Metabolic engineering of: Escherichia coli for production of non-natural acetins from glycerol

Article abstract of DOI:10.1039/d0gc02395g

Mono-, di- and triacetin are three glycerol esters which are usually synthesized via costly and environmentally unfriendly chemical synthesis methods. Here, Escherichia coli is metabolically engineered for the production of mono-, di- and triacetin using glycerol as a substrate. First, a novel biosynthetic route of mono- and diacetin is established by overexpression of a native enzyme, maltose O-acetyltransferase (MAA). Next, the biosynthetic pathway is extended to produce a mixture of mono-, di- and triacetin by overexpression of chloramphenicol-O-acetyltransferase (CAT). By successive strategies, including heterologous gene expression, metabolic engineering, and culture optimization, a recombinant E. coli is enabled to produce more than 27 g L-1 of a mixture of mono-, di- and triacetin in shake flask cultures, which is a >650-fold increase over the initial production of 0.04 g L-1. In vitro studies confirm the acetylation of glycerol to mono- and diacetin by MAA, and the additional acetylation to triacetin by CAT. When crude glycerol is used as a substrate, the engineered strain produced a total of 25.9 g L-1 of the acetin mixture, about the same as that achieved from pure glycerol. To our knowledge, this is the first successful report of microbial production of the artificial chemicals, acetins.

Full text of DOI:10.1039/d0gc02395g

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Metabolic engineering of Escherichia coli for
production of non-natural acetins from glycerol†
Cite this: DOI: 10.1039/d0gc02395g
a
Bakht Zada, ‡a Seongjoon Joo, ‡b Chonglong Wang, ‡c Tenzin Tseten,
Seong-Hee Jeong, a Hogyun Seo, b Jung-Hoon Sohn, d Kyung-Jin Kim
*
b
a
and Seon-Won Kim
*
Mono-, di- and triacetin are three glycerol esters which are usually synthesized via costly and environ-
mentally unfriendly chemical synthesis methods. Here, Escherichia coli is metabolically engineered for
the production of mono-, di- and triacetin using glycerol as a substrate. First, a novel biosynthetic route
of mono- and diacetin is established by overexpression of a native enzyme, maltose O-acetyltransferase
(MAA). Next, the biosynthetic pathway is extended to produce a mixture of mono-, di- and triacetin by
overexpression of chloramphenicol-O-acetyltransferase (CAT). By successive strategies, including heter-
ologous gene expression, metabolic engineering, and culture optimization, a recombinant E. coli is
enabled to produce more than 27 g L−1 of a mixture of mono-, di- and triacetin in shake flask cultures,
which is a >650-fold increase over the initial production of 0.04 g L−1. In vitro studies confirm the acetyl-
ation of glycerol to mono- and diacetin by MAA, and the additional acetylation to triacetin by CAT. When
crude glycerol is used as a substrate, the engineered strain produced a total of 25.9 g L−1 of the acetin
mixture, about the same as that achieved from pure glycerol. To our knowledge, this is the first successful
report of microbial production of the artificial chemicals, acetins.
Received 14th July 2020,
Accepted 11th September 2020
DOI: 10.1039/d0gc02395g
rsc.li/greenchem
developed,3–5 which can be called Zero Waste Biorefinery. This
biotechnology conserves and recovers all resources to elimin-
Introduction
Glycerol is a by-product accompanying the production of bio- ate the volume of waste materials produced by industry that
diesel. It is reported that 10 lb of crude glycerol is generated would be otherwise be burned or buried. The goal of Zero
for every 100 lb of biodiesel produced by trans-esterification of Waste Biorefinery is thus to process and convert waste streams
vegetable oils or animal fats.1 Thus, the worldwide production produced at various industrial sites into valuable chemical
of glycerol in 2020 is estimated to be six times more than its compounds, thereby closing the production loop with no net
demand,2 resulting in waste of glycerol. In recent years there waste. However, various products obtained from glycerol such
has been growing interest in biorefinery industries towards the as polymers (e.g. Sorona®, a co-polymer of 1,3-propanediol
utilization of surplus glycerol. Bioconversions of surplus gly- and terephthalates) and Susterra® propanediol are not associ-
cerol into 1,3-propandiol, propionic acid, citric acid, succinic ated with biodiesel and must be transported to their appli-
acid, butanol, single cell oil and hydrogen gas have been cation sites for further utilization.
Triacetin, also called glycerin triacetate, could be a poten-
tial alternative derived from waste glycerol. As a tri-ester of gly-
aDivision of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National
University, Jinju 52828, Republic of Korea. E-mail: swkim@gnu.ac.kr,
bakhtqau@gmail.com
bSchool of Life Sciences, KNU Creative BioResearch Group, Kyungpook National
University, Daegu41566, Republic of Korea. E-mail: kkim@knu.ac.kr,
wntjdwnswkd@naver.com
cSchool of Biology and Basic Medical Sciences, Soochow University, Suzhou, People’s
Republic of China. E-mail: clwang@suda.edu.cn
dSynthetic Biology and Bioengineering Research Center, Korea Research Institute of
Bioscience and Biotechnology, Daejeon 34141, Republic of Korea.
E-mail: sohn4090@kribb.re.kr
cerol, it can be used as a fuel additive to improve the freezing
and viscosity properties of biodiesel as well as an antiknock
agent which can reduce engine knocking in gasoline.6,7
Engine performance can be significantly improved by blending
10% triacetin with biodiesel.8 The production of triacetin
along with biodiesel production could serve as a complete on-
site Zero Waste Biorefinery (Fig. 1A). Triacetin also has appli-
cations in cosmetics, food additives, plasticizers and precur-
sors for isoamyl acetate synthesis.9–11 It is considered to be a
possible source of food energy in the artificial food regener-
ation system for long space missions.12 Of the same impor-
tance, mono- and diacetin have received a great deal of atten-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0gc02395g
‡These authors contributed equally to this work.
