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. 2025 Mar 12;12(1):19. doi: 10.1186/s40643-025-00858-9

System metabolic engineering modification of Saccharomyces cerevisiae to increase SAM production

Liangzhuang Tan 1,2,3, Yuehan Zhang 1,2,3, Ping Liu 1,2,3, Yihang Wu 1,2,3, Zuoyu Huang 1,2,3, Zhongce Hu 1,2,3, Zhiqiang Liu 1,2,3, Yuanshan Wang 1,2,3,✉, Yuguo Zheng 1,2,3
PMCID: PMC11904041  PMID: 40072693

Abstract

S-adenosyl-L-methionine (SAM) is an important compound with significant pharmaceutical and nutraceutical applications. Currently, microbial fermentation is dominant in SAM production, which remains challenging due to its complex biosynthetic pathway and insufficient precursor availability. In this study, a multimodule engineering strategy based on CRISPR/Cas9 was established to improve the SAM productivity of Saccharomyces cerevisiae. This strategy consists of (1) improving the growth of S. cerevisiae by overexpressing the hxk2 gene; (2) enhancing the metabolic flux toward SAM synthesis by upregulating the expression of the aat1, met17, and sam2 genes and weakening the synthesis pathway of L-threonine; (3) elevating precursor ATP synthesis by introducing the vgb gene; (4) blocking the SAM degradation pathway by knocking out the sah1 and spe2 genes. The SAM titer of the resulting mutant AU18 reached 1.87 g/L, representing an increase of 227.67% compared to the parental strain. With optimal medium, SAM titer of mutant AU18 reached 2.46 g/L in flask shake fermentation. The SAM titer of mutant AU18 further reached 13.96 g/L after 96 h incubation with a continuous L-Met feeding strategy in a 5 L fermenter. Therefore, with comprehensive optimization of both synthesis and degradation pathways of SAM, a multimodule strategy was established, which significantly elevated the SAM production of S. cerevisiae. This laid a foundation for the construction of hyperproducer for SAM and other valuable amino acids or chemicals.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40643-025-00858-9.

Keywords: S-adenosyl-L-Methionine, Saccharomyces cerevisiae, CRISPR/Cas9 system, Combinatorial metabolic engineering, Fed-batch fermentation, TCA cycle

Graphical Abstract

graphic file with name 40643_2025_858_Figa_HTML.jpg

Supplementary Information

The online version contains supplementary material available at 10.1186/s40643-025-00858-9.

Introduction

Ubiquitous presents in all organisms, S-adenosyl-L-methionine (SAM) is the second most abundant cofactor in nature. It plays a critical role in methylation, sulfurylation, and aminopropylation processes (Darroudi and Ziarani 2021; Huang et al. 2022; Pietrocola et al. 2019; Xuan et al. 2024). SAM exhibits significant efficacy in the treatment of diseases such as liver disease (Bian et al. 2021), depression (Girone et al. 2023), and cancer (Mathes et al. 2024). It has been also approved as a prescription dietary supplement (Chu et al. 2013). Therefore, the demand for SAM increases rapidly. Currently, as an environment benign, cost-effective, and sustainable method, microbial fermentation is dominant in SAM production compared chemical synthesis and enzymatic catalysis (Mamontova et al. 2018; Ravi et al. 2014; Wang et al. 2024a; Yu and Zhu 2017).

Due to its high SAM accumulation capacity and safety (classified as generally recognized as safe organism by Food and Drug Administration of USA) (Lian et al. 2018), Saccharomyces cerevisiae is widely used for SAM production. Various metabolic engineering strategies have been employed to enhance SAM titer in S. cerevisiae, including the improvement of biosynthetic pathways, the weakening of competing pathways, and the disruption of degradation pathways (Li et al. 2023). The details of these strategies are as follows: (1) Optimizing the synthesis pathway of SAM. This involves not only enhancing the SAM synthesis pathways but also attenuating competing pathways. Chen et al. (2016a) enhanced the SAM biosynthesis pathway in S. cerevisiae CGMCC2842 by co-expressing met6 and sam2 genes. The SAM titer of the resulting engineered strain YGSPM reached 1.55 g/L, increased by 234% compared to the parental strain. Chen et al. (2016b) also blocked the GYC (glyoxylate cycle) branch pathway by disrupting mls1 gene and enhanced conversion of acetic acid to acetyl-CoA by overexpressing acs2 gene in S. cerevisiae 2842. SAM titer of the resulting mutant Ymls1∆ reached 1.52 g/L. Fu et al. (2023) mutated the enzyme encoded by the sam2 gene and generated the variant SAM2S203F, W164R, T251S, Y285F, S365R. This variant exhibited almost 2-fold higher catalytic activity as the parental enzyme and was subsequently introduced into the S. cerevisiae strain BY4741. The SAM titer of the recombinant strain BSM7 reached 613.52 mg/L, which was 2.9-fold higher than that of the control. With further pathway optimization (knocking out the sah1 and glc3 genes) and fermentation process optimization, the SAM titer of the resulting mutant BSM8 in shake-flask fermentation reached 1.25 g/L, which was 7-fold higher than that of the control. (2) Increasing ATP supply: ATP plays a crucial role in biosynthesis, metabolic regulation, and cellular homeostasis. In addition, ATP serves as both a precursor and an energy donor for SAM synthesis (Chen 2020; Man et al. 2020). Therefore, enhancing ATP availability is essential for improving SAM production. Wang et al. (2024b) enhanced the acetyl-CoA supply of S. cerevisiae by knocking out the mls1 gene, resulting into an increase in ATP availability and an 8.92% increase in SAM titer of the resulting engineered strain. Hu et al. (2023) enhanced the respiratory activity of S. cerevisiae by knocking out the Cu/Zn superoxide dismutase encoding gene sod1, resulting in a 22.3% increase in SAM titer of the resulting engineered strain. (3) Blocking the catabolic pathway of SAM: Because of SAM is the primary methyl donor in living organisms, disrupting methylation reactions may promote the accumulation of SAM. The 24-sterol C-methyltransferase (encoded by erg6) catalyzes the formation of SAH (S-adenosyl-L-homocysteine) from SAM in S. cerevisiae. Xiao et al. (2024) knocked out erg6 gene in S. cerevisiae and resulted in a 10.39% increase in SAM titer of the resulting engineered strain C26P. Wang et al. (2024b) disrupted the sah1 gene of mutant SC04, led to the excessive accumulation of SAH, thereby enhanced feedback inhibition and reduced further metabolism of SAM. The SAM titer of mutant SC05 reached 1.55 g/L, resulting in a 313.64% increase compared to the parental strain.

