Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-a-decarboxylase  

作　　者：Mingyue Fei Xudan Mao Yiyang Chen Yalan Lu Lin Wang Jie Yang Juanping Qiu Dongchang Sun 

机构地区：[1]College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou 310014,China

出　　处：《Acta Biochimica et Biophysica Sinica》2020年第12期1420-1426,共7页生物化学与生物物理学报（英文版）

基　　金：supported by the grants from the Key Research and Development Program of Zhejiang Province(No.2020C02031);the Zhejiang Provincial Natural Science Foundation of China(No.L.Y16C010003);the National Natural Science Foundation of China(No.31670084).

摘　　要：β-Alanine (3-am inopropionic acid)holds great potential in industrial application.It can be obtained through a chemical synthesis route,which is hazardous to the environm ent.It is w ell known that L-aspartate-a-decarboxylase(ADC)can convert L-aspartate to β-alanine in bacteria.However,due to the low activity of ADC,industrial production of β-alanine through the green biological route remains unclear.Thus,im proving the activity of ADC is critical to reduce the cost of p-alanine production.In this study,we established a dual-fluorescence high-throughput system fo r efficient ADC screening.By measuring the am ount of β-alanine and the expression level of ADC using two different fluorescence markers,we can rapidly quantify the relative activity of ADC variants.From a mutagenesis library containing 2000 ADC variants,we obtained am utant w ith 33%increased activity.Further analysis revealed that m utations o f K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis.Compared with the previous singlefluorescence method,our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence,making it able to effectively screen out ADC variants with im proved relative activity.The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy fo r industrial production of β-alanine via the biological conversion route in the future.

关 键 词：L-aspartate-a-decarboxylase Β-ALANINE site-mutagenesis 

分 类 号：Q50[生物学—生物化学]