INTRODUCTION
Lycopene is a natural 40-carbon (C40H56) carotenoid widely found in fruits such as tomatoes and watermelons (Hernández-Almanza et al., 2016). As a tetraterpene compound, lycopene is composed of eight isoprene units containing 13 carbon-carbon double bonds. Lycopene mainly exists in nature in transconfiguration (Jing et al., 2022) and has great socioeconomic value in cosmetics, food, and medicine fields (Shi et al., 2019). The main methods of obtaining lycopene are natural plant extraction, chemical synthesis, and microbial fermentation currently. Because of plant extraction defects, such as low efficiency and slow growth cycle of raw materials, and chemical synthesis problems, such as complex synthesis processes and chemical reagent residue, microbial production has attracted increasing attention (Saini & Keum, 2018). As one of the most widely used microbial hosts in the fermentation industry, Saccharomyces cerevisiae has multiple advantages, such as simple nutritional requirements and mature gene editing technology (Han et al., 2023; Wang et al., 2020). Furthermore, its endogenous mevalonate (MVA) pathway offers sufficient precursors for terpenoid production. Consequently, S. cerevisiae is an ideal chassis strain for lycopene production (Xie et al., 2015).
In S. cerevisiae, as the precursor of lycopene, farnesyl pyrophosphate (FPP) is synthesized by the MVA pathway; it can be converted into lycopene by the exogenous introduction of geranylgeranyl pyrophosphate (GGPP) synthetase, phytoene synthetase, and phytoene desaturase (Hong et al., 2019). Over the past decade, the yield and efficiency of lycopene production in S. cerevisiae cell factories have greatly improved (Li et al., 2019; Ma et al., 2019; Zhou et al., 2023). For example, an S. cerevisiae strain was developed with a lycopene titer of 115.64 mg/L by enhancing the MVA metabolic pathway to increase the supply of the precursor GGPP (Li et al., 2019). By overexpressing lipid droplet seipin protein Fld1p and fatty acid desaturase Ole1p, an engineered strain was constructed with an elevated lycopene titer of 2.37 g/L (Ma et al., 2019). However, lycopene, a highly polyunsaturated lipophilic hydrocarbon, may cause membrane stress in S. cerevisiae and produce toxicity at high concentrations (Hong et al., 2019; Ma et al., 2019).
Transporter engineering is a valuable strategy for alleviating the problems of storage limitations, cytotoxicity, and feedback inhibition. In the past few years, this strategy has been used for improving the secretion of squalene, β-carotene, and other terpenoids (Bu et al., 2020; Liu et al., 2023; Xu et al., 2023). As a macromolecular terpene, lycopene is mostly stored in cells (Ma et al., 2019). Therefore, it was speculated that identifying lycopene transporters in the chassis strain and achieving its secretory production would be conducive to further improving lycopene production. ATP-binding cassette (ABC) transporters are a major family of transporters in S. cerevisiae, including exporters for product secretion and importers for nutrient absorption (Claus et al., 2019; Ford & Beis, 2019; Srikant & Gaudet, 2019). A previous study found that ABC transporters can promote the secretion of various terpenoids (Liu et al., 2023); therefore, they may be potential transporters of lycopene secretion.
In this work, a lycopene-producing S. cerevisiae strain L10 was constructed by adopting combination engineering strategies, including introducing the lycopene synthesis pathway, regulating and optimizing the copy number and integration sites of key genes, enhancing the supply of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH), and complementing the selective marker URA3. To identify transporters capable of secreting lycopene in S. cerevisiae, molecular docking simulation and overexpression verification of four endogenous transporters, Pdr5p, Snq2p, Yor1p, and Pdr10p, with lycopene were performed, and Snq2p was identified to promote lycopene secretion. The lycopene titer secreted by strain L10Z2 overexpressing SNQ2 reached 12.58 mg/L, which was 16.5-fold that of the control. After further optimization of the GAL regulatory system, the obtained strain L11Z2 was cultured in a 3-L bioreactor, and the intracellular and secreted lycopene titer reached 2113.78 and 26.28 mg/L, respectively. This study provided a novel idea to realize the secretory production of lycopene in S. cerevisiae cell factory.
