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Introduction of human m6Am methyltransferase PCIF1 facilitates the biosynthesis of terpenoids in Saccharomyces cerevisiae

Microbial Cell Factories volume 24, Article number: 78 (2025) Cite this article

Abstract

Background

The application of synthetic biology techniques has been recognized as an efficient alternative for the biosynthesis of high-value natural products, and various metabolic engineering strategies have been employed to develop microbial cell factories. However, exploration of more efficient metabolic pathway optimization strategies is still required to further improve the producing potential of microbial cell factories to meet the industrial requirements.

Results

In this study, we found that the introduction of human N6,2’-O-dimethyladenosine (m6Am) methyltransferase PCIF1 into Saccharomyces cerevisiae significantly promoted the biosynthesis of squalene, increased by 2.3-fold. Transcriptome analysis revealed that PCIF1 upregulated genes associated with glycolysis and acetyl-CoA biosynthesis pathways, and also activated the cell wall integrity mitogen-activated protein kinase (MAPK) pathway to improve the cell wall stress response. Importantly, PCIF1 expression notably enhanced squalene and sesquiterpenoid longifolene production in engineered yeast strains, with 2.3-fold and 1.4-fold higher increase, respectively.

Conclusion

This study presents a PCIF1-based metabolic engineering strategy that could serve as an effective approach for the optimization of terpene biosynthesis in yeast cell factories.

Introduction

Natural products represent a critical source of bioactive compounds utilized in various industries, such as artemisinin and taxol in drug development [1,2,3], santalol and longifolene in cosmetics industries [4,5,6], and pyrethrin and azadirachtin in contemporary agriculture [7,8,9]. The application of synthetic biology techniques in the development of microbial cell factories has been recognized as an efficient alternative to traditional methodologies for producing these high-value natural products [10]. Successful production of many important chemicals is achieved by the employment of metabolic engineering strategies [11,12,13,14]. The widely implemented classical metabolic engineering strategies encompass promoter engineering, the optimization of key rate-limiting enzymes, the fusion of upstream and downstream genes within metabolic pathways, and “push-pull-block” strategy [15,16,17,18,19].

Currently, ‌with the interdisciplinary collaboration between computer science and biotechnology, system level modelling, computational tools and machine learning approaches are frequently employed in metabolic engineering [20,21,22,23]. Although various metabolic engineering strategies have been developed for microbial biosynthesis of natural products, substantial improvement remains necessary to meet industrial production requirements. Consequently, further exploration of efficient metabolic pathway optimization strategies is still required to address future needs in the biosynthesis of natural products.

Post-transcriptional modifications of mRNA serve pivotal roles in regulating its splicing, transcription, translation, and stability [24,25,26]. N6-Methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ) modifications are found to be abundant in yeast mRNA [27,28,29,30], and among these modifications, the function of m6A is currently uncovered in detail. m6A modification can promote the decay and translation of mRNA, and regulates the meiosis process in diploid‌ yeast and metabolism in haploid yeast [31,32,33]. Besides the above mRNA modifications, N6,2’-O-dimethyladenosine (m6Am) represents a specific type of RNA modification, occurring when the first nucleotide following the N7-methylguanosine cap is 2’-O-methyladenosine, which subsequently undergoes methylation at the N6 position to generate m6Am [34]. In mammalian cells, phosphorylated CTD interacting factor 1 (PCIF1) is responsible for catalyzing the m6Am modification [35]. The m6Am modification represents a key epigenetic change in mRNA, exerting a vital influence on the regulation of cellular metabolic pathways. This modification is known to stabilize mRNA and enhance its translation efficiency, which in turn modulates the expression of essential genes and affects various cellular processes [34, 36, 37]. By playing a pivotal role in the regulation of gene expression, m6Am modification influences cellular metabolism by modulating specific metabolic pathways. For instance, it has been found to be enriched in multiple obesity- and metabolism-associated processes, with key metabolic genes undergoing m6Am methylation in obese mice [37, 38].

