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Biosynthesis of acetylacetone inspired by its biodegradation


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Background: Acetylacetone is a commercially bulk chemical with diverse applications. However, the traditional manufacturing methods suffer from many drawbacks such as multiple steps, harsh conditions, low yield, and environmental problems, which hamper further applications of petrochemical-based acetylacetone. Compared to conventional chemical methods, biosynthetic methods possess advantages such as being eco-friendly, and having mild conditions, high selectivity and low potential costs. It is urgent to develop biosynthetic route for acetylacetone to avoid the present problems. Results: The biosynthetic pathway of acetylacetone was constructed by reversing its biodegradation route, and the acetylacetone was successfully produced by engineered Escherichia coli (E. coli) by overexpression of acetylacetone-cleaving enzyme (Dke1) from Acinetobacter johnsonii. Several promising amino acid residues were selected for enzyme improvement based on sequence alignment and structure analysis, and the acetylacetone production was improved by site-directed mutagenesis of Dke1. The double-mutant (K15Q/A60D) strain presented the highest acetylacetone-producing capacity which is 3.6-fold higher than that of the wild-type protein. Finally, the strain accumulated 556.3 ± 15.2 mg/L acetylacetone in fed-batch fermentation under anaerobic conditions. Conclusions: This study presents the first intuitive biosynthetic pathway for acetylacetone inspired by its biodegradation, and shows the potential for large-scale production.
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Zhouetal. Biotechnol Biofuels (2020) 13:88
Biosynthesis ofacetylacetone inspired byits
Yifei Zhou1,2, Yamei Ding3, Wenjie Gao1, Jichao Wang1, Xiutao Liu1,2, Mo Xian1, Xinjun Feng1*
and Guang Zhao1,4*
Background: Acetylacetone is a commercially bulk chemical with diverse applications. However, the traditional
manufacturing methods suffer from many drawbacks such as multiple steps, harsh conditions, low yield, and environ-
mental problems, which hamper further applications of petrochemical-based acetylacetone. Compared to conven-
tional chemical methods, biosynthetic methods possess advantages such as being eco-friendly, and having mild
conditions, high selectivity and low potential costs. It is urgent to develop biosynthetic route for acetylacetone to
avoid the present problems.
Results: The biosynthetic pathway of acetylacetone was constructed by reversing its biodegradation route, and
the acetylacetone was successfully produced by engineered Escherichia coli (E. coli) by overexpression of acetylac-
etone-cleaving enzyme (Dke1) from Acinetobacter johnsonii. Several promising amino acid residues were selected
for enzyme improvement based on sequence alignment and structure analysis, and the acetylacetone production
was improved by site-directed mutagenesis of Dke1. The double-mutant (K15Q/A60D) strain presented the highest
acetylacetone-producing capacity which is 3.6-fold higher than that of the wild-type protein. Finally, the strain accu-
mulated 556.3 ± 15.2 mg/L acetylacetone in fed-batch fermentation under anaerobic conditions.
Conclusions: This study presents the first intuitive biosynthetic pathway for acetylacetone inspired by its biodegra-
dation, and shows the potential for large-scale production.
Keywords: Acetylacetone biosynthesis, Acetylacetone-cleaving enzyme, Rational design, Site-directed mutagenesis,
β-Dicarbonyl compounds
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Acetylacetone, also known as 2,4-pentanedione (CAS
No. 123-54-6), is an important commodity chemical and
widely used as a fuel additive, as dyeing intermediate, in
the fields of metal extraction, metal plating, and resin
modification [1]. Traditionally, acetylacetone is manufac-
tured through chemical routes using acetone and ketene,
which is produced by pyrolysis of acetone or acetic acid
at a temperature of 700–800°C, with carbon monoxide,
methane, hydrogen formed as by-products [2, 3]. In
specific, esterification of ketene and acetone forms iso-
propenyl acetate (IPA), in the presence of a strong acid
catalyst. en, IPA is transformed into acetylacetone
at 500–600°C with metallic molybdenum as a catalyst,
whereby the yield is only about 45%. In conclusion, the
chemical routes suffer from drawbacks such as multiple
steps, harsh conditions, low yield, and environmental
problems, which hamper further applications of petro-
chemical-based acetylacetone. To address the issue, new
methods need to be developed for acetylacetone prepara-
tion. Compared to conventional chemical methods, bio-
synthetic methods are expected to have advantages such
as being eco-friendly and having mild conditions, high
Open Access
Biotechnology for Biofuels
1 CAS Key Laboratory of Biobased Materials, Qingdao Institute
of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences,
Qingdao 266101, China
Full list of author information is available at the end of the article
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Zhouetal. Biotechnol Biofuels (2020) 13:88
selectivity and low potential costs [4], and have been used
to produce numerous products, e.g., bio-based chemicals
[5], pharmaceuticals [6], biopolymers [7]. For acetylac-
etone, the theoretical yield is as high as 1.5mol/mol glu-
cose by bioconversion. Predictably, low cost will also be
obtained thanks to the cheap carbon source and the high
Limited studies indicate that acetylacetone is biode-
gradable [8]. A strain of Acinetobacter johnsonii was
isolated and proved to have the ability to utilize acetylac-
etone as a sole carbon source. To reveal the decomposi-
tion mechanism, a novel C–C bond-cleaving enzyme,
acetylacetone-cleaving enzyme (Dke1, EC,
was found and purified from A. johnsonii [9]. e Dke1
enzyme can activate oxygen to cleave acetylacetone into
acetate and methylglyoxal, followed by the conversion
of methylglyoxal into lactate by glyoxalase. However, the
natural biosynthesis of acetylacetone has not yet been
reported. In previous studies, some non-natural prod-
ucts had been bio-synthesized by reversing their biodeg-
radation pathway. An artificial biosynthetic pathway to
methylacetoin was constructed by redirecting the methy-
lacetoin biodegradation, and the titer achieved at 3.4g/L
by enzyme screening and metabolic engineering [4]. e
direct biocatalytic route of 1,4-butanediol mirroring its
biodegradation pathway was reported with a produc-
tion of 18g/L [10]. In addition, the engineered reversal of
the β-oxidation cycle was adopted for fatty-acid-derived
compounds’ biosynthesis, and a series of short-,
medium-, and long-chain products were obtained at high
yields [1113]. ese successful cases provided a possible
strategy to our research for acetylacetone biosynthesis.
