利用新型基因重組與工程系統於大腸桿菌中生產 L-茶胺酸

Abstract

L-茶胺酸 (L-theanine) 是一種天然存在於茶葉中的非蛋白質胺基酸，是茶葉鮮味的主要來源。由於具有保護神經、緩解壓力、改善睡眠等多種生理功效，廣泛應用於食品、保健品和化妝品等產業。目前生產 L-茶胺酸的方法主要有茶葉萃取、化學合成、酵素催化和微生物發酵。其中，由於微生物發酵則能在大型生物反應器中穩定運行，適合大規模生產，成本更具競爭力。然而，目前利用微生物發酵生產 L-茶胺酸的詳細研究仍相對有限。相應地，L-茶胺酸的生產需要添加前驅物乙胺。雖然乙胺是L-茶胺酸合成的必要基質，但它價格昂貴、毒性大、易燃，對微生物和環境都有害。因此，已有研究開發出無需添加乙胺的微生物生產L-茶胺酸的生物方法。然而，迄今為止，尚未有詳細的實驗在單一微生物中同時測試所有可能影響L-茶胺酸最佳產量的因素。基於這些原因，本研究透過比較不同微生物的酵素活性，結合新的遺傳和代謝工程以及使用新的宿主菌株，在大腸桿菌中重新建立了一個優化的L-茶胺酸生產系統，以在不添加乙胺的情況下，以達到L-茶胺酸的最大產量。透過構建同時攜帶 CsAlaDC、BsAld 和 gmas 基因的雙質體菌株，並系統性優化宿主菌株、基因元件、代謝途徑與培養條件，有效提升 L-茶胺酸的產量。具體策略包括：選用 K-12 系列的 E. coli W3110::pI、使用突變型 CsAlaDC (L110F/P114A) 和來自 Pseudomonas syringae pv. syringae B728a 的 gmas基因，過度表達大腸桿菌內源性 gdha基因、補充多磷酸鹽並引入 Rsppk2基因增強 ATP 再生，以及刪除 L-丙胺酸外排基因 (alaE) 以提高前驅物供應。在最佳化條件 (0.1 mM IPTG 誘導、30°C 培養、30 g/L 葡萄糖) 下，經 24 小時培養後，L-茶胺酸產量由原先的 166.44 ± 3 mg/L 提升至 827.13 ± 25 mg/L，約為原來的 5 倍。本研究顯示，結合基因重組與代謝工程的優化，能有效強化 E. coli 合成 L-茶胺酸的能力，為後續開發高效的微生物發酵生產 L-茶胺酸方法提供了基礎和參考。

L-theanine is a non-proteinogenic amino acid naturally found in tea leaves and is the major contributor to the umami flavor of tea. Due to its multiple physiological benefits, including neuroprotection, stress relief, and sleep improvement, L-theanine is widely used in the food, health supplement, and cosmetics industries. Currently, L-theanine is produced via four main methods: tea leaf extraction, chemical synthesis, enzymatic catalysis, and microbial fermentation. Among these, microbial fermentation is particularly suitable for large-scale production because it can be stably operated in industrial bioreactors and offers a more cost-effective alternative. However, detailed studies on microbial fermentation for L-theanine production remain relatively limited. In most microbial processes, the addition of ethylamine is necessary as a precursor for L-theanine biosynthesis. Although ethylamine is an essential substrate, it is expensive, highly toxic, and flammable, posing hazards to both microorganisms and the environment. As a result, bio-based production strategies that do not require ethylamine supplementation have been developed. Nonetheless, to date, no single study has systematically evaluated all factors that may influence optimal L-theanine production in a single microbial host. In this study, we established an optimized ethylamine-free biosynthetic system for L-theanine production in Escherichia coli by comparing enzymatic activity from different microorganisms and integrating advanced genetic and metabolic engineering strategies, along with the use of a new host strain. A dual-plasmid system co-expressing CsAlaDC, BsAld, and gmas genes was constructed. Through systematic optimization of the host strain, genetic elements, metabolic pathway, and culture conditions, the production of L-theanine was significantly improved. Key strategies included the use of the E. coli K-12 strain W3110::pI, a mutant CsAlaDC (L110F/P114A), the gmas gene from Pseudomonas syringae pv. syringae B728a, overexpression of the endogenous gdhA gene, polyphosphate supplementation with overexpression of the Rsppk2 gene to enhance ATP regeneration, and deletion of the alaE gene (involved in L-alanine export) to improve precursor availability. Under optimized conditions (0.1 mM IPTG induction, 30°C cultivation, 30 g/L glucose), the L-theanine titer increased from 166.44 ± 3 mg/L to 827.13 ± 25 mg/L after 24 hours, representing approximately a 5-fold improvement. This study demonstrates that combining recombinant gene expression with metabolic engineering can effectively enhance L-theanine biosynthesis in E. coli, providing a foundation and reference for future development of efficient microbial fermentation strategies for L-theanine production.