During the fermentation, microbial cells experience stresses related to gene expression and metabolic flux. These stresses would cause negative effects to the growth and behavior of the engineered strains. For example, changes in environmental condition can cause the intracellular genetic perturbation and lower the accuracy of gene expression. Stabilizing the gene expression serves as an effective strategy to maintain cellular homeostatic state. Therefore, an approach is needed to stabilize the gene expression of these microbial cells. As PCIF1-mediated m6Am modification was reported to stabilize the mRNA expression and improve the transcription and translation in human cells, although it is absent in yeast [39, 40], we wondered whether the heterologous expression of PCIF1 would help to improve the performance of these microbial cell factories. To verify this hypothesis, PCIF1 was overexpressed in Saccharomyces cerevisiae to assess its impact on yeast gene expression and metabolism. Results indicated that PCIF1 introduction led to a promotion in squalene production, increase by 2.3-fold. Transcriptome sequencing revealed that PCIF1 upregulated genes associated with glycolysis and acetyl-CoA biosynthesis pathways. Subsequently, squalene and the sesquiterpene compound longifolene were chosen as the target compounds, and engineered strains were developed. PCIF1 expression enhanced the production of squalene to 274.10 ± 12.92 mg/L, representing a 2.3-fold increase, and promoted the yield of sesquiterpenoid longifolene to 16.23 ± 0.30 mg/L, a 1.4-fold improvement. This study presents a PCIF1-based metabolic engineering strategy that could serve as an effective approach for optimizing terpene biosynthesis in yeast.

Methods

Plasmid and strain construction

The genomic DNA (gDNA) of S. cerevisiae was extracted utilizing the FastPure Plant DNA Isolation Mini Kit (Vazyme Biotech, China). The endogenous yeast genes ERG12, ERG10, ERG13, ERG8, ERG19, IDI1, ERG20, and ERG9 were amplified from the gDNA by using 2 × Phanta Max Master Mix (Vazyme Biotech, China) and inserted into the pEASY®-Blunt cloning vector (TransGen Biotech, China) for sequence verification. The PCIF1, EfmvaE, and PsLS genes were synthesized by General Biol (China). Genes involved in the mevalonate (MVA) pathway were separately constructed into pESC-TRP/HIS/URA vectors. The expression cassette for PCIF1 was assembled by overlap PCR, linking the PCIF1 gene with PGK1p promoter, TDH1t terminator and the LEU selection marker. ERG20 was mutated to ERG20F96W/N127W using Fast Site-Directed Mutagenesis Kit (Tiangen, China) and subsequently integrated into the pESC-URA vector together with ERG9. The selection marker of pESC-HIS was substituted with KanMX, after which the PsLS gene was constructed into this modified vector.

The expression cassettes corresponding to each individual gene were amplified from their corresponding vectors and subsequently introduced into yeast cells by utilizing Frozen-EZ Yeast Transformation II Kit (Zymo, USA). Notably, ERG8, ERG19, and IDI1 were incorporated into the 308a locus of the yeast genome through the CRISPR/Cas9 technology, while the remaining genes were integrated at their designated locations within the yeast genome via homologous recombination. The plasmids and strains developed in this part are listed in Table 1, and the primers are listed in Table S1.

Table 1 Plasmids and strains constructed in this work
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Growth curve determination by microbial growth curve analyzer

Six single colonies of the positive yeast strain were inoculated into YPD yeast medium containing 20 g/L glucose as the carbon source, and the culture concentration was adjusted to an OD600 of 0.1. A volume of 500 µL of the yeast suspension was transferred to a 48-well plate and incubated at 30 °C with shaking at 700 rpm. And samples were periodically taken at designated time intervals using microbial growth curve analyzer (Scientz, China) to measure the OD600 values. And a growth curve was generated based on the accumulated data.

Semiquantitative RT-PCR

Total RNA was isolated from cultured yeast cells using Trizol reagent (Invitrogen, USA) and subsequently reverse-transcribed into cDNA utilizing the HiScript II Q RT SuperMix for qPCR kit (Vazyme Biotech, China). UBC6 was employed as the reference gene, and semiquantitative RT-PCR was conducted using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, China). The primer sequences used in this analysis are listed in Table S1.

Shake flask fermentation

A single colony of positive yeast strain was first introduced into 5 mL of SD medium, where they were incubated at 30 °C with shaking at 220 rpm for a period ranging between 12 and 16 h. Subsequently, a suitable volume of the culture was transferred into 50 mL of SD medium, adjusting the OD600 to 0.1.

