聚醯胺是一種常見的高分子材料，其可以用於紡織品、食物包裝、眼鏡零件、輪胎及防護裝備等用品的生產，這使得人們對聚醯胺的需求逐年上升。聚醯胺的原料可以透過化學法或生物法製造，化學合成需要在高溫、高壓的環境下與觸媒、金屬鹽類和有機溶劑反應，使得聚醯胺的製造過程必須消耗大量能源造成生產成本提高，而生物合成能夠在相對溫和的條件下進行，是對環境較為友善的製程。
本研究利用基因工程技術，以Escherichia coli作為全細胞生物催化劑生產聚醯胺原料:戊二胺以及5-胺基戊酸。將帶有賴氨酸脫羧酶(cadA)基因的質體轉入到宿主細胞Escherichia coli中表達，並利用pelB信號肽融合賴氨酸/戊二胺逆向轉運蛋白(cadB)，提高賴氨酸進入細胞及戊二胺排出胞外的速率。我們也將賴氨酸脫羧酶(cadA)基因與冰核蛋白(Ice-nucleation protein, INP)融合，使得大腸桿菌能在細胞膜上表達賴氨酸脫羧酶以提高菌株生產戊二胺的產率。為了生產5-胺基戊酸，我們將來自惡臭假單胞菌的賴氨酸2-單加氧酶（DavB）和 5-氨基戊醯胺水解酶 (DavA)基因轉至大腸桿菌中表達。
我們測試帶有pET24a-ptrc-cadA-ptac-pelB-cadB質體的菌株在24小時之內轉化反應溶液中87.5%的賴氨酸；同時帶有pCTCO33-INP-cadA與pET24a-ptrc-cadA質體的菌株在24小時之內轉化反應溶液中92.7%的賴氨酸，未來需要調整INP-cadA蛋白的誘導溫度以提升其轉位效率。
我們成功將賴氨酸2-單加氧酶（DavB）和 5-氨基戊醯胺水解酶 (DavA)基因轉至大腸桿菌中，並且從SDS-PAGE確認兩種基因能夠同時表達。但我們目前仍無法在HPLC上將5-胺基戊酸與賴氨酸分離，未來需要調整分離條件如流動相或更換管柱。

Nylon is a common polymer that can be used in the production of textiles, food packaging, eyewear components, tires, and protective equipment, among other products. This has led to a growing demand for nylon over the years. The raw materials for nylon can be manufactured through either chemical or biological methods. Chemical synthesis involves reactions with catalysts, inorganic salts, and organic solvents in high-temperature and high-pressure environments. This process consumes a significant amount of energy, resulting in increased production costs. On the other hand, bio-synthesis can take place under relatively mild conditions, making it a more environmentally friendly process.
This study utilizes genetic engineering techniques with Escherichia coli as a whole-cell biocatalyst for the production of nylon precursors: 1,5-diaminopentane and 5-aminovaleric acid. Plasmids containing the lysine decarboxylase (cadA) gene were introduced into the host cells of Escherichia coli for expression. The pelB signal peptide was fused with lysine/ 1,5-diaminopentane antiporter (cadB) to enhance the rate of lysine uptake into the cells and 1,5-diaminopentane export out of the cells. Additionally, we fused the lysine decarboxylase (cadA) gene with the ice-nucleation protein (INP) to enable the expression of lysine decarboxylase on the cell membrane of Escherichia coli, thereby increasing the yield of 1,5-diaminopentane production by the bacterial strain. To produce 5-aminovaleric acid, we expressed the lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA) genes from Pseudomonas putida in Escherichia coli.
We tested strains carrying the plasmid pET24a-ptrc-cadA-ptac-pelB-cadB, and within 24 hours, they transformed 87.5% of lysine in the reaction solution. Simultaneously, strains carrying both pCTCO33-INP-cadA and pET24a-ptrc-cadA plasmids transformed 92.9% of lysine in the reaction solution within 24 hours. In the future, it will be necessary to adjust the induction temperature of the INP-cadA protein to enhance its translocation efficiency.
We successfully transferred the genes for lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA) into Escherichia coli, and confirmed through SDS-PAGE that both genes can be expressed simultaneously. However, we are currently unable to separate 5-aminovaleric acid from lysine on HPLC. In the future, adjustments to separation conditions such as mobile phase or column replacement will be necessary.

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