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Article

Retinal Production by Precision Fermentation of Saccharomyces cerevisiae

by
Hye-Seon Hwang
1,
Kwang-Rim Baek
1 and
Seung-Oh Seo
1,2,*
1
Department of Food Science and Biotechnology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea
2
Research Institute of Food and Biotechnology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(4), 214; https://doi.org/10.3390/fermentation11040214
Submission received: 15 March 2025 / Revised: 7 April 2025 / Accepted: 10 April 2025 / Published: 14 April 2025
(This article belongs to the Special Issue Fermentation-Driven Biological Structural Modification of Natural Products)
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Abstract

Retinoids, including retinol, retinal, and retinoic acid, are a group of vitamin A derivatives with skin-improving effects. Retinoic acid is highly effective for skin anti-aging but can cause irritation, requiring a prescription. Retinol, a less irritating alternative, needs conversion to retinal and then retinoic acid in the skin, whereas direct absorption of retinal enhances efficacy by bypassing this conversion process. This study aimed to produce retinal through precision fermentation using metabolically engineered Saccharomyces cerevisiae. The introduction of heterologous retinal biosynthetic genes and overexpression of the truncated HMG-CoA reductase (tHMG1) and acetyl-CoA acetyltransferase (ERG10) genes in the mevalonate (MVA) pathway increased retinal production up to 10.2 mg/L. At the same time, ethanol was produced as a major byproduct in S. cerevisiae. To address this, a pyruvate decarboxylase (Pdc)-deficient S. cerevisiae strain, incapable of producing ethanol, was employed. Overexpression of ERG10 and tHMG1 in the Pdc-deficient S. cerevisiae harboring the retinal biosynthetic plasmids achieved a retinal production up to 117.4 mg/L in the dodecane layer without ethanol through a two-phase in situ fermentation and extraction. This study demonstrates that eliminating pyruvate decarboxylase activity effectively redirects carbon flux toward retinal biosynthesis in the recombinant S. cerevisiae, offering a promising approach for sustainable retinal production through precision fermentation.

1. Introduction

Retinoids, including retinol, retinal, and retinoic acid, are vitamin A derivatives commonly used in anti-aging cosmetics for their ability to reduce wrinkles and whiten skin through collagen synthesis and epithelial cell differentiation. These compounds interconvert via oxidation–reduction reactions, with retinoic acid being the active form on the skin [1,2]. Direct application of retinoic acid causes significant irritation; thus, it is restricted to prescription use, while retinol is used in cosmetics due to its lower toxicity. Retinol is converted into retinoic acid through a two-step process via retinal, but this conversion is relatively inefficient. Therefore, the direct use of retinal enhances efficacy by rapidly converting to retinoic acid, providing quicker skin improvements [3,4].
The production of commercial retinoids has predominantly relied on chemical synthesis using petroleum-based substances like acetone and acetylene, which are not sustainable [5,6,7,8]. Moreover, while the primary product of chemical synthesis is retinol, the oxidation from retinol to retinal presents further challenges in terms of control [9]. Alternatively, precision fermentation using genetically engineered microorganisms as a cell factory offers a more environmentally friendly and sustainable method for producing functional ingredients such as retinal [10,11]. Microbial production of retinal requires isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), synthesized via the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways in prokaryotes and eukaryotes, respectively [12]. Engineered Escherichia coli has been used for retinoid production, achieving 136 mg/L retinoids [13]. However, industrial production of retinal using E. coli might not be suitable due to the risk of endotoxin contamination and bacteriophage infection in large-scale fermentation.
Saccharomyces cerevisiae is a preferable host due to its generally regarded as safe (GRAS) status, advanced genetic manipulation tools, and native MVA pathway for retinal precursors, making it suitable for large-scale retinal production [14,15]. Increasing the pool of IPP, a key precursor, is necessary for the overproduction of the isoprenoid retinal. Specifically, previous studies have also focused on manipulating the MVA pathway in S. cerevisiae to overproduce valuable isoprenoids [16,17,18]. Despite manipulating the MVA pathway to divert carbon flux for the overproduction of isoprenoids, ethanol remains the primary product from glucose in S. cerevisiae. Pdc-deficient S. cerevisiae mutants, which lack pyruvate decarboxylase that converts pyruvate to ethanol [19], can help divert pyruvate from ethanol production to retinal synthesis. Overproduction of hydrophobic molecules like retinal is often hindered by low cell membrane permeability and solubility in the culture medium, limiting intracellular accumulation [8]. Thus, a two-phase extraction system can be implemented, allowing retinal to be automatically extracted into the extraction solvent during fermentation.
This study aims to overproduce retinal by engineered S. cerevisiae to overexpress ERG10 and tHMG1 genes in the MVA pathway and utilize a pyruvate decarboxylase (Pdc)-deficient S. cerevisiae mutant to reduce ethanol production. The heterologous retinal biosynthetic genes are introduced in the Pdc-deficient S. cerevisiae harboring the ERG10 and tHMG1 genes to increase the supply of retinal precursors from the mevalonate and ethanol pathways (Figure 1). Additionally, a two-phase extraction system is applied to overcome the limited storage capacity for intracellular hydrophobic substances and to enhance the efficient retinal production and extraction during fermentation. This study confirms the feasibility of producing retinal as a potential cosmetic ingredient through precision fermentation using metabolically engineered S. cerevisiae.

