Journal of Industrial Microbiology and Biotechnology, 2023, 50, kuad026

https://doi.org/10.1093/jimb/kuad026
Advance access publication date: 31 August 2023
Metabolic Engineering and Synthetic Biology—Original Paper

Production of S-methyl-methionine using engineered

Saccharomyces cerevisiae sake K6
Jun-Min Lee1 ,† , Min-Ho Park1 ,† , Bu-Soo Park1 ,2 ,† , Min-Kyu Oh 1

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1
Department of Chemical & Biological Engineering, Korea University, Seoul 136-763, Korea
2
Samyang Corp. 295 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, Republic of Korea
Correspondence should be addressed to: Min-Kyu Oh, Department of Chemical and Biological Engineering, Korea University, Anam-Ro 145 Sungbuk-Gu, 02841
Seoul, Korea. Phone: +82-2-3290-3308; Fax: +82- 2-926-6102. E-mail: mkoh@korea.ac.kr
†
These authors contributed equally to this work.

Abstract: S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants.
In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene
encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized
as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor
of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked
out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM
from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding
malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch
fermentation.
One-Sentence Summary: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered
Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

Keywords: S-methyl-methionine, Methioinine S-methyltransferase, S-adenosyl-methionine, Saccharomyces cerevisiae sake K6

Graphical abstract

Overall scheme of S-methyl-methionine (SMM) biosynthesis pathway for engineered Saccharomyces cerevisiae sake K6.
Abbreviations: MMT: Methionine S-methyltransferase; SAM: S-adenosyl-methionine; SMM: S-methyl-methionine; SAH: S-adenosyl-
homocysteine

Introduction promotes the growth of human fibroblasts and migration of hu-

man dermal fibroblasts, which are essential steps in treating skin
S-methyl-methionine (SMM) is commonly found in plants such
wounds (Kim et al., 2018, 2015, 2010). Given these benefits, the de-
as cabbage, broccoli, wheat, and sunflowers (Hattula & Granroth,
mand for SMM as a food supplement has continuously increased.
1974; Skodak et al., 1965; Turner & Shapiro, 1961). Although its
SMM is commonly produced in flowering plants up to 0.3–5
biological role in plants is not well understood, SMM is known
μmol/g in various organs, such as leaves, roots, etc. (Kovatscheva
to preserve methionine, serve as a methyl donor, and regulate
& Popova, 1977; Bourgis et al., 1999; Kim, 2003; Ludmerszki
S-adenosylmethionine (SAM) (Kocsis et al., 2003; Tan et al., 2010).
et al., 2014). It is mainly produced in leaves by methionine
In humans, SMM is sometimes referred to as vitamin U because
S-methyltransferase (MMT) and is transported to reproductive
of its therapeutic effects on gastrointestinal ulcers (Cheney, 1949;
tissues via the phloem. SMM is converted back to methionine
Samson, 1971; Kopinski et al., 2007; Sokmen et al., 2012; Gezginci-
through the SMM cycle by homocysteine S-methyltransferase
Oktayoglu et al., 2016; Kruchinina et al., 2018; Topaloglu et al.,
(HMT). This process is called the SMM cycle, and is used for the
2022). Recent studies have shown that SMM is effective in pro-
efficient transport and utilization of methionine during seed
tecting against ultraviolet (UV) exposure and that SMM treatment

Received: June 6, 2023. Accepted: August 29, 2023.

© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology. This is an Open Access article
distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
2 | Journal of Industrial Microbiology and Biotechnology, 2023, Vol. 50, No. 1

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Fig. 1. SMM biosynthesis pathway in S. cerevisiae sake K6. MMT gene encoding methionine S-methyltransferase was integrated to genome. Three genes
(MMP1, SAM4, and MHT1) were knocked out for accumulation of SMM. The native ACS2 promoter was changed to PGK1 promoter and MLS1 gene was
knocked out to enhance SAM concentration. The blue colors are up-regulated genes and red colors are knocked out genes. Glc-6p,
Glucose-6-phosphate; Fru-6p, Fructose-6-phosphate; Fru-1,6BP, Fructose-1,6-bisphosphate; SAM, S-adenosyl-methionine; SAH,
S-adenosyl-homocysteine; SMM, S-methyl-methionine.