For triterpene compound fermentation, the previously mentioned medium was cultured under the same conditions of 30 °C and 220 rpm for 120 h, after which the culture underwent harvesting. In the case of longifolene fermentation, the culture was initially incubated at 30 °C and 220 rpm for 24 h, following which 15% (v/v) sterile nonane was added. Fermentation then proceeded under the same conditions for a further 120 h.

Compound detection

Triterpene compounds detection: The culture was centrifuged at 3,000 rpm for 5 min. Yeast cells were harvested and subsequently boiled in a lysis buffer consisting of 20% (v/v) potassium hydroxide and 50% (v/v) ethanol for 10 min. The resulting lysate was then combined with the supernatant, and an equivalent volume of n-hexane was introduced, followed by a settling period lasting 48 h. The upper organic layer was then subjected to drying using a rotary evaporator. The dried sample was treated with 50 µL of N-Methyl-N-(trimethylsilyl) trifluoroacetamide at 80 °C for 30 min prior to gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS analysis utilized a 19,091 S-433UI gas chromatography column paired with a 5977B single quadrupole mass spectrometer equipped with an electron ionization source at 70 eV. Helium served as the carrier gas, with the flow rate maintained at 1.0 mL/min. The injection volume for the sample was 1 µL, operated in splitless or split mode at a 50:1 ratio. The initial temperature was programmed to 160 °C, held for 1 min, then raised to 280 °C at a rate of 30 °C/min and maintained for 10 min, with a subsequent final increase to 300 °C at a rate of 2 °C/min, where it was held for 5 min.

Longifolene detection: Following fermentation, 1 mL of the upper organic phase was extracted, filtered, and subsequently analyzed using GC-MS. The conditions for detection were as follows. Helium, serving as the carrier gas, maintained a flow rate of 1.0 mL/min. The sample injection volume was set to 1 µL, with the analysis conducted in splitless mode. The initial temperature was programmed at 50 °C and sustained for 3 min, after which it was elevated to 70 °C at a rate of 20 °C/min, where it was held for 1 min. Finally, the temperature was increased to 300 °C at a rate of 15 °C/min and held for 10 min.

RNA sequencing

Cells of CEN.PK2-1 C and TP01 strains, during their exponential phase, were collected through centrifugation at 3,000 rpm/min and 4 °C. Total RNA extraction was carried out using Trizol reagent (Invitrogen, USA), followed by quantification with a NanoDrop 2000 spectrophotometer. The integrity of the extracted RNA was evaluated using the Agilent RNA Nano 6000 kit. mRNA was isolated from the total RNA using Thermo Fisher Scientific DynabeadsTM Oligo(dT)25 mRNA purification beads. Transcriptome sequencing was subsequently performed on the Illumina Novaseq platform by Novogene Corporation (Beijing, China).

Statistical analysis

Data processing and statistical evaluations were conducted using GraphPad Prism 8 software. All experiments were carried out in triplicate, with the results presented as mean ± standard deviation (M ± SD). Statistical significance was defined as p < 0.05.

Results

PCIF1 introduction promotes the biosynthesis of squalene in S. cerevisiae

To explore the role of m6Am methyltransferase PCIF1 in S. cerevisiae metabolism, the PCIF1 gene expression module was integrated into the genome of S. cerevisiae strain CEN.PK2-1 C, generating strain TP01. The expression of the PCIF1 gene in this engineered yeast strain was first assessed, with semiquantitative RT-PCR results confirming its high expression in yeast cells (Fig. 1A). Growth curves were then monitored by using microbial growth curve analyzer and a fed-batch fermentation to evaluate the influence of PCIF1 on yeast growth. The data revealed that, compared to the WT strain, strain TP01 exhibited a growth advantage than the WT strain (Fig. 1B and Fig. S1). This suggests that the introduction of PCIF1 influenced the growth of yeast, which was consistent with the reports that PCIF1 impacted the growth of human cells [41, 42].

Further analysis involved the cultivation of both CEN.PK2-1C and TP01 strains in shake flasks to assess their metabolite profiles. GC-MS analysis demonstrated a significant enhancement of squalene biosynthesis in the TP01 strain (Fig. 1C and Fig. S2). By employing standard curves for squalene (Fig. S3), it was calculated that the TP01 strain produced 2.50 ± 0.07 mg/L of squalene, corresponding to 2.3-fold increases, compared to the WT strain (Fig. 1D). These findings indicate that the integration of the PCIF1 gene promotes the biosynthesis of triterpene compounds in yeast.