In addition to metabolic modification, mutagenesis breeding is also an important strategy for enhancing SAM production. Hu et al. (2024) obtained a mutant 616-19-5 through UV (ultraviolet) mutagenesis in conjunction with nystatin/sinefungin and high-throughput screening. The SAM titer of mutant 616-19-5 reached 1.39 g/L, resulting in a 329% increase compared to the parental strain. Furthermore, Weng et al. (2022) obtained a high SAM producing mutant T11-1 utilizing a combination of ARTP (atmospheric and room temperature plasma) and UV-LiCl mutagenesis, followed by droplet microfluidic cultivation (with ethionine, L-Met, nystatin and cordycepin as screening agents). The SAM titer of mutant T11-1 reached 10.72 g/L in a 5 L fermenter. Huang et al. (2012) obtained a mutant H5M147 by space mutagenesis of S. cerevisiae H5 and further constructed an engineered mutant H5MR83 by integrating sam2 gene into the genome of H5M147. The SAM titer of mutant H5MR83 reached 9.64 g/L in a 50 L fermenter, resulting in a 537% increase compared to the parental strain H5. Furthermore, it is well known that the components of fermentation medium play a significant role in the growth of the strain and the synthesis of products. Therefore, medium optimization is an effective approach to enhance SAM production. Fu et al. (2023) optimized the fermentation medium components using an orthogonal experimental strategy and elevated the SAM titer of strain BSM8 from 0.66 to 0.78 g/L in shake flask fermentation. Xiao et al. (2024) optimized the L-Met feeding strategy using a single-factor approach and elevated the SAM titer of strain C262P6S from 1.55 to 1.77 g/L. A Bayesian optimization strategy was further applied to optimize the medium composition and elevated the SAM titer to 2.97 g/L in shake flask fermentation. Wang et al. (2024b) used a single-factor experiment to optimize both the medium composition and L-Met feeding strategy, which increased the SAM titer of strain SC06 from 0.24 to 0.47 g/L in shake flask fermentation.

Despite these advancements (Table 1), microbial production of SAM still faces challenges. The metabolic pathway of SAM in S. cerevisiae is extremely sophisticated, often necessitating enhancement of primary biosynthetic routes. However, investigations on enhancing the synthesis pathway of SAM mainly focus on the metabolic pathway from L-aspartic acid to SAM, while neglecting the crucial node where this pathway competes with the TCA (tricarboxylic acid) cycle for oxaloacetate. Furthermore, modifications to competitive pathways have mainly focused on weakening the L-cystathionine branch with limited attention given to other branches, such as the ornithine and L-threonine pathways. In addition, the supply of ATP is a bottlenecking factor for the synthesis of SAM, while the introduction of Vitreoscilla hemoglobin (VHb, encoded by vgb) has been shown to increase intracellular oxygen level and promote ATP synthesis (Stark et al. 2012). However, introduction of vgb gene into S. cerevisiae, which might improve the SAM synthesis efficiency, has been seldomly investigated.

Table 1.

Research progress on the synthesis of SAM in S. cerevisiae over the past five years

Strains Strategies Titer (g/L) Source
S. cerevisiae 616-4-7 UV mutagenesis 10.92 (Hu et al. 2024)
S. cerevisiae SC06 Knocking out lsc2 and mls1 1.25 (Wang et al. 2024b)
S. cerevisiae C262P6S Combinatorial metabolic engineering and Bayesian optimization 2.97 (Xiao et al. 2024)
S. cerevisiae WT15-33 Knocking out sod1 and optimization of fermentation process 10.1 (Hu et al. 2023)
S. cerevisiae ZY1-5 ARTP and UV-LiCl mutagenesis, optimization of fermentation process 10.72 (Weng et al. 2022)
S. cerevisiae CGMCC 2842 Knocking out reg1 and overexpressing snf1 8.28 (Chen et al. 2022)
S. cerevisiae BY4742 Overexpressing snz3, rfc4, and rps18b 0.9 (Dong et al. 2021)
S. cerevisiae CGMCC 2842 Knocking out kcs1 and arg82 8.86 (Chen et al. 2021)
S. cerevisiae CGMCC 13,760 Optimization of fermentation medium 16.14 (Li et al. 2020)
S. cerevisiae AU18 System metabolic engineering modification and optimization of fermentation process 13.98 This study
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In this study, several metabolic engineering strategies were employed to enhance SAM production in S. cerevisiae AU (Fig. 1). The hxk2 gene was firstly overexpressed to improve growth of the strain. Subsequently, the L-threonine branch pathway was weakened, and the SAM synthesis pathway was strengthened to redirect carbon flux toward SAM biosynthesis. Additionally, the vgb gene was introduced to improve ATP supply. To further enhance SAM accumulation, the SAM degradation pathways were disrupted by knocking out the spe2 and sah1 genes. Finally, the shake flask fermentation medium was optimized to assess the nutritional preferences of the mutant AU18, and its performance was subsequently verified in a 5 L fermenter. This study systematically manipulated the metabolic pathways involved in SAM biosynthesis in S. cerevisiae, providing a foundation for the development of a hyperproducer of SAM.

