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(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.
Assuntos
Alpinia , Plantas Medicinais , Sesquiterpenos , Alpinia/metabolismo , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Plantas Medicinais/metabolismoRESUMO
Plant-derived natural active products have attracted increasing attention for use in flavors and perfumes. These compounds also have applications in insect pest control because of their environment-friendly properties. Holy basil (Ocimum sanctum), a famous herb used in Ayurveda in India, is a natural source of medical healing agents and insecticidal repellents. Despite the available genomic sequences and genome-wide bioinformatic analysis of terpene synthase genes, the functionality of the sesquiterpene genes involved in the unique fragrance and insecticidal activities of Holy basil are largely unknown. In this study, we systematically screened the sesquiterpenoid biosynthesis genes in this plant using a precursor-providing yeast system. The enzymes that synthesize ß-caryophyllene and its close isomer α-humulene were successfully identified. The enzymatic product of OsaTPS07 was characterized by in vivo mining, in vitro reaction, and NMR detection. This product was revealed as (-)-eremophilene. We created a mutant yeast strain that can achieve a high-yield titer by adjusting the gene copy number and FPP precursor enhancement. An optimized two-stage fed-batch fermentation method achieved high biosynthetic capacity, with a titer of 34.6 g/L cyclic sesquiterpene bioproduction in a 15-L bioreactor. Further insect-repelling assays demonstrated that (-)-eremophilene repelled the insect pest, fall leafworm, suggesting the potential of (-)-eremophilene as an alternative to synthetic chemicals for agricultural pest control. This study highlights the potential of our microbial platform for the bulk mining of plant-derived ingredients and provides an impressive cornerstone for their industrial utilization.
Assuntos
Ocimum sanctum , Sesquiterpenos , Reatores Biológicos , Fermentação , Saccharomyces cerevisiae/genéticaRESUMO
(+)-Nootkatone is a valuable, functional sesquiterpene that is widely used in food, cosmetics, pharmaceutical, agriculture, and other fields. However, only traces of it accumulate in plants, which is insufficient to meet the market demand. Therefore, commercial (+)-nootkatone is currently synthesized from (+)-valencene. Here, we engineered Saccharomyces cerevisiae to achieve high production of (+)-valencene. Employing gene screening, protein engineering and biosynthetic pathway optimization, we achieved 12.4 g/L (+)-valencene production with the mutant strain. This titer was further increased to 16.6 g/L, the highest titer reported to date, by coupling critical factors for cell growth and biochemical pathway induction. Subsequently, (+)-nootkatone was chemically synthesized from bio-fermented (+)-valencene with a yield of 80%. This study achieved efficient microbial synthesis of (+)-valencene, which may be utilized in industrial production and stabilize the supply of (+)-nootkatone.
Assuntos
Saccharomyces cerevisiae , Sesquiterpenos , Engenharia Metabólica , Sesquiterpenos Policíclicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismoRESUMO
Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.
Assuntos
Engenharia Metabólica , Plantas/química , Sesquiterpenos de Guaiano/química , Sistemas CRISPR-Cas , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Sesquiterpenos de Guaiano/síntese químicaRESUMO
Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil-triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2â¯mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5â¯mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37 g/L and 73.3 mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.
Assuntos
Metabolismo dos Lipídeos/genética , Licopeno/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/biossíntese , Fermentação , Deleção de Genes , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas/genética , NADP/metabolismo , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triglicerídeos/metabolismoRESUMO
To identify the microorganisms responsible for the formation of the main quality components of Qingzhuan brick tea (QZBT) during solid-state fermentation (SSF), predominant thermoduric strains were isolated from the tea leaves collected during SSF. According to their capability of releasing cellulase, pectase, protease, and polyphenol oxidase, four strains were selected as starter cultures to ferment sun-dried tea leaves during artificially inoculated SSF. According to the major enzymatic activities and quality components content (tea polyphenols, catechins, amino acids, soluble sugar, and theabrownin), it was found that Aspergillus fumigatus M1 had a significant effect on the transformation of polyphenols and Bacillus subtilis X4 could enhance the ability of bioconversion of strain M1. Strain X4 and M1 may be the core microbes responsible for developing these biochemical components of QZBT, as the values of quality components of tea leaves fermented by these two strains for 6 days was very close to that of the sample naturally fermented for 35 days in the tea factory. The results could be significant in developing industrial starters for the manufacture of QZBT and stabilizing the product quality of different batches.
