3A 9.9 7120 8.25 100 歪 6.6 80 4.95 60 3.3 40 1.65 20 10 15 20 TIme (days) ,3B Product yield C-Rich 640 480 320 180 30 40 Time (days) Figure 3.Variation in cellular ATP and protein content in (A)growth and (B)production media.In(B),Days 1-13 represent cultivation under control production conditions and subsequent cultivation is under C-rich conditions.Biomass concentration(dashed line)and product yield (dotted line)are indicated schematically.Numerical values may be found in Fig.2E.Additions of C-rich media are indicated by arrows.(Biomass in (A),both (ATP and (A)protein content in (A)and (B).All data are mean values from duplicate experiments. situation similar to cultivation in C-rich medium.However, metabolite production can occur simultaneously in this cell further studies revealed that cell growth and production line. could occur simultaneously.It can be seen (Fig.3B)that We have shown that addition of sucrose-rich medium shikonin production during cultivation under control con- to stationary phase cultures of L.erythrorhizon strongly ditions (days 1-13)started before protein synthesis had stimulates general secondary metabolic activity indicated by ceased.The same was also true for cultivation under C. the rapid accumulation of soluble phenolics and specifically rich conditions.In fact,in C-rich conditions,substantial the accumulation of shikonin derivatives.A relatively small accumulation of product was observed while cellular pro- increase in biomass was also observed.On the other hand, tein levels continued to rise.Thus,while protein synthesis N-rich medium stimulates biomass growth and represses the follows cell growth fairly closely and while elevated ni- accumulation of shikonin derivatives.Although most of the trogen levels were found to be inhibitory to shikonin product accumulated in the late-exponential and stationary production,it is evident that cell growth and secondary phases in control cultures,we did not observe a general 798 BIOTECHNOLOGY AND BIOENGINEERING,VOL.42,NO.7,SEPTEMBER 20,19933A .3B 0 5 10 15 20 25 Tlme (days) J - F E" 5 . - 640 9.9 1 120 t Product yield / C-Rich 't C-Rich / I 0 10 20 30 40 " Time (days) Figure 3. Variation in cellular ATP and protein content in (A) growth and (B) production media. In (B), Days 1-13 represent cultivation under control production conditions and subsequent cultivation is under C-rich conditions. Biomass concentration (dashed line) and product yield (dotted line) are indicated schematically. Numerical values may be found in Fig. 2E. Additions of C-rich media are indicated by arrows. (W) Biomass in (A), both (0) ATP and (A) protein content in (A) and (B). All data are mean values from duplicate experiments. situation similar to cultivation in C-rich medium. However, further studies revealed that cell growth and production could occur simultaneously. It can be seen (Fig. 3B) that shikonin production during cultivation under control con￾ditions (days 1 - 13) started before protein synthesis had ceased. The same was also true for cultivation under C￾rich conditions. In fact, in C-rich conditions, substantial accumulation of product was observed while cellular pro￾tein levels continued to rise. Thus, while protein synthesis follows cell growth fairly closely and while elevated ni￾trogen levels were found to be inhibitory to shikonin production, it is evident that cell growth and secondary metabolite production can occur simultaneously in this cell line. We have shown that addition of sucrose-rich medium to stationary phase cultures of L. erythrorhizon strongly stimulates general secondary metabolic activity indicated by the rapid accumulation of soluble phenolics and specifically the accumulation of shikonin derivatives. A relatively small increase in biomass was also observed. On the other hand, N-rich medium stimulates biomass growth and represses the accumulation of shikonin derivatives. Although most of the product accumulated in the late-exponential and stationary phases in control cultures, we did not observe a general 798 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 7, SEPTEMBER 20, 1993