In this work, we established for the first time the bio-
synthetic pathway of acetylacetone from fermentable
sugars (Fig.1) inspired by the known acetylacetone bio-
degradation pathway. It was proved that the acetylacetone
decomposition process catalyzed by Dke1 was revers-
ible. e Dke1 activity was improved by rational design,
resulting in enhanced acetylacetone production under
shake-flask conditions, and the underlying mechanism
was proposed. Fed-batch fermentation was conducted to
evaluate the potential for large-scale production.
Results anddiscussion
Design andverication oftheacetylacetone biosynthesis
Acetylacetone is not a naturally occurring metabolite;
however, it can be catabolized by Acinetobacter johnsonii
as a carbon source [8]. As reported, one molecule of acet-
ylacetone degrades into one acetate and one methylgly-
oxal catalyzed by acetylacetone-cleaving enzyme Dke1.
Methylglyoxal can be produced marginally through gly-
colysis [14], and acetate is a major product during sugar
fermentation especially under anaerobic conditions.
us, we proposed to achieve acetylacetone production
from methylglyoxal and acetate by redirecting the reac-
tion. As shown in Fig. 1, glyceraldehyde 3-phosphate
from glycolysis pathway was converted into glycerone
phosphate and methylglyoxal by the action of triose-
phosphate isomerase (TpiA) and methylglyoxal synthase
(MgsA), respectively. Acetyl-CoA was converted into
acetate by acetate kinase. Methylglyoxal and acetate were
finally transformed into acetylacetone by Dke1.
To test the hypothetical acetylacetone biosynthetic
pathway, acetylacetone-cleaving enzyme (Dke1) from
A. johnsonii (accession No. Q8GNT2.1) was cloned into
plasmid pETDuet-1, and then the recombinant plasmid
was introduced into E. coli BL21 (DE3) to construct an
acetylacetone producing strain. e expression of the
Dke1 was first checked by SDS-PAGE, and the corre-
sponding bands with molecular weights of 17kDa were
observed clearly (Additional file1: Fig. S1). e resulting
Fig. 1 Biodegradation-inspired biosynthetic pathway of acetylacetone. Acetylacetone biodegradation is presented with a dashed line, and the
constructed biosynthetic pathways are presented using a solid line. The enzymes used are as follows: AckA, acetate kinase; TpiA, triose-phosphate
isomerase; MgsA, methylglyoxal synthase; Dke1, acetylacetone-cleaving enzyme
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Zhouetal. Biotechnol Biofuels (2020) 13:88
strain Q3030 was cultured under shake-flask conditions,
and the fermentation products were analyzed by GC–
MS. However, no acetylacetone was detected. We then
referred back to the acetylacetone biodegradation path-
way. As the biodegradation of acetylacetone was acti-
vated by oxygen, we speculated the biosynthesis direction
was suppressed under aerobic conditions. us, anaero-
bic cultivation in a sealed serum bottle with nitrogen
was carried out for further verification. is time, the
production of acetylacetone was confirmed by GC–MS
analysis. As shown in Fig.2, a specific peak with a mass
of 100Da was detected, and dissociation of this ion led
to other masses such as 43 and 85Da, exactly the same
with acetylacetone standard. Under this condition, the
concentration of acetylacetone in fermentation broth was
32.6 ± 1.0mg/L. Furthermore, the production of acety-
lacetone was also performed in vitro in Tris buffer (pH
7.5) containing purified Dke1 protein, methylglyoxal and
ammonium acetate at 37°C, and 129.5 ± 8.9mg/L acety-
lacetone was accumulated in 24h. Compared with invivo
system, higher product titer was achieved with invitro
system, probably owing to a relatively simple pathway
without side reactions [15].
A sufficient supply of precursors is necessary for the
efficient synthesis of the final products [16]. As one of
the main metabolites in E. coli under anaerobic condi-
tion, acetate should be sufficient for acetylacetone bio-
synthesis. But in general, methylglyoxal synthesis is
inhibited in cell [14, 17]. So the methylglyoxal content in
the flask was improved by extra supplement with a con-
centration of 0.1mM or overexpression of the key genes
(tpiA and mgsA) for enhanced acetylacetone production
(Fig. 1). However, no more acetylacetone was accumu-
lated. Meanwhile, the invitro experiment with 20 mM
methylglyoxal was conducted, and the production of
acetylacetone (128.9 ± 8.6mg/L) did not show significant
difference with that with 10mM methylglyoxal (p = 0.94).
So, we speculated that low activity of Dke1 might be the
primary reason of low acetylacetone yield. en, the acet-
ylacetone-cleaving enzyme was first screened. Two more
genes encoding acetylacetone-cleaving enzyme from
Paraburkholderia xenovorans LB400 (P_Dke1, acces-
sion No. NC_007951.1) and Tistrella mobilis (T_Dke1,
accession No. NC_017966.1) were cloned and used for
acetylacetone production. Unfortunately, no acetylac-
etone was detected using engineered strains with P_Dke1
or T_Dke1. So, other strategies should be considered for
higher acetylacetone production.
Rational design ofDke1
Protein engineering using directed evolution or rational
design has been developed rapidly, and has acted as a
powerful tool for enzyme improvement [18, 19]. Espe-
cially, protein sequence information combined with
computational modeling tools can be used to iden-
tify promising amino acid sites and can be selected for
enzyme engineering [20]. us, rational designstrategies
were considered for increasing Dke1 activity.