Fig. 1
figure 1

PCIF1 promoted the biosynthesis of squalene in yeast. (A) Expression detection of PCIF1 by semiquantitative RT-PCR. (B) The growth curves of CEN.PK2-1 C and TP01 strain. (C) GC-MS detection of triterpene in yeast strain. (D) The production of squalene in different strains. The asterisks indicate significant differences (**p < 0.01, *p < 0.05)

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PCIF1 markedly affects the expression of genes involved in yeast metabolism and stress response

To further elucidate the mechanism by which PCIF1 enhances triterpene biosynthesis in yeast, a transcriptome sequencing analysis was conducted on the TP01 and WT strain. The analysis revealed 1,821 differentially expressed genes (DEGs), with 909 genes upregulated and 921 genes downregulated in comparison to the WT strain (Fig. S4A). Notably, 79 genes were uniquely expressed in the WT, whereas 60 genes were uniquely expressed in the TP01 strain (Fig. S4B). Gene Ontology (GO) enrichment analysis demonstrated that these DEGs were predominantly involved in oxidation-reduction processes, carbohydrate metabolism, phosphorus metabolic processes, transferase activity, and oxidoreductase activity (Fig. 2A). Importantly, the introduction of PCIF1 had a profound effect on the oxidation-reduction processes within yeast cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs were primarily enriched in pathways associated with the biosynthesis of secondary metabolites, carbon metabolism, amino acid biosynthesis, and glycolysis/gluconeogenesis (Fig. 2B). Of these, the biosynthesis of secondary metabolites pathway exhibited the highest degree of enrichment. Collectively, these findings underscore the pivotal role of PCIF1 in regulating oxidation-reduction processes, carbon metabolism, secondary metabolism, and amino acid synthesis in S. cerevisiae.

Fig. 2
figure 2

Transcriptome analysis of PCIF1 transformed strain and CEN.PK2-1 C. (A) GO analysis of DEGs in different yeast strains. (B) KEGG analysis of DEGs in different yeast strains

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During fermentation and cultivation, yeast cells are exposed to numerous stress factors, including nutrient depletion and the accumulation of toxic by-products, which result in the build-up of reactive oxygen species (ROS) and disturbances in energy metabolism, consequently restricting the biosynthesis of desired compounds. Previous reports have shown that the inhibition of ROS accumulation and the preservation of cell wall integrity are essential strategies employed by yeast cells to manage stress conditions [43,44,45]. Here, through transcriptome analysis, it was discovered that the overexpression of PCIF1 led to an enrichment of functional genes involved in cellular redox processes (Fig. 2A). Moreover, differential gene expression analysis revealed an upregulation in the expression of many genes encoding signaling molecules in the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway, including Pkc1, Mkk1 and Slt2 (Fig. 3B). These findings imply that PCIF1 might improve the stress tolerance of yeast cells by influencing the genes involved in the stress response.

Fig. 3
figure 3

The expression of genes involved in cell wall stress response in different yeast strains

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PCIF1 promoted squalene production through increasing the biosynthesis of acetyl-CoA

To determine the factors contributing to the elevated production of triterpene compounds in the TP01 strain, the squalene synthesis pathways were examined. A comparative analysis of the triterpene biosynthesis pathway and the upstream MVA pathway was carried out between the TP01 and WT strains. Unexpectedly, transcriptome data did not reveal significant increase in gene expression levels within these two pathways, and even some enzymes showed down-regulated expression levels (Table S2). As acetyl-CoA serves as the precursor of MVA, attention was subsequently directed toward the acetyl-CoA synthesis pathway. In yeast, pyruvate, a glycolytic product, can be converted into acetyl-CoA through either the pyruvate dehydrogenase (PDH) complex in mitochondria or PDH bypass in cytosol [46,47,48]. Also, acetyl-CoA can be derived via β-oxidation of fatty acids in peroxisomes [48]. Transcriptome sequencing demonstrated that genes in β-oxidation, including POX1 and POT1, were downregulated (Table S2), while most genes involved in the glycolytic pathway in the TP01 strain exhibited upregulated expression, including HXK1, TDH1, TDH3, PGK1, GPM1, and CDC19 (Fig. 4). In the two pathways leading from pyruvate to acetyl-CoA, genes coding for the pyruvate decarboxylase complex, such as PDA1, PDB1, and LAT1, were upregulated (Fig. 4). Similarly, aldehyde dehydrogenases involved in the PDH bypass, including Ald3, Ald4, and Ald5, were also found to be upregulated (Fig. 4). Meanwhile, CTP1, the transporter encoding gene responsible for the transportation of acetyl-CoA in the form of citrate from mitochondria to cytosol was also upregulated (Table S2). These findings indicate that PCIF1 promotes triterpene compound biosynthesis by upregulating both the glycolysis and acetyl-CoA synthesis pathways.