MATERIALS AND METHODS
Strains and media
All yeast strains are shown in Supporting Information S1: Table 1. S. cerevisiae CEN.PK2-1C (MATa; his3D1; leu2-3_112; ura3-52; trp1-289; MAL2-8c; SUC2) was used as the background strain for all engineering. Yeast extract-peptone-dextrose (YPD) medium (2% peptone, 1% yeast extract, and 2% glucose) or synthetic complete dropout (SD) medium (2% d-glucose, lacking uracil, leucine, histidine, or tryptophan and 0.67% yeast nitrogen base without amino acids) was used for the culturing and screening of yeast strains. YPD medium containing 5-fluoroorotic acid (1 mg/mL) was used to screen yeast strains (Zhou et al., 2023). Escherichia coli strains DH5α and JM109 were used for constructing plasmids. Luria-Bertani medium (1% NaCl, 1% peptone, and 0.5% yeast extract) containing appropriate ampicillin was used to culture E. coli strains. Synthetic fermentation medium (5% peptone from soya, 2.5% glycerol, 2.5% d-glucose, 2.5% sucrose, and 0.6‰ K2HPO4) optimized from a previous study was used for yeast strains' shake-flask culture. Enriched YPD medium (2% glucose, 2% peptone, 1% yeast extract, 50 mg/L thiamine-HCl, 1‰ MgSO4 and 0.5‰ ZnSO4) was used for yeast strains' fed-batch culture.
Recombinant plasmids and yeast strains construction
The primers are listed in Supporting Information S1: Table 2. The flowchart of the strain construction process is shown in Figure 1. All primers were purchased from Azenta. The plasmids are listed in Supporting Information S1: Table 3. The heterologous genes are listed in Supporting Information S1: Table 4. All heterologous genes were codon-optimized and chemically synthesized by Azenta or GenScript. The DNA polymerase was purchased from TaKaRa (Japan), and DNA fragments were amplified by polymerase chain reaction (PCR), and DNA fragments were purified using the EZ-10 Column DNA Purification Kit (Sangon Biotech). Using the Seamless Cloning Kit (Beyotime) and the SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech) for the construction and extraction of recombinant plasmids. Then, recombinant plasmids were validated by Sanger sequencing (Azenta). Yeast strains were transformed using the lithium acetate method for gene integration, overexpression, or deletion (Gietz & Schiestl, 2007). All S. cerevisiae genome integration sites were reported before (Reider Apel et al., 2017).
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Shake-flask fermentation of yeast strains
Yeast colonies were cultured in 1.5 mL YPD or SD medium for 24 h (30°C, 220 rpm). A 250 μL preculture was inoculated into shake flasks containing 25 mL fermentation medium for 96 h (30°C, 220 rpm). 2.5 mL extractive solvent dodecane was added for the two-phase fermentation.
Extraction and yield detection of lycopene and squalene
To analyze the secreted lycopene, the fermentation broth after two-phase fermentation was centrifuged at 12,000 × g for 10 min. Then the dodecane layer (containing lycopene) was detected by high-performance liquid chromatography (HPLC). Dodecane was added at 10%(v/v) of the fermentation medium, and the secretion lycopene concentration was divided by 10 times. To analyze the intracellular lycopene and squalene, yeast cells were washed and suspended and moved into 2 mL grinding tubes, and the same volume of ethyl acetate and 0.2 g glass beads (diameter: 0.5 mm) were added. Yeast cells were broken in the FastPrep®-24 homogenizer (MP Biomedicals, USA) for six cycles (40 s, 6.0 m/s). Then the samples were centrifuged at 12,000 × g for 10 min. The ethyl acetate layer (containing squalene) was detected by HPLC. The HPLC 1260 system (Agilent) was equipped with a variable-wavelength scanning ultraviolet detector and a C18 column (4.6 × 250 mm; 5 μm; Agilent). The detection wavelengths of lycopene and squalene were 450 and 195 nm, respectively. The mobile phase of lycopene (acetonitrile/methanol/isopropanol = 5:3:2, v/v) and mobile phase (100% acetonitrile) of squalene were eluted as 1 and 1.2 mL/min at 40°C, respectively. To analyze the concentration of acetate, fermentation supernatant detection was performed via HPLC (Waters) with an Aminex® HPX-87H column (7.8 × 300 mm; Bio-Rad) and refractive index detector. The mobile phase was 5 mM H2SO4 at a flow rate of 0.6 mL/min. The standards of lycopene, squalene and acetate were purchased from Solarbio Life Sciences. The standard curves are presented in Supporting Information S1: Figure 1.
Molecular docking of ABC transporters and lycopene
The predicted structures of four ABC transporters were acquired from the AlphaFold Protein Structure Database (Tunyasuvunakool et al., 2021). The structure of lycopene was obtained from the ZINC Database (Sterling & Irwin, 2015). Molecular docking was performed using AutoDockTools 1.5.6. The structures of lycopene and all transporters were preprocessed such as deleting water molecules and adding hydrogen. The docking box was adjusted to cover the intracellular domain of each transporter. Molecular docking setting information is listed in Supporting Information S1: Table 5. The genetic algorithm and Lamarckian GA were used as the search parameter and algorithm, respectively. The optimal docking poses of molecular docking were selected based on the lycopene binding positions and binding free energies. PyMOL 1.8.0.0 was used to visualize the transporter-lycopene poses.