To improve the Dke1 catalytic activity, multiple amino
acid sequence alignment was performed between Dke1
and 27 other acetylacetone-cleaving enzymes or hypo-
thetical proteins sharing more than 60% identity with
Dke1 (Additional file 2: TableS1). Among 153 amino
acid residues in Dke1, 52 residues were conserved in all
28 proteins, and referred to as definitely conservedsites
(highlighted in red in Fig. 3); 44 amino acid residues
appeared in more than 14 other proteins, and defined as
relatively conservedsites (highlighted in yellow in Fig.3);
Fig. 2 Verification of the production of acetylacetone by engineered E. coli strain using GC–MS. GC chromatography (a) and mass spectrometry (b)
results are shown for acetylacetone standard (lower panel) and Q3030 culture (upper panel)
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Zhouetal. Biotechnol Biofuels (2020) 13:88
and 19 amino acid residues rarely (less than 26%) showed
up in other proteins, and were called variable sites (high-
lighted in green in Fig. 3). Among the variation sites,
three amino acid residues were found conserved in all
proteins except Dke1, and site-directed mutagenesis was
performed to construct Dke1 single mutants harboring
each substitution as its homologs. en, shake flask cul-
tivation was performed to test the effect of each muta-
tion. e strain with Dke1 K15Q (Q3080) accumulated
60.6 ± 0.7mg/L acetylacetone, which is 1.9-fold higher
than that of the wild-type strain Q3030 (p < 0.01) (Fig. 4).
However, the strain with Dke1 Y21W (Q3082) only pro-
duced 28.8 ± 1.0mg/L acetylacetone, and the production
reduced dramatically to 7.0 ± 0.4mg/L when Dke1 S17D
was used in strain Q3081.
Meanwhile, 3D structure analysis of the wild-type
Dke1 was performed based on its crystal structure (PDB
ID: 3BAL). In the crystal structure of the ligand-free
Dke1, the Fe2+-coordinating His-62, His-64 and His-
104 comprise the substrate catalytic center [21]. e
definitely conserved sites occupy about three quarters
of the substrate channel, which are the most important
amino acid residues for maintaining structure and func-
tion of the proteins (Additional file 1: Fig. S2). ese
sites would not be modified in this study. Most of the
relatively conservedsites cover about one quarter of the
substrate channel, while a few sites are located close to
the catalytic center. We analyzed the possible impact
from the mutation of the relatively conservedsites near
the catalytic center and all the variation sites. By analy-
sis of the key influence factors including properties of
amino acids, hydrogen bonding, electron distribution,
and charge properties, 5 sites were selected as potential
mutation sites for improving Dke1 activity (Table1). For
Fig. 3 Alignment of multiple amino acid sequences of Dke1 and other proposed acetylacetone-cleaving enzymes. The definitely conserved sites
are labeled in red, the relatively conserved sites are labeled in yellow, the variable sites are labeled in green and the selected sites for mutation are
marked with red star
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Zhouetal. Biotechnol Biofuels (2020) 13:88
example, the carbonyl oxygen of A60 was interacted with
H62 by forming a hydrogen bond [22], and it will trans-
fer electrons to H62 after mutated to D60 or N60 which
might be helpful to enhance the catalytic activity. en,
10 mutants were constructed and compared with Dke1
wild-type protein. As shown in Fig. 4, the strain with
Dke1 A60D (Q3148) produced 80.2 ± 2.4 mg/L acety-
lacetone which is 2.5-fold higher than that of the strain
Q3030 (p < 0.01). e strain with Dke1 G101D (Q3151)
only produced 9.5 ± 1.4 mg/L acetylacetone while the
ability of other mutants was basically the same with wild-
type Dke1 protein (p > 0.1).
As the mutations K15Q and A60D had shown
improvement in acetylacetone production, the influ-
ence of the double mutant on acetylacetone produc-
tion was also assessed. As Fig. 4 demonstrated, the
strain carrying Dke1 K15Q/A60D (Q3170) exhibited
the highest acetylacetone synthesis efficiency, and
115.7 ± 5.1mg/L acetylacetone was accumulated in the
culture, which was 3.6-fold higher than that of strain
Q3030 (p < 0.01). Dke1 activity was assayed in vitro
using purified proteins and the activity of each enzyme
showed a similar trend with acetylacetone production
Fig. 4 Effect of Dke1 mutagenesis on acetylacetone production in flask cultivation. Data were obtained after each strain was induced for 24 h in
liquid LB medium. All the experiments were carried out in triplicate. All p values are based on two-tailed tests (wild type and mutant strain) and
presented over the bars
Table 1 Analysis oftheproposed sites forsite-directed mutagenesis
Proposed sites Mutation information Annotation
A60 N60 or D60 The carbonyl oxygen of A60 (Additional file 1: Fig. S3b) is hydrogen bonded to H62. The electrons could be
transferred to H62 after Ala mutated to Asp or Asn
G101 N101, D101 or S101 The N of the main chain of G101 (Additional file 1: Fig. S3c) is hydrogen bonded to the O of the main chain
of H64. When Gly was mutated to Asp, Asn or Ser, the electron distribution of site 101 will change and
thus affect the catalytic activity
L103 R103, Q103 or C103 L103 (Additional file 1: Fig. S3c) was located close to H104; the property of site 103 directly affects H104.
When the nonpolar amino acid (Leu) was mutated to polar amino acid (Gln, Cys) or alkaline amino acid
(Arg), the influence to H104 from site 103 may change and thus affect the catalytic activity
G105 D105 When G105 (Additional file 1: Fig. S3c) was mutated to D105, the electronegativity of the catalytic activity
center will be significantly increased
E140 V140 E140 (Additional file 1: Fig. S3d) with hydrophobic amino acids around was located on the outer edge of the
hydrophobic channel. When the acidic amino acid (Glu) was mutated to a hydrophobic amino acid (Val), it
might be more conducive to discharge the catalytic product
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Zhouetal. Biotechnol Biofuels (2020) 13:88
Structural simulation andmolecular docking ofDke1
To reveal the molecular basis for increased enzymatic
activity of Dke1 (K15Q/A60D), in silico structural mode-
ling and molecular docking were performed using the 3D
structure of Dke1 (PDB ID: 3BAL) as a model template.