Fig. 4
figure 4

The expression of genes involved in glycolysis. HXK1, hexokinase 1; TDH1/3, glyceraldehyde-3-phosphate dehydrogenase; PGK1, phosphoglycerate kinase; CDC19, pyruvate kinase; ALD3/4/5, aldehyde dehydrogenases; PDA1, pyruvate dehydrogenase; PDB1, pyruvate dehydrogenase; LAT1, dihydrolipoamide acetyltransferase

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PCIF1 increased squalene production in engineered yeast

To assess whether PCIF1 can sustain its capacity to elevate triterpene production in engineered strains and potentially serve as a viable metabolic engineering strategy for triterpene biosynthesis, engineered strains with enhanced triterpene output were developed, and triterpene yields were compared pre- and post-introduction of PCIF1. In S. cerevisiae, the MVA pathway initiates with acetyl-CoA. Acetyl-CoA is first converted by acetoacetyl-CoA thiolase Erg10 and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase Erg13 into HMG-CoA. This HMG-CoA is subsequently reduced by HMG-CoA reductase to generate MVA, which undergoes further conversion to isopentenyl pyrophosphate (IPP) through a series of phosphorylation and decarboxylation reactions catalyzed by Erg12, Erg8, and Erg19. IPP is then isomerized to dimethylallyl pyrophosphate (DMAPP) by IPP isomerase Idi1 [49,50,51]. Among them, previous studies have shown that EfmvaSA110G, a mutant of EfmvaS encoding HMG-CoA synthase from Enterococcus faecalis, alongside the HMG-CoA reductase EfmvaE, displays increased conversion efficiency [52,53,54]. Therefore, to optimize the MVA pathway, genes encoding the aforementioned enzymes were overexpressed, culminating in the engineered strain TP04 (Fig. 5A).

Within the subsequent steps of the triterpene biosynthesis pathway, IPP and DMAPP are first converted into farnesyl pyrophosphate (FPP) by the Erg20, after which FPP is further transformed into squalene by the squalene synthase Erg9 [55]. Previous research indicates that the Erg20F96W/N127W double mutant possesses greater catalytic efficiency and specificity [56]. To improve the triterpene biosynthesis pathway, both Erg20F96W/N127W and Erg9 were overexpressed (Fig. 5A), leading to the creation of the engineered strain TP05. Shake flask fermentation of this strain, alongside analysis using the squalene standard curve (Fig. S5), revealed that 120.59 ± 5.24 mg/L of squalene was produced (Fig. 5B). Following this, the PCIF1 expression module was inserted into the genome, resulting in strain TP06. Fermentation assays conducted with TP06 demonstrated a squalene production level of 274.10 ± 12.92 mg/L (Fig. 5B), representing a 2.3-fold increase compared to strain TP05. These findings illustrate that PCIF1 is capable of substantially enhancing squalene production in engineered yeast.

Fig. 5
figure 5

PCIF1 significantly promoted squalene production in engineered yeast. (A) The optimization of squalene biosynthesis pathway. Erg10, acetoacetyl-CoA thiolase; Erg13, HMG-CoA synthase; EfMvaE, HMG-CoA reductase from E. faecalis; Erg12, mevalonate kinase; Erg8, phosphomevalonate kinase; Erg19, mevalonate pyrophosphate decarboxylase; Idi1, isopentenyl diphosphate: dimethylallyl diphosphate isomerase; Erg20, farnesyl pyrophosphate synthetase; ERG9, squalene synthase. (B) The squalene production in different yeast strains. The asterisks indicate significant differences (**p < 0.01, *p < 0.05)

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PCIF1 enhances sesquiterpene biosynthesis in engineered yeast

