摘要
乳酰-N-新四糖(lacto-N-neotetraose,LNnT)是人乳寡糖(human milk oligosaccharides,HMOs)中的重要成分之一,已被美国和欧盟的食品药品监督管理局批准作为新资源食品配料添加到食品中。为实现乳酰-N-新四糖底盘菌株的构建和产物的高效合成,构建了含有关键酶基因lgtA、NmlgtB、glmS和galE过表达的重组菌株。将重组菌在(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导下摇瓶发酵培养,利用不同拷贝数的质粒对模块化LNnT合成通路进行组合筛选,结果表明,最佳质粒组合为pET-glmS-galE和pRSF-lgtA-NmlgtB,菌株SA1生产LNnT的产量达到1.168 g/L。进一步地,通过改变培养基种类、诱导温度、异丙基-β-D-硫代半乳糖苷浓度及诱导时刻来优化发酵条件,结果表明,重组菌株的最佳发酵培养基为DM培养基,诱导温度25℃,IPTG浓度0.2 mmol/L,诱导时间8 h,优化后工程菌SA1摇瓶发酵LNnT的产量为2.50 g/L,比优化前提高了114%。通过3-L发酵罐发酵,LNnT的产量达到了14.25 g/L,是目前报道的最高产量。该研究为LNnT工业化生产提供了方法学思路,为微生物发酵制备人乳寡糖提供了重要的参考价值。
Lacto-N-neotetraose(LNnT)is one of the important components of human milk oligosaccharides(HMOs),which has been approved by the Food and Drug Administration of the United States and the European Union to be added to food as a new resource food ingredient.To achieve the lacto-N-neotetraose producing strains and efficient synthesis of the products,recombinant strains containing overexpression of the key enzyme genes lgtA,NmlgtB,glmS and galE were constructed in this study.The recombinant bacteria were cultured in IPTG-induced shake flasks for fermentation and combinatorial screening of the modular LNnT synthesis pathway using plasmids of different copy numbers.The results showed that the optimal plasmid combination of pET-glmS-galE and pRSF-lgtA-NmlgtB resulted in a yield of 1.168 g/L of LNnT production by strain SA1.Further,the fermentation conditions were optimized by changing the type of medium,the induction temperature,the IPTG concentration and the moment of induction.The results showed that the optimal fermentation medium of the recombinant strain was DM medium,with an induction temperature of 25℃,IPTG concentration of 0.2 mmol/L,and induction time of 8 h.The yield of LNnT fermentation in shake flasks of the engineered bacterium SA1 was 2.50 g/L,an increase of 114%compared to the pre-optimization period.The yield of LNnT reached 14.25 g/L by fermentation in 3 L fermenters,which is the highest yield reported so far.This study provides a methodological idea for the industrial production of LNnT and provides an important reference value for the preparation of HMOs by microbial fermentation.
作者
骆叶姣
胡苗苗
李梦丽
张涛
LUO Yejiao;HU Miaomiao;LI Mengli;ZHANG Tao(State Key Laboratory of Food Science and Technology(Jiangnan University),Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2023年第21期23-29,共7页
Food and Fermentation Industries
基金
山东省科技攻关计划项目(2020CXGC010601)。
关键词
乳酰-N-新四糖
生物合成
基因敲除
模块化工程
发酵优化
lacto-N-neotetraose
biosynthesis
gene knockout
modular engineering
optimization of fermentation
