Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle
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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"Production of nonnatural straight-c..." refers background in this paper
...The chain extension of the aforementioned metabolic systems generally consists of a series of condensation, dehydration and reduction reactions (Chandran et al., 2011; Katz and Donadio, 1993; Textor et al., 2007)....
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...…α-isopropylmalate synthase (LeuA) in the leucine pathway (Hunter and Parker, 2014), the homocitrate synthase in the L-lysine α-aminoadipate pathway (Zabriskie and Jackson, 2000), the methylthioalkylmalate synthase (MAM) in the glucosinolate pathway (Mirza et al., 2016) and the (Homo)1→3aconitate…...
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...…sugars (Yang et al., 2017), α-ketoacids (Sonderby et al., 2010; Wen et al., 2013), fatty acids (Wu et al., 2014), and as well as several specialized metabolic pathways for the synthesis of polyketides (Gokhale et al., 2007; Miyanaga, 2017) and terpenoids (Gronenberg et al., 2013; Yu et al., 2012)....
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..., 102 2007; Miyanaga, 2017) and terpenoids (Gronenberg et al., 2013; Yu et al., 2012)....
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...As the transamination activity is limiting (Zhang et al., 2010), the final titer of 6ACA achieved in Turk et al.’s study was only 160 mg/L (Jorge et al., 2017; Turk et al., 2016)....
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...However, the reported strategy was bottlenecked at the transamination step (Turk et al., 2016; Zhang et al., 2010)....
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...With inadequate transamination efficiency previously recognized (Zhang et al., 2010), the final titer achieved by Turk et al. was only 160 mg/L (Jorge et al., 2017; Turk et al., 2016)....
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