Fed-batch fermentation of engineered yeast strains
Single colony of the strain was cultured in 2 mL YPD medium for 24 h (30°C, 220 rpm) as seed culture. Then 1 mL seed culture was transferred into 100 mL YPD medium and cultured for 24 h (30°C, 220 rpm) as the second culture. After centrifuging, the supernatant of the second culture was discarded, and cells were resuspended using 100 mL YPD medium and transferred to a 3-L bioreactor (T&J Bio-engineering) containing 900 mL YPD medium (containing 50 mg/L thiamine-HCl, 1 g/L MgSO4 and 0.5 g/L ZnSO4) for fermentation at 30°C. The entire culture process was divided into three stages. In the first stage, the strains consumed the initial nutrients in the bioreactor. In the second stage, feeding solution I containing 800 g/L glucose, 25 g/L (NH4)2SO4, and 5 g/L MgSO4 and feeding solution II containing 400 g/L yeast extract were added to facilitate fast cell growth. The feed rate was set to maintain glucose under 1 g/L during fed-batch culture. In the third stage, yeast cells produced lycopene using ethanol as the feeding solution III. Using an M-100 biosensor analyzer to measure glucose concentration (Sieman Technology, China). Ammonium hydroxide (50%, v/v) and 2 M hydrochloric acid were fed to maintain the pH between 5.90 and 6.10. The dissolved oxygen was maintained over 30% by adjusting the airflow (1–5 vvm) and stirring speed (300–1200 rpm). The extraction solvent dodecane (200 mL) was added at an appropriate time during the two-phase fermentation.
Gene expression level analysis
Total RNA of each sample was isolated from yeast cells after 16 h culture using the RNA Extraction Kit (Vazyme). Then, total RNA was reverse-transcripted to cDNA using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme). qPCR was performed on LightCycler 480 II Real-Time PCR instrument (Roche) with AceQ qPCR SYBR Green Master Mix (Vazyme, China). Primers for qPCR are listed in Supporting Information S1: Table S2. The 18S rDNA gene was used for normalizing the different samples, and the relative expression levels were verified using the 2−ΔΔCT method.
RESULTS AND DISCUSSION
Construction and optimization of the lycopene biosynthesis pathway
The conversion of FPP to lycopene requires the catalysis of exogenous geranylgeranyl diphosphate synthase CrtE, 15-cisphytoene synthase CrtB, and phytoene dehydrogenase CrtI in S. cerevisiae (Figure 2a). To construct a lycopene-producing strain, the MVA pathway-enhanced strain Y1 previously constructed in our laboratory was used as the starting strain. Genes crtE (Taxus x media), crtB (Pantoea agglomerans), and crtI (Blakeslea trispora) were selected to construct the lycopene synthesis pathway. Meanwhile, the GAL system was selected to regulate lycopene synthesis pathway gene expression to alleviate the possible adverse effects of lycopene synthesis on the yeast cell growth in the early stage.
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First, the GAL80 gene encoding the galactose metabolism negative regulator was knocked out so that the lycopene synthesis pathway genes were only regulated by glucose (Shi et al., 2019), and the engineered strain Y2 was constructed. Then, one copy of crtE and crtI was integrated into the genome of strain Y2. Simultaneously, FPP is the precursor of lycopene and endogenous product squalene, and reducing the squalene synthesis can alleviate the competitive consumption of FPP. Since squalene is the precursor of ergosterol, an important physiological component of yeast, squalene synthase gene ERG9 cannot be knocked out (Asadollahi et al., 2008), the natural promoter of ERG9 was substituted with a PHXT1 promoter to weaken its expression. Meanwhile, one copy of crtB was integrated into the genome, and constructed a lycopene-producing strain L1. After fermentation for 96 h, the lycopene titer of L1 was 8.44 mg/L (Figure 2b). Results also showed that although the squalene content of strain L1 was noticeably lower than that of strain Y2, it still reached 628.76 mg/L, indicating that a large amount of FPP was still converted to squalene (Figure 2c). In addition, OD600 value of strain L1 was also considerably higher than that of strain Y2. The construction of the lycopene synthesis pathway and replacement of the ERG9 natural promoter may alleviate the growth and metabolic burden caused by squalene accumulation.
