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Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

TL;DR: This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2,SCH3, -SOCH3), -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.
Abstract: Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended α-ketoacid is subsequently decarboxylated and oxidized by an α-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield the nonnatural straight-chain amino acid products. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 hours of enzyme catalysis starting from 2-keto-6-aminocaproate as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2,-SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.

Summary (2 min read)

2.1 Strains, plasmids and primers used in this study

  • The raiP from Scomber japonicus, kivD from Lactococcus lactis and padA from E. coli MG1655 were constructed in another operon in transcriptional order raiP-kivD-padA.
  • The engineered pET21a-raiP-kivD-padA was produced, also named as pETaRKP. BL21(DE3) was transformed with the plasmid pIVC03 or pIVC04 and pETaRKP, resulting in strain CJ03 or CJ04.

2.4 Enzyme assay

  • LeuA and LeuA* activities were assayed by measuring CoASH produced (Zhang et al., 2008) .
  • One unit of enzyme activity was defined as the amount of enzyme that catalyzes 1.0 μmol of CoASH produced per minute.
  • There were no other intermediates as substrates, so the enzyme activities of LeuB, LeuC, LeuD, KivD and PadA could not be detected.

2.6 Analytical methods

  • The optical density of the various E. coli cultures was detected using a UV/visible spectrophotometer (Ultrospec TM 2100 pro, GE Healthcare, UK).
  • The operating conditions were performed as 1.0 mL/min, column temperature 40 °C, wavelength 254 nm and analysis time 55 min.
  • For liquid chromatography-mass spectrometry (LC-MS) identification of 5AVA, 6ACA and 7AHA, exact mass spectra were explored with a Bruker micrOTOF-Q II mass spectrometer using the time of flight (TOF) technique, equipped with an ESI source operating in negative mode (Burker Co., Ltd, USA).

3.1 Construction of a L-lysine derived artificial iterative carbon-chain-extension cycle in vitro

  • To explore the feasibility of a RaiP-LeuABCD-KivD-PadA pathway, the necessary enzymes were expressed, purified and assayed against L-lysine for NNSCAAS LeuA exhibited low activity toward 2-keto-6-aminocaproate, whereas LeuA mutations (H97A/G462D, H97G/G462D, H97L/G462D, S139G/G462D and S139I/G462D) displayed higher activities.
  • Amano et al. and Arinbasarova et al. have previously characterized the recombinant enzyme of RaiP from Trichoderma viride (Amano et al., 2015; Arinbasarova et al., 2012) .
  • The reported specific activities of the purified enzyme was just 80 or 90 U/mg in the previous studies, which are about 11% of the specific activity the authors measured in this study.

3.2 Building a nonnatural iterative cycle for NNSCAA biosynthesis in vitro

  • To do this, the authors explored the promiscuity of LeuA mutants towards L-lysine-derived α-ketoacids with amino functional group, which is exemplified by LeuA # that can utilize primary amines such as 2-keto-6-aminocaproate and 2-keto-7-aminoheptanoate as substrate.
  • The malleability of the LeuABCD pathway remains to be further explored, as untargeted metabolomics of LeuA* expression in vivo may identify additional substrates.
  • Furthermore, directed evolution of LeuA or LeuA* may further broaden substrate profile.
  • In Brassicaceae plants, Methylthioalkylmalate synthases are evolutionary derived from an ancestral LeuA and catalyze carbon-chain-extension pathway in the biosynthesis of glucosinolates (de Kraker and Gershenzon, 2011; Mirza et al., 2016) .
  • The recruitment of LeuA for plant specialized metabolism suggests that the C-acetyltransferase family proteins can be further evolved to arrive at desirable activities starting from ancestral promiscuous activities (Weng and Noel, 2012) .

3.3 Dependence of 6ACA productivity on the supply of coenzyme

  • Moreover, KivD and PadA catalyze the conversion of 2-keto-7-aminoheptanoate to 6ACA, which requires coenzymes ThDP and NAD + , respectively (Fig. 1 ).
  • The effect of ThDP, the coenzyme of KivD, was also investigated in this work.
  • Without ThDP addition, no 6ACA was produced in this multi-enzyme cascade system.
  • The 0.5 mM of ThDP was set as the optimal dosage.