2. Material and Methods

2.1. Strains and Plasmids

The strains and plasmids used in this study are listed in Table 1. E. coli Top10 (Invitrogen, Carlsbad, CA, USA) was used for cloning and plasmid DNA amplification. The S. cerevisiae D452-2 [20] strain and the Pdc-deficient evolved S. cerevisiae S4H strain derived from the SOS4 [19] strain were used as a host strain for the production of retinal.

2.2. Media and Culture Conditions

E. coli was cultured in Lysogeny Broth (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) at 37 °C, supplemented with 100 ug/mL of ampicillin when required for gene cloning. S. cerevisiae strains were grown in YP (Yeast Peptone) medium (10 g/L yeast extract, 20 g/L peptone) with 20 g/L glucose at 30 °C and 220 rpm. The yeast transformants were selected on Synthetic Complete (SC) medium (1.7 g/L yeast nitrogen base without amino acids, 1.4 g/L yeast synthetic drop-out medium supplement, ammonium sulfate 5 g/L, and 20 g/L glucose; adjusted to pH 5.8), and amino acids were added if necessary. As required, 300 µg/mL G418 and/or 300 µg/mL hygromycin B were added for the selection of transformants.

2.3. Construction of Plasmids

The primers used in this study are listed in Table S1. All restriction enzymes were purchased from Takara (Takara Bio, Kusatsu, Japan). Target genes were amplified using PrimeSTAR HS DNA Polymerase (Takara Bio, Kusatsu, Japan). GoTaq DNA polymerase from Promega (Madison, WI, USA) was used for colony PCR. Plasmid preparation and gel extraction kits were obtained from Bionics (Seoul, Republic of Korea) and Favorgen (Taiwan, China), respectively, and sequencing was conducted by Bionics (Seoul, Republic of Korea).
To construct plasmids for retinal production, the CrtE, CrtYB, and CrtI genes derived from Xanthophyllomyces dendrorhous and the Blh gene from uncultured marine bacterium, Halobacterium salinarum NRC-1, were used. The Blh gene was codon-optimized for expression in S. cerevisiae and synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). The CrtE, CrtYB, CrtI, and Blh genes were amplified by polymerase chain reaction (PCR) with the corresponding primers listed in Table S1. The pRS425GPD, pRS423GPD, pRS426GPD, and pRS42KGPD plasmids were treated with the restriction enzyme BamHI to produce a linear vector. Each purified gene fragment was cloned into individual plasmids using Gibson Assembly Master Mix from New England Biolabs (NEB, Ipswich, MA, USA), resulting in the construction of pRS425GPD-crtE, pRS423GPD-crtYB, pRS426GPD-crtI, and pRS42KGPD-Blh plasmids.

