,A272R -TER22t-SmDAGK S47A,L124A -FBA1p (AtIPK mutants are S270P and A272R; SmDAGK mutants are S47A and L124A) was integrated into the XI2 site of the starting strain SS03, and the engineered strain SS011 was obtained after screening and verification with defective plates.

[0076] Experiment 4: Investigate the squalene content produced by engineered Saccharomyces cerevisiae SS010 and SS11.

[0077] 4.1 The engineered strains SS10 and SS11 were subjected to shake-flask fermentation. At 0 h, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol were added in a 1:1 ratio, with a total molar concentration of 30 mM. Samples were taken at 24, 48, 72, and 96 h, and the fermentation products were analyzed by HPLC.

[0078] Figure 6 This figure illustrates the effect of introducing the IU pathway gene on squalene production in Experiment 4 of this invention. As can be seen from the figure, compared to the control, the addition of both 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol significantly reduced the strain's ability to synthesize squalene, indicating that the substrates have certain growth toxicity to the strain.

[0079] 4.2 Engineered strains SS10 and SS11 were subjected to shake-flask fermentation. 3-Methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol were added at a 1:1 ratio at 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h of fermentation, respectively, with a total molar concentration of 30 mM. Samples were taken at 96 h, and the fermentation products were analyzed by HPLC. Figure 7 This figure shows the effect of substrate addition time on squalene yield in Experiment 4 of the present invention via the IU pathway. As can be seen from the figure, squalene yield increases significantly with delayed substrate addition time, reaching its highest level at 48 hours. This indicates that adding 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol in the later stages of fermentation is more beneficial for squalene synthesis via the IU pathway.

[0080] 4.3 Engineered strains SS10 and SS11 were subjected to shake-flask fermentation. 3-Methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol were added at 48 h of fermentation in gradient ratios, with a total molar concentration of 30 mM. The addition ratios of 3-methyl-2-buten-1-ol to 3-methyl-3-buten-1-ol were 10:0, 8:2, 10:0, 6:4, 4:6, 2:8, and 0:10, respectively. Samples were taken at 96 h, and the fermentation products were analyzed by HPLC. Figure 8 This figure illustrates the effect of the substrate molar ratio on squalene yield in Experiment 4 of this invention via the IU pathway. The figure shows that different addition ratios of 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol resulted in different squalene synthesis capabilities for strains SS10 and SS11. When the molar ratio of 3-methyl-3-buten-1-ol to 3-methyl-2-buten-1-ol was 4:6, strains SS10 and SS11 achieved the highest squalene yields, reaching 518.62 mg / L and 560.68 mg / L, respectively. Therefore, this substrate concentration was selected to regulate squalene synthesis.

[0081] Example 5 A type of engineered Saccharomyces cerevisiae strain SS12-SS13: Starting with strains SS10 and SS11, respectively, the expression cassettes of genes IDI1 and ERG20 were integrated into the XII4 site of the strain genome. After screening and verification by defective plates, engineered strains SS12-SS13 were obtained.

[0082] Construction of SS12 engineered strain: Starting with SS10 strain, the gene IDI1 and ERG20 expression cassette were integrated into the XII4 site of the strain genome. After screening and verification by defective plate, the engineered strain SS12 was obtained.

[0083] Construction of SS13 engineered strain: Starting with SS11 strain, the gene IDI1 and ERG20 expression cassette were integrated into the XII4 site of the strain genome. After screening and verification by defective plate, the engineered strain SS13 was obtained.

[0084] Experiment 5: Investigate the squalene content produced by engineered Saccharomyces cerevisiae SS12 and SS13.

[0085] Engineered strains SS12 and SS13 were subjected to shake-flask fermentation. At the 48th hour, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol were added at a molar ratio of 4:6. After 96 hours, the fermentation products were analyzed by HPLC.

