Production of L-alanyl-L-glutamine by immobilized Escherichia coli expressing amino acid ester acyltransferase

Pei, Xuze; Li, Yimin; Du, Cong; Yuan, Tangguo; Fan, Chengming; Hong, Hao; Yuan, Wenjie

doi:10.1007/s00253-020-10752-3

Cited by 5 publications

(5 citation statements)

References 33 publications

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“…With optimized media composition, a high-density culture in fermenter and immobilization of BPA has been already realized in our lab (Pei et al 2020 ). A large-scale production has been attempted in a 500 L reactor, and the cost of antibiotics and IPTG is acceptable.…”

Section: Discussionmentioning

confidence: 99%

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase

Jing

Liu

et al. 2023

Appl Microbiol Biotechnol

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L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA , pepB , pepD , pepN , dpp , and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)- ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, B pADN PA (BPA- ΔpepADN ) was built, and the production of Ala-Gln was 129% of the BPA’s accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. Key points • Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis • The balanced gene knockout combination is pepA, pepD, and pepN • The accumulation of Ala-Gln with B pADN PA was 129% of the control Graphical Abstract

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Section: Discussionmentioning

confidence: 99%

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase

Jing

Liu

et al. 2023

Appl Microbiol Biotechnol

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“…The solubility of glutamine monomer in water is only 36 g/L, whereas the solubility of dipeptide (Ala‐Gln) reaches an impressive 568 g/L (Jing et al., 2023). This significant difference effectively demonstrates the advantage of glutamine‐derived peptides over monomers in terms of physical properties.…”

Section: Functional Superiorities Of Glutamine‐derived Peptides Over ...mentioning

confidence: 99%

“…As shown in Figure 2b, in the cell factory production of Ala‐Gln, the carbon flux to the target product can be effectively increased by knocking out the peptidase genes ( Pep A, Pep B, Pep D, and Pep N) responsible for dipeptide degradation and the enzyme genes ( Gls A and Gls B). Moreover, inactivation of the dipeptide transport system ( Dpp ) helps to reduce the intracellular Ala‐Gln content and further increase its production (Jing et al., 2023; Zhu et al., 2020). However, the process of the biological manufacturing of peptides is extremely complex, involving multiple systems and multilevel regulation.…”

Section: Synthesis or Preparation Of Glutamine‐derived Peptidesmentioning

confidence: 99%

Glutamine‐derived peptides: Current progress and future directions

Yu,

Hu,

et al. 2024

Comp Rev Food Sci Food Safe

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Glutamine, the most abundant amino acid in the body, plays a critical role in preserving immune function, nitrogen balance, intestinal integrity, and resistance to infection. However, its limited solubility and instability present challenges for its use a functional nutrient. Consequently, there is a preference for utilizing glutamine‐derived peptides as an alternative to achieve enhanced functionality. This article aims to review the applications of glutamine monomers in clinical, sports, and enteral nutrition. It compares the functional effectiveness of monomers and glutamine‐derived peptides and provides a comprehensive assessment of glutamine‐derived peptides in terms of their classification, preparation, mechanism of absorption, and biological activity. Furthermore, this study explores the potential integration of artificial intelligence (AI)‐based peptidomics and synthetic biology in the de novo design and large‐scale production of these peptides. The findings reveal that glutamine‐derived peptides possess significant structure‐related bioactivities, with the smaller molecular weight fraction serving as the primary active ingredient. These peptides possess the ability to promote intestinal homeostasis, exert hypotensive and hypoglycemic effects, and display antioxidant properties. However, our understanding of the structure–function relationships of glutamine‐derived peptides remains largely exploratory at current stage. The combination of AI based peptidomics and synthetic biology presents an opportunity to explore the untapped resources of glutamine‐derived peptides as functional food ingredients. Additionally, the utilization and bioavailability of these peptides can be enhanced through the use of delivery systems in vivo. This review serves as a valuable reference for future investigations of and developments in the discovery, functional validation, and biomanufacturing of glutamine‐derived peptides in food science.

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“…OPA should exhibit an excellent ability to produce Ala-Gln, 17,18 even in large-scale fermentations, but the potential biosafety hazards caused by bacterial endotoxins and special reagents (e.g., isopropyl-β-D-thiogalactopyranoside) severely hinder the widespread application of E. coli-derived products. Therefore, Komagataella phaffii, which was granted a qualified presumption of safety (QPS) status by European Food Safety Authority (EFSA), 19 and Saccharomyces cerevisiae, which was determined to be generally recognized as safe (GRAS) by the Food and Drug Administration (FDA), 20 were used as safer chassis cells to design new biocatalysts.…”

Section: ■ Introductionmentioning

confidence: 99%

“…OPA should exhibit an excellent ability to produce Ala-Gln, , even in large-scale fermentations, but the potential biosafety hazards caused by bacterial endotoxins and special reagents (e.g., isopropyl-β- d -thiogalactopyranoside) severely hinder the widespread application of E. coli-derived products.…”

Section: Introductionmentioning

confidence: 99%

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

Jing

et al. 2023

J. Agric. Food Chem.

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L-Alanyl-L-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the Nglycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g•(L•min) −1 , respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.

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Production of L-alanyl-L-glutamine by immobilized Escherichia coli expressing amino acid ester acyltransferase

Cited by 5 publications

References 33 publications

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase

Glutamine‐derived peptides: Current progress and future directions

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

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