2.4. Construction of Recombinant Strains

To overexpress the ERG10 and tHMG1 genes in the MVA pathway using CRISPR/Cas9, the plasmids pRS426TEFp-ERG10-CYCt and pRS426PGKp-tHMG1-TEFt were constructed to provide donors. The PGK1p, tHMG1, TEF1t, TEF1p, ERG10, and CYC1t parts from S. cerevisiae were amplified by PCR with the corresponding primers listed in Table S1. The pRS426GPD plasmid was treated with the restriction enzymes SacI and KpnI to remove the GPD promoter and CYC1 terminator gene sequences. Each purified promoter, gene, and terminator fragment was cloned into the pRS426 vector, leading to the construction of the pRS426TEFp-ERG10-CYCt and pRS426PGKp-tHMG1-TEFt plasmids. All gene sequences used in this study are listed in Table S1.
Using the CRISPR/Cas9 system, the TEFp-ERG10-CYCt and PGKp-tHMG1-TEFt cassettes were targeted for integration into the cryptic subrepeats 5 (CS5) and cryptic subrepeats 6 (CS6) regions of the S. cerevisiae genome, respectively [23]. The pRS42K-CS5 and pRS42K-CS6 plasmids, which contain the guide RNA (gRNA) expression cassette, were constructed by modifying the 20 bp target sequence of the previous gRNA cassette [22]. Target sequences of gRNA used for CRISPR/Cas9 in this study are listed in Table S2.
To evaluate the effects of enhancing the MVA pathway, the ERG10 and tHMG1 genes were overexpressed using CRISPR/Cas9 in the S. cerevisiae D452-2 strain and the Pdc-deficient evolved S. cerevisiae S4H strain. The plasmids pCas-hyg, pRS42K-CS5, and pRS42K-CS6, along with donor DNA (ERG10 and tHMG1 expression cassettes), were transformed into S. cerevisiae D452-2 and S4H, respectively. Transformed cells were selected on YPD (Yeast Peptone Dextrose) plate supplemented with 300 ug/mL geneticin (G418) and 300 ug/mL hygromycin B. Positive colonies were confirmed by colony PCR using the primer sets F_check_CS5 and R_check_CS5, as well as F_check_CS6 and R_check_CS6 (Table S1), and they were designated as S. cerevisiae DET and S. cerevisiae SET, respectively. To produce retinal, four expression plasmids (pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, and pRS42KGPD-Blh) were transformed into D452-2, DET, S4H, and SET, respectively. Transformed cells were selected on SCD (Synthetic Complete Dextrose) plate supplemented with 300 ug/mL G418, and they were designated as D452-RE, DET-RE, S4H-RE, and SET-RE, respectively. The empty plasmids (pRS423GPD, p425GPD, p426GPD, and pRS4KGPD) were transformed into D452-2 and S4H as a control, and they were designated as D452-CON and S4H-CON, respectively. The transformation method followed the high-efficiency yeast transformation method using the LiAc/SS carrier DNA/PEG [24].

2.5. Fermentation Condition

The engineered yeast cells were pre-cultured in 5 mL of SC medium with 20 g/L glucose for 3–4 days. The pre-cultured cells were harvested and washed twice with distilled water before being inoculated into the main fermentation. The main fermentations were inoculated at an initial optical density (OD600) of around 1.0. Main fermentations were conducted in 250 mL baffled flasks containing 100 mL of YP medium with 20 g/L glucose and 10 mL dodecane at 30 °C and 220 rpm. Dodecane was used as the extraction solvent in the two-phase extraction system during fermentation. All fermentations were repeated independently in triplicate.