Fig. 1.

Fig. 1

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SAM biosynthetic pathway from glucose in S. cerevisiae. The orange background represents TCA cycle, while the blue background indicates the steps of SAM biosynthesis. Black solid arrows indicate native pathways, dashed arrows denote multistep reactions, red font arrows show gene knockout, and green font arrows represent gene overexpression

Materials and methods

Strains and culture conditions

The mutant S. cerevisiae AU was maintained in our laboratory and served as the parental strain for further genome editing. E. coli DH5α was used as the cloning host for plasmid and fragment construction. Details of all strains used in this study were listed in Table 2. S. cerevisiae was incubated in YPD (yeast peptone dextrose) agar (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, 20 g/L agar) plate at 30 ℃ for 48 h. Single colonies were inoculated into test tubes containing 5 mL of YPD medium and incubate at 30 ℃ and 220 rpm for 12 h. E. coli DH5α was incubated in LB (Luria Bertani) medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract) at 37 ℃ and 220 rpm for 8 h.

Table 2.

Strains used or constructed in this study

Strains Descriptions Source
E. coli DH5α Cloning host Tsingke, China
AU Starting strain S. cerevisiae Lab collection
AU01 AU harboring plasmid pESC-hxk2 This study
AU02 AU01 deleting ura4 This study
AU03 AU01, Δthr1∷PTEF-hxk2-TTEF This study
AU04 AU01 deleting cys4 This study
AU05 AU03 harboring plasmid pESC-aat1 This study
AU06 AU03 harboring plasmid pESC-hom3 This study
AU07 AU03 harboring plasmid pESC- hom2 This study
AU08 AU03 harboring plasmid pESC- hom6 This study
AU09 AU03 harboring plasmid pESC-met2 This study
AU10 AU03 harboring plasmid pESC-met 17 This study
AU11 AU03 harboring plasmid pESC- met6 This study
AU12 AU03 harboring plasmid pESC-sam2 This study
AU13 AU03, ΔGal80∷PTEF-sam2-TTEF-PTEF-met17-TTEF-TTEF-PTEF-aat1-TTEF This study
AU14 AU13, Δthr1∷PTEF-vgb-TTEF This study
AU15 AU14, Δerg6 This study
AU16 AU14, Δsah1 This study
AU17 AU14, Δspe2 This study
AU18 AU14, Δsah1∷PTEF-vgb-TTEF, Δspe2∷PTEF-aat1-TTEF This study
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Knocking out genes

The CRISPR/Cas9 gene editing system (containing Cas9, sgRNA, and donor DNA) was used to induce gene deletion and substitutive mutations in the strains (Laughery and Wyrick 2019). Here, the process of gene thr1 deletion was presented as an example. Construction of sgRNA: Firstly, the PAM site of the thr1 gene was identified using the Benchling website (https://www.benchling.com/). The primers were designed using SnapGene software and the PAM site in the sgRNA was replaced by PCR. The PCR product was then transformed into E. coli DH5α, which were plated onto LB agar plates supplemented with 100 µg/mL ampicillin. After 14 h of incubation, positive sgRNAs were identified by colony PCR and sequencing. Construction of donor DNA: Homologous arms (500 bp) on either side of the thr1 gene were amplified based on the gene sequence, and these fragments were then fused to create the donor-thr1 (1000 bp) (Fig. S1). Subsequently, the Cas9, sgRNA, and donor-thr1 were transformed into S. cerevisiae via the LiAc/SS carrier DNA/PEG transformation method (Gietz 2014). The transformed cells were plated onto YPD agar plates containing G418 (500 µg/mL) and Hygromycin B (300 µg/mL) to screen positive transformants. Then colony PCR with validation primers were performed to screen thr1 gene knocking out strains. A list of all primers used in this study is provided in Table S1 of the supporting information.

Overexpressing genes

Gene overexpression can be achieved through plasmid-based overexpression and integration of the target gene into the host strain. Using the overexpression of sam2 as example, the specific steps for both methods were described as follows: (1) For plasmid-based overexpression: The sam2 gene was first amplified from the genome of S. cerevisiae BY4741. The amplified sam2 fragment was then ligated into a linearized pESC plasmid using the ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). This ligation mixture was transformed into E. coli DH5α to construct the pESC-TEF-sam2 plasmid (Fig. S2). The resulting plasmid was subsequently transformed into S. cerevisiae via the LiAc/SS carrier DNA/PEG method (Gietz 2014). The transformed cells were plated onto YPD agar plates containing 300 µg/mL Hygromycin B to select positive transformants. (2) For genomic integration: The method used to integrate the sam2 gene into the host strain is the same as that used for gene knockout. The only difference is that when constructing the donor DNA, the sam2 fragment needs to be fused into the middle of the homologous arm of the insertion site.