To reduce the conversion of FPP to squalene in strain L1, the flow of more FPP to the lycopene synthesis pathway was promoted by increasing the copy number of crtE. Studies have demonstrated that lycopene synthesis in S. cerevisiae may be limited by the desaturation step of phytoene; therefore, the copy number of crtI was optimized at the same time (Verwaal et al., 2007; Xie et al., 2015). The engineered strain L2 was constructed by adding one copy of crtE and crtI genes to the genome of strain L1. However, the intracellular lycopene titer of strain L2 was only slightly increased to 9.59 mg/L, and the OD600 decreased to 49.36. At the same time, due to the degeneration of strain L2 in subsequent batch shake-flask culture, it is speculated that the integration site of crtB gene in strain L2 may make it genetically unstable during the passage of strains. Therefore, the crtB gene was integrated into the 1021b locus of strain Y2, and two copies of crtE and crtI genes were further integrated into the 308a and 416d loci, respectively, and obtained the engineered strain L3. Results showed that the lycopene titer of strain L3 was improved to 37.35 mg/L, and the OD600 value was 89.90 (Figure 2d).
Subsequently, the copy numbers of crtE and crtI were further increased in strain L3. The OD600 value of strain L4 (integrated 3 copies of crtE and crtI genes) was significantly lower than that of strain L3, but the intracellular lycopene titer further increased to 63.14 mg/L (Figure 2e). The OD600 value of strain L5 (integrated 4 copies of crtE and crtI genes) was further decreased to 19.49, and the intracellular lycopene titer of strain L5 was also significantly decreased compared with strain L4 (Figure 2e). It indicated that integrating three copies of crtE and crtI genes may be the most favorable option for lycopene synthesis, and further increase in crtE and crtI expression levels may lead to the accumulation of intermediates, thus inhibiting cell growth and disrupting metabolic flux balance. Because the increase in crtE and crtI gene copy numbers failed to further increase lycopene titer, it was speculated that 15-cisphytoene synthase CrtB deficiency may be the key step for limiting lycopene production. To promote the efficient conversion of GGPP to phytoene, a mutant of phytoene synthase CrtYB (Phaffia rhodozyma), CrtYB01M (W61R), with higher catalytic activity (Xie et al., 2015), was selected to construct the engineered strain L6. Results showed that the lycopene titer of strain L6 substantially elevated to 234.40 mg/L (Figure 2e).
Regulating cofactor supply, reducing squalene accumulation and URA3 complementation to enhance lycopene production
In S. cerevisiae, there was a strong correlation between engineered metabolic pathways and intracellular metabolism (Shi et al., 2019). Due to NADPH consumption by the MVA pathway, a cofactor and metabolic flux imbalance may be caused in engineered strains. Therefore, one copy of the NADH kinase coding gene POS5 was added to strain L6 to enhance the NADPH supply. Studies have shown that the knockout of glucan 1,3-β-glucosidase coding gene EXG1 can effectively improve lycopene production; hence, the EXG1 was simultaneously knocked out, and the strain L7 was constructed (Figure 3a). Results showed that the lycopene titer of strain L7 improved to 303.72 mg/L, and the OD600 value of strain L7 was significantly higher compared with strain L6 (Figure 3b). To determine the carbon flux distribution, the squalene titer in strain L7 was detected. Interestingly, unlike strain L1, the intracellular squalene titer of strain L7 did not decrease but increased to 702.01 mg/L (Supporting Information S1: Figure 2). It was speculated that the increase in NADPH supply can also promote squalene synthesis.
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Because CrtE is a crucial rate-limiting enzyme for the conversion of FPP to GGPP, this study attempted to further optimize the copy number of crtE to promote an extra increase in lycopene production (Zhou et al., 2023). TY1 is a transposon present in S. cerevisiae, and its replication cycle could be used in metabolic engineering to achieve multicopy integration of genes (Qu et al., 2022). Therefore, the crtE gene was inserted into the intron site of TY1 in strain L7, and the constructed strain was named L8. The lycopene titer of strain L8 was further elevated to 475.53 mg/L, whereas the squalene titer also significantly decreased to 183.52 mg/L, indicating that the crtE copy number increase effectively reduced the carbon flux flow toward squalene synthesis (Figure 3c,d).