2.6. Extraction and Quantitative Analysis Methods

Cell growth was determined by measuring the optical density at 600 nm with a spectrophotometer (SHIMADZU, Kyoto, Japan, UV-1900i). Metabolites such as glucose and ethanol were quantified by a high-performance liquid chromatography (HPLC) system (Agilent Technologies 1260 Infinity II, Santa Clara, CA, USA) equipped with a refractive index detector and an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad Laboratories, Hercules, CA, USA). The column was eluted with 0.005 N of H2SO4 at a flow rate of 0.6 mL/min at 60 °C.
Intracellular β-carotene and retinal were extracted using acetone with modifications to the method described in a previous study [8] and then quantified. Cultured cells (1 mL) were subjected to centrifugation, and the supernatant was decanted. The resulting cell pellets were re-suspended with 1 mL of acetone in a 2 mL screw-cap tube containing 0.5 mm zirconia beads and crushed using a vortexer. After centrifugation, the clear supernatant was analyzed by HPLC. To quantify extracellular β-carotene and retinal, the dodecane layer was separated from the supernatant of the culture medium after centrifugation at 4 °C for 30 min at 13,000 rpm. The clear supernatant, which was dodecane, was used for HPLC analysis.
β-carotene and retinal were analyzed using modified methods reported in previous studies [25,26]. β-carotene was quantified using an HPLC (Agilent Technologies 1100 Series, USA) system equipped with a UV detector and a YMC Carotenoid column (250 × 4.6 mm, YMC, Devens, MA, USA). The mobile phase was composed of solvent A, methanol/Methyl tert-butyl ether (MTBE)/water (92:5:3, v/v/v), and solvent B, methanol/MTBE/water (8:90:2, v/v/v), delivered in a gradient elution mode. From 0 to 12 min, solvent B increased linearly from 10% to 45%. From 12.01 to 24 min, solvent B increased linearly from 45% to 100%. From 24.01 to 28 min, solvent B was maintained at 100%. From 28.01 to 32 min, solvent B decreased from 100% to 10%. The flow rate was 0.6 mL/min at 35 °C, and β-carotene was detected at 450 nm. Retinal and its derivatives, including retinoic acid and retinol, were separated with a YMC-Pack ODS-AQ device (250 × 4.6 mm, YMC). The mobile phase was a mixture of 92.5% acetonitrile and 7.5% acetic acid solution (2% v/v). The flow rate was 1 mL/min at 40 °C, and retinal was detected at 352 nm. Standard curves were established using β-carotene (C4582, Sigma-Aldrich, St. Louis, MO, USA) and retinal (R2500, Sigma-Aldrich) at known concentrations.

3. Results and Discussion

3.1. Introduction of Retinal Biosynthetic Pathway into S. cerevisiae

To construct the retinal biosynthetic pathway, the CrtE, CrtYB, and CrtI from X. dendrorhous and the Blh genes from H. salinarum NRC-1 were cloned into the expression plasmids, resulting in pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, and pRS42KGPD-Blh. The four expression plasmids were transformed to the wild-type S. cerevisiae D452-2, resulting in the D452-RE strain. The empty plasmids were transformed into D452-2 to construct the D452-CON strain as a control strain. To compare the production of retinal between D452-CON and D452-RE, batch fermentations were conducted in YP medium with 20 g/L glucose and 10% (v/v) dodecane (Figure 2A,B). Dodecane is frequently used as an organic phase in two-phase fermentation and extraction due to its relatively low toxicity to S. cerevisiae. It has been demonstrated that dodecane does not significantly inhibit the growth of cells [1,27]. In the two-phase fermentation system, the S. cerevisiae D452-CON strain did not produce retinal but produced 12.6 g/L of ethanol. The D452-RE strain harboring the retinal biosynthesis pathway produced 2.7 mg/L of retinal and 12.7 g/L of ethanol at 12 h when the glucose was fully consumed. These findings indicate that the overexpression of the CrtYB, CrtE, CrtI, and Blh genes enables retinal production in S. cerevisiae. Also, the expression plasmids containing the heterologous retinal biosynthetic genes constructed in this study were functional. Furthermore, native dehydrogenases in S. cerevisiae, such as Env9, have been reported to possess activity that converts retinal to retinol [28]. Therefore, retinol and retinoic acid were also analyzed. However, these compounds were not detected (Figure S1). The D452-RE strains showed similar cell growth, glucose consumption, and ethanol production profiles with the control strain D452-CON. The small amount of retinal production may be due to an insufficient supply of retinal precursors in S. cerevisiae.