Given the ability of PCIF1 to stimulate the biosynthesis of triterpene compounds in yeast, it is important to explore whether it can similarly enhance the biosynthesis of other terpenes. Longifolene, a tricyclic sesquiterpene obtained from Pinus palustris, is an aromatic compound with notable antibacterial and antioxidant activities and is widely utilized in the pharmaceutical, fuel, fragrance, and cosmetic industries [8, 57, 58]. As such, longifolene was chosen as a represented sesquiterpene to assess the effect of PCIF1 on its biosynthesis. In strain TP04, where the MVA pathway was optimized, the native promoter of ERG9 was substituted with the glucose-repressible HXT1p promoter, producing strain TP07 (Fig. 6A). Subsequently, the longifolene synthase gene PsLS from P. sylvestris [7] was transformed into strain TP07, resulting in strain TP08. Using the longifolene standard curve (Fig. S6), it was determined that strain TP08 produced 2.35 ± 0.39 mg/L of longifolene during shake flask fermentation (Fig. 6B and Fig. S7).

To further improve the efficiency of longifolene production, a fusion protein consisting of Erg20F96W/N127W and PsLS, linked by a peptide linker GSG, was constructed and introduced into TP07, resulting in strain TP09. Fermentation analysis of TP09 showed that longifolene production reached 11.46 ± 1.01 mg/L (Fig. 6C). Following this, the PCIF1 expression cassette was inserted into the genome of strain TP09, generating strain TP10. Fermentation results revealed that this strain produced 16.23 ± 0.30 mg/L of longifolene (Fig. 6C), representing a 1.4-fold increase over strain TP10. These results suggest that the PCIF1-based metabolic engineering approach enhances sesquiterpene production in engineered yeast strains.

Fig. 6
figure 6

PCIF1 promoted longifolene production in engineered yeast. (A) The optimization of longifolene biosynthesis pathway. Erg10, acetoacetyl-CoA thiolase; Erg13, HMG-CoA synthase; EfMvaE, HMG-CoA reductase from E. faecalis; Erg12, mevalonate kinase; Erg8, phosphomevalonate kinase; Erg19, mevalonate pyrophosphate decarboxylase; Idi1, isopentenyl diphosphate: dimethylallyl diphosphate isomerase; Erg20, farnesyl pyrophosphate synthetase; ERG9, squalene synthase; PsLS, codon-optimized longifolene synthase. (B) GC-MS detection of longifolene standard and its production in engineered yeast. (C) The longifolene production in different yeast strains. The asterisks indicate significant differences (**p < 0.01, *p < 0.05)

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Discussion

Previously, m6Am modification has been implicated in cellular adaptation to hypoxia and glucose metabolism [34, 37]. Transcriptome studies have demonstrated that this modification markedly alters the metabolic pathways of cancer cells, such as those in gastric and colorectal cancers, by regulating genes critical to cancer cell metabolism, thereby promoting cellular growth and proliferation [59, 60]. These findings underscore the importance of m6Am modification not only in transcriptional regulation but also in its broader impact on cellular functions through modulation of specific metabolic pathways. In this study, it was observed that when the human m6Am methyltransferase PCIF1 was expressed in S. cerevisiae, it markedly influenced cellular redox processes and was involved in yeast carbon metabolism, secondary metabolism, and amino acid synthesis (Fig. 2). Additionally, PCIF1 was found to activate the MAPK pathway, which is capable of enhancing cell wall integrity, thereby increasing yeast resistance during fermentation (Fig. 3).

Significantly, this research reveals for the first time that PCIF1 is involved in the regulation of glycolysis and acetyl-CoA synthesis pathways, leading to the upregulation of key enzymes in these pathways and the enrichment of the acetyl-CoA pool. It is widely acknowledged that acetyl-CoA acts as a precursor in the biosynthesis of numerous compounds, including isoprenoids, terpenes, fatty acids and their derivatives, and polyketides [61]. Several prior studies have documented enhanced production of these target compounds by augmenting acetyl-CoA synthesis [62]. For instance, the enhanced supply of acetyl-CoA and NADPH by inhibiting the acetyl-CoA competing pathway promoted the betulinic acid titer from 88.07 ± 5.83 mg/L to 166.43 ± 1.83 mg/L, increased by 1.89-fold in engineered yeast strain [63]. An improved acetyl-CoA supply by the combination of phosphoketolase (PK) and phosphotransacetylase (PTA) pathways led to the production of 50.29 mg/L β-carotene, improved by 80% [64]. The introduction of an alternative cytoplasmic acetyl-CoA pathway to Yarrowia lipolytica resulted in a 32% enhanced β-ionone titer [65]. The present study confirms that the PCIF1-induced enrichment of acetyl-CoA can stimulate terpene biosynthesis, 2.3-fold and 1.4-fold increase in squalene and longifolene production, respectively, in engineered yeast strains (Fig. S8). Thus, the regulation of acetyl-CoA biosynthesis through PCIF1 presents a promising metabolic engineering strategy for acetyl-CoA-derived compounds, particularly terpenes, to enhance the efficiency of target compound synthesis. Future research will focus on further investigating the role of PCIF1 in promoting the production of other acetyl-CoA-derived compounds in microbial cell factories to broaden its applications in natural product biosynthesis.

