Construction of Anthocyanin Biosynthesis System Using Chalcone as a Substrate in Lactococcus lactis NZ9000

Abstract

ABSTRACTAnthocyanins are high‐value natural compounds, but to date, their production still mainly relies on extraction from plants. A five‐step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin‐rich blueberry: chalcone isomerase (CHI), flavanone 3‐hydroxylase (F3H), dihydroflavonol 4‐reductase (DFR), anthocyanin synthase (ANS), and UDPG‐flavonoid 3‐O‐glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A‐Cm‐repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H‐DFR‐ANS and 3GT under NZProm 2 and 3, respectively. Using LC‐MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O‐hexoside, malvidin 3,5‐diglucoside, and pelargonidin 3‐O‐malonyl‐malonylhexoside, increased in the supernatant of the co‐culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five‐step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.