As shown in Fig.5, no remarkable differences in the over-
all structure have been observed between wild-type and
mutated Dke1 protein, implying that the variant is cor-
rectly folded. Interestingly, with the mutation of K15Q,
the channel, through which substrates enter the enzyme
reactive center, was widened. Furthermore, a neutral resi-
due glutamine is less attractive to negative-charged ace-
tate than lysine which is positive charged. Taken together
both factors make it easier for acetate to arrive at the
reactive site of Dke1. e residue A60, as a hydrophobic
amino acid, is located close to the reactive center and
its side chain is oriented toward the interior of the pro-
tein. e substitution of alanine by a hydrophilic residue,
aspartate, changed the orientation of the side chain at
position 60, resulting in a reactive center with increased
volume, which may be more conducive for substrate
binding. In summary, these changes brought about by the
double mutant increased substrate access to the enzyme
active site, resulting in enhanced enzyme activity.
Fed‑batch fermentation
As the strain Q3170 presented the highest acetylacetone
production in flask cultivation, fed-batch fermentation
was conducted in a 5-L bioreactor. e concentrated glu-
cose was used for cell growth and controlled between 10
and 20g/L during fermentation. 0.2mM IPTG was added
to induce the recombinant proteins when the cell den-
sity reached to about 30 OD600 at 12h. Simultaneously,
the sterilized air was switched to high-purity nitrogen
for anaerobic environments. Cell growth, residual glu-
cose and acetylacetone accumulation were monitored
over the course of the fermentation. As shown in Fig.6,
the cell mass reached to a maximum 16.8g DCW/L at
28h and decreased slightly after 36h. A small amount of
acetylacetone had already been synthesized before induc-
tion possibly due to Dke1 basal expression. After induc-
tion, acetylacetone rapidly accumulated and reached the
Table 2 Activity of the Dke1 and its mutants (p < 0.01
compared withthewild type)
Enzyme Specic activity
WT 0.8 ± 0.01 × 103
K15Q 1.7 ± 0.07 × 103
A60D 2.5 ± 0.02 × 103
K15Q/A60D 3.0 ± 0.07 × 103
Fig. 5 Analysis of the Dke1 structure with molecular docking. a Wild-type. b K15Q/A60D mutant. The β-pleated sheet shown in purple, the α-helix
shown in light blue, and the substrate channel shown in grey
Fig. 6 The time profiles of cell growth, residual glucose and
acetylacetone accumulation in fed-batch fermentation performed in
a 5-L laboratory bioreactor. Dry cell weight was marked with circle,
residual glucose was marked with triangle, and acetylacetone was
marked with asterisk
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Zhouetal. Biotechnol Biofuels (2020) 13:88
maximum of 556.3 ± 15.2 mg/L at 48h with a yield of
5.1mmol/mol glucose. e productivity of 21.2mg/L/h
was achieved during 16–36h while only 1.1mg/L/h dur-
ing the latter 24h.
Although the yield of acetylacetone from recombinant
E. coli in this study was still relatively low, E. coli is a com-
petitive species for industrial applications with short mul-
tiplication time, growth with inexpensive carbon sources
and amenability to genetic modification [23]. Many strat-
egies can be used for improving the acetylacetone yield
in future study, such as metabolic flow regulation [24]
and key genes expression level modulation [25]. Previous
study has shown that acetylacetone has toxic side effects
on the immune system of mammals [26], various aquatic
organisms [27] and microorganisms [28]. e toxicity
threshold of acetylacetone is 67mg/L for Pseudomonas
putida [28], and an excess of acetylacetone (> 1.5g/L) can
completely inhibit the cell growth of A. johnsonii [9]. We
speculated that the acetylacetone toxicity might be one of
the main reasons for the low yield. e toxicity of acety-
lacetone to E. coli cells was tested (Additional file1: Fig.
S4). e results showed that 100mg/L acetylacetone has
obvious inhibition effect on the growth of E. coli, and
the cell was almost completely inhibited at a concentra-
tion of 5g/L. e mechanism of toxicity, however, has
still not been revealed. Further research is needed on
the underlying mechanism to guide the metabolic engi-
neering for higher production. Adaptive evolution is also
aneffective measure to improve thebacterial tolerance
and enhance the production [29]. As E. coli naturally pro-
duces a mixture of ethanol, hydrogen, organic acids, such
as lactic acid, succinic acid, acetic acid under anaerobic
conditions [30], metabolic flow analysis and regulation
are necessary to conduct for acetylacetone production
enhancement. In addition, as the cultivation conditions
play an important role on cell growth, product synthesis,
and conversion efficiency, it is also worthy to optimize
the medium components, substrate addition strategy, fer-
mentation mode, etc. in future research.
e biosynthetic pathway of acetylacetone was con-
structed, and the acetylacetone was produced
successfully from glucose by engineered E. coli by over-
expression of acetylacetone-cleaving enzyme (Dke1).
e production was improved by site-directed mutagen-
esis of Dke1 and the double mutant (K15Q/A60D) ena-
bled the highest acetylacetone-producing capacity.
Finally, 556.3 ± 15.2 mg/L acetylacetone was obtained
at 36h post-induction in fed-batch fermentation under
anaerobic condition. is study presents the first intui-
tive biosynthetic pathway of acetylacetone inspired by
its biodegradation, and shows its potential for large-scale
production. As reported, Dke1 is not absolutely specific
for acetylacetone. Many related β-dicarbonyl compounds
such as 3,5-heptanedione, 2,4-octanedione, 2-acetylcy-
clohexanone, ethylacetoacetate, etc. can be accepted as
substrate by Dke1 [8, 9]. As a consequence, the results of
this study also provide a possible biosynthesis method for
other β-dicarbonyl compounds.
Materials andmethods
Strains andplasmids
All strains and plasmids used in this study are listed in
Table3. e primers used for plasmid construction and
allele verification are listed in Additional file2: TableS2.
