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

Abstract

Backgroundl-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.ResultsIn 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.ConclusionsCharacterized 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1077-1) contains supplementary material, which is available to authorized users.

Clean Production of l-Alanyl-l-glutamine by an Efficient Yeast Biocatalyst Expressing α-Amino Acid Ester Acyltransferase without N-Glycosylation

“…In K. phaffii and S. cerevisiae, it was previously observed that expressed SsAet was secreted with lower activity; thus, we inferred that glycosylation occurred and decreased the activity. 21 Considering the tolerance and robustness of K. phaffii with a relatively low glycosylation level, we selected K. phaffii KM71 as the chassis host to investigate the effect of N-linked glycosylation on SsAet, avoiding the interference of other types of glycosylation, such as O-glycosylation and C-glycosylation. The codon-optimized SsAET K gene was expressed with (Aet9), or without (Aet3) an α-factor signal peptide, and then the secreted Aet9 and nonsecreted Aet3 were purified and characterized by SDS-PAGE and western blotting.…”

“…Therefore, Komagataella phaffii, which was granted a qualified presumption of safety (QPS) status by European Food Safety Authority (EFSA), and Saccharomyces cerevisiae, which was determined to be generally recognized as safe (GRAS) by the Food and Drug Administration (FDA), were used as safer chassis cells to design new biocatalysts. Unexpectedly, obviously lower molar yields were obtained, which were approximately 70 and 50% of that of OPA in the recombinant Pichia pastoris strain GS115-pPIC9- SsAET p (GPA p ) and the recombinant S. cerevisiae strain with a surface-displayed SsAet (unpublished data).…”

Magnetic Immobilization of Pichia pastoris Cells for the Production of Recombinant Human Serum Albumin

“…This is the first time that we can achieve a higher production in the immobilized P. pastoris. In previous experiments, cells were immobilized in a polymeric matrix, and a significant reduction in cell performance and production was inevitable [12,13,15,16].…”

“…Hence, there is a vast attempt to develop cell immobilization techniques as an efficient alternative for centrifugation. The P. pastoris system is not exceptional and several attempts were done to efficiently immobilize this cell for the production of valuable products, such as HSA, alcohol oxidase, l-alanyl-l-glutamine, and invertase [11][12][13][14].…”

Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture

“…Recently, cell immobilization techniques such as entrapment in polymers have been extensively used in many applications ranging from wastewater treatment ( 11 , 12 ) to the production of valuable metabolites such as alcohol ( 13 , 14 ), vitamins ( 15 ), antibiotics ( 16 ), and amino acids ( 17 ). The method of cell encapsulation in a matrix can provide an array of beneficial features ( 18 ), the most considerable being that metabolite production and purification processes would become simpler.…”