3.4 The confirmation of the rate-limiting enzyme in this artificial iterative cycle

  • No further titer improvement was observed when the enzyme concentrations reached 2.0 μM for LeuC, LeuD and PadA, 4.0 μM for LeuB, whereas 5.0 μM of KivD inhibited 6ACA production.
  • The optimal molar ratio of RaiP: LeuA # :LeuB: LeuC: LeuD: KivD: PadA was determined as 1:20:4:2:2:5:2 in this artificial iterative pathway, which was inferred from the titration studies, as seen in Fig. 4 .

3.5 Assembling a nonnatural NNSCAA biosynthetic pathway in E. coli

  • The natural substrates of LeuA are 2-ketoisovalerate and 2-ketobutyrate (Shen and Liao, 2008) .
  • Their engineered E. coli strain could use 2-keto-6-aminocaproate as the alternative substrate to simultaneously produce 5AVA, 6ACA and 7AHA from Llysine with a titer of total at 2.18 g/L, as seen in Fig. 7 .

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1
Production of nonnatural straight-chain amino acid 6-aminocaproate
1
via an artificial iterative carbon-chain-extension cycle
2
3
Jie Cheng
1,2
, Tingting Song
1
, Huayu Wang
1
, Xiaohua Zhou
1
, Michael P. Torrens-
4
Spence
3
, Dan Wang
1,
*, Jing-Ke Weng
3,4,*
, Qinhong Wang
2,
*
5
6
1
Department of Chemical Engineering, School of Chemistry and Chemical Engineering,
7
Chongqing University, Chongqing 401331, P. R. China
8
2
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial
9
Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China
10
3
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA,
11
02142, United States
12
4
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA,
13
United States
14
Corresponding author: Tel: +86-23-65678926
15
E-mail: dwang@cqu.edu.cn (D. Wang), wang_qh@tib.cas.cn (Q.H. Wang)
16
E-mail address: 55 Daxuecheng South Road, Shapingba District, Department of
17
Chemical Engineering, School of Chemistry and Chemical Engineering, Chongqing
18
University, Chongqing, 401331, P. R. China.
19
20
21
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted March 5, 2019. ; https://doi.org/10.1101/568121doi: bioRxiv preprint

2
Abstract
22
Bioplastics produced from microbial source are promising green alternatives to
23
traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids,
24
especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential
25
monomers for the synthesis of polymeric bioplastics as their primary amine and
26
carboxylic acid are ideal functional groups for polymerization. Previous pathways for
27
5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from
28
L-lysine catabolism and citric acid cycle, respectively. Here, we show the construction
29
of an artificial iterative carbon-chain-extension cycle in Escherichia coli for
30
simultaneous production of a series of nonnatural amino acids with varying chain length.
31
Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate as a
32
non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-
33
extended α-ketoacid is subsequently decarboxylated and oxidized by an α-ketoacid
34
decarboxylase and an aldehyde dehydrogenase, respectively, to yield the nonnatural
35
straight-chain amino acid products. The engineered system demonstrated simultaneous
36
in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate
37
and 4.78 mg/L of 7-aminoheptanoate after 8 hours of enzyme catalysis starting from 2-
38
keto-6-aminocaproate as the substrate. Furthermore, simultaneous production of 2.15
39
g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-
40
aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising
41
metabolic-engineering strategy to access other medium-chain organic acids with -NH
2
,
42
-SCH
3
, -SOCH
3
, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-
43
elongation chemistry.
44
Keywords: Nonnatural straight chain amino acid, 6-Aminocaproate, Carbon chain
45
elongation, Synthetic biology, Iterative cycle
46
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted March 5, 2019. ; https://doi.org/10.1101/568121doi: bioRxiv preprint