E. coli DH5α (Invitrogen) was used for plasmids’ prepa-
ration and BL21 (DE3) was used for recombinant pro-
tein expression and acetylacetone production. e genes
encoding acetylacetone-cleaving enzyme from A. john-
sonii, Paraburkholderia xenovorans LB400, and Tistrella
mobilis were codon optimized and chemically synthe-
sized by BeijingLiuheBGI, and cloned into pETDuet-1
between EcoRI and BamHI sites to construct plasmids
pETDuet-Dke1, pETDuet-P_Dke1, and pETDuet-T_
Dke1, respectively. Mutations were introduced into the
Dke1 gene in pETDuet-Dke1 using an overlap extension
PCR method [31]. e resulting plasmids were named
pM1 to pM19, respectively. All the recombinant plasmids
were verified by colony PCR and nucleotide sequencing.
Protein expression andgel electrophoresis analysis
To check the expression of the recombinant proteins,
single colonies of E. coli BL21 (DE3) harboring differ-
ent recombinant plasmids were cultured in LB medium
containing appropriate antibiotics at 37°C overnight and
then diluted 1:100 into fresh LB medium and induced
with 0.2 mM isopropyl-β--thiogalactopyranoside
(IPTG) at an OD600 of 0.6–0.8. e cells were collected
from 10 mL bacteria cultures 4 h post-induction and
washed with phosphate buffer (pH 6.8). e washed cells
were suspended in 1mL buffer and subjected to ultra-
sonication (SCIENTZ JY92-IIN, 300W, 3s pulse on and
3s pulse off for 5min). e cell lysates were centrifuged
and the protein expression was analyzed by SDS-PAGE.
Dke1 activity assay
e invitro reaction system (1mL) for the activity assay
of Dke1 contained 0.1 M purified enzyme, 0.5 mM
FeSO4·7H2O, 10mM MgCl2·6H2O, 10mM KCl, 1mM
DTT, 10 mM methylglyoxal and 10 mM ammonium
acetate in 20mM Tris/HCl buffer (pH 7.5) and was incu-
bated at 37°C for 24h. e reaction mixture was centri-
fuged at 5000×g for 5min, and then the supernatant was
subjected to HPLC or GC–MS analysis to verify acetylac-
etone production.
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Shake‑ask cultivation
To evaluate the acetylacetone production using different
engineered strains, shake-flask cultivations were carried
out with 50mL of liquid LB medium containing 20g/L
glucose in 250-mL non-baffled flasks or serum bottles
with appropriate antibiotics. When necessary, ampicil-
lin and chloramphenicol were added at a final concen-
tration of 100 g/mL and 50 g/mL, respectively. e
strains were inoculated to the medium and incubated in
an orbital shaker incubator at 37°C and 180rpm. 0.2mM
Table 3 Strains andplasmids used inthis study
Strains orplasmids Genotype/description Source
E. coli DH5 F recA endA1 Φ80dlacZΔM15 hsdR17(rK
E. coli BL21 (DE3) F ompT hsdSB (rB
) gal dcm λ (DE3) Invitrogen
Q2837 E. coli BL21(DE3) carrying pETDuet-Dke1 and pACYCDuet-MgsA-TpiA This study
Q3028 E. coli BL21(DE3) carrying pETDuet-Tmo_Dke1 This study
Q3029 E. coli BL21(DE3) carrying pETDuet-Pxe_Dke1 This study
Q3030 E. coli BL21(DE3) carrying pETDuet-Dke1 This study
Q3080 E. coli BL21(DE3) carrying pM1 This study
Q3081 E. coli BL21(DE3) carrying pM2 This study
Q3082 E. coli BL21(DE3) carrying pM3 This study
Q3085 E. coli BL21(DE3) carrying pM6 This study
Q3086 E. coli BL21(DE3) carrying pM7 This study
Q3148 E. coli BL21(DE3) carrying pM9 This study
Q3149 E. coli BL21(DE3) carrying pM10 This study
Q3150 E.coli BL21(DE3) carrying pM11 This study
Q3151 E. coli BL21(DE3) carrying pM12 This study
Q3152 E. coli BL21(DE3) carrying pM13 This study
Q3153 E. coli BL21(DE3) carrying pM14 This study
Q3154 E. coli BL21(DE3) carrying pM15 This study
Q3155 E. coli BL21(DE3) carrying pM16 This study
Q3170 E. coli BL21(DE3) carrying pM17This study
pETDuet-1 AmpR, reppBR322, lacI PT7 Novagen
pACYCDuet-1 CmR, p15A origin, lacI PT7 Novagen
pETDuet-Dke1 pETDuet-1 harboring acetylacetone-cleaving enzyme (Dke1) from A. johnsonii This study
pETDuet-Tmo_Dke1 pETDuet-1 harboring acetylacetone-cleaving enzyme from T. mobilis This study
pETDuet-Pxe_Dke1 pETDuet-1 harboring acetylacetone-cleaving enzyme from P. xenovorans This study
pACYCDuet-MgsA-TpiA pACYCDuet-1 harboring methylglyoxal synthase (MgsA) and triose-phosphate isomerase
(TpiA) from E. coli
This study
pM1 reppBR322 AmpR lacI PT7 Dke1K15Q This study
pM2 reppBR322 AmpR lacI PT7 Dke1S17D This study
pM3 reppBR322 AmpR lacI PT7 Dke1Y21W This study
pM6 reppBR322 AmpR lacI PT7 Dke1L103R This study
pM7 reppBR322 AmpR lacI PT7 Dke1G105D This study
pM9 reppBR322 AmpR lacI PT7 Dke1A60D This study
pM10 reppBR322 AmpR lacI PT7 Dke1A60N This study
pM11 reppBR322 AmpR lacI PT7 Dke1G101N This study
pM12 reppBR322 AmpR lacI PT7 Dke1G101D This study
pM13 reppBR322 AmpR lacI PT7 Dke1G101S This study
pM14 reppBR322 AmpR lacI PT7 Dke1L103C This study
pM15 reppBR322 AmpR lacI PT7 Dke1L103Q This study
pM16 reppBR322 AmpR lacI PT7 Dke1E140V This study
pM17 reppBR322 AmpR lacI PT7 Dke1K15Q/A60D This study
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 10
Zhouetal. Biotechnol Biofuels (2020) 13:88
IPTG was added into the medium to induce the recombi-
nant protein expression when the cells reached at about
0.6–0.8 OD600. Nitrogen protection was introduced to
create an anaerobic environment in the serum bottle.