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Sesquiterpene synthases in Trichoderma viride have been seldom studied, despite the efficiency of filamentous fungi for terpenoid production. Using the farnesyl diphosphate-overexpressing Saccharomyces cerevisiae platform to produce diverse terpenoids, we herein identified an unknown sesquiterpene synthase from T. viride by genome mining and determined the structure of its corresponding products. One new 5/6 bicyclic sesquiterpene and its esterified derivative were characterised by GC-MS and 1D and 2D NMR spectroscopy. To the best of our knowledge, this is the first well-identified sesquiterpene synthase from T. viride to date.
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The chemical 3-hydroxypropionate (3HP) is an important starting reagent for the commercial synthesis of specialty chemicals. In this study, a part of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula was utilized for 3HP production. To study the basic biochemistry of this pathway, an in vitro-reconstituted system was established using acetyl-CoA as the substrate for the kinetic analysis of this system. The results indicated that 3HP formation was sensitive to acetyl-CoA carboxylase and malonyl-CoA reductase, but not malonate semialdehyde reductase. Also, the competition between 3HP formation and fatty acid production was analyzed both in vitro and in vivo. This study has highlighted how metabolic flux is controlled by different catalytic components. We believe that this reconstituted system would be valuable for understanding 3HP biosynthesis pathway and for future engineering studies to enhance 3HP production.
Assuntos
Ácido Láctico/análogos & derivados , Oxibato de Sódio/metabolismo , Sulfolobaceae/metabolismo , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilase/metabolismo , Vias Biossintéticas , Ciclo do Carbono , Cinética , Ácido Láctico/biossíntese , Oxirredutases/metabolismo , Sulfolobaceae/enzimologiaRESUMO
Recently, mycotoxin risks in fermented tea have received high attention, but mycotoxin transfer rates from tealeaf to infusion during brewing were rarely considered. In addition, the assessment data (i.e., mycotoxin occurrences and tea consumption) in previous assessments were usually limited. Here, a comprehensive and cumulative risk assessment of aflatoxins and ochratoxin A was performed using a tea assessment model, by which mycotoxin transfer rates were included and the assessment data were collected worldwide. By 10 times of brewing, the aflatoxin transfer rate was only 2.94% and OTA was 63.65%. Besides the extreme case, hazard quotients (HQs) from all consumers were lower than the threshold of 1.0, indicating no noncarcinogenic risk; the P95 cumulative margin of exposure (1/MoET) values were 2.52E-04 (30-39 years of age) and 2.42E-04 (≥50 years of age) for two high exposure groups under the upper bound scenario, which a little higher than the carcinogenic risk threshold of 1.00E-04. Notably, the P95 cumulative 1/MoET values (3.24E-03 -7.95E-03) by food assessment model were ten times higher than those of by tea assessment model. The comparative results showed that mycotoxin dietary risks on tea consumption by food assessment model were much overestimated. The result of this study indicated that the contaminants transfer rates should be considered for risk assessment on tea consumption in future work.
Assuntos
Aflatoxinas , Micotoxinas , Ocratoxinas , Aflatoxinas/toxicidade , Exposição Dietética , Contaminação de Alimentos/análise , Micotoxinas/toxicidade , Micotoxinas/análise , Medição de Risco , CháRESUMO
Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.
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Sesquiterpenes have been identified as promising ingredients for aviation fuels due to their high energy density and combustion heat properties. Despite the characterization of numerous sesquiterpene structures, studies testing their performance properties and feasibility as fuels are scarce. In this study, 122 sesquiterpenoid skeleton compounds, obtained from existing literature reports, are tested using group contribution and gaussian quantum chemistry methods to assess their potential as high-energy aviation fuels. Seventeen sesquiterpene compounds exhibit good predictive performance and nine compounds are further selected for overproduction in yeast. Through fed-batch fermentation, all compounds achieve the highest reported titers to date. Subsequently, three representative products, pentalenene, presilphiperfol-1-ene, and α-farnesene, are selected, produced, purified in large quantities, and tested for use as potential fuels. The performance of pentalenene, presilphiperfol-1-ene, and their derivatives reveals favorable prospects as high-energy aviation fuels.
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Interferometry has been widely used in biosensing due to its ability to acquire molecular affinity and kinetics in real-time. However, interferometric-based sensors are susceptible to environmental disturbances, including temperature and non-specific binding of target molecules, which reduces their detection robustness. To address this shortcoming, this paper proposes a self-referencing interference sensor based on coherence multiplexing to resist environmental disturbances. The proposed sensor can address temperature and non-specific binding, but it is not limited only to these types of disturbances. In the proposed sensor design, each sensor signal is encoded using a specific optical path difference determined by the optical thickness of a sensor chip. In addition, two sensor signals for disturbances tracking and biomolecule detection are detected simultaneously without additional cost to the second spectrometer and then differenced to achieve real-time self-reference. The temperature fluctuations experiments and specific binding experiments of protein A to IgG are performed to verify the performance of the proposed sensor. The results demonstrate that the proposed sensor can eliminate non-specific binding and temperature disturbances in real-time during biomolecule detection, achieving higher detection robustness. The proposed sensor is suitable for applications that require large-scale testing of biomolecular interactions, such as drug screening.