3.2. Enhancement of Mevalonate Pathway for Increasing Retinal Production

Previous studies have reported that HMG-CoA reductase is a rate-limiting enzyme in the MVA pathway that provides retinal precursors in S. cerevisiae [29]. To improve metabolic flux in the MVA pathway, the tHMG1 gene, which encodes only the catalytic domain of HMG1 by truncation, can be overexpressed [18,30,31]. Additionally, overexpression of both the tHMG1 and ERG10 genes in the MVA pathway can enhance isoprenoid production in S. cerevisiae [30]. Thus, both the tHMG1 and ERG10 genes were overexpressed in the retinal-producing recombinant S. cerevisiae strains to increase retinal production in this study. To achieve more stable expression of the genes, the ERG10 and tHMG1 expression cassettes were integrated into the S. cerevisiae D452-2 chromosome by CRISPR/Cas9-based genome editing, resulting in the DET strain. The four expression plasmids, pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, and pRS42KGPD-Blh, were transformed into DET to construct the DET-RE strain. To investigate the effect of ERG10 and tHMG1 overexpression on retinal biosynthesis, the batch fermentation of the DET-RE strain was conducted in YP medium with 20 g/L glucose and 10% (v/v) dodecane (Figure 2C). As a result, the DET-RE strain produced 10.2 mg/L of retinal and 13.9 g/L of ethanol at 12 h when the glucose was fully consumed. The retinal yield in the DET-RE strain was 3.3 times higher compared to the D452-RE strain. This result suggests that the overexpression of the ERG10 and tHMG1 genes leads to improved retinal production in S. cerevisiae. This will be an effective strategy to increase the intracellular pool of precursors for other valuable carotenoids in S. cerevisiae. Furthermore, retinal production could be further enhanced by increasing the copy number of rate-limiting enzymes or by modulating transcriptional regulation. As demonstrated in previous studies, the overexpression of tHMG1 and the deletion of transcriptional repressors in the MVA pathway, such as ROX1 and MOT3, have resulted in substantial improvements in retinoid production in S. cerevisiae [28]. In addition, the precursor synthesis step involving geranylgeranyl pyrophosphate (GGPP) has been identified as another rate-limiting point in the pathway. To overcome this bottleneck, a positive mutant of CrtE (CrtE03M) was introduced, resulting in an enhanced GGPP supply and, consequently, a further increase in retinoid titers [28]. One study also systematically evaluated multiple heterologous Blh genes for retinoid biosynthesis in S. cerevisiae and found that one variant exhibited superior performance compared to others from different species [32]. However, despite these improvements, a significant amount of ethanol production still needs to be addressed to further increase retinal production in the recombinant S. cerevisiae.