After induction, the temperature was set at 30°C for fur-
ther 24h cultivation. All shake-flask experiments were
performed in triplicate.
Fed‑batch fermentation
For large-scale production, fed-batch fermentation was
carried out in a Biostat B plus MO5L bioreactor (Sarto-
rius Stedim Biotech GmbH, Germany) containing 2 L
growth medium (20g/L tryptone, 10g/L yeast extract,
20 g/L NaCl, 3 g/L KH2PO4, 0.26g/L MgSO4, 1.0g/L
NH4Cl, 15.2 g/L Na2HPO4, 20 g/L glucose and 2 mL
of trace element solution). e trace element solution
contained (per liter) 3.7 g (NH4)6Mo7O24·4H2O, 2.47g
H3BO3, 1.58 g MnCl2·4H2O, 0.29 g ZnSO4·7H2O, and
0.25 g CuSO4·5H2O. Two hundred milliliters of over-
night seed culture was inoculated into the bioreactor to
start the fermentation at 37°C. During the fermentation,
sterilized air was supplied at 1 vvm and ammonia was
added automatically to control the pH 7. e agitation
speed was set at 400 rpm and then associated with the
dissolved oxygen to maintain the concentration at 20%
saturation. Fed-batch mode was commenced by feeding
60% glucose when the dissolved oxygen increased. When
the cell density reached to an OD600 of 30, the recombi-
nant proteins were induced by 0.2mM IPTG along with
0.5mM FeSO4·7H2O added, and nitrogen was used for
anaerobic conditioning after induction. e temperature
was adjusted to 30°C for further cultivation. e agita-
tion speed was kept at a constant rate of 200rpm dur-
ing anaerobic fermentation. More details of the fed-batch
fermentation are presented in Additional file1: Fig. S5
and Additional file2: Fed-batch fermentation. 5mL of the
fermentation broth was withdrawn periodically to deter-
mine the cell density, residual glucose and product titer.
e fed-batch fermentation was performed in triplicate.
Molecular docking
Molecular docking of acetylacetone with Dke1 was car-
ried out with AutoDock 4.2.6 program. e initial struc-
ture was prepared using AutoDockTools 1.5.6 [32],
preserving the original charge of the protein and generat-
ing a pdbqt file for docking. e 3D structure of the acet-
ylacetone was downloaded from the PubChem database.
MOPAC program was then used to optimize the struc-
ture and calculate the PM3 atomic charge. e structure
of acetylacetone was also prepared by AutoDock Tools
1.5.6, and the corresponding pdbqt file was generated
for docking. e active site of Dke1 was chosen as the
binding pocket for docking. e number of grid points
in the XYZ of grid box was set to 40 × 40 × 40, the grid
spacing was 0.375Å, the number of Genetic Algorithm
(GA) run was set to 100, and the rest parameters were set
to default. Finally, the structure with the lowest docking
energy was carried out with energy minimization. e
optimization process is carried out in two steps: first, the
steepest descent method optimization of 2000 steps, then
the structure was further optimized by the 2000 steps
with conjugate gradient method.
Analytic methods
e OD at 600nm was routinely used to monitor cell
growth via ultraviolet spectrophotometer (Varian Cary
50 UV–Vis, US), and one unit of OD600 was equivalent
to 0.43g DCW/L [33]. e residual glucose was deter-
mined by an SBA-40D biosensor analyzer (Institute of
Biology, Shandong Academy of sciences, China). e
culture samples were centrifuged at 10,000×g for 10min;
the supernatants were filtered through a 0.2-M Tuffryn
membrane (China) and used for HPLC analysis (Waters
1525, 300 mm × 7.8 mm Aminex HPX-87H, UV–Vis
detector at 280 nm, for more detailed information see
Additional file1: Fig. S6 and Additional file2: Standard
curve establishment and TableS3). To further confirm
acetylacetone production in our cultures, the same sam-
ple was analyzed by GC–MS after HPLC determination.
e GC–MS analysis was performed with an Agilent
GC quadrupole instrument. e analysis conditions
were as follows: a 30-m HP-5 column (internal diameter
0.25mm, film thickness 0.25m), the column tempera-
ture program was composed of an initial hold at 50°C for
5min, ramping at 15°C per min to 240°C and holding
for 5min. e injector and transfer line temperature were
240 and 250°C, respectively. e mass spectrometry full
scan was from 30 to 400, the ion source and quadrupole
temperature were 230 and 150°C, respectively.
Supplementary information
Supplementary information accompanies this paper at https ://doi.
org/10.1186/s1306 8-020-01725 -9.
Additional le1. Additional figures.
Additional le2. Additional tables.
Dke1: Acetylacetone-cleaving enzyme; TpiA: Triose-phosphate isomerase;
MgsA: Methylglyoxal synthase; E. coli: Escherichia coli; A. johnsonii: Acinetobacter
johnsonii; GC–MS: Gas chromatography–mass spectrometer; HPLC: High-
performance liquid chromatography; OD600: Optical density at wavelength
600 nm; DTT: Dithiothreitol; IPTG: Isopropyl-β-
-thiogalactoside; DCW: Dry cell
We thank Miss Yingxin Fu (QIBEBT, CAS) for her work on the additional acety-
lacetone tolerance experiment, especially she has overcome many difficulties
that is being caused by the COVID-19 pandemic.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 10
Zhouetal. Biotechnol Biofuels (2020) 13:88
Authors’ contributions
XF and GZ designed the study. YZ, WG, JW, and XL carried out the experi-
ments. GZ, XF, YD, XM, and YZ contributed to discuss and analyze the data. XF,
GZ, YZ, and MX wrote the manuscript. All authors read and approved the final
This work was supported by the National Defense Science and Technology
Innovation Zone Foundation of China, National Natural Science Foundation
of China (31722001, 31670089, and 31800081), Taishan Scholars Program of
Shandong Province (ts201712076), Natural Science Foundation of Shandong
(JQ201707 and ZR2019QB015), and the Youth Innovation Promotion Associa-
tion at the Chinese Academy of Sciences.