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Vitamin E is one of the most widely used vitamins. In the classical commercial synthesis of vitamin E (α-tocopherol), the chemical synthesis of isophytol is the key technical barrier. Here, we establish a new process for isophytol synthesis from microbial fermented farnesene. To achieve an efficient pathway for farnesene production, Saccharomyces cerevisiae was selected as the host strain. First, ß-farnesene synthase genes from different sources were screened, and through protein engineering and system metabolic engineering, a high production of ß-farnesene in S. cerevisiae was achieved (55.4 g/L). This farnesene can be chemically converted into isophytol in three steps with approximately 92% yield, which is economically equal to that from the best total chemical synthesis. Furthermore, we co-produced lycopene and farnesene to reduce the cost of farnesene. A factory based on this new process was successfully operated in Hubei Province, China, in 2017, with an annual output of 30,000 tons of vitamin E. This new process has completely changed the vitamin E market due to its low cost and safety.
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Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.
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A specialized method for ochratoxin A (OTA) determination on fermented teas was developed and validated using ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Methodology results showed that recovery, relative standard deviation, accuracy, and precision were qualified. The limits of detection and quantification were 0.32 and 0.96 µg/kg, respectively. Two of 158 collected samples were screened for OTA contamination. Comprehensive risk assessment based on OTA contaminations of this study and other peer-reviewed publications was performed. The highest hazard quotient (HQ) value (8.86 × 10-2) and the highest 1/MoE value (8.61 × 10-5) in probabilistic assessment were equally below the recommended non-neoplastic and neoplastic thresholds, indicating no health risks. However, the HQ and 1/MoE values of the 95th percentiles in 20-39 and ≥50 years of age were close to thresholds of 1.0 and 1.0 × 10-4, respectively. Under the extreme case, there were only a few scenarios (e.g., 40-49 years of age) of HQ values below the non-neoplastic threshold, but the 1/MoE value of each group exceeded the neoplastic threshold. This is the first extensive risk assessment on OTA from fermented teas worldwide, but the sample size is still limited, and a large number of samples is encouraged in a future study for a more accurate assessment.
Assuntos
Exposição Dietética , Ocratoxinas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Contaminação de Alimentos/análise , Ocratoxinas/análise , Medição de Risco , Espectrometria de Massas em TandemRESUMO
Biallelic mutations in the sorbitol dehydrogenase (SORD) encoding gene were recently identified as a common genetic cause in autosomal-recessive CMT patients. Here, we investigated the clinical, genetic, and electrophysiological characteristics of three CMT patients with biallelic SORD mutations from a Chinese cohort. Two patients harbored c.757delG (p.A253Qfs*27) homozygous mutations, and one patient carried both c.757delG (p.A253Qfs*27) and c.625C>T (p.R209X) compound heterozygous mutations. Interestingly, the two patients homozygous for the c.757delG mutation exhibited positive responses for pinprick test. In conclusion, we confirmed SORD mutations as causative for CMT and further expanded the mutational and phenotypic spectrum of SORD-related CMT.
Assuntos
Doença de Charcot-Marie-Tooth/genética , L-Iditol 2-Desidrogenase/genética , Adulto , Povo Asiático/genética , Feminino , Humanos , Masculino , Mutação , Adulto JovemRESUMO
Recently, mycotoxins safety in Chinese post-fermented teas has attracted much attention because it is still controversial whether environmental fungi in the teas are able to produce aflatoxins. In this study, a rapid and selective analytical method based on immunoaffinity column (IAC) cleaning coupled with liquid chromatography-mass spectrometry (LC-MS) was developed to quantify four aflatoxins (B1, B2, G1 and G2) in post-fermented teas. Recoveries ranged from 80.8 to 92.2% with relative standard deviation less than 3%. The limits of detection and quantification were 0.109-0.348 µg kg-1 and 0.364-1.159 µg kg-1, respectively. Two out of 158 samples were positive for aflatoxins examination (occurrence rate 1.27%). The deterministic assessment for the maximal aflatoxins exposure under upper bound was 9.19 × 10-6 µg kg-1 day-1 from heavy exposure consumers (≥50 year age group), but the exposure was lower than the JECFA acceptable value of 1.0 ng kg-1 day-1 on liver risk. Probabilistic assessment showed that the upper bound 95th percentile carcinogenic risk value for the 318 consumers (Yunnan China and Ulan Bator Mongolia) was 1.75 × 10-7, and for heavy exposure consumers was 2.4 × 10-7, and the values equally below the acceptable carcinogenic risk level.
