Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures

The activities of the biosynthetic enzymes phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) were measured in cells transferred from growth to production medium in a two-stage batch culture. It was found that both these enzymes showed transient increases, PAL (three- to fourfold) and HMGR (two- to four-fold), at or near the point of exhaustion of nitrogen source (NO(3)). Production of shikonin derivatives also started at this time. The addition of excess nitrate to the medium shortly before nitrate exhaustion (days 6-8) markedly reduced the final product yield (by 70-80%) while addition of excess nitrate in the later stationary growth phase (days 14-16) had no significant effect. When the production rate of shikonin derivatives was correlated with PAL activity, it was observed that production rate is very low (less than 1 mg/L . day) at low levels of PAL activity (below 0.1 unit/mg protein). Once a threshold level of PAL activity (about 0.15 unit/mg protein) is reached, the biosynthetic rate of shikonin derivatives increases. Such a relationship could not be deduced for HMGR activity. It was concluded that the production of shikonin derivatives may be limited at the phenylalanine deaminating step at low levels of PAL activity.