Genetic engineering in Yarrowia lipolytica for overproducing gamma-glutamylcysteine
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Abstract
This study aimed to engineer the yeast Yarrowia lipolytica for the enhanced accumulation of the dipeptide γ-glutamylcysteine (γ-GC). The research methodology involved the disruption of the ylGSH2 gene, which encodes glutathione synthetase, to block the second step of the glutathione (GSH) synthesis pathway. Subsequently, the ylGSH1 gene was overexpressed in either single or double copies under the control of the constitutive pTEF promoter. The successful synthesis of γ-GC instead of GSH in the resulting mutant strains (RIY455 & RIY445) was confirmed through advanced analytical techniques, including NMR spectroscopy & LC-QTOF/MS analysis. Results indicated that the engineered strains achieved intracellular thiol content of approximately 102 nmol/mg protein, while the presence of γ-GC was confirmed by NMR and LC-QTOF/MS analyses. Furthermore, supplementation with the precursors cysteine & glutamate significantly improved yields, reaching a maximum of 239.9 nmol/mg protein in strain RIY445, representing a 177% increase. The findings conclude that inactivation of the ylGSH2 gene, which encodes glutathione synthetase, together with overexpression of the ylGSH1 gene (one or two copies), resulted in increased accumulation of intracellular γ-glutamylcysteine (γ-GC). This research provides a promising biotechnological framework for the efficient production of γ-GC, a compound of significant medical & nutritional interest.
Tóm tắt
Nghiên cứu này được thực hiện nhằm thiết kế chủng nấm men Yarrowia lipolytica có khả năng tích lũy cao dipeptide gamma-glutamylcysteine (γ-GC). Phương pháp nghiên cứu bao gồm việc xóa bỏ gene ylGSH2 mã hóa enzyme glutathione synthetase để chặn con đường tổng hợp glutathione, kết hợp với việc biểu hiện quá mức gene ylGSH1 (một hoặc hai bản sao) dưới sự kiểm soát của promoter mạnh pTEF. Các phương pháp phân tích hiện đại như NMR và LC-QTOF/MS đã được sử dụng để xác nhận sự hiện diện của γ-GC thay cho glutathione. Kết quả cho thấy các chủng đột biến (RIY455 và RIY445) ghi nhận hàm lượng thiol nội bào khoảng 102 nmol/mg protein; sự hiện diện của γ-GC được xác nhận bằng NMR và LC-QTOF/MS. Việc bổ sung cysteine và glutamate vào môi trường giúp nâng hiệu suất lên tối đa 239,9 nmol/mg protein (tăng 177%). Bên cạnh đó, kết quả nghiên cứu còn cho thấy đối với Y. lipolytica, việc bất hoạt gene ylGSH2 mã hóa enzyme glutathione synthetase và biểu hiện quá mức gene ylGSH1 (một hoặc hai bản sao) làm tăng tích lũy γ-GC nội bào. Ý nghĩa của nghiên cứu giúp mở ra hướng sản xuất γ-GC bằng công nghệ sinh học hiệu quả, phục vụ cho các ứng dụng y dược và thực phẩm chức năng.
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