Availability of data and materials
We provide all the necessary data for the publication of this article. All addi-
tional data are present in the article and the additional material documents.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy
and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101,
China. 2 University of Chinese Academy of Sciences, Beijing 100049, China.
3 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071,
China. 4 State Key Laboratory of Microbial Technology, Shandong University,
Qingdao 266237, China.
Received: 12 March 2020 Accepted: 7 May 2020
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... Biocatalysis has drawn increasing attention as a powerful alternative to traditional, complicated, and dangerous chemical synthesis methods because of its environmental friendliness and safety. 1,2 Specifically, an enzyme is a catalyst in the biocatalytic reactions. Microbial-based metabolic engineering, which relies on wild-type or artificial metabolic pathways, has enabled the production of many high-value compounds through intracellular enzyme catalysis. ...
The novel Ni-based coordination polymer [Ni(H2edda) (bpe)] (CP 1) was successfully synthesized by employing 5,5′ (ethane-1,2-diylbis(oxy)) diisophthalic acid (H4edda) and 1,2-bis (4-pyridyl) ethylene (bpe) under hydrothermal conditions. Structural analysis indicates that CP 1 presents a 2-fold-interpenetrated two-dimensional (2 D) framework with a uninodal sql topology. Further investigation reveals that the newly obtained CP can be stable in water or even exposed in a certain acid/base aqueous solution (pH = 3-11) for a long period of time. Remarkably, CP 1 can further serve as a bifunctional sensor for ascorbic acid (AA) and acetylacetone (acac) in aqueous media according to fluorescence enhancing and quenching effects, respectively. Notably, both sensing processes are labeled with exceptional anti-interference property, high sensitivity and selectivity, wide linear range (0-50 μM for AA and 0.2-1.8 mM for acac) and low detection limit (1.37 nM for AA and 1.58 μM for acac). Theoretical simulations and experimental findings point out that in the process of AA detection by CP 1, the significant fluorescence enhancement could be caused by the photo-induced electron transfer (PET) process, while the inner filter effect (IFE) mechanism combined with PET should be responsible for the fluorescence quenching of CP 1 by acac.
Full-text available
Renewable and abundant carbohydrates are promising feedstocks for producing valuable chemicals. Here we report a highly efficient Zr-catalysed conversion of xylose and acetylacetone (acac) to a new type of bisfuranic monomer, 1-(4-((4-acetyl-5-methylfuran-2-yl)methyl)-2-methylfuran-3-yl)ethenone (MFE). The formation of MFE stems from the intermediate obtained through the nucleophilic addition of acac to xylose. Under optimized conditions (microwave irradiation, 140 °C, 24 min, NaI as an additive), MFE is obtained in near-quantitative yield (98%). Importantly, the reaction selectivity can be tuned by the inclusion of an additive. When NaCl is used, the reaction gives 3-(furan-2-ylmethylene)pentane-2,4-dione (FMPD, 55%), a jet-fuel precursor, and MFE (30%) with a total carbon yield of 85%. To the best of our knowledge, this is the first report on straightforward xylose transformation to a bisfuranic compound with excellent carbon efficiency. This Garcia Gonzalez (GG) reaction inclusive strategy is remarkable and could lead to many innovations in bio-based polymer synthesis.
A family of novel lanthanide-based metal-organic frameworks (Ln-MOFs) with bifunctional fluorescence sensing properties, namely, {[(CH3)2NH2]5[Ln5(TBAPy)5]·solvent}n (Ln = Gd (1), Tb (2), and Dy (3); H4TBAPy = 1,3,5,7-tetra(4-carboxybenzene)pyrene), was synthesized and structurally characterized. Structural analysis reveals that Ln-MOFs1-3 are isomorphic, and Gd-MOF1 has a three-dimensional structure with a porosity of 20.40%. Due to the existence of the pyrene ring featuring a conjugated system, Ln-MOFs1-3 exhibit strong fluorescence emission. Interestingly, Ln-MOFs1-3 could be considered as good bifunctional fluorescence sensors toward acetylacetone and aspartic acid via a turn-on effect. The detection limits of Tb-MOF2 toward acetylacetone and aspartic acid are 0.129 and 0.025 ppm, respectively. Furthermore, the probable sensing mechanisms for target analytes were also discussed in detail.
The synthesis of new types of furan-based compounds other than 5-hydroxymethylfurfural from glucose is a very attractive yet underexploited strategy. We report here a catalytic conversion of glucose with acetylacetone (acac) to furan-centered chemicals, 2-methyl-3-acetylfuran (MAF) and 1-(5-(1,2-dihydroxyethyl)-2-methylfuran-3-yl)ethan-1-one (DMAF), which are potential building blocks for the synthesis of fine chemicals. The experimentally supported reaction mechanism is cascade-type, including glycolaldehyde (GA) formation by H2MoO4-catalysed retro-aldol condensation (C2 + C4) of glucose and immediate capture of transient C2 and C4 intermediates by acac to yield MAF and DMAF. To the best of our knowledge, this is the first report on the straightforward synthesis of MAF and DMAF from glucose, providing a new but generic synthesis strategy for GA-based C2 and erythrose-based C4 chemistry in biorefining.
Two new ternary coordination polymers (CPs), [Cd0.5(TBTA)0.5(L1)]n (1) and [Cd(TBTA)(L2)(H2O)]n (2) (L1 = 1,3-bis(2-methylbenzimidazol-1-yl)-2-propanol, L2 = 1,2-bis(benzimidazol-1-ylmethyl)benzene, H2TBTA = tetrabromoterephthalic acid) were prepared under the hydrothermal condition. 1 possesses a 1D infinite chain. 2 features a 4-connect sql layer with point symbol of {4⁴.6²}. Both 1 and 2 are further extended into the 3D supramolecular networks via OH⋯N and OH⋯O hydrogen-bonding interactions, respectively. The structural variety of the Cd(II) ternary CPs demonstrates that different N-containing ligands play great influence on the final topological network. Luminescence titration results display that 1 and 2 are dual-responsive probes for detection of acetylacetone (acac) and Fe³⁺ ions.