S. cerevisiae produces a large amount of ethanol as a by-product during fermentation, owing to the Crabtree effect (Yao et al., 2023). Thus, the coding gene of acetyl-CoA hydrolase Ach1p was knocked out, and ethanol dehydrogenase Adh2p, acetyl-CoA synthetase Acs1p, and acetaldehyde dehydrogenase Ald6p were enhanced to improve ethanol consumption and increase acetyl-CoA supply in strain L8 (Supporting Information S1: Figure 3a) (Ma et al., 2019; Su et al., 2022). Nevertheless, the fermentation results showed that the lycopene titer and OD600 of the obtained strain L9 were drastically reduced ((Supporting Information S1: Figure 3b), and the fermentation liquid had a distinct sour taste. As ethanol can be converted to acetate by Adh2p and Ald6p, the acetate titers of strains L8 and L9 were detected, and the results showed that the acetate titer in the fermentation supernatant of L9 was significantly increased to 10.42 g/L ((Supporting Information S1: Figure 3c). It was speculated that the overexpression of Adh2p and Ald6p promoted the conversion of ethanol to acetate, but the insufficient supply of ATP restricted the conversion of acetate to acetyl-coA, and the accumulated acetate inhibited the cell growth and synthesis of lycopene.
The riboside 5′-phosphate (OMP) decarboxylase Ura3p is responsible for the conversion of OMP to uracil nucleotide (UMP). Because UMP is an essential nucleotide for the growth of S. cerevisiae, the growth of yeast strains with URA3 deletion requires a medium containing uracil or uridine. Therefore, URA3 is typically used as a genetic marker for yeast (Li et al., 2023; Moon et al., 2022; Zhou et al., 2023). Previous studies have shown that Ura3p can also increase a strain's tolerance to ethanol, which may positively affect S. cerevisiae fermentation to produce secondary metabolites (Guo et al., 2022; Pais et al., 2013). Based on strain L8, strain L10 was obtained by URA3 complementation. Results revealed that the lycopene titer of engineered strain L10 was further enhanced than that of control strain L8 after fermentation for 96 h, reaching 597.80 mg/L, and its OD600 value also increased to 55.94 (Figure 3e). It also suggested that the complementation of the URA3 gene could also facilitate yeast cell growth and promote lycopene synthesis to a greater extent as expected.
Prediction and identification of the lycopene transporters
After constructing the lycopene-producing strain L10, the potential transporters of lycopene were screened. The currently reported S. cerevisiae transporter information was obtained from the TransportDB2 database (Elbourne et al., 2017). Results showed that 343 transporters were identified, belonging to 51 subtypes in five classes. A previous study found that ABC transporters promote the secretion of diterpenoids, triterpenoids, and tetraterpenoids (Liu et al., 2023). Four ABC transporters Pdr5p, Snq2p, Yor1p, and Pdr10p located in the plasma membrane of S. cerevisiae were chosen as potential transporters of lycopene for molecular docking prediction. The consequences of molecular docking showed that the internal channels of all the above four transporters could accommodate and bind to lycopene, with Snq2p having the lowest binding free energy with lycopene (only −7.30 kcal/mol). The binding free energies of Pdr5p, Yor1p, and Pdr10p with lycopene were −6.00, −4.21, and −5.76 kcal/mol, respectively, indicating that Snq2p was the most likely to bind and secrete lycopene among the four transporters (Figure 4a).
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To verify the lycopene secretion function of the above transporters, the four predicted transporter coding genes were overexpressed in strain L10, and the engineered strains L10Z1–L10Z4 were obtained. The improved expression levels of these genes were confirmed by qPCR ((Supporting Information S1: Figure 4). Because lycopene is a hydrophobic compound with easy degradation properties and its solubility is low in the aqueous environment of the fermentation solution, two-phase fermentation was performed by adding dodecane as the extractant to detect the extracellular lycopene content. After two-phase fermentation for 96 h, the results showed that strain L10Z2, which overexpressed SNQ2, had the most obvious improvement on lycopene secretory production, and its extracellular lycopene secretion reached 12.58 mg/L, 16.5 times that of strain L10 (0.76 mg/L), which could also be observed from the color of the dodecane layer (Figure 4b–d). There was no significant increase in extracellular lycopene in engineered strains that overexpressed the other three transporters. Results were also consistent with molecular docking predictions that Snq2p can promote lycopene secretion. There was a slight decrease in the OD600 value of strain L10Z2 compared with L10.