3.3. Increased Retinal Production in S. cerevisiae by Elimination of Ethanol Pathway

Since pyruvate is used for both retinal and ethanol production in S. cerevisiae (Figure 1), the ethanol pathway competes with the retinal biosynthesis pathway. Therefore, the evolved Pdc-deficient S. cerevisiae S4H strain harboring deletions of the PDC1 and PDC5 genes was used in this study to enhance retinal production by eliminating ethanol production. The retinal biosynthetic plasmids were transformed into the S4H strain, resulting in the S4H-RE strain. As a control, the S4H-CON strain was constructed by transformation of the empty plasmids. To evaluate retinal production by the recombinant Pdc-deficient S. cerevisiae, batch fermentations of the S4H-CON and S4H-RE strains were conducted in YP medium with 20 g/L glucose and 10% (v/v) dodecane (Figure 3A,B). The S4H-RE strain produced 39.2 mg/L of retinal without ethanol production, while S4H-CON produced neither retinal nor ethanol. When compared to the D452-RE strain, the S4H-RE strain exhibited a 13.3 times higher retinal yield without ethanol production. This result demonstrates that eliminating ethanol production by using a Pdc-deficient S. cerevisiae strain is an effective strategy for enhancing retinal production in S. cerevisiae.
To further increase the retinal production in Pdc-deficient S. cerevisiae, overexpression cassettes of the ERG10 and tHMG1 genes were introduced in the genome of the S4H strain, resulting in the SET strain. The retinal biosynthetic plasmids were transformed into the SET strain to construct the SET-RE strain. The batch fermentation of the SET-RE strain was performed under the same conditions as the other recombinant strains. As a result, the SET-RE strain produced 117.4 mg/L of retinal without ethanol production (Figure 3C). Notably, the SET-RE strain exhibited the highest retinal titer among the recombinant strains (Table 2), indicating that Pdc gene deletion with overexpression of the ERG10 and tHMG1 genes in S. cerevisiae was the most effective strategy for retinal enhancement in this study (Figure 4A). In addition, the use of promoters with varying strengths to regulate the expression of retinal biosynthetic genes, as well as increasing gene copy numbers through high-copy plasmids, could be considered as additional strategies. Such approaches have been successfully applied in previous studies to enhance the production of valuable compounds. For instance, modifying the promoter strength of selection markers has been shown to significantly increase the episomal plasmid copy number, thereby improving expression levels and enhancing metabolic output in S. cerevisiae [33]. In another study, various promoter engineering strategies in S. cerevisiae were reviewed, emphasizing how fine-tuning promoter strength can effectively modulate gene expression and increase product yield [34]. Additionally, the CRISPR-δ-integration method, which involves CRISPR-mediated cleavage of δ-sequences on the yeast chromosome followed by δ-integration of the target gene, has been shown to effectively increase the integrated gene copy number and the expression level [35]. These strategies are expected to improve the overall flux through the retinal biosynthesis pathway. Interestingly, the retinal produced by the SET-RE strain was mainly accumulated in dodecane layer, while retinal in both the intracellular fraction and the aqueous fermentation medium was not detected (Figure 4B). This result suggests that a two-phase extraction system was successfully applied to continuously extract intracellular retinal during fermentation, leading to the efficient production and recovery of retinal. This was also confirmed by the visual observation that the dodecane layer of the SET-RE strain turned yellow compared to the control strain, indicating that the retinal was effectively extracted into the dodecane (Figure 5).

4. Conclusions

In this study, metabolically engineered S. cerevisiae producing retinal was successfully constructed by the introduction of heterologous retinal biosynthetic genes from yeast and bacteria. The overexpression of the endogenous ERG10 and tHMG1 genes involved in the MVA pathway increased retinal production in S. cerevisiae. Subsequently, the Pdc-deficient S. cerevisiae strain was employed to produce retinal without ethanol production, resulting in the highest retinal production up to 117.4 mg/L in a dodecane layer. This is the first report to produce retinal without ethanol using Pdc-deficient S. cerevisiae. The two-phase extraction system used in this study successfully accumulated retinal into the solvent layer without accumulating in the intracellular and fermentation medium. Our findings demonstrate that eliminating pyruvate decarboxylase activity effectively redirects carbon flux toward retinal biosynthesis in recombinant S. cerevisiae, offering a promising approach for sustainable retinal production through precision fermentation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11040214/s1, Figure S1: HPLC chromatograms for the detection of retinal derivatives under the same analytical condition; Table S1: Primer sequences used in this study; Table S2: Target sequences of gRNA used for CRISPR/Cas9 in this study.