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Fig. 1 Schematic overview of Zero Waste Biorefinery and acetin biosynthesis in recombinant E. coli. (A) Approx. 10% surplus glycerol is produced by
the trans-esterification of triacylglycerol (TAG) and methanol during biodiesel synthesis. A biological pathway is devised to convert surplus glycerol
to the biofuel additive acetins that are used for blending with biodiesel to improve freezing and viscosity properties. The on-site application of
surplus glycerol suggests a Zero Waste Biorefinery with no net waste. (B) Monoacetin is synthesized via co-condensation of glycerol and acetyl-CoA
catalyzed by E. coli native enzyme, MAA. The monoacetin is further acetylated to diacetin by the action of the same enzyme, MAA. The CAT enzyme
further transforms diacetin into triacetin by involving a third molecule of acetyl-CoA. The acetyl-CoA as a co-substrate is generated via the EMP
pathway. The abbreviations of enzymes are as follows: GlpF, glycerol facilitator; GlpK, glycerol kinase; MAA, maltose O-acetyltransferase; CAT, chlor-
amphenicol O-acetyltransferase.
tion in recent years as high-value specialty chemicals. They are Moreover, the trans-esterification reaction catalyzed by com-
currently being used in pharmaceuticals, food industries and mercial lipase is reversible, and the complex of the final reac-
cosmetics, as well as in the chemical industry as plasticizers, tion mixture, comprising reactants (glycerol and alkyl acetate)
softening and tanning agents, explosives and raw materials for and alcohol byproducts beyond the target acetins, increases
biodegradable polyesters.13,14 Like triacetin, diacetin could the difficulty and cost of further processing to get pure acetins.
also be used as a biofuel additive.15
The co-substrate, alkyl acetate, for acetin synthesis would
An acetin mixture of mono-, di-, and triacetin has been pro- incur an extra cost in the lipase-based approach. Perhaps this
duced by the chemical esterification of glycerol with acetic approach might also be restricted to pure glycerol as a sub-
acid or acetic anhydride using inorganic catalysts strate because most enzymes are highly sensitive to impurities
(Fig. S1A†).16–18 However, the chemical method suffers from and crude glycerol contains various impurities. Therefore, the
the requirements for expensive catalysts, a high reaction temp- microbial production of acetins using glycerol as a substrate
erature and pressure, the use of hazardous materials, and the remains the method of choice due to its economic feasibility
release of toxic intermediates causing environmental pol- as well as environmental friendliness.
lution. Besides, the chemically synthesized acetins contain
In the last few decades, paramount interest is being paid to
toxic byproducts and radicals, which limits their applications the use of microbes for the production of high-value specialty
in the pharmaceutical, food, and cosmetic industries. The bio- chemicals. Recent advances in metabolic engineering, protein
logical production of acetins using a commercial lipase, engineering and synthetic biology have revolutionized the
Novozyme 435 (CalB), has been adopted to catalyze the trans- ability to design and engineer biosynthetic pathways in
esterification reaction of glycerol with alkyl acetate to acetins microbial hosts for the production of a wide variety of com-
(Fig. S1B†).19,20 However, the CalB enzyme is too costly to modity products, such as silk proteins,21 pharmaceuticals,22
support the feasible and economical production of acetins. bio-plastics,23 platform chemicals24 and biofuels.25,26
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Therefore, it will be an immense benefit to the industrial DH-MD8 produced 31.8 mg L−1 and 8.9 mg L−1 of mono- and
world to design and establish a novel biological route in host diacetin, respectively (Fig. 2B). The broad substrate specific Ec-
microorganisms for the production of non-natural acetins. MAA acetylates various sugars, including maltose and glucose,
Among the limited number of microorganisms harnessed as and the most effective substrate is glucose which acetylates at
cell factories, E. coli is the most popular platform cell factory the C6 position.34–36 Strain DH-MD8 did not produce any
because of its high growth rate, well-known physiology, and acetin when glucose was substituted instead of glycerol as the
easy culture and genetic manipulation.27 E. coli has been meta- substrate (data not shown). Thus, this study newly reveals that
bolically engineered for the production of many useful chemi- E. coli maltose O-acetyltransferase (Ec-MAA) can acetylate even
cals and biofuels from glycerol as a carbon source.28–32 As glycerol (C3) to monoacetin (C5) and diacetin (C7) besides its
acetins are glycerol-based compounds, E. coli could be an ideal native substrates of maltose and glucose. For the first time, we
host for the production of acetins, if a biosynthetic pathway have successfully created a biosynthetic pathway through the
for non-natural acetins can be successfully created and engin- overexpression of MAA to produce mono- and diacetin in
eered. Only a few microorganisms such as Klebsiella oxytoca, E. coli using glycerol as a substrate (Fig. 1B), whereas a fully
Enterobacter aerogenes and some other Enterobacter sp. have acetylated product of glycerol, triacetin, which was among our
been reported to produce monoacetin naturally in a trace target products but not produced by this engineered strain.
amount.33 However, a synthetic pathway for monoacetin (or di-
Structural basis for acetins synthesis by MAA
and triacetin) has not been elucidated in any living organisms.
In this study, for the first time, we have designed and estab- To provide the structural basis for mono- and diacetin biosyn-
lished a novel biosynthetic pathway for mono-, di- and triace- thesis, we analyzed the crystal structures of maltose
tin from glycerol in E. coli.
O-acetyltransferase from E. coli (Ec-MAA). First, we attempted
to determine the crystal structure of Ec-MAA in complex with
glycerol (Fig. S4 & Table S2†). However, neither co-crystalliza-
tion nor a soaking experiment with glycerol was successful
because the tris(hydroxymethyl)aminomethane (Tris) molecule
was bound at the substrate binding site of Ec-MAA (Fig. 3A).
Fortunately, the Tris-complexed structure provides a more
Results and discussion
Formation of mono- and diacetin in E. coli overexpressing
O-acetyltransferase
So far, no report has been available on the production of explicit explanation for the acetylation of glycerol. In fact, Tris
mono- and diacetin in living organisms, and annotated meta- and glycerol have quite similar structures; Tris contains an
bolic pathways do not exist for a reference. As acetins are amine-group head and three hydroxymethyl-group arms, and
acetyl-esterified compounds of glycerol, it was hypothesized glycerol is composed of a hydroxyl-group head and two hydro-
that some O-acetyltransferases could catalyze the acetylation of xymethyl-group arms. In our structure, the Tris molecule is
glycerol to acetins. In E. coli, there are several mainly stabilized with residues of Asp69, Asn83, His113 and
O-acetyltransferases catalyzing the acetyl-CoA dependent trans- Glu125 through hydrogen bonds (Fig. 3B). The first hydroxy-
formation of a diverse set of substrates, including carbo- methyl-group arm (position I) is stabilized with the catalytic
hydrates and amino acids. However, the functional activities of His113 residue by a hydrogen, suggesting that the hydroxy-
most of these endogenous acetyltransferases have not methyl-group might be acetylated by the enzyme (Fig. 3B).
been fully explored. Five candidate genes of lacA, maa, cysE, Asp69 forms hydrogen bonds with the second (position II) and
wecH, and nhoA encoding galactoside O-acetyltransferase, third hydroxymethyl-group (position III) arms, and Asn83 and
maltose
O-acetyltransferase,
serine
acetyltransferase, Glu125 stabilize the second hydroxymethyl-group arm and the
O-acetyltransferase and arylamine N-acetyltransferase, respect- amine-group head, respectively (Fig. 3B). Based on these obser-
ively, as well as three putative acetyltransferase genes of yjgM, vations, we suggest that glycerol might bind in two different
yjaB and yiiD are screened by function-mediated mining and orientations so both 1′- and 3′-aceylated monoacetins can be
proposed to test acetin synthesis from glycerol in E. coli. These synthesized. 1′-Acetylated monoacetin can be synthesized
candidate genes of Ec-lacA, Ec-cysE, Ec-yjgM, Ec-yjaB, Ec-yiiD, when glycerol binds in a mode with two hydroxymethyl-groups
Ec-wecH, Ec-nhoA and Ec-maa were cloned into a medium copy at position I and position II (Fig. 3C), while 3′-acetylated mono-
vector pTrc99A with a strong Ptrc promoter and transformed acetin can be synthesized by binding in a mode with two
into E. coli DH5α to create recombinant strains DH-MD1 to hydroxymethyl-groups at position I and position III (Fig. 3D).