Fed-batch culture of engineered strains
Next, to evaluate its production performance, strain L10Z2 was cultured in a 3-L bioreactor. However, strain L10Z2 could not achieve a significant increase in biomass and lycopene yield during fed-batch fermentation. The intracellular lycopene yield only slightly improved to 610.36 mg/L, and the secreted lycopene titer was only 7.32 mg/L, and OD600 reached 109.07 ((Supporting Information S1: Figure 5). Because lycopene accumulation could be detected in the sample taken at 6 h, it was speculated that the leakage expression of PGAL1,10 and PGAL7 promoters after GAL80 knockout caused the poor decoupling effect between yeast cell growth and lycopene synthesis, which may be the reason for limiting the lycopene production increase of L10Z2 in fed-batch fermentation. To cut off galactose consumption and restore the galactose induction performance in strain L10Z2, coding genes of galactokinase Gal1p, galactose-1-phosphate uridylyl-transferase Gal7p, and UDP-glucose-4-epimerase Gal10p were knocked out in strain L10Z2, and the GAL80 gene was also complemented. Meanwhile, to improve the induction effect of galactose, an additional copy of the regulatory protein gene GAL3 was integrated into the genome of L10Z2. Subsequently, the obtained strain L11Z2 was cultured in a 3-L bioreactor. After 6 h of culture, glucose in the initial medium was exhausted. At 8 h, feeding solutions I and II were added to maintain the rapid yeast cell growth, and the OD600 exceeded 200 at 30 h. At 33 h, the galactose concentration was adjusted to 25 g/L using the preprepared galactose solution (500 g/L) to activate lycopene synthesis, and the feeding solution was switched to solution III at 42 h to facilitate further production of lycopene. Meanwhile, 200 mL dodecane was added as an extraction solvent. During the whole fermentation process, the lycopene yield continued to increase, and the maximum value of OD600 (247.93) was obtained at 54 h. As the total volume of fermentation liquid increased due to the adding of feeding solutions, the OD600 decreased slightly but was maintained at >200. After 150 h of fermentation, the total volume of fermentation liquid in the bioreactor reached 2.2 L, and the intracellular and secreted lycopene content reached 2113.78 and 26.28 mg/L, respectively (Figure 5a–c). As a macromolecular lipophilic product, lycopene is stored in lipid droplets in S. cerevisiae to avoid its associated toxicity (Ma et al., 2019). It was speculated that the storage of lycopene by lipid droplets may limit lycopene secretion to extracellular space. Additionally, the transporter Snq2p requires energy through ATP hydrolysis in the process of binding and secreting the substrate (Godinho et al., 2018; Mahe et al., 1996). Moreover, the production of acetyl CoA from ethanol is also accompanied by the consumption of ATP (Zhang et al., 2023). Insufficient ATP supply may be another factor affecting the lycopene secretion level in L11Z2. However, given the current low level of lycopene secretion, ATP restriction may not be the main factor. Therefore, in future studies, enhancing lycopene transport in lipid droplet-cytoplasm-membrane and increasing intracellular ATP supply may be conducive to further improving the secretion of lycopene.
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CONCLUSIONS
A new platform yeast cell factory was established to produce lycopene using metabolic engineering strategies, such as regulating the copy number of key enzymes, introducing key enzyme mutants, and increasing NADPH supply in this work. In combination with molecular docking and overexpression verification, the endogenous transporter Snq2p was identified to promote lycopene secretion. The intracellular and secreted lycopene titer of the optimized strain reached 2113.78 and 26.28 mg/L, respectively, in the two-phase fermentation of a 3-L bioreactor. In summary, this work offers guidance and a basis for industrial production and further increase in the natural tetraterpene lycopene.
AUTHOR CONTRIBUTIONS
Jiaheng Liu: Methodology; investigation; writing—original draft; software; data curation. Minxia Song: Methodology; investigation. Xianhao Xu: Formal analysis; investigation. Yaokang Wu: Supervision. Yanfeng Liu: Supervision. Guocheng Du: Supervision; project administration. Jianghua Li: Project administration. Long Liu: Conceptualization; project administration; funding acquisition. Xueqin Lv: Writing—review and editing; project administration; supervision; conceptualization.
ACKNOWLEDGMENTS
The authors are thankful for the support from the National Key Research and Development Program of China (2022YFC3401303).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
ETHICS STATEMENT
None declared.
Asadollahi, M. A., Maury, J., Møller, K., Nielsen, K. F., Schalk, M., Clark, A., & Nielsen, J. (2008). Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnology and Bioengineering, 99(3), 666–677.
Bu, X., Lin, J., Cheng, J., Yang, D., Duan, C., Koffas, M., & Yan, G. (2020). Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of beta‐carotene in Saccharomyces cerevisiae. Biotechnology for Biofuels, 13(168), 1–14.
Claus, S., Jezierska, S., & Van Bogaert, I. N. A. (2019). Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane. FEBS Letters, 593(13), 1508–1527.
Elbourne, L. D. H., Tetu, S. G., Hassan, K. A., & Paulsen, I. T. (2017). TransportDB 2.0: a database for exploring membrane transporters in sequenced genomes from all domains of life. Nucleic Acids Research, 45, D320–D324.