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Background Squalene is currently used widely in the food, cosmetics, and medicine industries. It could also replace petroleum as a raw material for fuels. Microbial fermentation processes for squalene production have been emerging over recent years. In this study, to study the squalene-producing potential of Escherichia coli (E. coli), we employed several increasing strategies for systematic metabolic engineering. These include the expression of human truncated squalene synthase, the overexpression of rate-limiting enzymes in isoprenoid pathway, the modification of isoprenoid-feeding module and the blocking of menaquinone pathway. Results Herein, human truncated squalene synthase was engineered in Escherichia coli to create a squalene-producing bacterial strain. To increase squalene yield, we employed several metabolic engineering strategies. A fivefold squalene titer increase was achieved by expressing rate-limiting enzymes (IDI, DXS, and FPS) involved in the isoprenoid pathway. Pyridine nucleotide transhydrogenase (UdhA) was then expressed to improve the cellular NADPH/NADP⁺ ratio, resulting in a 59% increase in squalene titer. The Embden–Meyerhof pathway (EMP) was replaced with the Entner–Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) to feed the isoprenoid pathway, along with the overexpression of zwf and pgl genes which encode rate-limiting enzymes in the EDP and PPP, leading to a 104% squalene content increase. Based on the blocking of menaquinone pathway, a further 17.7% increase in squalene content was achieved. Squalene content reached a final 28.5 mg/g DCW and 52.1 mg/L. Conclusions This study provided novel strategies for improving squalene yield and demonstrated the potential of producing squalene by E. coli. Electronic supplementary material The online version of this article (10.1186/s13068-019-1415-x) contains supplementary material, which is available to authorized users.
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Background Kluyveromyces marxianus, the known fastest-growing eukaryote on the earth, has remarkable thermotolerance and capacity to utilize various agricultural residues to produce low-cost bioethanol, and hence is industrially important to resolve the imminent energy shortage crisis. Currently, the poor ethanol tolerance hinders its operable application in the industry, and it is necessary to improve K. marxianus’ ethanol resistance and unravel the underlying systematical mechanisms. However, this has been seldom reported to date. Results We carried out a wild-type haploid K. marxianus FIM1 in adaptive evolution in 6% (v/v) ethanol. After 100-day evolution, the KM-100d population was obtained; its ethanol tolerance increased up to 10% (v/v). Interestingly, DNA analysis and RNA-seq analysis showed that KM-100d yeasts’ ethanol tolerance improvement was not due to ploidy change or meaningful mutations, but founded on transcriptional reprogramming in a genome-wide range. Even growth in an ethanol-free medium, many genes in KM-100d maintained their up-regulation. Especially, pathways of ethanol consumption, membrane lipid biosynthesis, anti-osmotic pressure, anti-oxidative stress, and protein folding were generally up-regulated in KM-100d to resist ethanol. Notably, enhancement of the secretory pathway may be the new strategy KM-100d developed to anti-osmotic pressure, instead of the traditional glycerol production way in S. cerevisiae. Inferred from the transcriptome data, besides ethanol tolerance, KM-100d may also develop the ability to resist osmotic, oxidative, and thermic stresses, and this was further confirmed by the cell viability test. Furthermore, under such environmental stresses, KM-100d greatly improved ethanol production than the original strain. In addition, we found that K. marxianus may adopt distinct routes to resist different ethanol concentrations. Trehalose biosynthesis was required for low ethanol, while sterol biosynthesis and the whole secretory pathway were activated for high ethanol. Conclusions This study reveals that ethanol-driven laboratory evolution could improve K. marxianus’ ethanol tolerance via significant up-regulation of multiple pathways including anti-osmotic, anti-oxidative, and anti-thermic processes, and indeed consequently raised ethanol yield in industrial high-temperature and high-ethanol circumstance. Our findings give genetic clues for further rational optimization of K. marxianus’ ethanol production, and also partly confirm the positively correlated relationship between yeast’s ethanol tolerance and production. Electronic supplementary material The online version of this article (10.1186/s13068-019-1393-z) contains supplementary material, which is available to authorized users.
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1,2,4‐butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4‐butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl‐coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA over‐expression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl‐CoA production by overexpression of the acyl‐CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed‐batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.<PE‐FRONTEND> This article is protected by copyright. All rights reserved
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Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed.
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Polyhydroxyalkanoates (PHA) are a family of diverse biopolyesters synthesized by bacteria. PHA diversity, as reflected by its monomers, homopolymers, random and block copolymers, as well as functional polymers, can now be generated by engineering the three basic synthesis pathways including the acetoacetyl-CoA pathway, in situ fatty acid synthesis, and/or β-oxidation cycles, as well as PHA synthase specificity. It is now possible to tailor the PHA structures via genome editing or process engineering. The increasing PHA diversity and maturing PHA production technology should lead to more focused research into their low-cost and/or high-value applications.
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Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l(-1) for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.
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Triacetic acid lactone is demonstrated to be a versatile biorenewable molecule with potential as a platform chemical for the production of commercially valuable bifunctional chemical intermediates and end products, such as sorbic acid.
Engineering cell metabolism for bioproduction not only consumes building blocks and energy molecules (e.g., ATP) but also triggers energetic inefficiency inside the cell. The metabolic burdens on microbial workhorses lead to undesirable physiological changes, placing hidden constraints on host productivity. We discuss cell physiological responses to metabolic burdens, as well as strategies to identify and resolve the carbon and energy burden problems, including metabolic balancing, enhancing respiration, dynamic regulatory systems, chromosomal engineering, decoupling cell growth with production phases, and co-utilization of nutrient resources. To design robust strains with high chances of success in industrial settings, novel genome-scale models (GSMs), (13)C-metabolic flux analysis (MFA), and machine-learning approaches are needed for weighting, standardizing, and predicting metabolic costs.
Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, dep>99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (kcat/Km) toward 1a up to 12.37s(-1)mM(-1), which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7mM with dep value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis.
An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6- to 10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C6-C10 dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C6-C10 ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.