DH-MD8 (Fig. 2A & Table S1†). E. coli DH5α harboring the Like the fashion for glycerol binding, the binding of mono-
empty plasmid pTrc99A (DH-MD0) was used as a control. All acetin isomers would show two different orientations in the
the strains were cultivated at 37 °C in a shake flask with 50 mL diacetin synthesis, where 1′- or 3′-acetyl-hydroxymethyl-groups
of M9 medium containing 5 g L−1 yeast extract (M9YE), 2% of monoacetins are located at position III and position II,
(v/v) glycerol, and 0.5 mM IPTG. Compared to strains DH-MD0 respectively. In both cases, the free-hydroxymethyl-groups are
to DH-MD7 that failed to produce either mono- or diacetin, located to position I to be acetylated. It should be noted that
strain DH-MD8, overexpressing Ec-maa produced mono- and binding of the 3′-acetyl-hydroxymethyl-group to position III
diacetin, as detected by GC-FID (Fig. S2†) and consequently might be much weaker than that of the 1′-acetyl-hydroxy-
confirmed by GC-MS (Fig. S3†). After 48 h of culture, strain methyl-group to position II, because position II does not seem
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Fig. 2 Production of mono- and diacetin from recombinant E. coli. (A) The construction scheme of E. coli endogenous O-acetyltransferases. Eight
O-acetyltransferases genes lacA, cysE, yjgm, yjaB, yiiD, wecH, nhoA, and maa are constructed under a strong promoter trc. (B) Production of mono-
and diacetin from the recombinant E. coli overexpressing endogenous O-acetyltransferases. The strains DH-MD1 to DH-MD8 are E. coli DH5α trans-
formed with the plasmids pMD1 to pMD8, respectively. The control strain DH-MD0 is E. coli DH5α harboring the empty vector pTrc99A. (C) The con-
struction scheme of heterologous maltose O-acetyltransferase (MAA) from different organisms. The genes Sc-maa, Hj-maa, Lb-maa, Pp-maa, and
Bs-maa are derived from S. carnosus, H. jeotgali, L. brevis, P. putida and B. subtilis, respectively. (D) Production of mono- and diacetin from strains
DH-MD9 to DH-MD13 transformed with plasmids pMD9 to pMD13, respectively. The means and standard divisions are from two biological
replicates.
to have enough space to accommodate the 3′-acetyl-group of 3′- (https://www.genscript.com/codon_opt_pr.html) with
a codon
acetylated monoacetin (Fig. 3A). We suspect that this phenom- adaptation index (CAI) of more than 0.8 (Table S3†). The optimized
enon can cause incomplete conversion of 3′-acetylated mono- genes were cloned into pTrc99A (pMD9 to pMD13) and trans-
acetin to diacetin, which might explain the accumulation of a formed in E. coli DH5α to create recombinant strains DH-MD9 to
large portion of monoacetin in this in vivo acetin biosynthesis DH-MD13 (Fig. 2C & Table S1†). Mono- and diacetin were conse-
and serve as the rational basis for protein engineering to quently evaluated from all these newly constructed strains
produce specific acetins.
(Fig. 2D). Strains DH-MD11, DH-MD12 and DH-MD13 show
higher titers of mono- and diacetin when compared to strain
DH-MD8. The highest titer of 214.9 mg L−1, equivalent to a 5.2-
fold increase over strain DH-MD8, was achieved from strain
DH-MD13, which overexpressed an efficient catalyzer, Bs-MAA, for
mono- and diacetin production.
Screening of efficient heterologous maltose O-acetyltransferase
To screen an efficient heterologous MAAs for acetin production,
MAAs from other microbial species were retrieved by similarity
searches of amino acid sequences using Ec-maa. Five MAA candi-
dates from Staphylococcus carnosus, Halalkalicoccus jeotgali,
Lactobacillus brevis, Bacillus subtilis and Pseudomonas putida were
selected by a BLAST search (Fig. S5 & S6†). To gain the optimal
Extension of mono- and diacetin biosynthetic pathway to
triacetin in E. coli
expression of the heterologous genes in E. coli, the ORF sequences Various metabolic engineering strategies have been developed
of all these genes were optimized by using the GenScript algorithm and applied to increase the production of chemicals of inter-
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Fig. 3 Structural basis for acetin biosynthesis by Ec-MAA and CAT. (A) Substrate binding pocket of Ec-MAA. The Ec-MAA structure is presented as
an electrostatic potential surface model. The Tris molecule bound in Ec-MAA is shown as a stick model with a cyan color. The purple circles show
the positions of the three hydroxymethyl groups of Tris. (B–D) Binding mode of the Tris and glycerol molecules. The Ec-MAA structure is presented
as a cartoon diagram with a gray color. The Tris and glycerol molecules are shown as sticks with cyan and light-blue colors, respectively. Residues
involved in the stabilization of the Tris and glycerol molecules in Ec-MAA are shown as sticks. The purple circles show the position of the hydroxy-
methyl group of Tris. (E) The binding mode of chloramphenicol in CAT and docked diacetin. The CAT structure is presented as a cartoon diagram
with a gray color. The chloramphenicol molecule is shown as sticks with a cyan color. Residues forming the chloramphenicol binding pocket are
shown as gray-colored sticks. (F) The binding mode of simulated diacetin. The simulated diacetin molecules are shown as sticks with a light-blue
color.
est. A canonical strategy is to balance the expression level of The gene Bs-maa was thus cloned into pET-28a(+) and pSTV28,
protein constituents of biosynthetic pathways, which can be respectively, resulting in pMD14 and pMDT1 plasmids
easily addressed by harnessing promoters of various strengths. (Fig. 4A). E. coli DH5α (DE3) and DH5α harboring pMD14 and
Thus, a stronger PT7 promoter and a weaker Plac promoter than pMDT1 are accordingly called DH-MD14 and DH-MDT1. Strain
Ptrc were applied to optimize the expression level of Bs-MAA. DH-MD14 produced 125.5 mg L−1 of mixed mono- and diace-
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Fig. 4 Production of mono-, di-, and triacetin from recombinant E. coli. (A) The construction scheme of pMD13, pMD14, and pMDT1. (B)
Production of acetins and (C) cell growth of recombinant E. coli strains DH-MD13, DH-MD14, and DH-MDT1. The means and standard divisions are
from two biological replicates.
