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Production of nonnatural straight-chain amino acid 6-aminocaproate
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via an artificial iterative carbon-chain-extension cycle
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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,
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Chongqing University, Chongqing 401331, P. R. China
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2
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial
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Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China
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3
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA,
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02142, United States
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4
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA,
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United States
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Corresponding author: Tel: +86-23-65678926
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E-mail: dwang@cqu.edu.cn (D. Wang), wang_qh@tib.cas.cn (Q.H. Wang)
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E-mail address: 55 Daxuecheng South Road, Shapingba District, Department of
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Chemical Engineering, School of Chemistry and Chemical Engineering, Chongqing
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University, Chongqing, 401331, P. R. China.
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20
21
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2
Abstract
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Bioplastics produced from microbial source are promising green alternatives to
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traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids,
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especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential
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monomers for the synthesis of polymeric bioplastics as their primary amine and
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carboxylic acid are ideal functional groups for polymerization. Previous pathways for
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5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from
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L-lysine catabolism and citric acid cycle, respectively. Here, we show the construction
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of an artificial iterative carbon-chain-extension cycle in Escherichia coli for
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simultaneous production of a series of nonnatural amino acids with varying chain length.
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Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate as a
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non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-
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extended α-ketoacid is subsequently decarboxylated and oxidized by an α-ketoacid
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decarboxylase and an aldehyde dehydrogenase, respectively, to yield the nonnatural
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straight-chain amino acid products. The engineered system demonstrated simultaneous
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in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate
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and 4.78 mg/L of 7-aminoheptanoate after 8 hours of enzyme catalysis starting from 2-
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keto-6-aminocaproate as the substrate. Furthermore, simultaneous production of 2.15
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g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-
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aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising
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metabolic-engineering strategy to access other medium-chain organic acids with -NH
2
,
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-SCH
3
, -SOCH
3
, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-
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elongation chemistry.
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Keywords: Nonnatural straight chain amino acid, 6-Aminocaproate, Carbon chain
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elongation, Synthetic biology, Iterative cycle
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.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
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Abbreviations:
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NNSCAA, Nonnatural straight chain amino acid; 5AVA, 5-Aminovalerate; 6ACA, 6-
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Aminocaproate; 7AHA, 7-Aminoheptanoate; RaiP, L-lysine α oxidase; LeuA, α-
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Isopropylmalate synthase; LeuA*, LeuA mutants; LeuA
#
, LeuA with
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H97L/S139G/G462D mutations; LeuB, 3-Isopropylmalate dehydrogenase; LeuC, 3-
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Isopropylmalate dehydratase; LeuD, 3-Isopropylmalate dehydratase; KivD, α-
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Ketoacid decarboxylase; PadA, Aldehyde dehydrogenase; ThDP, Thiamine
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diphosphate; TCEP, Tris (2-carboxyethyl) phosphine; KPB, Potassium phosphate
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buffer; LC-MS, Liquid chromatography-mass spectrometry; SDS-PAGE, Sodium
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dodecyl sulfate polyacrylamide gel electrophoresis; 4AAP, 4-Aminoantipyrine
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58
59
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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4
1. Introduction
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Microbial polyimide bioplastics present a class of green materials with broad
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applications in many downstream industries, and can potentially replace the traditional
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petrochemical-derived polymers. Consequently, platform chemicals containing suitable
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functional groups necessary for polyimide polymerization have attracted significant
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attention as targets for metabolic engineering. These compounds include diamines such
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as putrescine (Del Rio et al., 2018) and cadaverine (Kim et al., 2018), amino acids such
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as lysine (Borri et al., 2018) and glutamate, organic acids such as succinate (Jantama et
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al., 2008) and lactate (Pang et al., 2010), diols such as butanediol and hexanediol
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(Muller et al., 2010). Nonnatural straight-chain amino acids (NNSCAAs), especially 5-
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aminovalerate (5AVA) and 6-aminocaproate (6ACA) are important platform chemicals
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for the synthesis of polyimides, which are widely used as raw materials for automobile
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parts, clothes, backpacks and disposable goods such as nylon 5, nylon 6 and nylon 5,6
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(Haushalter et al., 2017). In addition to its utility in bioplastics, 6ACA was also
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implicated to promote blood clotting, suggesting potential applications as an
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antifibrinolytic agent (Lu et al., 2015; Schou-Pedersen et al., 2015). Whereas 5AVA
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biosynthesis is a viable approach for industrial production, effective methods to
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biosynthesize other NNSCAAs at scale has yet to be established (Jorge et al., 2017;
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Turk et al., 2016). Biosynthesis of 6ACA was first demonstrated to occur through the
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condensation of acetyl-CoA and succinyl-CoA (Turk et al., 2016). The second
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biosynthetic route utilizes -ketoadipate as the starter molecule, which is chain-
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extended by (homo)
1 → 3
aconitate synthase (AksA), (homo)
1 → 3
aconitate isomerase
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complex (AksD, AksE), iso(homo)
1 → 3
citrate dehydrogenase (AksF) to give the
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intermediate α-ketopimelate (AKP). AKP is decarboxylated and transaminased to yield
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
6ACA (Chae et al., 2017). The precursors of the two pathways are all derived from the
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tricarboxylic acid (TCA) cycle which are scarce in cells. With inadequate
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transamination efficiency previously recognized (Zhang et al., 2010), the final titer
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achieved by Turk et al. was only 160 mg/L (Jorge et al., 2017; Turk et al., 2016).
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L-lysine is the second most-produced amino acid worldwide after glutamate.
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Currently, L-lysine is mainly produced through microbial fermentation, and is
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commonly used as an additive to poultry and swine feed (Wang et al., 2016). Annual
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world L-lysine production is estimated to exceed 2.5 million tons by 2020 (Vassilev et
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al., 2018). Due to the market competition in industrial capacity and demand, the price
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of L-lysine as a commodity chemical has dropped significantly in recent years (Xu et
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al., 2018). As a result, developing high-value chemicals derived from L-lysine presents
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an emerging opportunity in the field of metabolic engineering (Cheng et al., 2018a).
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Novel L-lysine-derived products may contribute to an environmentally friendly
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chemical industry (Hoffmann et al., 2018; Sgobba et al., 2018).
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Nonpolymeric carbon-chain-extension pathways occur broadly in many primary
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metabolism pathways for the synthesis of rare sugars (Yang et al., 2017), α-ketoacids
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(Sonderby et al., 2010; Wen et al., 2013), fatty acids (Wu et al., 2014), and as well as
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several specialized metabolic pathways for the synthesis of polyketides (Gokhale et al.,
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2007; Miyanaga, 2017) and terpenoids (Gronenberg et al., 2013; Yu et al., 2012). The
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chain extension of the aforementioned metabolic systems generally consists of a series
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of condensation, dehydration and reduction reactions (Chandran et al., 2011; Katz and
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Donadio, 1993; Textor et al., 2007). The first carbon-chain-extension step of the
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pathway is catalyzed by a C-acetyltransferase, such as the citramalate synthase in the
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citramalate pathway (Drevland et al., 2007), the citrate synthase in the TCA (Harder et
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al., 2018), the α-isopropylmalate synthase (LeuA) in the leucine pathway (Hunter and
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