Transcriptome sequencing of the PCIF1-transformed strain revealed that the expression levels of enzymes involved in the two pathways responsible for converting the glycolytic product pyruvate into acetyl-CoA, including the PDH complex and the PDH bypass, were markedly upregulated. Consequently, these two pathways present potential targets for further enhancement in future engineering efforts aimed at increasing acetyl-CoA production. For example, building on a previously reported strategy for optimizing the PDH bypass, overexpression of Ald6 and the mutant acetyl-CoA synthetase SeACSL641P from Salmonella enteric could be employed to further promote acetyl-CoA synthesis [66], which would further improve the biosynthesis of its derived compounds.

In one of our previous reports, we overexpressed another similar methyltransferase, the m6A modification methyltransferase Ime4, in yeast, and the overexpression of this methyltransferase significantly increased the biosynthesis of isoprenoids and aromatic compounds, by significantly elevating the expression of genes in the glycolysis, acetyl-CoA synthesis (PDH bypass) and shikimate/aromatic amino acid synthesis modules [33]. While in this work, the overexpression of PCIF1 increased the production of squalene, and the upregulation of glycolysis was observed, which was different to that in Ime4-overexpressed strain. Thus, the observed effects in PCIF1-overexpressed yeast strain might be specifically due to PCIF1’s m6Am methyltransferase activity, and we will further verify this in the future.

Conclusion

Here, we reported an efficient engineering method for the biosynthesis of terpenoid in yeast based on m6Am modification. The introduction of human m6Am methyltransferase PCIF1 into yeast significantly promoted the production of squalene to 2.3-fold increase. Transcriptome analysis revealed that PCIF1 upregulated genes associated with glycolysis and acetyl-CoA biosynthesis pathways. Meanwhile, PCIF1 expression significantly enhanced squalene and sesquiterpenoid longifolene production in engineered yeast strains, with 2.3-fold and 1.4-fold higher increase, respectively. This work provided a PCIF1-based metabolic engineering strategy for the efficient biosynthesis of terpenoids in yeast cell factories.

Data availability

The data supporting this study can be found in this article and supplementary materials. The transcriptome data have been submitted to the NCBI Sequence Read Archive (SRA) database under the accession code PRJNA1155854. All datasets are available from the corresponding author upon reasonable request.

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Funding

This work was supported by Shandong Provincial Natural Science Foundation (Grant number ZR2021QC097 and ZR2024QH067) and Introduction and Cultivation Project for Young Creative Talents of Higher Education of Shandong Province.

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  1. Guoli Wang, Mingkai Li and Bengui Fan contributed equally to this work.

Authors and Affiliations

  1. Featured Laboratory for Biosynthesis and Target Discovery of Active Components of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Binzhou Medical University, Yantai, 264003, China

    Guoli Wang, Mingkai Li, Bengui Fan, Xiqin Liang, Jun Wang, Yanbing Shi, Qiusheng Zheng, Defang Li & Tianyue An

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  1. Guoli Wang
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Contributions

QZ, DL and TA designed the experiment. GW, ML and BF performed the experiments and analyzed the data. XL, JW and YS helped to analyzed the data. GW and TA supervised the work and wrote the manuscript. QZ, DL and TA revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qiusheng Zheng, Defang Li or Tianyue An.

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Wang, G., Li, M., Fan, B. et al. Introduction of human m6Am methyltransferase PCIF1 facilitates the biosynthesis of terpenoids in Saccharomyces cerevisiae. Microb Cell Fact 24, 78 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02701-4

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Keywords

Microbial Cell Factories

ISSN: 1475-2859

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