development (Cohen et al., 2017; Menegus et al., 2004; Ranocha
et al., 2001). Similar to other vitamins, SMM is produced by
high-temperature extraction from plants or enzyme synthesis. To
date, no studies have investigated conducted on SMM production
from microorganisms. In the present study, we introduced MMT
gene, which encodes a methionine S-methyltransferase from
Arabidopsis thaliana, into Saccharomyces cerevisiae to produce SMM.
Saccharomyces cerevisiae has a long history of use in various
industries, such as wine making, baking, and brewing. It is a
well-studied model organism in eukaryotic biology and the most
commonly used microorganism in fermentation processes. The
SMM production pathway involves the transfer of a methyl group
from SAM to methionine via methionine S-methyltransferase
(Fig. 1). The intracellular concentrations of the methionine and
SAM precursors play an important role in SMM production. Sake
yeast accumulates SAM intracellularly at higher concentrations
than other commonly used S. cerevisiae strains (Shiozaki et al.,
1984). Although there are differences in nutrient characteris-
tics between S. cerevisiae sake strains and laboratory strains like
S288C, their open reading frames share a high degree of similarity,
with an average BLAST similarity of over 95% (Akao et al., 2011).
Therefore, we selected the S. cerevisiae sake K6 strain for metabolic
engineering to produce SMM. In a previous study, an auxotrophic
S. cerevisiae sake K6 was constructed, making it a host strain for
metabolic engineering (Choi et al., 2009). In this study, a MMT gene
was introduced into the strain to enable the production of SMM.
Several steps were performed to increase SMM produc-
tion and prevent its degradation by microorganisms. First, we
deleted the MHT1 and SAM4 genes encoding homocysteine
S-methyltransferase to prevent the decomposition of SMM from
being decomposed. Additionally, we deleted MMP1, which encodes
SMM permease, to prevent SMM from being imported into the
cells. To further increase the pool of SAM, we upregulated ACS2,
which encodes acetyl-CoA synthase, by changing the promoter
and deleting MLS1, which encodes malate synthase (Chen et al.,
2016, 2021; Nielsen, 2014). We cultivated this engineered strain to
improve SMM production and optimize culture conditions for fed-
batch fermentation. Finally, for the first time, high levels of SMM
production were achieved using an engineered microorganism.
Materials and Methods
Strains and Plasmids
Table 1 provides a list of the strains and plasmids utilized in this
study. The S. cerevisiae sake K6 (Sake Kyokai No. 6) was purchased
from korean collection for type cultures (Daejeon, South Korea).
We previously developed a leucine auxotroph for S. cerevisiae sake,
K6-1 (Choi et al., 2009). Escherichia coli DH5α was used for plasmid
construction. To construct p425-MMT, a codon-optimized MMT
gene from A. thaliana was introduced into the p425GPD plasmid.
The amplification of the MMT gene and vector backbone frag-
ments was performed using Q5 DNA polymerase (New England
Biolabs, MA, USA). The plasmid backbone was assembled by
utilizing Gibson Assembly Master Mix (New England Biolabs, MA,
USA). To construct the plasmid for CRISPR-Cas9, the pML104-Leu
plasmid was used as the backbone. All oligonucleotide sequences
 Lee et al. | 3

Table 1. Strains and plasmids used in this study

Strains and plasmids Description Reference

Strains
BY4742 MATα his3 leu2 lys2 ura3 Lab stock
CEN.PK MATa, his3 leu2 trp1 ura3 Lab stock
K6 S. cerevisiae Sake Kyokai No. 6, MATa/α (Choi et al., 2009)
K6-1 K6 derivative, Leucine auxotroph (Choi et al., 2009)
BYM BY4742 harboring p425-MMT This study
CENM CEN.PK harboring p425-MMT This study

