Functional expression of l-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of l-pipecolic acid from dl-lysine

Tani, Yasuhiro; Miyake, Ryoma; Yukami, Ryoichi; Dekishima, Yasumasa; China, Hideyasu; Saito, Shigeki; Kawabata, Hiroshi; Mihara, Hisaaki

doi:10.1007/s00253-014-6308-0

Cited by 34 publications

(21 citation statements)

References 47 publications

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“…Additional overexpression of the E. coli gene for lysine uptake, lysP, accelerated L-PA formation from L-lysine about five fold (Fujii et al 2002). Biotransformation of racemic DL-lysine with E. coli overproducing L-lysine α-oxidase from S. japonicus yielded high titers (about 45 g L −1 ) of optically pure L-PA (Tani et al 2015). The functionality of L-lysine cyclodeaminase from Actinoplanes friuliensis has been shown in C. glutamicum (Wagner et al 2010), and the enzyme from Streptomyces.…”

Section: Discussionmentioning

confidence: 94%

“…L-PA accumulated intracellularly when Escherichia coli overproducing L -lysine 6-dehydrogenase from S. pomeroyi was grown in L-lysine-containing media (Neshich et al 2013). L-PA was produced from L-lysine in biotransformations with E. coli overproducing either L-lysine α-oxidase from Scomber japonicus (Tani et al 2015) or LAT (Fujii et al 2002).…”

Section: Introductionmentioning

confidence: 99%

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Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Pérez-García

Peters‐Wendisch

Wendisch

2016

Appl Microbiol Biotechnol

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The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

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Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Pérez-García

Peters‐Wendisch

Wendisch

2016

Appl Microbiol Biotechnol

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“…l -PA, an important chiral precursor for the synthesis of optically pure piperidine rings, forms core structure of many naturally occurring alkaloids and drugs in clinical use [9, 18]. l -PA can be biosynthesized using crude enzymes or whole-cell biocatalysts [14, 15, 19], and several of the different catalytic steps involved have been investigated, including some that are unstable and accompanied by high manufacturing costs that limit their applications. However, few researchers have focused on the direct fermentation of l -PA, even though fermentation potentially offers an alternative, efficient, low cost method [22].…”

Section: Discussionmentioning

confidence: 99%

“…The second involves the loss of the ε-nitrogen and incorporation of an α-nitrogen into l -PA (P6C pathway). Several studies on the enzymatic production of l -PA using enzymes from both pathways have been reported [14, 15]. For example, Neshich et al, obtained l -pipeclic acid on lysine containing medium (about 0.6 μg/mg protein) using recombinant E. coli cells over-expressing the l -lysine 6-dehydrogenase gene in P6C pathway [16]; Fujii et al reported that about 3.9 g/L of optically pure l -PA was biosynthesised using the enzymes from the P2C pathway [17].…”

Section: Introductionmentioning

confidence: 99%

Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate l-pipecolic acid in Escherichia coli

et al. 2017

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BackgroundThe six-carbon circular non-proteinogenic compound l-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of l-pipecolic acid from glucose.ResultsThe metabolic pathway from l-lysine to l-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, l-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor l-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD+, the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD+ and enhanced l-pipecolic acid production significantly. Further, optimization of Fe2+ and surfactant in the fermentation process resulted in 5.33 g/L l-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation.ConclusionsWe expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate l-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of l-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0666-0) contains supplementary material, which is available to authorized users.

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“…The UV absorbance at 280 nm was mensurated as the protein concentration by SpectraMax M2 e (Molecular Devices, American) (Annamalai et al, 2011; Zhang et al, 2008). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified enzymes with a 12 % acrylamide gel (Tani et al, 2015a)2…”

Section: Methodsmentioning

confidence: 99%

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

Cheng

Song²,

Wang³

et al. 2019

Preprint

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22Bioplastics 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 -NH2, 42 -SCH3, -SOCH3, -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 3 47 Abbreviations: 48 NNSCAA, Nonnatural straight chain amino acid; 5AVA, 5-Aminovalerate; 6ACA, 6-49

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Functional expression of l-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of l-pipecolic acid from dl-lysine

Cited by 34 publications

References 47 publications

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate l-pipecolic acid in Escherichia coli

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

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