Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production

Abstract

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bodies (CatIBs) of the constitutive L-lysine decarboxylase from E. coli (EcLDCc-CatIBs) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum. EcLDCc-CatIBs can easily be produced in E. coli followed by a simple purification protocol yielding up to 43% dry CatIBs per dry cell weight. The stability and recyclability of EcLDCc-CatIBs was demonstrated in (repetitive) batch experiments starting from L-lysine concentrations of 0.1 M and 1 M. EcLDC-CatIBs exhibited great stability under reaction conditions with an estimated half-life of about 54 h. High conversions to DAP of 87-100% were obtained in 30-60 ml batch reactions using approx. 180-300 mg EcLDCc-CatIBs, respectively. This resulted in DAP titres of up to 88.4 g l^-1 and space-time yields of up to 660 g_DAP l^-1 d^-1 per gram dry EcLDCc-CatIBs. The new process for DAP production can therefore compete with the currently best fermentative process as described in the literature.

Figure 1

LDC-catalysed decarboxylation of L-lysine to DAP.

Figure 2

Live cell images of E. coli BL21(DE3) cells containing EcLDCc-CatIBs. For details see Supplementary “Live cell imaging.”

Figure 3

Left: Production and purification of EcLDCc-CatIBs produced in E. coli BL21(DE3). Right: SDS-PAGE analysis of the EcLDCc-CatIB preparation (calculated molecular weight: 87.8 kDa, arrow); CCE = crude cell extract, which was centrifuged to separate the supernatant (S1) from the pellet (P1). The pellet P1 was washed once with MilliQ water by resuspension and subsequent centrifugation, resulting in S2 and P2; the protein concentration was measured using the Bradford assay (see Methods). For SDS-PAGE, samples were diluted with water to a protein concentration of 1 mg ml⁻¹ by the following dilution factors: 4 for CCE, 2 for S1 and P1, 4.5 for P2; 1 for S2; M = Marker. For details see Methods.

Figure 4

pH optimum of EcLDCc-CatIBs for the decarboxylation of L-lysine in the presence (0.1 mM) and without additional PLP. For assay conditions see Methods. 100% relative activity refers to 0.34 U mg⁻¹_CatIBs, which corresponds to 0.52 U mg⁻¹_protein.

Figure 5

Conversion curves of the EcLDCc-CatIB-catalysed decarboxylation of different L-lysine concentrations to DAP in CGXII medium. Empty symbols indicate the point in time at which full conversion was reached. 2 mg ml⁻¹ lyophilised EcLDCc-CatIBs. For details see Methods.

Figure 6

Repetitive batches for the production of DAP with EcLDCc-CatIBs with pH-control. Experimental conditions: 3 mg ml⁻¹ lyophilised EcLDCc-CatIBs, 0.1 M L-lysine, 0.1 mM PLP in 60 ml cell-free culture supernatant (CGXII medium, pH 8). Two 4 h batches (batch 1, 2, 4, 5, 7, 8) were followed by 15 h overnight batches (3, 6, 9) on three subsequent days.

Figure 7

Conversion curve for the production of DAP with EcLDCc-CatIBs in a 30 ml batch reactor with pH control. Experimental conditions: 10 mg ml⁻¹ lyophilised EcLDCc-CatIBs, 1 M L-lysine, 0.1 mM PLP, in 30 ml cell-free culture supernatant (CGXII medium, pH 8). For the dosage profile with NaOH and HCl, respectively, to keep the pH constant see Supplementary Fig. S6.