Real-time fluorescence quantitative PCR analysis

RNA extraction and reverse transcription were performed using Bacterial RNA extraction kit R403 and HiScript II Q RT SuperMix, respectively, (Vazyme Biotech Co., Ltd.). The system for real-time qPCR is ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.). The reaction procedure of qPCR involved initial denaturation at 95 °C for 30 s, followed by 40 cycles, each cycle consisting of 10 s at 95 ° C and 15 s at 60 ° C. The mRNA level was analyzed with housekeeping gene act1 as internal reference and calculated by the 2−ΔCt method (ΔCt = Ct (test) - Ct (act1) (Pang et al. 2024).

Glucose determination

Glucose in the fermentation broth was measured using the DNS method (Hagishita et al. 1996). 1 mL fermentation broth was centrifuged at 12,000 rpm for 2 min, then 200 µL supernatant was mixed with 400 µL of DNS solution. The mixture was boiled in water for 5 min, followed by the addition of 1.4 mL of dH2O. After thoroughly mixing, the absorbance of the solution at 540 nm (OD540) was measured (Wang et al. 2024b).

SAM production in shake flask fermentation and 5 L fermenter fermentation

For shake flask fermentation, the mutants were grown in test tubes containing 5mL YPD medium at 37 °C and 220 rpm for 12 h to prepare primary seed. The seed was then inoculated (6% v/v) into 250 mL shake flasks containing 50 mL shake flask fermentation medium (30 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, 4 g/L KH2PO4, 2 g/L K2HPO4, and 0.5 g/L MgSO4·H2O), and cultured for 24 h at 30 °C and 220 rpm. Then 2 mL 50 g/L L-Met was supplemented and cultured for another 24 h.

For 5 L fermenter fermentation, 300 mL seed culture was prepared in six 250 ml shake flasks containing 50 mL YPD medium, and the flasks were incubated for 12 h at 30 °C and 220 rpm. Then the seed culture were inoculated into a 5 L fermenter (BIOTECH-5BG, Shanghai Baoxing Bio-Engineering Equipment Co., Ltd, China) containing 2.7 L fermentation medium (20 g/L glucose, 10 g/L yeast extract, 25 g/L peptone, 4 g/L KH2PO4, 2 g/L K2HPO4, 0.1 g/L MnSO4·7H2O, 0.6 g/L ZnSO4·7H2O, 0.5 g/L MgSO4, 0.5 g/L CaCl2, 1.6 mg/L CuSO4, 4.8 mg/L ammonium molybdate, 0.5 g/L NaCl, 0. 3 mg/L biotin, 3.6 mg/L calcium pantothenate, 3.6 mg/L vitamin B1, 3.6 mg/L vitamin B6, 0.4 g/L sodium citrate). The fermentation was conducted at 30 °C with 10–20% DO (dissolved oxygen) and 500 rpm. The pH was maintained at 5.5 by automatically feeding 40% NH3·H2O. The glucose was maintained at approximately 1–4 g/L by supplementing the feeding broth (500 g/L glucose and 12 g/L yeast extract). When biomass was stable (about 36 h), 8 g L-Met was supplemented every 4 h (Weng et al. 2022).

SAM, ATP, and L-Met analysis

For SAM analysis, 1 mL fermentation broth was centrifuged at 12,000 rpm for 2 min. The supernatant was discarded. Then 200 µL ethyl acetate and 200 µL dH2O were added to the resulting cells and thoroughly mixed. The mixture was then incubated in a metal bath at 35 °C and 1500 rpm for 30 min. Subsequently, 500 µL of 0.35 mol/L H2SO4 was added, and the reaction continued for 1.5 h. After the reaction, the mixture was centrifuged at 12,000 rpm for 3 min. The supernatant was then filtered through a 0.22 μm membrane and quantified using HPLC (high-performance liquid chromatography) (Agilent 1260 HPLC system, Agilent, USA) with an UV detector (wavelength 254 nm) and a C18 column (4.6 mm × 250 mm × 5 μm, Welch Materials (Shanghai) Co., Ltd, China). The mobile phase, a mixture of 82% (v/v) salt solution (2 mmol/L sodium 1-heptanesulfonate and 40 mmol/L NH4H2PO4), and 18% (v/v) methanol solution, was run at 1 mL/min at 30 °C (Weng et al. 2022).

For L-Met analysis, 1 mL fermentation broth was centrifuged at 12, 000 rpm for 2 min. The supernatant was discarded, and 2 mL 1.5 mol/L perchloric acid was added and thoroughly mixed. The mixture was then incubated in a metal bath at 30 °C and 1500 rpm for 2 h. HPLC analysis was performed using a mobile phase consisting of 10% methanol and 90% H2O at 210 nm. All other conditions were consistent with those used for SAM detection (Wang et al. 2024b).

For intracellular ATP analysis, the samples were treated as described for L-Met analysis, except that the reaction temperature was adjusted to 40 °C. For HPLC analysis, the mobile phase consisted of a mixture of 95% 50 mmol/L phosphate buffer (pH 6.0, containing 1 mmol/L EDTA) and 5% methanol. All other detection conditions were identical to those used for SAM analysis (Wang et al. 2024b).

