摘要
小白链霉菌(Streptomyces albulus)是ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)的工业生产菌,其在发酵生产过程中遭受低p H胁迫,易造成ε-PL合成能力下降。因此,该文采用一种逐级增加选择压力的低p H适应性进化策略,经过连续93 d的适应性进化,实现S.albulus GS114耐受p H值由4.0降低到3.6。经过大量筛选,获得了低p H适应性进化菌株S.albulus ALE4.0、S.albulus ALE3.8和S.albulus ALE3.6;结合低p H耐受性和摇瓶发酵实验,确定最优适应性进化菌株为S.albulus ALE3.6。进一步考察不同p H值对S.albulus ALE3.6合成ε-PL的影响发现,S.albulus ALE3.6在不同p H值条件下,较出发菌株S.albulus GS114均表现出发酵优势。最后,采用恒定p H 4.0补料分批发酵方式,在5 L发酵罐水平实现S.albulus ALE3.6的ε-PL产量达到43.7 g/L,较出发菌株提高了63%。研究结果一方面表明通过适应性进化方法可以显著提高S.albulus的低p H耐受能力,另一方面也说明通过增强S.albulus低p H耐受性是提高其合成ε-PL能力的有效途径。
Streptomyces albulus is an industrial strain for-poly-L-lysine(-PL)production.-PL is a homopoly(amino acids)produced by S.albulus,which is currently used as an excellent natural food preservative in many countries due to its eminent antibacterial activity.It often suffers low-pH stress during fermentation,which easily cause a decrease in the capacity of-PL biosynthesis.As a result,the adaptive evolution strategy with increasing the selection pressure step by step was introduced to improve the low-pH tolerance of S.albulus GS114 in this study.We first determine the initial transfer pH(pH 4.0)and transfer time(36 h)in adaptive evolution strategy.After 93 days of adaptive evolution,the tolerance pH value of S.albulus GS114 is reduced from 4.0 to 3.6.After a lot of strain screening in each selection pH(i.e.pH 4.0,3.8,3.6)in acid tolerance and-PL yield,we obtained S.albulus ALE4.0,S.albulus ALE3.8 and S.albulus ALE3.6.We chose spore plate coating and-PL shake flask fermentation to evaluate the characteristics of the strains after adaptive evolution.Combined with low pH tolerance and shaker fermentation experiments,it was determined that the optimal adaptive evolutionary strain is S.albulus ALE3.6.Further study investigated the influence of pH on the production of-PL by S.albulus ALE3.6.Results showed that S.albulus ALE3.6 had significant fermentation advantages under different pH values compared with parent strain S.albulus GS114.In terms of-PL production,dry cell weight,-PL yield,and-PL productivity,S.albulus ALE3.6 had different degrees of advantages.The-PL production of S.albulus ALE3.6 in batch fermentation at pH 4.0,3.8,and 3.6 were reached 4.63,6.14,and 7.2 g/L,respectively,which were enhanced by 13.5%,8.9% and decreased by 7.7%,compared with the original strain S.albulus GS114.Meanwhile,the dry cell weight of S.albulus ALE3.6 was achieved by 16.8,11.8,and 14.7 g/L,respectively,which were decreased to varying degrees compared to the original strain.After comparing the fermentation processes of different pH,we chose pH 4.0 for follow-up study from the point of view of the average specific synthesis rate of the product.Finally,the-PL production of S.albulus ALE3.6 in fed-batch fermentation at pH 4.0 were investigated.In a 5 L scale fermentor,-PL production of S.albulus ALE3.6 was reached by 43.7 g/L in 192 h,which is 63% higher than the original strain S.albulus GS114.The dry cell weight was attained by 39.8 g/L,which was 30% lower than the original strain.S.albulus ALE3.6 had higher ability of cell synthesis per unit and average specific synthesis rate of products in this fermentation process than the original strain.The acquisition of the S.albulus ALE3.6 provided an object for the analysis of the mechanism of acid tolerance.Furthermore,this result showed that the low-pH tolerance of S.albulus can be significantly improved through the adaptive evolution method,and enhancing the low-pH tolerance of S.albulus is an effective way to improve its-PL production.
作者
柳天一
张越
王靓
张宏建
张建华
陈旭升
LIU Tianyi;ZHANG Yue;WANG Liang;ZHANG Hongjian;ZHANG Jianhua;CHEN Xusheng(School of Biotechnology,Jiangnan University,Wuxi 214122,China;The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CSCD
北大核心
2024年第1期14-21,共8页
Food and Fermentation Industries
基金
国家自然科学基金面上项目(31671846)
江苏省重点研发计划项目(BE2022703)。