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K6M-1 K6-1 harboring p425-MMT This study
K6U-1 K6-1 derivative, URA3:: PGPD _MMT This study
K6U1-1 K6U-1 MHT1, SAM4 This study
K6U2-1 K6U-1 MMP1 This study
K6U3-1 K6U-1 MHT1, SAM4, MMP1 This study
K6U3-1p K6U3-1 PACS2 :: PPGK1 This study
K6U4-1p K6U3-1p MLS1 This study
Plasmids
p425 GPD Plasmid with 2μ origin, GPD promoter, CYC1 terminator and LEU2 selectable marker Lab stock
p425-MMT p425GPD harboring MMT This study
pML104 Plasmid with 2μ origin, GAP promoter, CYC1 terminator and URA3 selectable marker Lab stock
pML104-Leu pML104 derivative, LEU2 selectable marker This study
pML-MMT pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(MMT)_gRNA scaffold This study
pML-MHT1 pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(MHT1)_gRNA scaffold This study
pML-SAM4 pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(SAM4)_gRNA scaffold This study
pML-MMP1 pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(MMP1)_gRNA scaffold This study
pML-ACS2 pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(ACS2)_gRNA scaffold This study
pML-MLS1 pML104-Leu derivative, PSNR52_ tRNA(Gly)_target(MLS1)_gRNA scaffold This study