Ford, R. C., & Beis, K. (2019). Learning the ABCs one at a time: structure and mechanism of ABC transporters. Biochemical Society Transactions, 47, 23–36.
Gietz, R. D., & Schiestl, R. H. (2007). Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2(1), 35–37.
Godinho, C. P., Dias, P. J., Ponçot, E., & Sá‐Correia, I. (2018). The paralogous genes PDR18 and SNQ2, encoding multidrug resistance ABC transporters, derive from a recent duplication event, PDR18 being specific to the Saccharomyces genus. Frontiers in Genetics, 9, 476.
Guo, W., Ai, L., Hu, D., Chen, Y., Geng, M., Zheng, L., & Bai, L. (2022). [URA3 affects artemisinic acid production by an engineered Saccharomyces cerevisiae in pilot‐scale fermentation]. Sheng wu gong cheng xue bao = Chinese Journal of Biotechnology, 38(2), 737–748.
Han, L., Wu, Y., Xu, Y., Zhang, C., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). Engineered Saccharomyces cerevisiae for de novo δ‐tocotrienol biosynthesis. Systems Microbiology and Biomanufacturing, 4, 150–164.
Hernández‐Almanza, A., Montañez, J., Martínez, G., Aguilar‐Jiménez, A., Contreras‐Esquivel, J. C., & Aguilar, C. N. (2016). Lycopene: progress in microbial production. Trends in Food Science & Technology, 56, 142–148.
Hong, J., Park, S. H., Kim, S., Kim, S. W., & Hahn, J. S. (2019). Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production. Applied Microbiology and Biotechnology, 103(1), 211–223.
Jing, Y., Wang, Y., Zhou, D., Wang, J., Li, J., Sun, J., Feng, Y., Xin, F., & Zhang, W. (2022). Advances in the synthesis of three typical tetraterpenoids including β‐carotene, lycopene and astaxanthin. Biotechnology Advances, 61, 108033.
Li, S., Fu, W., Su, R., Zhao, Y., & Deng, Y. (2023). Producing malonate in Saccharomyces cerevisiae via the β‐alanine pathway. Systems Microbiology and Biomanufacturing, 3, 328–338.
Li, X., Wang, Z., Zhang, G., & Yi, L. (2019). Improving lycopene production in Saccharomyces cerevisiae through optimizing pathway and chassis metabolism. Chemical Engineering Science, 193, 364–369.
Liu, J., Wang, X., Jin, K., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). In silico prediction and mining of exporters for secretory bioproduction of terpenoids in Saccharomyces cerevisiae. ACS Synthetic Biology, 12(3), 863–876.
Ma, T., Shi, B., Ye, Z., Li, X., Liu, M., Chen, Y., Xia, J., Nielsen, J., Deng, Z., & Liu, T. (2019). Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high‐yield production of lycopene. Metabolic Engineering, 52, 134–142.
Mahé, Y., Parle‐McDermott, A., Nourani, A., Delahodde, A., Lamprecht, A., & Kuchler, K. (1996). The ATP‐binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: A novel target for the transcription factors Pdr1 and Pdr3. Molecular Microbiology, 20(1), 109–117.
Moon, H. Y., Sim, G. H., Kim, H. J., Kim, K., & Kang, H. A. (2022). Assessment of Cre‐lox and CRISPR‐Cas9 as tools for recycling of multiple‐integrated selection markers in Saccharomyces cerevisiae. Journal of Microbiology, 60(1), 18–30.
Pais, T. M., Foulquié‐Moreno, M. R., Hubmann, G., Duitama, J., Swinnen, S., Goovaerts, A., Yang, Y., Dumortier, F., & Thevelein, J. M. (2013). Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol levels of cell proliferation in yeast. PLoS Genetics, 9(6), e1003548.
Qu, L., Xiu, X., Sun, G., Zhang, C., Yang, H., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2022). Engineered yeast for efficient de novo synthesis of 7‐dehydrocholesterol. Biotechnology and Bioengineering, 119(5), 1278–1289.
Reider Apel, A., d'Espaux, L., Wehrs, M., Sachs, D., Li, R. A., Tong, G. J., Garber, M., Nnadi, O., Zhuang, W., Hillson, N. J., Keasling, J. D., & Mukhopadhyay, A. (2017). A Cas9‐based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Research, 45(1), 496–508.
Saini, R. K., & Keum, Y. S. (2018). Carotenoid extraction methods: a review of recent developments. Food Chemistry, 240(2), 90–103.