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Aflatoxinas/química , Aflatoxinas/toxicidade , Chá/química , Adulto , China , Feminino , Fermentação , Contaminação de Alimentos , Humanos , Masculino , Pessoa de Meia-Idade , Medição de Risco , Adulto JovemRESUMO
Chinese dark tea is widely enjoyed for its multiple health-promoting effects and pleasant taste. However, its production involves fermentation by microbiota in raw tea, some of which are filamentous fungi and thus potential mycotoxin producers. Accordingly, whether mycotoxins pose health risk on dark tea consumption has become a public concern. In this study, a cleaning method of multi-functional column (MFC) and immunoaffinity column (IAC) in tandem combined to HPLC detection was developed and validated for determining ten mycotoxins of six groups (i.e., aflatoxins of B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, fumonisins of B1, B2, and T-2) in dark teas. The interferences from secondary metabolites were effectively reduced, and the sensitivities and recoveries of the method were qualified for tea matrices. Six groups mycotoxins were determined in 108 samples representing the major Chinese dark teas by using the new method. Subsequently, the dietary exposure and health risks were evaluated for different age and gender groups in Kunming and Pu'er in China and Ulan Bator in Mongolia. The occurrence of zearalenone was 4.63% and that of ochratoxin A was 1.85%, with the other four groups mycotoxins were below the limits of quantification. The hazard index values for the five groups' non-carcinogenic mycotoxins were far below 1.0. The deterministic risk assessment indicated no non-carcinogenic risks for dark tea consumption in the three areas. Probabilistic estimation showed that the maximum value of 95th percentile carcinogenic risk value for the aflatoxins was 2.12 × 10-8, which is far below the acceptable carcinogenic risk level (10-6). Hereby, six groups mycotoxins in Chinese dark tea showed no observed risk concern to consumers.
Assuntos
Micotoxinas , China , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Mongólia , Medição de Risco , Espectrometria de Massas em Tandem , CháRESUMO
Dry tea matrix contains an abundance of caffeine and polyphenols which are different from the food matrix (e.g., protein, lipid, and carbohydrates), and only a few studies have tried aflatoxins determination with tea samples. Here, a specific, accurate, and sensitive method was developed and validated for the simultaneous determination of aflatoxin B1, B2, G1, and G2 in dark teas. Aflatoxins were extracted by acetonitrile/water, press-filtered, and cleaned by multifunctional purification column (MFC) and immunoaffinity column (IAC) in tandem. The cleaned extract was analyzed by liquid chromatography tandem mass spectrometry. The matrix interference was effectively reduced by MFC-IAC cleaning method. Recoveries at the spiking concentrations of 5-60 µg/kg ranged from 77.5 to 93%, with relative standard deviations <11.0%. The correlation coefficients of aflatoxins standard were >0.998. The limits of detection were 0.024-0.21 µg/kg and the limits of quantification were 0.08-0.74 µg/kg. The intra- and interday accuracy ranged from 74 to 87%, and the intra- and interday precisions ranged from 0.4 to 3.1%. After the method validation, the aflatoxins contaminations in 100 collected dark teas were detected, and the results were compared with those of other methods.
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Aflatoxinas/química , Cromatografia Líquida de Alta Pressão/métodos , Contaminação de Alimentos/análise , Imunoensaio/métodos , Espectrometria de Massas em Tandem/métodos , Chá/química , Camellia sinensis/química , Folhas de Planta/químicaRESUMO
Lycopene is widely used in foods, cosmetics, nutritional supplements, and pharmaceuticals. Microbial production of lycopene has been intensively studied. However, there are few systematic engineering studies on Saccharomyces cerevisiae aimed at achieving high-yield lycopene production. In the current study, by employing a systematic optimization strategy, we screened the key lycopene biosynthetic genes, crtE, crtB, and crtI, from diverse organisms. By adjusting the copy number of these three key genes, knocking out endogenous bypass genes, increasing the supply of the precursor acetyl-CoA, balancing NADPH utilization, and regulating the GAL-inducible system, we constructed a high-yield lycopene-producing strain BS106, which can produce 310 mg/L lycopene in shake-flask fermentation, with gene expression controlled by glucose. In optimized two-stage fed-batch fermentation, BS106 produced 3.28 g/L lycopene in a 7 L fermenter, which is the highest concentration achieved in S. cerevisiae to date. It will decrease the consumption of tomatoes for lycopene extraction and increase the market supply of lycopene.