Author Contributions

Conceptualization, S.-O.S.; Validation, H.-S.H. and K.-R.B.; Investigation, H.-S.H.; Writing—original draft, H.-S.H.; Writing—review & editing, K.-R.B. and S.-O.S.; Supervision, S.-O.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) and funded by the Korean government (MSIT) (No. 2022M3E5E6080049), and it was supported by the Research and Publication Support Program of the Ottogi Ham Taeho Foundation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 11 00214 g001
Figure 1. Metabolic pathway of retinal production from glucose in engineered S. cerevisiae. The abbreviations of enzyme and metabolite names are as follows: pyruvate decarboxylase, PDC; alcohol dehydrogenase, ADH; isopentenyl pyrophosphate, IPP; geranyl pyrophosphate, GPP; farnesyl pyrophosphate, FPP; geranylgeranyl pyrophosphate, GGPP; pyruvate dehydrogenase complex, PDHc; acetyl-CoA acetyltransferase, ERG10; truncated HMG-CoA reductase, tHMG1; mevalonate kinase, ERG12; phosphomevalonate kinase, ERG8; diphosphomevalonate decarboxylase, MVD1; farnesyl diphosphate synthase, ERG20; GGPP synthase, CrtE; lycopene cyclase, CrtYB; phytoene desaturase, CrtI; β-carotene 15, 15′-dioxygenase, Blh.
Figure 1. Metabolic pathway of retinal production from glucose in engineered S. cerevisiae. The abbreviations of enzyme and metabolite names are as follows: pyruvate decarboxylase, PDC; alcohol dehydrogenase, ADH; isopentenyl pyrophosphate, IPP; geranyl pyrophosphate, GPP; farnesyl pyrophosphate, FPP; geranylgeranyl pyrophosphate, GGPP; pyruvate dehydrogenase complex, PDHc; acetyl-CoA acetyltransferase, ERG10; truncated HMG-CoA reductase, tHMG1; mevalonate kinase, ERG12; phosphomevalonate kinase, ERG8; diphosphomevalonate decarboxylase, MVD1; farnesyl diphosphate synthase, ERG20; GGPP synthase, CrtE; lycopene cyclase, CrtYB; phytoene desaturase, CrtI; β-carotene 15, 15′-dioxygenase, Blh.
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Figure 2. Fermentation profiles of the S. cerevisiae D452-CON (A), D452-RE (B), and DET-RE (C). The results are represented as the mean value and standard deviation of each treatment (n = 3). Symbols: gray square (OD600), white circle (glucose), blue diamond (ethanol), red triangle (intracellular β-carotene), yellow downward triangle (extracellular retinal).
Figure 2. Fermentation profiles of the S. cerevisiae D452-CON (A), D452-RE (B), and DET-RE (C). The results are represented as the mean value and standard deviation of each treatment (n = 3). Symbols: gray square (OD600), white circle (glucose), blue diamond (ethanol), red triangle (intracellular β-carotene), yellow downward triangle (extracellular retinal).
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Figure 3. Fermentation profiles of the Pdc-deficient S. cerevisiae S4H-CON (A), S4H-RE (B), and SET-RE (C). The results are presented as the mean value and standard deviation of each treatment (n = 3). Symbols: gray square (OD600), white circle (glucose), blue diamond (ethanol), red triangle (intracellular β-carotene), yellow downward triangle (extracellular retinal).
Figure 3. Fermentation profiles of the Pdc-deficient S. cerevisiae S4H-CON (A), S4H-RE (B), and SET-RE (C). The results are presented as the mean value and standard deviation of each treatment (n = 3). Symbols: gray square (OD600), white circle (glucose), blue diamond (ethanol), red triangle (intracellular β-carotene), yellow downward triangle (extracellular retinal).
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Figure 4. Summary of retinal titer and yield in the dodecane layer by recombinant S. cerevisiae strains (A) and investigation of extraction capacity for dodecane solvent in the SET-RE strain with retinal concentrations of intracellular fraction, aqueous fermentation medium, and dodecane solvent layer (B). −, negative; +, positive.
Figure 4. Summary of retinal titer and yield in the dodecane layer by recombinant S. cerevisiae strains (A) and investigation of extraction capacity for dodecane solvent in the SET-RE strain with retinal concentrations of intracellular fraction, aqueous fermentation medium, and dodecane solvent layer (B). −, negative; +, positive.