[0086] Figure 9 The effect of pathway precursor optimization on squalene yield was investigated. The figure shows that overexpression of both IDI1 and ERG20 enhanced the squalene synthesis capacity of strains SS12 and SS13 to varying degrees. Squalene yield in strain SS12 was 608.80 mg / L, an increase of 14.81%, while the yield in strain SS13 was 687.93 mg / L, an increase of 18.50%.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A brewer's yeast strain that synergistically produces squalene through endogenous and artificial synthesis pathways, characterized in that, The engineered Saccharomyces cerevisiae strain used ZS00 as the starting strain and overexpressed the acetyl-CoA acetyltransferase gene, the hydroxymethylglutaryl-CoA synthase gene, and the N-terminally truncated hydroxymethylglutaryl-CoA reductase gene. The acetyl-CoA acetyltransferase gene is ERG10, and the amino acid sequence of ERG10 is shown in SEQ ID NO: 1; The hydroxymethylglutaryl coenzyme A synthase gene is ERG13, and the amino acid sequence of ERG13 is shown in SEQ ID NO: 2; The N-terminal truncated hydroxymethylglutaryl-CoA reductase gene is tHMGR, and the amino acid sequence of tHMGR is shown in SEQ ID NO:

3.

2. The engineered brewer's yeast strain according to claim 1, characterized in that, In the engineered Saccharomyces cerevisiae strain, mevalonate kinase gene and mevalonate pyrophosphate decarboxylase gene from Enterococcus faecalis were introduced, and genes that promote the conversion of precursors to squalene were overexpressed. The mevalonate kinase gene is MvaE, and the amino acid sequence of MvaE is shown in SEQ ID NO: 4; The mevalonate pyrophosphate decarboxylase gene is MvaS, and the amino acid sequence of MvaS is shown in SEQ ID NO:

5.

3. The engineered brewer's yeast strain according to claim 2, characterized in that, The gene that promotes the conversion of the precursor into squalene is the squalene synthase gene ERG9, and the amino acid sequence of the squalene synthase gene ERG9 is shown in SEQ ID NO:

6.

4. The engineered brewer's yeast according to claim 2, characterized in that, Introduce the choline kinase gene and the isopentenyl phosphokinase gene, or introduce a mutant of the choline kinase gene and a mutant of the isopentenyl phosphokinase gene into the engineered Saccharomyces cerevisiae. The choline kinase gene is CK, and the amino acid sequence of CK is shown in SEQ ID NO: 7; The isopentenyl phosphokinase gene is IPK, and the amino acid sequence of IPK is shown in SEQ ID NO: 8; The mutant of the choline kinase gene is CK. mut The CK mut The amino acid sequence is shown in SEQ ID NO: 9; The mutant of the isopentenyl phosphokinase gene is IPK. mut The IPK mut The amino acid sequence is shown in SEQ ID NO:

10.

5. The engineered brewer's yeast strain according to claim 4, characterized in that, The expression cassettes of genes IDI1 and ERG20 were integrated into the XII4 site of the strain genome; The amino acid sequence of IDI1 is shown in SEQ ID NO: 11; The amino acid sequence of ERG20 is shown in SEQ ID NO:

12.

6. The application of the engineered Saccharomyces cerevisiae according to claim 1 or 2 in the production of squalene, characterized in that, The method of application includes: placing engineered Saccharomyces cerevisiae in a culture medium, fermenting, and synthesizing squalene.

7. The application of an engineered Saccharomyces cerevisiae strain according to any one of claims 3 to 5 in the production of squalene, characterized in that, The method of application includes: placing engineered Saccharomyces cerevisiae in a culture medium for fermentation, adding a substrate during the fermentation process, and synthesizing squalene; the substrate is 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol.

8. The application according to claim 7, characterized in that, The molar ratio of 3-methyl-3-buten-1-ol to 3-methyl-2-buten-1-ol is 6:4 to 4:6; and / or the substrate is added between 24 and 48 hours after fermentation.

9. The application according to claim 7, characterized in that, The molar ratio of 3-methyl-3-buten-1-ol to 3-methyl-2-buten-1-ol is 4:

6.

10. The application according to any one of claims 6 to 8, characterized in that, The culture medium contains yeast extract, peptone, and glucose; the fermentation conditions are: 30°C, 220 rpm in a shaker for 72 h to 120 h.

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