used in this study are listed in Table S1. To construct a single-
guide RNA (sgRNA) of MHT1, SAM4, MMP1, URA3, ACS2, and MLS1
genes, the amplification of each gene fragment was carried out
using Q5 DNA polymerase. NEB restriction enzymes (BclI and
SwaI) (New England Biolab, MA, USA) were added and incubated
at 37 °C for 2 hr. Then, 3 hr of ligation was performed for using a
DNA Ligation Kit (Takara, Kusatsu, Japan).
Growth Medium and Culture Conditions
Escherichia coli DH5α was cultured for gene cloning purposes using
Luria-Bertani (LB) broth. The cultivation temperature was main-
tained at 37 °C. For selection, 50 μg/L carbenicillin was added.
The initial culture of S. cerevisiae K6-1 was performed in 5 mL
of yeast extract peptone dextrose medium, comprising 10 g/L of
yeast extract, 20 g/L of peptone, and 10 g/L of D-glucose. Strains
carrying p425-MMT plasmid were cultured in synthetic defined
(SD) medium supplemented with specific amino acids and yeast
extract. The SD medium was composed of magnesium sulfate
heptahydrate (0.4 g/L), potassium dihydrogen phosphate (4 g/L),
dipotassium phosphate (2 g/L), yeast nitrogen base without
amino acids (6.7 g/L), and D-glucose (10 g/L). The selected amino
acid mixture included L-histidine, L-uracil, and L-tryptophan at a
concentration of 10 mg/L. Additionally, 3 g/L of yeast extract was
added to the medium. Then, the seed strains were used for inoc-
ulation of 5 mL SD medium containing 0.5% (w/v) of glycine and
0.5 g/L of methionine and cultured for 1.5 days. After pre-culturing
in the SD medium, 50 mL of fresh SD medium was re-inoculated
for the main culture. The entire culture procedure was performed
aerobically at 30 ˚C in a shaking incubator at 250 rpm.
Fed-batch fermentation of the SMM-overproducing strain,
K6U4-1p, was conducted in 3-L bioreactors, where the working
volume of SD medium was set at 1 L, and was continued for
3 days. The medium for fed-batch fermentation is composed of
3% (w/v) of yeast extract, 0.4 g/L of MgSO4 *7H2 O, 4 g/L of KH2 PO4 ,
2 g/L of K2 HPO4 , 10 g/L of peptone, 5 g/L of methionine, 5% (w/v)
of glycine and 10 g/L of D-glucose. The fermentation process
commenced with an initial OD600 value of 0.125. During the fed-
batch fermentation process, the temperature was maintained at
30 ˚C, with an aeration rate of 1 L/min and an agitation speed of
300 rpm. The aeration rate and agitation speed were increased up
to 5 L/min, 500 rpm to maintain the dissolved oxygen over 30%. A
solution of 60% (w/v) of D-glucose was used to maintain glucose
concentration above 3 g/L.
Metabolite Assays and RT-PCR
The biomass determination in the cell broth was carried out by
measuring the optical density at 600 nm using a DU730 spectrom-
eter (Beckman Coulter, Brea, CA, USA). To analyze the extracel-
lular metabolites, the culture supernatant was obtained by cen-
trifugation. A high-performance liquid chromatography (HPLC)
system equipped with a Waters 2414 refractive index detector
(Milford, MA, USA) with SH1011 column (Shodex, Tokyo, Japan),
was used for the analysis. The detector and oven temperature
was set to 45 and 75 °C, respectively, for the quantification of sug-
ars, organic acids, and alcohols. The mobile phase consisted of a
10 mM sulfuric acid solution with a liquid flow rate of 0.6 mL/min.
For the determination of SMM and methionine titers, an
Agilent 1260 HPLC system, HPLC UV detector system (Agilent
Technology, Santa Clara, CA, USA), was utilized. An amino acid
analysis column (Agilent Technology) at an oven temperature
of 40 °C was employed, and the HPLC UV system was used for
analysis. The mobile phases A and B were used for a gradient
flow method, as outlined in Table S2. Mobile phase A consisted of
20 mM Na2 HPO4 with 0.11% (v/v) phosphoric acid, while mobile
phase B comprised 45% (v/v) acetonitrile, 45% (v/v) methanol, and
10% (v/v) water. Prior to analysis, the samples were derivatized
4 | Journal of Industrial Microbiology and Biotechnology, 2023, Vol. 50, No. 1
using OPA reagent (Sigma-Aldrich, St. Louis, MO, USA), and the
UV detectors measured the samples at 338 nm.
Similarly, for the determination of SAM titer, an Agilent 1260
HPLC system, HPLC UV detector system was utilized with Eclipse
Plus C18 column (Agilent Technology) at oven temperature of
40 °C. The mobile phase A, consisting of 97% 0.5 M ammonium
formate with 3% (v/v) methanol, was adjusted for a constant flow
rate. The samples were analyzed at 254 nm using an automated
injection program with a UV detector.
To compare the expression levels of the ACS2 gene, real-time
PCR analysis was conducted. Strains K6U3-1 and K6U3-1p were
cultured in SD medium supplemented with yeast extract for
48 hr. Following the harvest of each strain through centrifugation
at 3 500 rpm for 5 min, the purification of total RNA was carried
out using the RNeasy Purification Kit (Qiagen, Hilden, Germany)
in accordance to the manufacturer’s instructions. Subsequently,
cDNA synthesis was performed using the iScriptTM cDNA synthe-
sis kit (Bio-Rad, Hercules, CA, USA) with 500 ng of total RNA. The
resulting cDNA was then utilized as a template for real-time PCR