tin after 48 h of culture, which is 42% lower than that of strain products.40–43 The esterification of diacetin to triacetin by CAT
DH-MD13 (Fig. 4B). The decrease in acetin production from can be speculated from the broad substrate specificity of this
the PT7 promoter is likely to be due to a metabolic burden on enzyme.
the cells, since the cell growth of DH-MD14 was much lower
In order to elucidate whether CAT does three consecutive
than that of DH-MD13 (Fig. 4C). An unexpected peak was acetylations of free glycerol to triacetin or recruits the mono-
observed in the GC chromatogram (Fig. S2†) of strain or diacetin generated by MAA, the DH-CAT strain harboring
DH-MDT1 which was consequently identified as triacetin by pT-CAT (pTrc99A containing cat under Ptrc, Table S1†) was cul-
GC-MS (Fig. S3†). After 48 h of culture, strain DH-MDT1 pro- tured in M9YE medium supplemented with 2% (v/v) glycerol.
duced 581.1 mg L−1 of total acetins, consisting of 300.8 mg L−1 No acetin was observed from the culture of strain DH-CAT
of monoacetin, 75.5 mg L−1 of diacetin and 204.8 mg L−1 of (data not shown). Strain DH-CAT was also cultured in M9YE
triacetin (Fig. 4B). The total acetin production of DH-MDT1 medium containing 2% (v/v) of glycerol and 5000 mg L−1 of
was 2.7-fold higher than that of DH-MD13 (214.9 mg L−1), the acetin mixture, which is composed of 3800 mg L−1 of
accompanied by better cell growth of DH-MDT1 than monoacetin and 1200 mg L−1 of diacetin (Fig. S7†). Strain
DH-MD13. The remaining key task of screening DH-MD0 harboring an empty plasmid pTrc99A was used as a
O-acetyltransferase for triacetin formation was fortunately control. After 36 h of incubation, diacetin was completely
attained from strain DH-MDT1. The triacetin generation from transformed to triacetin by strain DH-CAT while there was no
strain DH-MDT1 was suspected to be due to the cat gene of conversion of diacetin to triacetin by strain DH-MD0. There
pMDT1, encoding chloramphenicol O-acetyltransferase (CAT). was no significant variation in monoacetin in cultures of
It generally functions as an antibiotic marker to detoxify chlor- either strain. Thus, these results indicate that CAT contributes
amphenicol to either chloramphenicol 3-acetate or chloram- to the acetylation of diacetin to triacetin, and a schematic
phenicol 1-acetate, by covalently attaching an acetyl group to diagram of mono-, di- and triacetin biosynthesis is presented
hydroxyl groups of the antibiotic.37 Nevertheless, CAT is in Fig. 1B.
known to have a broad substrate specificity and catalyzes the
non-specific esterification of various alcohols to ester deriva-
Structural basis for acetin synthesis by CAT
tives such as 3-methyl-1-butyl acetate, isobutyl isobutyrate, and To understand the molecular mechanism of acetylation of dia-
2-phenylethyl acetate.38,39 CAT has also been reported for the cetin to triacetin by CAT, we performed a molecular docking
esterification of isoprenoid alcohols, such as isopentenol, peri- simulation of diacetin into the CAT structure and compared it
llyl alcohol, geraniol and retinol, to corresponding acetylated with the structure of CAT in complex with chloramphenicol
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Fig. 5 Strong expression of CAT increasing acetin productivity. (A) The construction scheme of pMDT2 plasmid expressing cat and Bs-maa. (B)
Production of acetins and (C) percentage composition between strains DH-MDT1 and DH-MDT2. (D) Time course production of acetins from strain
DH-MDT2. The means and standard divisions are from two biological replicates.
(PDB: 3U9F) (Fig. S8†). In the chloramphenicol-complexed CAT that the high-level expression of CAT via using a strong promo-
structure, the V-shaped chloramphenicol molecule is mainly ter and a high copy vector could increase triacetin production.
stabilized by hydrophobic residues, and the hydroxymethyl- Thus, the cat gene was cloned into the upstream of Bs-maa
group to be acetylated is located in the vicinity of the catalytic under a strong trc promoter in a high copy vector pMD13
site (Fig. 3E). Our docking simulation result showed that diace- (pTrc99A based vector with 20–30 copies), resulting in the
tin binds to the substrate binding site in a mode similar to pMDT2 plasmid (Fig. 5A). The resulting strain DH-MDT2 was
that of chloramphenicol (Fig. S8†). The simulated diacetin cultured on M9YE medium with 2% (v/v) glycerol. After 48 h of
molecule also exhibits a V-shape with an acetylated arm at culture, DH-MDT2 produced 2.62 g L−1 of total acetins, which
either side (Fig. 3F). The hydroxyl-group to be acetylated is also represented an increase of 4.5-fold over the production of
located near the hydroxymethyl-group of chloramphenicol to DH-MDT1 (Fig. 5B). Among the total acetins produced from
be acetylated (Fig. 3F), indicating that acetylation of diacetin strain DH-MDT2, 56.1% is the triacetin constituent which is a
can occur at the 2′-hydroxyl-group in this binding mode. We 62.7% improvement over strain DH-MDT1 (Fig. 5C). This
suspect that the two acetylated arms play a crucial role in the result shows that the expression of CAT from the pMDT2
binding of diacetin into CAT, explaining why the enzyme plasmid not only increases the production titer of total acetins
cannot utilize glycerol or monoacetin as a substrate.
but also enhances the proportion of triacetin among the total
acetins. Thus, the increased production of triacetin in
DH-MDT2 is ascribed to the improved expression of CAT as it
Overexpression of CAT to improve triacetin production
We succeed in the production of triacetin catalyzed by CAT is under a strong promoter and in a high copy vector.
that acts as an antibiotic marker in the pMDT1 plasmid.
