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

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.