analysis. The PCR was performed using a MiniOpticon (Bio-Rad).
Results and Discussion
Identification of Methionine S-methyltransferase
and Suitable Yeast Strain
To produce SMM in microorganisms, methionine S-
methyltransferase, which is responsible for transferring a methyl
group from SAM to methionine, thereby producing SMM and
S-adenosylhomocysteine (SAH), is needed. MMT genes encoding
methionine S-methyltransferases have been identified in several
plants, such as mouse ear cress, maize, sunflower, and barley.
We synthesized three representative MMT genes derived from A.
thaliana, Wedelia bifolora, and Hordeum vulgare, codon-optimized,
and expressed them in E. coli DH5α. The cultivation of E. coli
strains was carried out in M9 medium supplemented with 0.5 g/L
of methionine for 24 hr. The titer of SMM with E. coli containing A.
thaliana-derived MMT was more than twofold higher than that of
the other strains (Fig. S1). Based on these observations, the MMT
gene from A. thaliana was selected for subsequent experiments.
To using GRAS a generally regarded as safe (GRAS) host, we
attempted to produce SMM using the MMT gene from A. thaliana
in yeast. To select an appropriate host, three different yeast
strains–BY4742, CEN-PK, and K6-1–were used to produce SMM.
The strains carrying p425-MMT, named as BYM, CENM, and
K6M-1, were grown in SD medium supplemented with 0.5 g of
methionine for 48 hr. As shown in Fig. 2, K6M-1 strain produced
considerable amounts of SMM (0.07 g/L), whereas the other two
strains did not produce SMM. This was probably due to the ability
of the K6-1 strain to produce high levels of SAM, as confirmed in
a previous study (Choi et al., 2009).
When heterologous genes are expressed in yeast using plas-
mids, a defined medium is often used to select the strains with
auxotrophic markers. This can cause problems, such as a slow
growth rate of the cells and/or low production of target chem-
icals. To address these issues, the MMT gene from A. thaliana
was integrated into the genome of K6-1 strain, specifically at
the URA3 locus. The resulting strain was named as K6U-1.
Through this integration, a specific selection medium is not
needed. Additionally, the URA auxotrophic phenotype of K6U-1
allows for further genetic engineering. When K6U-1 was cultured
in SD medium with yeast extract to alleviate the low specific
growth rate (Table S3), a significant increase in SMM production
(0.26 g/L) was observed compared to the strain, K6M-1 (Fig. 2).
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Fig. 2. SMM and SAM titer of MMT gene introduced by vector for SMM
production in BY4742, CEN.PK and K6-1 strains, MMT gene integrated in
K6-1 strain and various strains that have undergone engineering to
increase SMM titer when cultured in a 50 mL flask for 48 hr. Vec(+)
indicates overexpressed by vector. gDNA(+) indicates the gene
integrated into gDNA for overexpression. (-) indicates the deletion of
genes related to SMM production. Error bars represent standard
deviations of triplicated experiments.
This was more than a threefold increase in SMM production by
the integration of genes and by using a complex medium.
Knock-out of Genes Involved in SMM
Degradation Pathway Enhances SMM Titer
Several studies have investigated the SMM degradation pathway
in yeast (Rouillon et al., 1999; Thomas et al., 2000; Eder et al.,
2022). These studies have successfully identified the genes in-
volved in the uptake of sulfur complexes and those responsible
for decomposing and utilizing SMM within the cell. The identi-
fication of these genes revealed the biochemical pathways that
govern the utilization of sulfur compounds in yeast. Specifically,
these studies have shown that yeasts possess specialized trans-
port and degradation pathways for various sulfur compounds,
including SMM. Once inside the cell, SMM is degraded by a series
of enzymes that produce several compounds that can be used for
cell growth and survival.
Two genes in the yeast genome, MHT1 and SAM4 that encode
homocysteine S-methyltransferases, have been identified in the
SMM degradation pathway. Another gene, MMP1, which encodes
S-methyl-methionine permease, is involved in SMM uptake
(Fig. 1). To alleviate SMM degradation, MHT1 and SAM4 were
knocked out in the K6U-1 strain to produce K6U1-1. In addition,
MMP1 was knocked out to generate K6U2-1 cells. Deletion of
Homocysteine S-methyltransferase and S-methyl-methionine
permease improved SMM production (Fig. 2). Therefore, the strain
with all three genes deleted, K6U3-1, was generated and produced
0.58 g/L of SMM (Fig. 2). We confirmed that knocking out genes
related to SMM degradation in yeast effectively enhanced SMM
production in yeast.
Optimizing Precursor Biosynthesis for Improved
SMM Production
Because SAM is a precursor of SMM, its accumulation can be a key
factor in achieving high yields of SMM while minimizing its impact
on other SAM-dependent biological processes. Several engineer-
ing studies have been carried out to augment the acetyl-CoA
pool, which is related to methionine metabolism and to increase
the production of SAM (Chen et al., 2016; Kanai et al., 2017).