3
47
Abbreviations:
48
NNSCAA, Nonnatural straight chain amino acid; 5AVA, 5-Aminovalerate; 6ACA, 6-
49
Aminocaproate; 7AHA, 7-Aminoheptanoate; RaiP, L-lysine α oxidase; LeuA, α-
50
Isopropylmalate synthase; LeuA*, LeuA mutants; LeuA
#
, LeuA with
51
H97L/S139G/G462D mutations; LeuB, 3-Isopropylmalate dehydrogenase; LeuC, 3-
52
Isopropylmalate dehydratase; LeuD, 3-Isopropylmalate dehydratase; KivD, α-
53
Ketoacid decarboxylase; PadA, Aldehyde dehydrogenase; ThDP, Thiamine
54
diphosphate; TCEP, Tris (2-carboxyethyl) phosphine; KPB, Potassium phosphate
55
buffer; LC-MS, Liquid chromatography-mass spectrometry; SDS-PAGE, Sodium
56
dodecyl sulfate polyacrylamide gel electrophoresis; 4AAP, 4-Aminoantipyrine
57
58
59
60
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted March 5, 2019. ; https://doi.org/10.1101/568121doi: bioRxiv preprint

4
1. Introduction
61
Microbial polyimide bioplastics present a class of green materials with broad
62
applications in many downstream industries, and can potentially replace the traditional
63
petrochemical-derived polymers. Consequently, platform chemicals containing suitable
64
functional groups necessary for polyimide polymerization have attracted significant
65
attention as targets for metabolic engineering. These compounds include diamines such
66
as putrescine (Del Rio et al., 2018) and cadaverine (Kim et al., 2018), amino acids such
67
as lysine (Borri et al., 2018) and glutamate, organic acids such as succinate (Jantama et
68
al., 2008) and lactate (Pang et al., 2010), diols such as butanediol and hexanediol
69
(Muller et al., 2010). Nonnatural straight-chain amino acids (NNSCAAs), especially 5-
70
aminovalerate (5AVA) and 6-aminocaproate (6ACA) are important platform chemicals
71
for the synthesis of polyimides, which are widely used as raw materials for automobile
72
parts, clothes, backpacks and disposable goods such as nylon 5, nylon 6 and nylon 5,6
73
(Haushalter et al., 2017). In addition to its utility in bioplastics, 6ACA was also
74
implicated to promote blood clotting, suggesting potential applications as an
75
antifibrinolytic agent (Lu et al., 2015; Schou-Pedersen et al., 2015). Whereas 5AVA
76
biosynthesis is a viable approach for industrial production, effective methods to
77
biosynthesize other NNSCAAs at scale has yet to be established (Jorge et al., 2017;
78
Turk et al., 2016). Biosynthesis of 6ACA was first demonstrated to occur through the
79
condensation of acetyl-CoA and succinyl-CoA (Turk et al., 2016). The second
80
biosynthetic route utilizes -ketoadipate as the starter molecule, which is chain-
81
extended by (homo)
1 3
aconitate synthase (AksA), (homo)
1 3
aconitate isomerase
82
complex (AksD, AksE), iso(homo)
1 3
citrate dehydrogenase (AksF) to give the
83
intermediate α-ketopimelate (AKP). AKP is decarboxylated and transaminased to yield
84
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted March 5, 2019. ; https://doi.org/10.1101/568121doi: bioRxiv preprint

