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標題: Development of food-grade expression system to express heterologous proteins in Lactic acid bacteria
作者: 劉錦峰
Liu, Chin-Feng
關鍵字: food-grade expression system;食品級表現系統;lactic acid bacteria;nisin;nisI;nisin resistance (nsr);reporter gene;artificial expression signals;乳酸菌;乳酸鏈球菌素;nisin抗性基因;食品級表現載體;報導基因;人工合成表現元件
出版社: 食品科學系
The antimicrobial peptide nisin belongs to the family of lantibiotics and is produced by several strains of Lactococcus lactis. Lactic acid bacteria (LAB) have been used for centuries in the preparation and processing of foods, beverages and animal feed due to its food-grade status. In this study, We aimed to develop a food-grade expression system in lactic acid bacteriaby taking advantage of the nisin immunity (nisI) and nisin resistance (nsr) gene from L. lactis BCRC10791, BCRC14016 and pFM011, respectively instead of using the common antibiotic resistance genes as selection markers.
In previous study, we had chemically synthesized a promoter derived from B. subtilis veg promoter. A hagp Up element consensus -35 (TTGACA) and -10 (TATAAT) hexamers, and a TG motif were designed to construct the expression signals. In order to analysis the function of the expression signals, gusA was used as a reporter gene. Functional analysis of expression signals showed that the fusion of endogenous gusA to an artificial σA-type promoter constitutively expressed in Lactococcus lactis NZ9000; however, lower GusA activities was found for Lactobacillus paracasei BCRC14023. The secretable reporter levanase (sacC) controlled by the artificial σA-type promoter is able to be expressed extracellularly in L. lactis NZ9000 and Lb. paracasei BCRC14023. To develop a new selection system for lactic acid bacteria, the food grade selection markers, nisI and nsr were amplified from L.lactis BCRC10791 as well as BCRC14016 and from pFM011 by PCR. The amplified product were then cloned into pET29a and pHAσAGus and transformed into B.subtilis DB104. Both nisI and nsr gene elevated the nisin resistance level in Bacillus subtilis DB104. From the nisin tolerance tests of L. lactis NZ9000 and Lb. paracasei BCRC14023, 50 IU and 500 IU nisin concentrations was chosen for food grade vecter selection in L. lactis NZ9000 and Lb. paracasei BCRC14023, respectively. Food grade vector with nsr gene enhances the nisin tolerance of L. lactis NZ9000 and Lb. paracasei BCRC14023 efficiently. Food grade vector with nisI gene are not so effective, maybe due to the low expression level.

乳酸鏈球菌素 (nisin)為Lactococcus lactis 所生產,屬於抑菌胜,為FDA所認可之食品添加劑,常應用於醱酵食品之添加,藉以提高產品的保存期限,而乳酸菌屬於食品級之菌株,長期應用於醱酵食品、乳品及動物飼料之製造上。於本研究中以nisin抗性基因nisin immunity (nisI)及nisin resistance (nsr)基因,作為食品級篩選標記,取代遺傳工程中常用之抗藥性篩選模式,以建立乳酸菌食品級表現系統,進一步改善抗生素濫用而造成環境及生物上之危害。
本實驗室先前設計與改造枯草桿菌veg啟動子,並利用重疊延展聚合酶連鎖反應合成此改造之σA-type啟動子,其-10區域由原先TACAAT改為TATAAT,-16區域導入TG motif,由於該-10與-35區域與乳酸菌之共識序列相同,故進行其於乳酸菌中之功能性測試。在表現元件之測試上,以本實驗室合成之σA型啟動子融合胞內gusA報導基因,並於乳酸菌中觀察其表現情形,結果顯示L. lactis NZ9000中GusA之活性隨著培養時間之增加而增加,可進行持續表現,而Lactobacillus paracasei BCRC 14023則是集中於對數生長前期,後期之表現與控制組相差無幾。另一胞外分泌性蛋白質SacC表現可分泌至胞外,但表現量不大。食品級大腸桿菌-乳酸菌穿梭載體之構築方面,在食品級抗性基因(nisI)之選殖上,以nisin生產株L. lactis subsp. lactis BCRC 10791以及BCRC 14016皆可順利增幅該基因,另自載體pFM011上增幅食品級nisin抗性基因(nsr),初步在Bacillus subtilis DB104宿主顯示其具抗nisin效果。乳酸菌宿主對於nisin之耐受度測試結果顯示,L. lactis NZ9000與Lb. paracasei BCRC 14023分別在50 IU及500 IU之nisin濃度下具有抑制效果,故以此濃度作為遺傳工程之篩選濃度,所建構之帶有nsr基因之食品級載體,於L. lactis NZ9000及Lb. paracasei BCRC 14023均可有效的提升菌體對於nisin耐受之程度。帶有nisI之質體,抗性較差,可能因其表現量較低所致。
Appears in Collections:食品暨應用生物科技學系

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