Results and discussions

hxk2 overexpression improved growth and SAM titer of S. cerevisiae

In S. cerevisiae metabolism, glucose serves not only as a direct energy source but also as the carbon backbone for the synthesis of biomacromolecules (Olivares-Marin et al. 2018). Therefore, promoting cellular glucose uptake and utilization can theoretically enhance the growth and SAM production of S. cerevisiae. Hexokinase isoenzyme 2 (encoded by hxk2) is primarily localized in the cytoplasm and plays a crucial role in glucose uptake. This enzyme is also essential for the phosphorylation of intracellular glucose and functions as a critical enzyme in the glycolytic pathway (Gancedo 1998; Rodríguez et al. 2001).

To evaluate the effect of hxk2 overexpression on growth and SAM production in S. cerevisiae, a mutant (AU01) overexpressing hxk2 was constructed. The growth of AU01 was monitored by measuring OD600 of the fermentation broth. The results showed a significant increase in cell growth of mutant AU01 (Fig. 2a). Subsequently, shake flask fermentation was carried out, and the SAM titer and OD600 of AU01 reached 0.65 g/L and 15.15, respectively, resulting into a 16.27% and 11.48% increase compared to the parental strain AU (Fig. 2b). This result may be due to the overexpression of hxk2 gene, which promotes the fructose phosphorylation reaction in glycolysis, enhances glucose utilization and optimizes metabolism (Pérez et al. 2014), ultimately increases biomass and SAM titer of the strain. Therefore, the hxk2 gene was integrated into the genome of the mutant in subsequent experiments to further enhance SAM production.

Fig. 2.

Fig. 2

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The effect of gene overexpression on growth and SAM titer of the mutants. (a) Growth curve of mutant overexpressing hxk2 gene. (b) The SAM titer and growth of mutant overexpressing hxk2 gene

Weakening competitive pathways

Redirecting carbon flux away from competing pathways to enhance the titer of target products is a widely used strategy (Choi et al. 2019). The synthesis pathways of orotate, L-threonine, and L-cystathionine compete with the biosynthesis of SAM. Previous studies have successfully developed high SAM-producing strains by deleting key genes involved in the L-cystathionine biosynthesis pathway (Qin et al. 2020). In this study, to direct more carbon flux toward SAM synthesis, several genes involved in the orotate, L-threonine, and L-cystathionine biosynthesis pathways (ura4, thr1, and cys4, respectively) were knocked out, resulting in the mutants AU02, AU03, and AU04 (Fig. 3a).

Fig. 3.

Fig. 3

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Effect of weakening the competitive pathways and catabolic pathways on SAM production. (a) Knocking out key genes in the competitive pathways of SAM synthesis. (b) Knocking out key genes in the catabolic pathways of SAM synthesis. (c, d) SAM titer and growth of mutants in shaking flask fermentation

The shake flask fermentation results of these mutants showed that only knocking out the thr1 gene increased SAM titer (Fig. 3c). Specifically, the mutant AU02 produced 0.33 g/L SAM, the mutant AU03 produced 1.03 g/L SAM, resulting in a 58.46% increase compared to mutant AU01, the mutant AU04 produced 0.69 g/L SAM. These findings suggested that reasonable regulation of competitive pathways can improve SAM production. Although L-threonine is an essential amino acid for S. cerevisiae, no significant difference in growth of mutant AU03 and AU01 was observed (Fig. 3c). This is likely due to the sufficient supply of L-threonine from the yeast extract (Tao et al. 2023) in the medium, which meets the metabolic demands of mutant AU03. Therefore, the L-threonine synthesis pathway can be blocked to prevent competition for carbon flux with the SAM biosynthesis pathway. However, the knockout of ura4 gene in the orotate biosynthesis pathway significantly inhibited strain growth and notably reduced SAM synthesis. This can be attributed to the role of orotate as a precursor in the synthesis of purine and pyrimidine nucleotides. The depletion of orotate severely impairs nucleic acid synthesis in S. cerevisiae and thus negatively affects growth (Löffler et al. 2016). Additionally, the block of the L-cystathionine biosynthesis pathway did not result in a significant increase in SAM production as reported in previous studies (He et al. 2006; Qin et al. 2020). This suggests that the L-cystathionine biosynthesis pathway is not a major pathway which competes for carbon flux with SAM in this engineered strain.