The production of acetins was evaluated over culture time
Originally the cat gene in the pMDT1 plasmid (pSTV28 based in strain DH-MDT2 in order to find out the culture time for
vector) is under a weak CAT promoter, which expresses CAT maximum production. The production reached its maximum
just for the detoxification of the antibiotic, chloramphenicol. at 48 h of culture and there was no further increase after that
Moreover, pMDT1 is a p15A Ori based vector (Fig. 4A) with a (Fig. 5D). To find out the reason for no increase in production
low copy number (10–15 copies). Therefore, we hypothesized after 48 h, the cells were collected at 12 h intervals from the
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culture of strain DH-MDT2, resuspended to an OD600 of 1 in DH5α to create recombinant strains DH-MDT3 to DH-MDT7
fresh M9 medium with 2% (v/v) glycerol, and incubated for (Fig. 6A & Table S1†). Triacetin was indeed produced from
12 h at 37 °C with 250 rpm shaking. The production of acetins strains DH-MDT3, DH-MDT4 and DH-MDT5 overexpressing
by the harvested cells was compared at different culture times Bc-Oat, Pa-Oat, and Ca-Oat, respectively, while there was no
(Fig. S9†). The maximum acetin production was achieved from production from strains DH-MDT6 and DH-MDT7 with Ma-Oat
the harvesting at 36 h while a trace amount of acetins was and Lb-Oat (Fig. 6B). This suggests that Ma-Oat and Lb-Oat are
observed in the harvest at 48 h (Fig. S9†). It is speculated that unable to convert diacetin to triacetin. Disappointingly, the
the aged E. coli cells at 48 h could not generate acetyl-CoA, newly recruited O-acetyltransferases did not outperform CAT
resulting in no increase in acetin production after 48 h of in triacetin biosynthesis and produced less triacetin than CAT,
culture.
although no significant difference in cell growth was observed
among the recombinant strains (Fig. 6C). Moreover, no colo-
nies were observed, when strains DH-MDT3 to DH-MDT7 were
plated on an LB solid medium containing 50 mg L−1 chloram-
phenicol (data not shown). This result indicates that the newly
recruited O-acetyltransferases lack the chloramphenicol (anti-
biotic) resistance activities.
Screening of CAT-like O-acetyltransferases
In order to find a better O-acetyltransferase than CAT that
could efficiently catalyze the acetylation of diacetin to triacetin,
a BLAST search was performed with the amino acid sequences
of CAT, by which five O-acetyltransferases from Bacillus cereus
(Bc-Oat), Pseudomonas aeruginosa (Pa-Oat), Clostridium aceto-
butylicum (Ca-Oat), Mycobacterium abscessus (Ma-Oat), and
Lactobacillus brevis (Lb-Oat) were retrieved from the GenBank
Optimization of host strain and culture condition for acetin
production
database (Fig. S10 & S11†). For optimal expression of the heter- To achieve a high titer of acetins, the screening of an appropri-
ologous genes in E. coli, all these genes were codon-optimized ate host strain and culture condition other than the incessant
to achieve a CAI of more than 0.8 (Table S3†). The optimized pathway optimization was performed. Production of acetins
genes were cloned in front of Bs-maa in pMD13, resulting in was firstly assessed from the genetic background of E. coli
plasmids of pMDT3 to pMDT7, and transformed into E. coli BW25113, W3110, MG1655 and AceCo (a talented strain in
Fig. 6 Screening of CAT-like O-acetyltransferases. (A) The construct scheme of CAT-like O-acetyltransferases from different organisms. Genes of
Bc-Oat, Pa-Oat, Ca-Oat, Ma-Oat, and Lb-Oat are derived from B. cereus, P. aeruginosa, C. acetobutylicum, M. abscessus and L. brevis, respectively.
(B) Production of acetins and (C) cell growth from strains DH-MDT2 to DH-MDT7 harboring plasmids pMDT2 to pMDT7, respectively. The means
and standard divisions are from two biological replicates.
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acetyl-CoA generation44) in comparison with the control strain
The supplied glycerol serves both as a precursor of acetins
E. coli DH5α (Fig. 7A). The highest production of 3.92 g L−1 of as well as a carbon and energy source of cell mass. The effect
total acetins is obtained from strain MG-MDT2, which shows a of glycerol concentration was investigated on the production of
1.4-fold increase over strain DH-MDT2 (Fig. 7A), and no signifi- acetins using the best strain MG-MDT2 to find an optimal gly-
cant difference in cell growth was observed for any of the cerol supply for the production (Fig. 7C). The highest pro-
recombinant strains (Fig. 7B). As the acetins are biosynthe- duction of 16.5 g L−1 of total acetins is obtained with the
sized from glycerol and acetyl-CoA, we expected a higher titer addition of 10% (v/v) glycerol, which is an increase of 4.2 fold
of acetins from AceCo than from the other strains. However, over the addition of 2% (v/v) glycerol. Production of acetins
the obtained results are contrary to expectations, suggesting can be enhanced with an increase in glycerol supply below
that the availability of acetyl-CoA is not the limiting factor of 15% (v/v) glycerol. A significant retardation of cell growth
acetin biosynthesis in E. coli.
appeared at 15% (v/v) glycerol, which was probably owing to
Fig. 7 Effect of different host strains, glycerol concentrations, and culture media on acetin production and cell growth. (A) Production of acetins
and (B) cell growth from E. coli strains with different genetic backgrounds. Strains DH-MDT2, AC-MDT2, BW-MDT2, W3-MDT2 and MG-MDT2 are
created by transformation of pMDT2 to E. coli DH5α, AceCo, BW25113, W3110, and MG1655, respectively. (C) Production of acetins and (D) cell
growth of strain MG-MDT2 fed on varying glycerol concentrations of 2% to 15% (v/v). (E) Production of acetins and (F) cell growth of strain
MG-MDT2 cultivated in different culture media. The means and standard divisions are from two biological replicates.
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severe osmotic stress generated from the extraordinarily high portion of crude glycerol is generated for every 10 portions of
glycerol concentration (Fig. 7D). Thus, glycerol should not be biodiesel.1 Accordingly, more than 12 000 tons of glycerol are
supplied beyond the level causing osmotic stress.
generated from a 30-million-gallon plant per year. Such a fact
Strain MG-MDT2 was cultivated in four different media of has a negative influence on the biodiesel prices as well as on
M9, M9 with 5 g L−1 of yeast extract (M9YE), LB and 2YT with the environment. To increase the value of the biodiesel indus-
the addition of 10% (v/v) glycerol (Fig. 7E). The highest pro- try, one solution is to employ the microbial conversion of
duction of 27.4 g L−1 of total acetins was obtained from the crude glycerol into essential chemicals, such as 1,3-propane-
2YT medium supporting the best cell growth (Fig. 7F). The diol, hydrogen, butanol, citric acid, polyhydroxyalkanoates,
2YT medium enables E. coli MG-MDT2 to produce 2.5 fold docosahexaenoic acid and succinic acid. However, the crude
more acetins than the M9YE medium. After the analysis of glycerol needs to be transported from the biodiesel production
residual glycerol in 2YT medium culture, it was found that site to various industrial sites in order to produce other chemi-
E. coli MG-MDT2 consumed 4.1% (v/v) (52.7 g L−1) of glycerol cals. Since di- and triacetin are used as biodiesel additives, it
for the production of 27.4 g L−1 of total acetins. Based on the would be more convenient and economical to use crude gly-
stoichiometric calculation (Fig. S12†), strain MG-MDT2 theor- cerol obtained from the biodiesel refinery for the production
etically requires 43.0 g L−1 of glycerol to produce 27.4 g L−1 of of acetins, as shown in Fig. 1A.
total acetins. This means that 81.6% of consumed glycerol is
E. coli MG-MDT2 was fed with crude glycerol composed of
converted to acetins, which is a significant conversion rate. 88.9% glycerol, 3.4% water, 4.7% methanol, and 3% other con-
However, the conversion rate is lowered to 34.1% due to the tents (salts, fatty acids, and ash). After 48 h of culture,
5.9% (v/v) of residual glycerol, provoking us to continuously MG-MDT2 produced 25.9 g L−1 of total acetins from 10% (v/v)
develop a more efficient strategy for an improved total conver- of crude glycerol (Fig. 8A). This result shows that the engin-
sion rate.
eered E. coli strain is capable of utilizing crude glycerol directly
derived from the biodiesel side stream for acetin production.
