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

Abstract

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.

Function of L-Pipecolic Acid as Compatible Solute in Corynebacterium glutamicum as Basis for Its Production Under Hyperosmolar Conditions

“…et al, 2016; Li and Gänzle, 2016) the biotechnological production of compatible solutes has gained increasing momentum recently (Sauer and Galinski, 1998; Jensen and Wendisch, 2013; Tan et al, 2016; Chen et al, 2017). This included the establishment of strains that produce and secrete compatible solutes such as ectoine, L -PA or α- D -glucosylglycerol that are not synthesized by the wild-type strains (Ning et al, 2016; Pérez-García et al, 2017a,b; Ying et al, 2017; Roenneke et al, 2018). Production of L -PA by recombinant E. coli expressing the gene for lysine cyclodeaminase from Streptomyces hygroscopicus was established with a titer of 5.33 g L -1 L -PA and a yield of 0.13 g L -1 of glucose obtained in fed-batch cultivation and a titer of 0.64 g L -1 L -PA in shake flasks (Ying et al, 2017).…”

“…This included the establishment of strains that produce and secrete compatible solutes such as ectoine, L -PA or α- D -glucosylglycerol that are not synthesized by the wild-type strains (Ning et al, 2016; Pérez-García et al, 2017a,b; Ying et al, 2017; Roenneke et al, 2018). Production of L -PA by recombinant E. coli expressing the gene for lysine cyclodeaminase from Streptomyces hygroscopicus was established with a titer of 5.33 g L -1 L -PA and a yield of 0.13 g L -1 of glucose obtained in fed-batch cultivation and a titer of 0.64 g L -1 L -PA in shake flasks (Ying et al, 2017). Our previous work on establishing L -PA production in C. glutamicum led to superior values: 14.4 g L -1 L -PA and a yield of 0.20 g g -1 in fed-batch cultivation and a titer of 3.9 g L -1 L -PA in shake flasks (Pérez-García et al, 2017a).…”

Methanol fermentation increases the production of NAD(P)H-dependent chemicals in synthetic methylotrophic Escherichia coli

“…Methanol concentrations were measured using an Agilent 1290 Infinity System (Santa Clara, CA, USA) equipped with an Aminex HPX-87H column. l -Lysine was determined using SBA-40E immobilized enzyme biosensor (Shandong, China) [21]. NADH and NADPH were assayed by means of the NAD/NADH quantitation Kit and the NADP/NADPH quantitation Kit (Sigma-Aldrich).…”

“…With the recombinant E. coli BL21/ΔfrmA-NudF-Mdh2-Hps-Phi, no lysine was detected in the medium. To improve lysine production, the key genes involved in lysine biosynthesis, including dapA , dapB , PPC, and lysC fbr (a lysine-insensitive aspartokinase) [20, 21], were co-overexpressed in the recombinant E. coli BL21/ΔfrmA-NudF-Mdh2-Hps-Phi to generate E. coli BL21/ΔfrmA-ML. Thereafter, 0.06 g/L lysine was produced after fermenting for 24 h, with glucose as the sole carbon source (Fig.…”

Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment

“…In fact, the 5AVA production is closely related to the biological metabolism of L-lysine 7 . 5AVA is mainly synthetized through the coupled system of L-lys 2-monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) 8 .…”