Strengthening the synthetic pathway of SAM

In S. cerevisiae, two enzymes are responsible for SAM synthesis, encoded by the sam1 and sam2 genes. The enzyme encoded by sam1 is inhibited by excess L-Met, whereas sam2 is not (Kodaki et al. 2003). To enhance SAM production, the sam2 gene was overexpressed in mutant AU02, generating a mutant AU12. In addition, to further investigate the key rate limiting enzymes in the SAM biosynthesis pathway, genes involved in the TCA cycle to L-Met synthesis include aat1, hom3, hom2, hom6, met2, met17, and met6 were also overexpressed (Fig. 4a). This resulted in the mutants AU05, AU06, AU07, AU08, AU09, AU10, and AU11. After culturing these mutants in YPD for 12 h, their intracellular L-Met levels were measured (Fig. 4b). The results showed that overexpression of aat1, met17, and met6 significantly increased L-Met synthesis. Shake flask fermentation was further performed to evaluate the SAM production of these mutants. The results demonstrated that overexpressing aat1, met17, met6, and sam2 significantly increased SAM titer, reached 1.21 g/L, 1.33 g/L, 1.18 g/L, and 1.23 g/L, respectively, resulting in a 17.48%, 29.13%, 14.56%, and 19.42% increase compared to mutant AU02 (Fig. 4d). Subsequently, the genes for these key enzymes were combined, and the optimal combination (overexpressing aat1, met17, and sam2) was integrated into the genome, resulting in mutant AU13. Following successful gene insertion, real-time fluorescence quantitative PCR was performed on mutant AU13 to measure the expression levels of the overexpressed genes. As shown in Fig. 4c, the expression levels of all overexpressed genes were significantly elevated. Shake flask fermentation of mutant AU13 was then carried out. Obviously, after enhancing the supply of precursor L-Met and strengthening the synthesis of SAM, the SAM titer significantly increased to 1.4 g/L (Fig. 4d), resulting into a 35.92% increase compared to mutant AU03. The results indicate that, consistent with previous studies (Chen et al. 2016a; Wang et al. 2024b; Xiao et al. 2024), SAM synthetase is a key limiting enzyme in SAM synthesis. Overexpression of the sam2 gene effectively enhanced SAM production and resulted in a significant increase in SAM production. Furthermore, mitochondrial aspartate aminotransferase (encoded by aat1) is also a rate-limiting enzyme in the SAM biosynthesis pathway which catalyzes the conversion of oxaloacetate to aspartate (Morin et al. 1992). Overexpression of this enzyme can increase the conversion of oxaloacetate to aspartate in the TCA cycle and thereby redirect more carbon flux towards SAM production. Additionally, the conversion of O-acetyl-L-homoserine and hydrogen sulfide to L-homocysteine represents another key rate-limiting node in SAM biosynthesis. The O-acetylhomoserine (thiol)-lyase (encoded by met17) plays a crucial role in L-Met and cysteine biosynthesis and inorganic sulfur assimilation (Brzywczy and Paszewski 1993; Yamagata et al. 1994). Therefore, overexpression of this enzyme effectively alleviated this bottleneck and thereby enhanced SAM production.

Fig. 4.

Fig. 4

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Effect of Overexpressing aat1, met17, and sam2 on SAM production of mutants. (a) Biosynthetic pathway of SAM. (b) Intracellular L-Met concentration in mutants. (c) The transcript levels of aat1, met17, and sam2 genes in mutants. (d) SAM titer of mutants in shaking flask fermentation

Enhancing supply of precursors

Ensuring an adequate supply of precursor is critical for enhancing product titer (Yang et al. 2020). As one of the precursors for SAM synthesis, ATP not only serves as both a precursor but also an energy source for SAM synthesis. Therefore, increasing ATP availability is essential for improving SAM production. VHb, enables Pseudomonas aeruginosa to thrive under microaerobic conditions. This protein modulates cellular metabolism in oxygen-limited environments and promotes cell growth and protein synthesis (Stark et al. 2012). It is hypothesized that introducing vgb could alleviate this limitation by increasing intracellular oxygen levels and ultimately improving SAM titer. To verify this hypothesis, the vgb gene was introduced into mutant AU13, resulting in mutant AU14. The relative expression level of vgb in mutant AU14 was assessed by real-time quantitative PCR (Fig. 5a). The intracellular ATP levels were also measured. The results indicated the successful expression of vgb in mutant AU14 with ATP concentrations reaching 80 mg/L (Fig. 5a), resulting in an 84.76% increase compared to mutant AU13. To further investigate the influence of vgb expression on SAM production, shake flask fermentation was performed. The SAM titer of mutant AU14 reached 1.63 g/L, resulting in a 19.29% increase compared to mutant AU13 (Fig. 5b). The increase in ATP content following vgb overexpression is likely due to VHb providing storage site for oxygen and facilitates its transfer to the terminal respiratory oxidase, and thereby enhances ATP production, reduces NADH levels, and increases the cell membrane potential (Webster et al. 2021). The resulting over accumulation of ATP further promotes SAM synthesis. With this study and previous research (Chen and Tan 2018), it has been demonstrated that ATP is one of the bottlenecking factors in SAM synthesis, and improving the supply of ATP can effectively enhance SAM production. As another precursor, L-Met, can be transported into S. cerevisiae via two methionine permeases encoded by the mup1 and mup3 genes (Isnard et al. 1996). In this research, to verify whether methionine permeases are a limiting factor for SAM synthesis, the mup1 and mup3 genes were overexpressed in mutant AU14 using the pESC-TEF overexpression plasmid. As shown in Fig. S3, the SAM titers of strains overexpressing mup1 and mup3 reached 1.65 and 1.60 g/L, respectively. There is no significant difference between the engineered strains and mutant AU14. This indicates that methionine permease is not a limiting factor for SAM production in the AU14 strain which is consistent with the findings of Ravi et al. (2014).

Fig. 5.

