Selection of constitutive expression signals for high level expression of antifreeze protein analogous in Lactococcus lactis and Lactobacillus paracasei

Abstract

中文摘要 乳酸菌為安全級食用菌株，為食品工業重要微生物之ㄧ。近年來於遺傳工程研究上也有相當進展。在本研究中，建立乳酸菌Lactococcus lactis及Lactobacillus paracasei之持續型表現系統，以表現重組抗凍蛋白類似物，並偵測重組抗凍蛋白類似物之活性。 質體選擇方面，首先進行質體穩定度測定，比較pHY與pNZ之系列質體，於乳酸菌中之穩定性。結果發現這兩種系列質體，均可穩定存在於乳酸菌中。但因為pHY系列質體無法經由鹼裂解法抽取出質體，而pNZ系列質體可以抽出，故推測可能是pHY系列質體於乳酸菌中copy number較低之緣故，基於實驗操作方便性，故選擇pNZ系列質體作後續研究之載體。 持續型啟動子方面，包括：slpA gene promoters ( pSA1,pSA2), nisin resistance gene ( pNSR ), usp45 gene promoter 與 synthesize promoter ( pσA )等五種啟動子，並利用大腸桿菌之β- glucuronidase 基因 ( gusA )做為報導基因，來選擇出於Lactococcus lactis 與Lactobacillus paracasei中最佳之啟動子，以5-bromo-4-chloro-3-indolyl-β-D- glucuronide ( X-glu )之平板培養基及利用para-nitrophenyl-β-D- glucuronide ( PNPG )作基質測其活性加以分析，結果顯示於Lc. lactis中，帶有pSA1 啟動子之質體可表現出最高活性(1891 U OD600-1 )。而在Lb. paraacasei部分，由於轉形入之pNZ系列質體，易發生突變現象，推測可能因此菌株為wild-type菌株，具有較強之修飾能力，若要於此菌株進行遺傳工程操作，必須先進行宿主特性之修飾。 此外，本實驗將選擇出之最強啟動子pSA1，於其下游加入YaB訊息胜肽，以表現不同重複片段之重組抗凍蛋白類似物，結果發現Lc. lactis可成功表現並分泌具抗凍活性之重組抗凍蛋白類似物至菌體外。

Abstract Lactic acid bacteria ( LAB ) are generally regarded as safe ( GRAS ) and important industrial micro-organisms in dairy and food fermentation. In recent years, a lot of efforts have been directed to the molecular-genetic characterization of LAB. In this study, the constitutive expression system of Lactococcus lactis and Lactobacillus paracasei were developed to express recombinant antifreeze protein analog. The antifreeze activity was examined as ice recrystallization inhibition. To choose a proper plasmid, series of pHY plasmids and pNZ plasmids were examined. The segregation stability of all plasmids are stable in two hosts. But the series of pHY plasmids can't be isolated by traditional alkaline lysis method while the series of pNZ plasmids can be easily isolated. So the pNZ plasmid was chose to develop expression vectors. Five constitutive promoters (slpA gene promoters ( pSA1,pSA2), nisin resistance gene promoter ( pNSR ), usp45 gene promoter and artificially synthesized promoter ( pσA )) and reporter Escherichia coli β-glucuronidase gene ( gusA ) were transcriptionally fused to choose the best constitutive promoter in Lactococcus lactis and Lactobacillus paracasei. The β-glucuronidase characterization were assayed as GM17/MRS plate containing 5-bromo-4-chloro-3-indolyl-β-D-glucuronide ( X-glu ) and hydrolysis of para-nitrophenyl-β-D-glucuronide ( PNPG ). The vector pNZSA1-GUS exhibited highest GusA activities ( 1891 U OD600-1) in Lc. lactis. In wild-type Lb. paracasei, mutations occurred from time to time. Probably, the wild-type Lb. paracasei has a strong modification activity which may modify the transformed plasmid. The best constitutive promoter, pSA1 was fused with Subtilisin YaB signal peptide to express the recombinant antifreeze protein ( rAFP ) analog in Lc. lactis. High-level extracellular expression of bioactive rAFP was achieved in Lc. lactis.