Fig. 3. The native ACS2 promoter was replaced to PGK1 promoter to
up-regulate ACS2 gene for enhanced acetyl-CoA level. The gene MLS1
was deleted for enhanced acetyl-CoA level. The following graph
illustrates the comparison of SMM production among the strains
expected to have increased acetyl-CoA levels. Error bars represent
standard deviations of triplicated experiments.
To enhance acetyl-CoA accumulation, the expression of ACS2
gene encoding acetyl-coenzyme A synthetase was modulated by
replacing the native promoter of the ACS2 gene with the PGK1
promoter, a strong promoter (Fig. S2). RT-PCR analysis revealed an
approximately 1.3fold increase in the expression of ACS2 (Fig. S2).
However, the resulting strain, K6U3-1p, did not exhibit a mean-
ingful increase in SMM production (Fig. 3). We aimed to further
increase the pool of acetyl-CoA by blocking its entry into the
glyoxylate cycle with deletion of the MLS1 gene encoding malate
synthase in the K6U3-1p strain. According to the previous study
(Chen et al., 2016), it was expected that the up-regulation of ACS2
gene expression and deletion of MLS1 gene together would lead to
a relatively higher intracellular level of acetyl-CoA compared to
the wild-type strain, K6-1. The anticipated elevation in acetyl-CoA
levels is expected to enhance the efficient accumulation of SAM,
subsequently resulting in an increased production of SMM. The
resulting strain, K6U4-1p, produced 0.71 g/L of SMM, which is sig-
nificant increase from that of K6U3-1 (Fig. 3), with slight decrease
of the specific growth rate (Table S3). Increasing the intracellular
acetyl-CoA pool showed a positive effect in SMM production.
Overproduction of SMM through Fed-Batch
Fermentation
The resulting strain, K6U4-1p, which showed the highest SMM
titer, was selected for fermentation to maximize SMM production.
For the fed-batch fermentation, a minimal medium, SD media,
supplemented with 3% (w/v) yeast extract was utilized. In order
to enhance the intracellular production of SMM, the folate cycle,
which plays a vital role in SAM biosynthesis, was effectively
stimulated by supplementing of 0.5% (w/v) glycine. Methionine
(5 g/L) was added to promote SMM production. A 1 L medium
was used in a 3 L-jar for fed-batch fermentation. d-Glucose, with
an initial concentration of 10 g/L, was monitored every hour to
maintain at least 3 g/L of glucose by feeding.
The maximum optical density (OD600 ) of the fermentation
reached 16.8 at 72 h. The highest SMM titer in fermentation
was 1.92 g/L, which was achieved through periodic feeding of D-
glucose during fed-batch fermentation (Fig. 4). This is significant,
considering that this is the first attempt at SMM production by
microorganisms.
Conclusion
We attempted the heterologous production of SMM in a GRAS
microorganism by introducing a key enzyme, MMT, from A.
thaliana. The MMT gene was synthesized by codon optimization
in S. cerevisiae and integrated into the URA3 locus using CRISPR-
Lee et al. | 5
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Fig. 4. Results of 72 hr fed-batch fermentation with 1 L working volume.
D-glucose (10 g/L), 3% (w/v) of yeast extract, 0.5% (w/v) of glycine and
5 g/L of methionine were used for initial media. The SMM overproducing
strain, K6U4-1p, was used for SMM synthesis. Black circle, cell growth;
Black square, SAM (g/L); Black triangle, SMM (g/L); and Black reverse
triangle, Glucose (g/L)
Cas9. S. cerevisiae K6-1 strain, with enhanced intracellular SAM
production, successfully produced a significant amount of SMM.
To enhance SMM production, genes involved in SMM recycling
(MMP1, MHT1, and SAM4) were knocked out in the host strain.
Furthermore, the expression of ACS2 was enhanced by substi-
tuting its promoter, and the removal of MLS1 was carried out to
augment the intracellular pool of acetyl-CoA, the precursor of
SAM. The final strain, K6U4-1p, was used in fed-batch fermenta-
tion, which resulted in an SMM titer of approximately 2 g/L. This
is the first report of SMM production from a microorganism and
demonstrates the potential of genetic engineering to enhance
SMM production in S. cerevisiae.
Supplementary Material
Supplementary material is available online at JIMB
(www.academic.oup.com/jimb).
The following information is included in the Supporting In-
formation: All primers and sequences used in this study and
flow rate for each mobile phase as a function of time in HPLC for
measuring SMM production. Specific growth rates and titers of
the engineered strains in different media. The results of the SMM
titers using various plant-derived MMT genes expressed in E. coli
are presented in the data. The data included in this study com-
prised the growth curves for each strain. Additionally, the results
of RT-PCR analysis after replacing the native ACS2 promoter with
the well-known strong PGK1 promoter are provided.
Funding
This work was supported by the Korea University funding.
Competing Interest
The authors declare that they have no competing financial inter-
ests or personal relationships that could have influenced the work
reported in this study.
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