Screen Of High Acid-tolerant SRNAs In Lactococcus Lactis And Investigation Of Their Effect On Improving Nisin Yield

Nisin is secreted by certain Lactococcus lactis.In L.lactis,carbohydrates are mainly converted into lactic acid.The accumulation of lactic acid influences strain growth and nisin production.Bacteria small non-coding RNA(sRNA),as an important post transcription regulator,plays a key role in response to many stress conditions.Therefore,the research of sRNA related to acid resistance in L.lactis is very important to increase nisin production.Previous experiments have identified several acid resistance related s RNAs including s004,s144 and c263 in L.lactis F44 and proteomic analysis of L.lactis F44 under acid stress has also been performed.Target genes of s004,s144 and c263 were predicted by proteomic combined with computational methods.Applying eGFP validation,we found that s144 inhibited the expression of yjdC fusion protein,c263 promoted the expression of add and inhibited the expression of groES.To attain the interaction mechanism between sRNAs and their taget genes,the secondary structure analysis was applied and the binding sites were predicted.We overexpressed 5 sRNAs which were earlier identified by RNA-seq and qRT-PCR in L.lactis.Fermentation results showed that the overexpression of s013,s087 and s224 could significantly increase survival rate and nisin titer.Then co-expression of these 3 sRNAs combined with previously screened 5 sRNAs(s004,s015,s144,c263 and s042)was conducted.The resulting 3 engineered strains exhibited an improved acid resistance ability.The survival rate of s004-s015-s144-c263 co-expressing strain under pH 4.0 after 3 h increased 83.15 times compared to that of original strain.Then s004-s015-s144-c263 module and 3 single sRNAs including s004,s144 and c263 were introduced into a high nisin producing strain L.lactis xl1.Fermentation phenotype showed that nisin titer of those sRNA over-expression stains were higher than that of control strain.The highest nisin yield of 6959.70 IU/ml was attained which was 10% higher than xl1.This study aimed to improve acid resistance of L.lactis using sRNA,which filled the gap in the research of L.lactis sRNA.Exploring the targets of sRNA expands our knowledge of molecular mechanism of sRNA function under acid stress condition.This study revealed the complexity and hierarchy of the sRNA regulation and provided various important clues for understanding the survival mechanism of L.lactis under acid stress.