Fig. 5

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The effect of expression vgb gene on ATP and SAM titer of the mutants. (a) The ATP concentration and mRNA expression level of mutant expressing the vgb gene. (b) The SAM titer and OD600 of mutant expressing vgb gene

Blocking the catabolic pathway of SAM

In S. cerevisiae, two distinct metabolic pathways are involved in metabolism of SAM: (1) SAM is converted to SAH by SAM: C-24 sterol methyltransferase (encoded by erg6) (Qu et al. 2019). SAH is subsequently hydrolyzed by S-adenosyl-L-homocysteine hydrolase (encoded by sah1 gene), producing L-homocysteine and thereby completing the SAM cycle (Visram et al. 2018). (2) SAM is decarboxylated by S-adenosylmethionine decarboxylase (encoded by spe2 gene) to form S-adenosyl 3-propylamine. This intermediate is further metabolized into L-Met, polyamines, and pantothenic acid (Balasundaram et al. 1994). Therefore, theoretically, knocking out the erg6, sah1 and spe2 genes could promote SAM accumulation.

To investigate the effect of blocking SAM catabolic pathway on SAM accumulation, the erg6, sah1, and spe2 genes were deleted in mutant AU14, resulting in mutant AU15, AU16, and AU17 (Fig. 3b). The fermentation performance of mutant AU14, AU15, AU16, and AU17 was evaluated in shake flask. As shown in Fig. 3d, except for mutant AU15, all other mutants with downregulated genes exhibited increased SAM titer. Specifically, mutant AU16 produced 1.73 g/L SAM, while mutant AU17 produced 1.77 g/L SAM. Subsequently, the spe2 gene was knocked out in mutant AU16, resulting in mutant AU18. Although biomass significantly decreased, the SAM titer of mutant AU18 reached 1.86 g/L, representing a 14.11% increase compared to mutant AU14. The differences in SAM titers among these mutants may be ascribed to: (1) Knockout of the erg6 gene in the first SAM metabolic pathway not only reduced the SAM titer but also significantly affected strain growth, likely due to the disruption of ergosterol biosynthesis. Ergosterol is a major sterol in fungal membranes and plays a crucial role in maintaining membrane fluidity and regulating various cellular processes (Choy et al. 2023). (2) In contrast, knocking out the sah1 gene in this pathway did not affect strain growth, but significantly increased SAM titer. This is likely because deletion of the sah1 gene does not directly disrupt the methylation reaction itself, but instead blocks the further metabolism of SAH, the product of SAM demethylation. SAH acts as a competitive inhibitor in the transmethylation reaction (Tehlivets et al. 2004), and when it accumulates to a certain level, it can inhibit the activity of methyltransferases, thereby promoting SAM accumulation. Therefore, knocking out the sah1 gene prevents the degradation of SAM and enhances its accumulation without affecting normal strain growth. (3) Although the deletion of spe2 has been reported to cause a deficiency in spermidine (Chattopadhyay et al. 2006), as shown in Fig. 3d, no significant differences in growth were observed between AU17, AU18, and AU14. As previously reported (Zhao et al. 2016), spermidine deficiency does not affect the normal strain growth, likely due to trace amounts of spermidine derived from yeast extract, which are sufficient to meet the growth requirements of the host. These results suggest that appropriately blocking the further metabolic pathways of SAM is also a promising strategy for developing high SAM-producing yeast strains.

Fermentation optimization

Shake flask optimization

The carbon source, nitrogen source, and yeast extract in the medium are critical for yeast growth and SAM formation. Additionally, the addition of L-Met is essential for SAM production during fermentation, as high concentrations of L-Met can inhibit yeast growth, while low concentrations of L-Met are insufficient for SAM synthesis. Therefore, in this study, a series of optimizations were performed using a single-factor strategy to adjust the types and concentrations of carbon and nitrogen sources, yeast extract, and L-Met feeding strategy in the fermentation medium. Specifically, carbon sources are not only essential components of yeast cells but also provide energy for yeast cell growth and regulate cellular metabolism. Therefore, carbon sources are indispensable nutrients for the growth of S. cerevisiae and SAM synthesis. In studies on SAM production using S. cerevisiae, glucose or sucrose is commonly used as the carbon source (Hu et al. 2023; Wang et al. 2024b; Weng et al. 2022; Xiao et al. 2024). Therefore, glucose and sucrose were selected for the exploration of the optimal carbon source for AU18 in this study. From Fig. 6a and b, it can be seen that using glucose as the sole carbon source and 20 g/L glucose was optimal for SAM production in mutant AU18. This supports the notion that the hxk2 gene enhances glucose uptake into the cell, eliminating the need for high concentrations of glucose in the medium. Nitrogen sources are another essential component for the growth and SAM synthesis of S. cerevisiae. In previous studies (Hu et al. 2023; Qin et al. 2020; Weng et al. 2022; Xiao et al. 2024), peptone or (NH4)2SO4 has typically been used as the nitrogen source. Therefore, in this study, the optimal type and concentration of nitrogen source for AU18 were explored. The data (Fig. 6c and d) revealed that the highest SAM titer was obtained when peptone was used as the sole nitrogen source at 25 g/L. The main components of yeast extract include peptides, amino acids, and flavor nucleotides, all of which play a crucial role during fermentation. Yeast extract not only provides essential nutrients but also improves fermentation quality, simplifies the extraction process, and enhances overall efficiency (Tao et al. 2023). Therefore, yeast extract is a fundamental component of the fermentation medium for S. cerevisiae (Hu et al. 2023; Qin et al. 2020; Wang et al. 2024b; Weng et al. 2022; Xiao et al. 2024). Thus, in this research, the optimal concentration of yeast extract was investigated. From Fig. S4, it can be seen that although higher yeast extract concentrations led to increased biomass, no further improvement in SAM titer was observed when yeast extract exceeded 10 g/L. L-Met, as one of the precursors for SAM synthesis, is an essential component for SAM biosynthesis. It is typically added during fermentation to meet the demand for SAM production. However, excessive L-Met can suppress the growth of yeast (Kodaki et al. 2003). Therefore, to further improve the SAM titer, the supplementation time and concentration of L-Met were explored. The results showed that supplementation of L-Met at 12 h, during the rapid growth phase, enhanced SAM synthesis (Fig. 6e), with the highest SAM titer obtained at 4 g/L L-Met (Fig. 6f). Under the optimal medium and feeding strategy, the SAM titer and OD600 of mutant AU18 in shake flask fermentation reached 2.46 g/L and 13.93, respectively. The optimal medium composition was determined to be 20 g/L sucrose, 10 g/L yeast extract,25 g/L peptone, 4 g/L KH2PO4, 2 g/L K2HPO4, and 0.5 g/L MgSO4·H2O.