Shi, B., Ma, T., Ye, Z., Li, X., Huang, Y., Zhou, Z., Ding, Y., Deng, Z., & Liu, T. (2019). Systematic metabolic engineering of Saccharomyces cerevisiae for lycopene overproduction. Journal of Agricultural and Food Chemistry, 67(40), 11148–11157.
Srikant, S., & Gaudet, R. (2019). Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters. Nature Structural & Molecular Biology, 26(9), 792–801.
Sterling, T., & Irwin, J. J. (2015). ZINC 15‐Ligand discovery for everyone. Journal of Chemical Information and Modeling, 55, 2324–2337.
Su, B., Lai, P., Yang, F., Li, A., Deng, M. R., & Zhu, H. (2022). Engineering a balanced acetyl coenzyme A metabolism in Saccharomyces cerevisiae for lycopene production through rational and evolutionary engineering. Journal of Agricultural and Food Chemistry, 70(13), 4019–4029.
Tunyasuvunakool, K., Adler, J., Wu, Z., Green, T., Zielinski, M., Žídek, A., Bridgland, A., Cowie, A., Meyer, C., Laydon, A., Velankar, S., Kleywegt, G. J., Bateman, A., Evans, R., Pritzel, A., Figurnov, M., Ronneberger, O., Bates, R., Kohl, S. A. A., … Hassabis, D. (2021). Highly accurate protein structure prediction for the human proteome. Nature, 596, 590–596.
Verwaal, R., Wang, J., Meijnen, J. P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High‐level production of beta‐carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350.
Wang, Z., Sun, J., Yang, Q., & Yang, J. (2020). Metabolic engineering Escherichia coli for the production of lycopene. Molecules, 25(14), 3136.
Xie, W., Lv, X., Ye, L., Zhou, P., & Yu, H. (2015). Construction of lycopene‐overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metabolic Engineering, 30, 69–78.
Xu, Y., Han, L., Cheng, Y., Zhang, Y., Wu, Y., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2023). ATP‐binding cassette exporter PDR11‐mediated terpenoid secretion in engineered yeast. ACS Synthetic Biology, 12(4), 1146–1153.
Yao, Z., Guo, Y., Wang, H., Chen, Y., Wang, Q., Nielsen, J., & Dai, Z. (2023). A highly efficient transcriptome‐based biosynthesis of non‐ethanol chemicals in Crabtree negative Saccharomyces cerevisiae. Biotechnology for Biofuels and Bioproducts, 16(1), 37.
Zhang, Y., Wang, W., Wei, W., Xia, L., Gao, S., Zeng, W., Liu, S., & Zhou, J. (2023). Regulation of ethanol assimilation for efficient accumulation of squalene in Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 71, 6389–6397.
Zhou, K., Yu, C., Liang, N., Xiao, W., Wang, Y., Yao, M., & Yuan, Y. (2023). Adaptive evolution and metabolic engineering boost lycopene production in Saccharomyces cerevisiae via enhanced precursors supply and utilization. Journal of Agricultural and Food Chemistry, 71, 3821–3831.
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Abstract
Lycopene is a high‐value‐added tetraterpenoid, which is widely used in cosmetics, medicine, food, and dietary supplements. The intracellular mevalonate pathway of Saccharomyces cerevisiae provides natural precursors for terpenoid product synthesis, so it is an excellent host for the heterologous production of lycopene. In this study, a recombinant strain named L10 with efficient lycopene production capability was constructed through multiple strategies, such as regulating the gene copy number of key enzymes, increasing nicotinamide adenine dinucleotide phosphate supply, and reducing squalene accumulation. Then, considering that intracellular lycopene accumulation can cause cytotoxicity to S. cerevisiae, we attempted to identify a transporter that can efficiently transport lycopene from intracellular to extracellular space. Molecular docking simulations predicted that the ATP‐binding cassette transporter Snq2p may be a potential transporter of lycopene, and its function in promoting lycopene secretion was further determined by overexpression verification. The lycopene secretion titer of the strain L10Z2 overexpressing Snq2p increased to 16.5 times that of the control at the shake‐flask level. After optimizing the galactose regulation system, the intracellular and secreted lycopene production of L11Z2 reached 2113.78 and 26.28 mg/L, respectively, after 150 h fed‐batch culture in a 3‐L bioreactor. This work provides a new research direction for efficient lycopene synthesis in S. cerevisiae cell factory.
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1 Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China, Science Center for Future Foods, Ministry of Education, Jiangnan University, Wuxi, China, Food Laboratory of Zhongyuan, Jiangnan University, Wuxi, China
2 Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China, Science Center for Future Foods, Ministry of Education, Jiangnan University, Wuxi, China