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

ABSTRACT Anthocyanins 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.