There was no difference in acetin production or in cell growth
of strain MG-MDT2 while feeding on crude glycerol or pure gly-
cerol (Fig. 8). These results indicate that the crude glycerol
containing impurities is not toxic to strain MG-MDT2 and can
Acetin production using crude glycerol
Biodiesel along with bioethanol constitutes more than 90% of
commercial biofuels. During biodiesel production, a huge
amount of waste glycerol is generated. It is reported that one
Fig. 8 Comparable production of acetins using pure and crude glycerol. (A) Acetin production and (B) cell growth of E. coli MG-MDT2 strain fed on
pure and crude glycerol. The means and standard divisions are from two biological replicates.
Table 1 The titers, yields and conversion rates from the engineered strains for acetins biosynthesis
Strains
Media
Acetin titer (g L−1
)
Yield (mg g−1 glycerol)
Conversion rate (%)
DH-MD8
M9YE medium, 2% (v/v) glycerol
M9YE medium, 2% (v/v) glycerol
M9YE medium, 2% (v/v) glycerol
M9YE medium, 2% (v/v) glycerol
M9ME medium, 2% (v/v) glycerol
M9ME medium, 10% (v/v) glycerol
2YT medium, 10% (v/v) glycerol
2YT medium, 10% (v/v) crude glycerol
0.039
0.214
0.582
2.629
3.927
16.518
27.385
25.975
1.5
8.5
23.1
104.3
155.8
131.1
217.3
206.2
0.2
1.2
3.5
16.4
24.6
20.5
34.1
32.5
DH-MD13
DH-MDT1
DH-MDT2
MG-MDT2
MG-MDT2
MG-MDT2
MG-MDT2
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be applied for the efficient production of acetins. Generally,
The seed culture preparation was performed in LB medium.
the production of chemicals from crude glycerol is lower than For the production of acetins, strains are grown either in
that of pure glycerol. For example, the production of 1,3-propa- M9 minimal salt medium (Na2HPO4·7H2O 12.8 g L−1, KH2PO4
nediol using pure glycerol is 93.7 g L−1 while it is 76.2 g L−1 3 g L−1, NaCl 0.5 g L−1, NH4Cl 1 g L−1, MgSO4 0.12 g L−1
,
from crude glycerol.45 Similarly, the production of citric acid CaCl2 0.01 g L−1), M9YE (M9 medium containing 5 g L−1 yeast
from pure glycerol is 98 g L−1 when the titer declined to 71 g extract), LB (peptone 10 g L−1, yeast extract 5 g L−1, NaCl 10 g
L−1 with a 27.4% reduction from crude glycerol.46 The lowered L−1) or 2YT (peptone 16 g L−1, yeast extract 10 g L−1, NaCl 5 g
production from crude glycerol is generally considered to be L−1) containing 2% (v/v) of glycerol as a carbon source, unless
due to the presence of toxic impurities, especially a high con- otherwise specified. Antibiotics were added to culture media at
centration of salts which cause cell growth inhibition. a final concentration of 100 mg L−1 ampicillin, 50 mg L−1
Therefore, the crude glycerol used herein contains a trace kanamycin and 50 mg L−1 chloramphenicol as required. To
amount of salts and does not inhibit cell growth and acetin make the seed culture, colonies were picked for each construct
production.
and inoculated in 5 mL of LB medium with appropriate anti-
biotics and grown overnight at 37 °C with 250 rpm shaking.
The production culture was made by inoculating the seed
culture into a 300 mL flask with 50 mL of media to an OD600
of 0.1. The production culture was initially induced with
0.5 mM IPTG and incubated for 48 h with 200 rpm shaking in
a rotary shaker at 37 °C, unless otherwise specified. Cellular
growth of culture was measured in terms of OD600 by UV
spectrophotometer. A complete list of strains used in this
study is provided in Table S1.†
Conclusions
To our knowledge, this work provides for the first time an
efficient biosynthetic route in a living cell for the large-scale
production of acetins, hitherto inaccessible molecules from a
renewable resource, glycerol. The use of microbial hosts for
the production of acetins offers several advantages over chemi-
cal synthesis methods because it provides safe products in a
sustainable and environmentally friendly way. Our strategy of
Zero Waste Biorefinery would benefit the biodiesel industry by
utilizing waste glycerol that is generated during biodiesel pro-
duction. By a myriad of approaches, including gene mining,
homolog substitution, and optimization of the gene expression
and culture media, the titer, yields, and conversion rate were
improved in a stepwise fashion (Table 1). A lucrative titer of
27.4 g L−1 is achieved from the engineered strain MG-MDT2,
which is promising for commercialization to rival the biopro-
cess producing 1,2-propanediol or 1,3-propanediol. We expect
that further efforts to improve the host strain along with fer-
mentation process optimization will guarantee a higher pro-
duction rate and titer of acetins.