Fig. 6.

Fig. 6

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The effect of fermentation media and feeding on SAM titer and growth of mutant AU18. (a) The effect of carbon source concentrations. (b) The effect of glucose concentrations. (c) The effect of nitrogen source concentrations. (d) The effect of peptone concentrations. (e) The effect of L-Met adding time. (f) The effect of L-Met concentrations. The “Mixture” refers to the blend of the remaining two components in a 1:1 ratio in the same experiment

5 L fermenter fermentation

The fermentation was scaled up in a 5 L fermenter to further validate the SAM production of mutant AU18. Regulation of L-Met concentration during fermentation in the 5 L fermenter is critical. To maintain a relatively stable L-Met concentration and minimize fluctuations, the strategy of L-Met addition was improved based on previous study (Weng et al. 2022) and employed a continuous feeding strategy to reduce fluctuations as much as possible. To evaluate the effect of L-Met feeding strategy on SAM synthesis, two strategies were examined: (1) Intermittent feeding: in which 8 g L-Met was added every 4 h, and (2) Continuous feeding: in which 50 g/L L-Met was added at a rate of 0.5 mL/min. As shown in Fig. 7, continuous feeding of L-Met resulted in a maximum SAM titer of 13.96 g/L at 92 h, whereas intermittent feeding achieved a maximum titer of 11.53 g/L at 100 h. These results indicated that continuous feeding of L-Met is more beneficial for the synthesis of SAM, not only achieving higher titer, but also shortening the fermentation time, meanwhile reduces the amount of L-Met supplementation. This is because intermittent feeding may cause a decrease in L-Met levels during the final phase of each 4 h period, potentially insufficient for SAM synthesis. The continuous feeding of L-Met addresses this issue by maintaining an excess of L-Met in the fermentation broth, enables continuous SAM synthesis and reduces its degradation. However, the excess L-Met inhibited strain growth and leading to slower biomass formation.

Fig. 7.

Fig. 7

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Time course of SAM production and growth of mutant AU18 in 5 L fermenter with different L-Met feeding strategies. (a) Intermittent L-Met feeding strategy. (b) Continuous L-Met feeding strategy

Conclusion

The growing demand for SAM underscores the need for more efficient production methods. In this study, the growth characteristics of the strain AU were first optimized, followed by a comprehensive optimization of its SAM biosynthetic pathway using a modular strategy. This approach involved enhancing the supply of precursors, such as L-Met and ATP, while minimizing the degradation of SAM. The final mutant, AU18, achieved a SAM titer of 1.78 g/L in shake flask fermentation, resulting in a 227.67% increase compared to the parental strain AU. In this study, the overexpression of the hxk2 and aat1 genes was firstly employed to promote glucose metabolism and to competitively divert more oxaloacetate from the TCA cycle, thereby enhancing SAM synthesis. Additionally, a comprehensive analysis and optimization of the synthetic and degradation pathways of SAM were conducted. The composition of the fermentation medium was further optimized to meet the nutritional requirements of mutant AU18. With the optimal medium, the SAM titer reached 2.46 g/L. Moreover, various L-Met feeding strategies for SAM production of mutant AU18 were examined in a 5 L fermenter. The continuous feeding of L-Met resulted in a SAM titer of 13.89 g/L, which is relatively high among the reported yields so far (Table 1). Therefore, in this research the SAM biosynthesis of S. cerevisiae was significantly enhanced with the multimodule strategy based on comprehensive optimization of both synthesis and degradation pathways of SAM. The established multimodule strategy study provides a foundation for the development of S. cerevisiae with improved productivity of SAM and other target metabolites.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1MB, docx)

Acknowledgements

Not applicable.

Abbreviations

SAM

S-adenosyl-L-methionine

SAH

S-adenosyl-L-homocysteine

GYC

Glyoxylate cycle

UV

Ultraviolet

ARTP

Atmospheric and room temperature plasma

TCA

Tricarboxylic acid

YPD

Yeast peptone dextrose

LB

Luria–Bertani

HPLC

High-performance liquid chromatography

VHb

Vitreoscilla Hemoglobin

DO

Dissolved oxygen

Author contributions

YW, YZ, and ZL conceived and designed research. LT, YZ, and YW conducted experiments. PL, and ZH analyzed data. YW, and LT wrote the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (22478351).

Data availability

All data generated during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no confects of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Material 1 (1MB, docx)

Data Availability Statement

All data generated during this study are included in this published article.

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