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Figure 5. A picture of the two-phase extraction system of the SET-RE (A) and S4H-CON (B).
Figure 5. A picture of the two-phase extraction system of the SET-RE (A) and S4H-CON (B).
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Table 1. Strains and plasmids used in this study.
Table 1. Strains and plasmids used in this study.
NameDescriptionReference
Strains
Escherichia coli TOP10F−mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupGInvitrogen
(Carlsbad, CA, USA)
Saccharomyces cerevisiae D452-2MATα, his3, leu2, ura3, can1[20]
Saccharomyces cerevisiae SOS4S. cerevisiae D452-2 Δpdc1, Δpdc5[19]
Saccharomyces cerevisiae S4HS. cerevisiae SOS4, evolved for cell growthIn this study
Saccharomyces cerevisiae DETS. cerevisiae D452-2 CS5::PGKp-tHMG1-TEFt, CS6::TEFp-ERG10-CYCtIn this study
Saccharomyces cerevisiae SETS. cerevisiae SOS4 CS5::PGKp-tHMG1-TEFt, CS6::TEFp-ERG10-CYCtIn this study
D452-CONS. cerevisiae D452-2 (pRS423GPD, pRS425GPD, pRS426GPD, pRS42KGPD)In this study
D452-RES. cerevisiae D452-2 (pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, pRS42KGPD-Blh)In this study
DET-RES. cerevisiae DET (pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, pRS42KGPD-Blh)In this study
S4H-CONS. cerevisiae S4H (pRS423GPD, pRS425GPD, pRS426GPD, pRS42KGPD)In this study
S4H-RES. cerevisiae S4H (pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, pRS42KGPD-Blh)In this study
SET-RES. cerevisiae SET (pRS423GPD-crtYB, pRS425GPD-crtE, pRS426GPD-crtI, pRS42KGPD-Blh)In this study
Plasmids
pRS423GPDGPDp, CYCt, 2u origin, HIS3, Ampr[21]
pRS425GPDGPDp, CYCt, 2u origin, LEU2, Ampr[21]
pRS426GPDGPDp, CYCt, 2u origin, URA3, Ampr[21]
pRS42KGPDGPDp, CYCt, 2u origin, KanMX, AmprIn this study
pRS423GPD-crtYBpRS423GPD harboring the crtYB geneIn this study
pRS425GPD-crtEpRS425GPD harboring the crtE geneIn this study
pRS426GPD-crtIpRS426GPD harboring the crtI geneIn this study
pRS42KGPD-BlhpRS42HGPD harboring the Blh geneIn this study
pRS426PGKp-tHMG1-TEFtPGKp, TEFt, URA3, Ampr, harboring the tHMG1 geneIn this study
pRS426TEFp-ERG10-CYCtTEFp, CYCt, URA3, Ampr, harboring the ERG10 geneIn this study
pCas-HygCas9 expression plasmid, Hygromycin B marker[22]
pRS42K-CS5pRS42K, gRNA expression cassette targeting the intergenic site on Chr XVIn this study
pRS42K-CS6pRS42K, gRNA expression cassette targeting the intergenic site on Chr VIIIn this study
Table 2. Summary of retinal production by the recombinant S. cerevisiae strains in batch fermentation containing 20 g/L glucose and 10% (v/v) dodecane.
Table 2. Summary of retinal production by the recombinant S. cerevisiae strains in batch fermentation containing 20 g/L glucose and 10% (v/v) dodecane.
StrainRetinal
Biosynthetic Genes		Overexpression of
ERG10 and tHMG1		Pdc DeletionRetinal
(mg/L)		Ethanol
(g/L)		Yield of Retinal
(mg Retinal/g Glucose)
D452-CON−−−N.D.12.6N.D.
D452-RE+−−2.712.70.1
DET-RE++−10.213.90.3
S4H-CON−−+N.D.N.D.N.D.
S4H-RE+−+39.2N.D.1.4
SET-RE+++117.4N.D.5.7
−, negative; +, positive; N.D., not detected.
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Hwang, H.-S.; Baek, K.-R.; Seo, S.-O. Retinal Production by Precision Fermentation of Saccharomyces cerevisiae. Fermentation 2025, 11, 214. https://doi.org/10.3390/fermentation11040214

AMA Style

Hwang H-S, Baek K-R, Seo S-O. Retinal Production by Precision Fermentation of Saccharomyces cerevisiae. Fermentation. 2025; 11(4):214. https://doi.org/10.3390/fermentation11040214

Chicago/Turabian Style

Hwang, Hye-Seon, Kwang-Rim Baek, and Seung-Oh Seo. 2025. "Retinal Production by Precision Fermentation of Saccharomyces cerevisiae" Fermentation 11, no. 4: 214. https://doi.org/10.3390/fermentation11040214

APA Style

Hwang, H.-S., Baek, K.-R., & Seo, S.-O. (2025). Retinal Production by Precision Fermentation of Saccharomyces cerevisiae. Fermentation, 11(4), 214. https://doi.org/10.3390/fermentation11040214

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