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