5
6ACA (Chae et al., 2017). The precursors of the two pathways are all derived from the
85
tricarboxylic acid (TCA) cycle which are scarce in cells. With inadequate
86
transamination efficiency previously recognized (Zhang et al., 2010), the final titer
87
achieved by Turk et al. was only 160 mg/L (Jorge et al., 2017; Turk et al., 2016).
88
L-lysine is the second most-produced amino acid worldwide after glutamate.
89
Currently, L-lysine is mainly produced through microbial fermentation, and is
90
commonly used as an additive to poultry and swine feed (Wang et al., 2016). Annual
91
world L-lysine production is estimated to exceed 2.5 million tons by 2020 (Vassilev et
92
al., 2018). Due to the market competition in industrial capacity and demand, the price
93
of L-lysine as a commodity chemical has dropped significantly in recent years (Xu et
94
al., 2018). As a result, developing high-value chemicals derived from L-lysine presents
95
an emerging opportunity in the field of metabolic engineering (Cheng et al., 2018a).
96
Novel L-lysine-derived products may contribute to an environmentally friendly
97
chemical industry (Hoffmann et al., 2018; Sgobba et al., 2018).
98
Nonpolymeric carbon-chain-extension pathways occur broadly in many primary
99
metabolism pathways for the synthesis of rare sugars (Yang et al., 2017), α-ketoacids
100
(Sonderby et al., 2010; Wen et al., 2013), fatty acids (Wu et al., 2014), and as well as
101
several specialized metabolic pathways for the synthesis of polyketides (Gokhale et al.,
102
2007; Miyanaga, 2017) and terpenoids (Gronenberg et al., 2013; Yu et al., 2012). The
103
chain extension of the aforementioned metabolic systems generally consists of a series
104
of condensation, dehydration and reduction reactions (Chandran et al., 2011; Katz and
105
Donadio, 1993; Textor et al., 2007). The first carbon-chain-extension step of the
106
pathway is catalyzed by a C-acetyltransferase, such as the citramalate synthase in the
107
citramalate pathway (Drevland et al., 2007), the citrate synthase in the TCA (Harder et
108
al., 2018), the α-isopropylmalate synthase (LeuA) in the leucine pathway (Hunter and
109
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted March 5, 2019. ; https://doi.org/10.1101/568121doi: bioRxiv preprint

Citations
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Journal ArticleDOI
TL;DR: The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts, which can be applied to the biosynthesis of other valuable chemicals.
Abstract: Microorganisms can utilize biomass to produce valuable chemicals, showing sustainable, renewable and economic advantages compared with traditional chemical synthesis. As a potential five-carbon platform polymer monomer, 5-aminovalerate has been widely used in industrial fields such as clothes and disposable goods. Here we establish an efficient whole-cell catalysis for 5-aminovalerate production with ethanol pretreatment. In this study, the metabolic pathway from L-lysine to 5-aminovalerate was constructed at the cellular level by introducing L-lysine α-oxidase. The newly produced H2O2 and added ethanol both are toxic to the cells, obviously inhibiting their growth. Here, a promising strategy of whole-cell catalysis with ethanol pretreatment is proposed, which greatly improves the yield of 5-aminovalerate. Subsequently, the effects of ethanol pretreatment, substrate concentration, reaction temperature, pH value, metal ion additions and hydrogen peroxide addition on the whole-cell biocatalytic efficiency were investigated. Using 100 g/L of L-lysine hydrochloride as raw material, 50.62 g/L of 5-aminovalerate could be excellently produced via fed-batch bioconversion with the yield of 0.84 mol/mol. The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts. This strategy, combined with ethanol pretreatment, can not only greatly enhance the yield of 5-aminovalerate but also be applied to the biosynthesis of other valuable chemicals.

17 citations

Journal ArticleDOI
TL;DR: Experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.
Abstract: Nylon is a polyamide material with excellent performance used widely in the aviation and automobile industries, and other fields. Nylon monomers such as hexamethylene diamine and other monomers are in huge demand. Therefore, in order to expand the methods of nylon production, we tried to develop alternative bio-manufacturing processes which would make a positive contribution to the nylon industry. In this study, the engineered E. coli-overexpressing Lysine decarboxylases (LDCs) were used for the bioconversion of l-lysine to cadaverine. An integrated fermentation and microfiltration (MF) process for high-level cadaverine production by E. coli was established. Concentration was increased from 87 to 263.6 g/L cadaverine after six batch coupling with a productivity of 3.65 g/L-h. The cadaverine concentration was also increased significantly from 0.43 g cadaverine/g l-lysine to 0.88 g cadaverine/g l-lysine by repeated batch fermentation. These experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.

4 citations

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TL;DR: The feasibility of an approach to build artificial metabolism beyond the natural metabolic network is demonstrated by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol, which is included in the metabolite family of living systems.
Abstract: Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.

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"Production of nonnatural straight-c..." refers background or methods or result in this paper

  • ...The UV absorbance at 280 nm was mensurated as the protein concentration by SpectraMax M2e(Molecular Devices, American) (Annamalai et al., 2011; Zhang et al., 2008)....