Plasmid construction
The polymerase chain reaction (PCR) primers used in this
study are listed in Table S4.† PCR was performed using Pfu
DNA polymerase (SolGent, Daejeon, Korea) according to the
manufacturer’s instructions for amplification of DNA frag-
ments. The genes encoding O-acetyltransferases, Ec-lacA
[GenBank: NC_000913.3 (361249..361860)], Ec-cysE [GenBank:
NC_000913.3
NC_000913.3
NC_000913.3
NC_000913.3
NC_000913.3
(3781741..3782562)],
(4479034..4479537)],
(4213680..4214123)],
(4077449..40784380],
(3725887..3726882)],
Ec-yjgM
Ec-yjaB
Ec-yiiD
Ec-wecH
Ec-nhoA
[GenBank:
[GenBank:
[GenBank:
[GenBank:
[GenBank:
NC_000913.3 (1534024..1534869)], and Ec-maa [GenBank:
NC_000913.3 (479367..479918)] were PCR amplified from
genomic DNA of E. coli MG1655 with primer sets of Ec-lacA-F/
Ec-lacA-R, Ec-cysE-F/Ec-cysE-R, Ec-yjgm-F/Ec-yjgM-R, Ec-yjaB-F/
Ec-yjaB-R, Ec-yiiD-F/Ec-yiiD-R Ec-wecH-F/Ec-wecH-R, Ec-
nhoA-F/Ec-nhoA-R, and Ec-MAA-F/Ec-MAA-R, respectively. The
PCR products were digested with BamHI and XbaI restriction
Materials and methods
Strains, culture media and culture conditions
Escherichia coli DH5α was used for cloning purposes. For the enzymes, and ligated between the corresponding restriction
construction of plasmids, cells were grown at 37 °C overnight sites of pTrc99A to create pMD1, pMD2, pMD3, pTMD4,
in LB (Luria Bertani) medium (10 g of tryptone, 5 g of yeast pMD5, pMD6, pMD7, and pMD8 plasmids, respectively. Genes
extract and 10 g of sodium chloride per 1 L). E. coli DH5α, of Sc-maa [GenBank: AM295250.1 (284780..285337)], Hj-maa
DH5α (DE3), AceCo, BW25113, W3110, and MG1655 were used [GenBank:
as host strains for the production of acetins (Table S1†). E. coli [GenBank:
NC_014297.1
NC_008497.1
(2655659..2656213)],
(1691113..1691676)],
Lb-maa
Pp-maa
DH5α (DE3) strain was constructed using the λDE3 lysogeniza- [GenBank: NC_002947.4 (5887693..5888259)], and Bs-maa
tion method (Novagen, Darmstadt, Germany). Briefly, E. coli [GenBank: NC_000964.3 (4195778..4196332)] were optimized
DH5α was grown in LB medium supplemented with 0.2% according to codon usage of E. coli and synthesized at
maltose and 10 mM MgSO4 at 37 °C to an OD600 of 0.5. A 5 µL GenScript Corporation (860 Piscataway, NJ). For the construc-
of culture were mixed with 108 pfu λDE3, 108 pfu helper phage tion of pMD9, pMD10, pMD11, pMD12, and pMD13 plasmids,
and 108 pfu selection phage, and then incubated at 37 °C for the genes were PCR amplified with primer sets of Sc.maa-F/Sc.
20 min before plating on LB agar. After overnight cultivation, maa-R, Hj.maa-F/Hj.maa-R, Lb.maa-F/Lb.maa-R, Pp.maa-F/Pp.
the λDE3 lysogen was verified using T7 Tester phage.
maa-R and Bs.maa-F/Bs.maa-R, respectively, digested with
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BamHI and XbaI restriction enzymes, and ligated between the fied protein was concentrated to 50 mg mL−1 in Buffer
corresponding restriction sites of pTrc99A. For the construc- A. Crystallization of the purified protein was initially per-
tion of pMD14 and pMDT1, Bs-maa fragment was PCR ampli- formed with the following crystal screening kits: Index, PEG/
fied with primers of Bs-maa-F and Bs-maa-R1, digested with Ion (Hampton Research) and Wizard I and II (Rigaku) using
BamHI and SalI restriction enzymes, and inserted between the the hanging-drop vapor-diffusion technique at 18 °C. Each
corresponding restriction sites of pET28a (+) and pSTV28, experiment comprised mixing 1.0 μL protein solution with
respectively. The DNA fragment of cat [GenBank: NC_023277.2 1.0 μL reservoir solution and then equilibrating against 50 μL
(187866..188525)]
encoding
chloramphenicol of the reservoir solution. The best quality Ec-MAA crystals
O-acetyltransferase (CAT) was PCR amplified from pSTV28 with appeared in 20% polyethylene glycol 3350, 0.1 M Tris (pH 8.0)
primers of Cat-F and Cat-R, digested with SacI and BamHI and 0.2 M NaCl. The crystals were transferred to a cryoprotec-
restriction enzymes, and inserted between the corresponding tant solution containing 20% (v/v) polyethylene glycol 3350,
restriction sites of pMD13 to create pMDT2. Plasmids contain- 0.1 M Tris (pH 8.0), 0.2 M NaCl and 30% (v/v) glycerol,
ing CAT-like O-acetyltransferases and Bs-maa were constructed extracted with a loop larger than the crystals and flash-frozen
like as follows. The Bs-maa along with pTrc99A backbone was by immersion in liquid nitrogen.
first amplified from pMD13 with primers of Trc-Bs.maa-F and
X-ray diffraction data collection and structure determination
Trc-Bs.maa-R.
The
genes
Bc-Oat
Pa-Oat
Ca-Oat
encoding
[GenBank:
CAT-like
O-acetyltransferases,
(2311034..2311684)],
(347344..347778)],
CP001407.1 Data were collected at the 100 K beamline 7A in the Pohang
CP000744.1 Accelerator Laboratory (Pohang, Korea) with a Quantum 270
NC_001988.2 CCD detector (ADSC, USA). The data were then indexed, inte-
[GenBank:
[GenBank:
(61507..62169)], Ma-Oat [GenBank: NZ_NQUY01000003.1 grated, and scaled using the HKL2000 software suite.47 The Ec-
(1..690)],
and
Lb-Oat
[GenBank:
NC_008497.1 MAA crystals belonged to the space group P3, with unit cell
(1786705..1787373)] were also codon-optimized and syn- parameters of a = 62.104 Å, b = 62.104 Å and c = 81.128 Å. With
thesized by GenScript Corporation, and PCR amplified with two molecules of Ec-MAA per asymmetric unit, the Matthews
primer sets of Bc.Oat-F/Bc.Oat-R, Pa.Oat-F/Pa.Oat-R, Ca.Oat-F/ coefficient was 2.26 Å3 Da−1, which corresponds to a solvent
Ca.Oat-R, Ma.Oat-F/Ma.Oat-R and Lb.Oat-F/Lb.Oat-R, respect- content of approximately 45.57%.48 The structure of Ec-MAA
ively. The DNA fragment of Bs-maa-containing pTrc99A was was determined by molecular replacement with the CCP4
assembled with each of Bc-Oat, Pa-Oat, Ca-Oat, Ma-Oat, and version of MOLREP49 using the structure of maltose transace-
Lb-Oat DNA fragments via HiFi DNA assembly method tylase from Geobacillus kaustophilus (GkMAA, PDB: 2IC7, 67%
(NEBuilder HiFi DNA Assembly Master Mix, New England sequence identity) as a search model. The model building was
BioLab, catalog no. E2621S) to produce pMDT3, pMDT4, performed using the WinCoot program50 and the refinement
pMDT5, pMDT6 and pMDT7 plasmids, respectively. A com- was performed with REFMAC5.51 The data statistics are sum-
plete list of plasmids used in this study is provided in marized in Table S2.† The refined model of Ec-MAA has been
Table S1.†
deposited in the Protein Data Bank with PDB code 6AG8.
