Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase

Abstract

Background: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln.

Results: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe³⁺ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles.

Conclusions: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Keywords: Codon optimization; Immobilization; L-Alanyl-L-glutamine; Recycle; α-Amino acid ester acyltransferase.

Fig. 1

Codon usage bias adjustment of SsAET gene. Colors of codons indicated the frequency in host P. pastoris. Rarely used codons were shown in cyan and frequently used codons were shown in red. A codon with more red indicated higher frequency in P. pastoris for enhanced expression

Fig. 2

Comparison of catalytic activity between GPA and GPAp to screen for the optimum Ala-Gln producer. GPA and GPAp were denoted in grey and orange, respectively. Samples at two time points were detected by HPLC

Fig. 3

Effects of induction conditions on the catalytic activities of GPAp. a Induction temperatures of 18 °C, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, and 30 °C. b Induction time from 2 to 6 days. Blue line showed the different supplemented methanol concentrations of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5%

Fig. 4

Effects of reaction conditions on the Ala-Gln production. a Reaction temperature of 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, and 30 °C at pH 8.5. b Reaction pH of 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5 at 24 °C, adjusting with 6 mol/L NaOH. c Different amino acid esters, including AlaOMe, AlaOEt, AlaOiPr, AlaOtBu, and AlaOBzl. d Different ratios of AlaOMe/Gln of 1/1, 1/1.5, 1/2, 1.5/1, and 2/1

Fig. 5

The curves of Ala-Gln concentrations with time in different substrate concentrations

Fig. 6

Application of immobilized GPAp by recycling to maintain the stability of Ala-Gln production. a The catalytic activities of GPAp were detected daily by HPLC during the 5 days which GPAp was stored at the optimal reaction pH and temperature (8.5 at 28 °C). b The catalytic activities were detected and compared among free GPAp with one unit of OD₆₀₀, immobilized GPAp with one unit of OD₆₀₀ and immobilized GPAp with double OD₆₀₀. c The reusability of immobilized GPAp was detected at optimum conditions for recycling 10 times