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  • ...Zhang et al. reported that the LeuA G462D mutant displays a low Kcatof 0.018 s-1 for (S)-2-keto-3-methylvalerate (Zhang et al., 2008)....

    [...]

  • ...This result suggests that LeuA#is a rate-limiting enzyme in the system, consistent with the previous findings from the iterative nonnatural alcohol system (Zhang et al., 2008)....

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  • ...Another LeuA* G462D/S139G mutant could improve the titer of 1-pentanol to 204.7 mg/L (Zhang et al., 2008)....

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  • ...Overexpression of LeuABCD, α-ketoacid decarboxylase (KivD) and alcohol dehydrogenase (ADH6) in a modified threonine-overproduction strain of Escherichia coli (E. coli) ATCC98082/ThrABC-TdcB-IlvGMCD led to the production of (S)-3-methyl-1-pentanol with a final titer of 6.5 mg/L (Zhang et al., 2008)....

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TL;DR: This work systematically improved the synthesis of 1-propanol and 1-butanol through deregulation of amino-acid biosynthesis and elimination of competing pathways.

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"Production of nonnatural straight-c..." refers background or methods in this paper

  • ...However, several mutants derived from LeuA exhibit interesting substrate promiscuity and catalyzes flexibility, which we use LeuA* to refer to (Shen and Liao, 2008; Umbarger, 1978)....

    [...]

  • ...The natural substrates of LeuA are 2-ketoisovalerate and 2-ketobutyrate (Shen and Liao, 2008)....

    [...]

  • ...LeuA* displays an enhanced degree of substrate promiscuity and is capable of catalyzing the condensation reaction on multiple α-ketoacid substrates (Shen and Liao, 2008)....

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  • ...Metabolic engineering of L-leucine biosynthesis using LeuABCD has been thoroughly explored in E. coli (Shen and Liao, 2008; Xiong et al., 2012)....

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  • ...LeuA could also catalyze the condensation of acety-CoA and α-ketobutyrate to produce α-ketovalerate as a precursor of the non-natural amino acid (Shen and Liao, 2008)....

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Journal ArticleDOI
TL;DR: Mutations rendering certain domains nonfunctional have been introduced into genes for complex polyketides, resulting in the production of novel molecules.
Abstract: Polyketides fall into two structural classes: aromatic and complex. The former are built mainly from acetate units through a reiterative process wherein the beta-carbonyl groups formed after each condensation cycle are left largely unreduced. Complex polyketides are composed of acetates, propionates, or butyrates, and the extent of beta-carbonyl reduction varies from one cycle to the next. Two themes for polyketide synthases are emerging. Aromatic PKSs are determined by four to six genes encoding mono- or bifunctional enzymes; one PKS complex is used for all synthesis steps. Complex PKSs are composed of several multifunctional polypeptides that contain enzymatic domains for the condensation and reduction steps; each domain is used at a unique step in the pathways, and the extent of beta-carbonyl processing depends on the functional domains operating at that cycle. Mutations rendering certain domains nonfunctional have been introduced into genes for complex polyketides, resulting in the production of novel molecules.

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TL;DR: Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes by combination of gene deletions and metabolic evolution.
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"Production of nonnatural straight-c..." refers background in this paper

  • ...…diamines such as putrescine (Del Rio et al., 2018) and cadaverine (Kim et al., 2018), amino acids such as lysine (Borri et al., 2018) and glutamate, organic acids such as succinate (Jantama et al., 2008) and lactate (Pang et al., 2010), diols such as butanediol and hexanediol (Muller et al., 2010)....

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291 citations


"Production of nonnatural straight-c..." refers background in this paper

  • ...…diamines such as putrescine (Del Rio et al., 2018) and cadaverine (Kim et al., 2018), amino acids such as lysine (Borri et al., 2018) and glutamate, organic acids such as succinate (Jantama et al., 2008) and lactate (Pang et al., 2010), diols such as butanediol and hexanediol (Muller et al., 2010)....

    [...]