Protein expression and crystallization
Molecular docking simulation
The gene coding for Ec-MAA was amplified by PCR using Molecular docking of diacetin to CAT structure was carried out
genomic DNA from E. coli strain MG1655 as a template. The by using AutoDock Vina.52 The ligand of CAT was prepared
PCR product was then subcloned into pET30a, and the result- with WinCoot50 and PRODRG53 and nonpolar H atoms were
ing expression vector pET30a-Ec-maa was transformed into merged onto both the ligands and the targets using AutoDock
E. coli strain BL21 (DE3)-T1R, which was grown in 5 L of Tools prior to performing these docking simulations. The grid
Lysogeny broth medium containing kanamycin at 37 °C. After box was designated with (x: 20.681, y: 15.693, z: 50.01) and the
induction by the addition of 0.5 mM IPTG, the culture was size of the grid was 24.0 × 18.0 × 18 Å. The model structure of
maintained for a further 16 h at 18 °C. The culture was then diacetin was calculated and the best models of each com-
harvested by centrifugation at 4000g for 12 min at 20 °C. The pound were selected by the theoretical affinity of the binding
cell pellet was re-suspended in buffer A (50 mM Na2HPO4, pH (ΔGbind). The ΔGbind of the docked molecule was −4.6 kcal
7.0) and then disrupted by ultra-sonication. The cell debris mol−1. The final conformation produced in this simulation
was removed by centrifugation at 13 500g for 30 min and the was checked using PyMOL software.
lysate was applied to a Ni-NTA agarose column (Qiagen). After
washing with buffer A containing 36 mM imidazole, the
Phylogenetic tree analysis
bound proteins were eluted with 300 mM imidazole in buffer Iterative searching for maltose O-acetyltransferases and chlor-
A. Finally, trace amounts of contaminants were removed by amphenicol O-acetyltransferases was performed with the Basic
size-exclusion chromatography using a Superdex 200 prep- Local Alignment Search Tool (BLAST) in the National Center
grade column (320 ml, GE Healthcare) equilibrated with buffer for Biotechnology Information (NCBI) server using the posi-
A. All purification experiments were performed at 4 °C. The tion-specific iterated BLAST (PSI-BLAST) method. Multiple
degree of protein purification was confirmed by sodium alignments were performed with Clustal Omega. The evol-
dodecyl sulfate polyacrylamide gel electrophoresis. The puri- utionary history was inferred by using the maximum likeli-
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hood method and Le_Gascuel_2008 model.54 The bootstrap stoichiometric calculation (Table S5 & Fig. S12†). The total con-
consensus tree inferred from 100 replicates is taken to rep- version rate is the percentage of the glycerol converted to
resent the evolutionary history of the taxa analyzed.55 Branches acetins, which is calculated from the total production of
corresponding to partitions reproduced in less than 50% boot- acetins according to each theoretical yield. The equations are
strap replicates are collapsed. Initial tree(s) for the heuristic as follows:
search were obtained automatically by applying Neighbor-Join
Total production of acetins ðmgÞ
Yield ¼
;
and BioNJ algorithms to a matrix of pairwise distances esti-
mated using the JTT model, and then selecting the topology
with a superior log-likelihood value. A discrete gamma distri-
bution was used to model evolutionary rate differences among
sites (5 categories (+G, parameter = 0.6051)) and (5 categories
(+G, parameter = 1.6680)), respectively. The rate variation
model allowed for some sites to be evolutionarily invariable
([+I], 1.91% sites) in the phylogenetic tree of chloramphenicol
O-acetyltransferases. These analyses involved 61 and 56 amino
acid sequences, respectively. There were a total of 289 and 236
positions in the final dataset, respectively. Evolutionary ana-
lyses were conducted in MEGA X.56
Total glycerol ðgÞ
and
Glycerol converted to acetins ðgÞ
Total glycerol ðgÞ
ꢀ 100%:
Conversion rate ð%Þ ¼
Feeding mono- and diacetin to culture
A mixture consisting of 3.8 g L−1 of monoacetin and 1.2 g L−1
of diacetin, which was extracted from the culture of strain
DH-MD13, was added into 5 mL of M9YE medium containing
2% (v/v) of glycerol as a carbon source, which was inoculated
with strains DH-MD0 and DH-CAT. The culture was initially
induced with 0.5 mM IPTG and incubated at 37 °C with 250
rpm shaking in a rotary shaker for 48 h. Conversion of mono-
acetin or diacetin to triacetin was analyzed during the culture
by using GC-FID.
Analysis of acetins by GC-FID and GC-MS
Acetins were extracted from the culture broth with ethyl
acetate, and subjected to instrumental analysis. Mono-, di-,
and triacetin produced from engineered strains were identified
by GC-MS (GC-MS-QP2010, Shimadzu, Japan). All products
were quantitatively analyzed with GC, equipped with a flame
ionization detector (FID) and an auto-sampler (GC-FID, Agilent
Technologies 7890A, California 95051, USA). Each sample of
1 μL was injected into an HP-INNOWax column (19091N-133,
30 m in length, 0.250 mm in internal diameter and 0.25 μm
film thicknesses). Nitrogen was used as the carrier gas at an
inlet pressure of 39 psi. The GC oven temperature started at
50 °C and was raised with a gradient of 20 °C min−1 until
90 °C. It was then raised at a rate of 15 °C min−1 until 150 °C,
at rate of 20 °C min−1 until 190 °C, and at rate of 15 °C min−1
until 230 °C, where it was held for 2 min. The detector temp-
erature was maintained at 280 °C. Standard curves of mono-,
di-, and triacetin were prepared for quantitative calculation of
the production (Fig. S13†).
Source of crude glycerol
The crude glycerol was obtained from a 300 L pilot-scale of
enzymatic biodiesel production unit using an immobilized
recombinant CalB (Candida antarctica lipase B) produced in
Saccharomyces cerevisiae. Soybean oil was used as a substrate
in the process. The crude glycerol was simply separated by a
gravity separation method without any prior purification
process. The composition of the crude glycerol was analyzed
as 88.9% glycerol, 3.4% water, 4.7% methanol, and 3% other
contents (salts, fatty acids and ashes).
Conflicts of interest
Quantification of residual glycerol
There are no conflicts of interest to declare.
Concentration of residual glycerol was determined by high per-
formance liquid chromatography (HPLC) (Shimadzu, Kyoto,
Japan) with a refractive index detector and Kromasil KR100-
5NH2 column (250 mm × 4.6 mm, Eka Chemicals, Bohus,
Sweden). The culture broth was first centrifuged at 5000g for
10 minutes. The supernatant obtained was filtered through a
0.22 μm membrane filter (Minisart) and then injected into the
HPLC for the quantification of residual glycerol. The mobile
phase was 75% acetonitrile in water at 35 °C and was run at a
flow rate of 1.5 mL min−1. External standards of pure glycerol
were applied for the identification and quantification of peak
areas.
Acknowledgements
This work was supported by a grant from the Next-Generation
BioGreen 21 Program (SSAC, grant#: PJ01326501), RDA, Korea.
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