Improvement of Shikonin Productivity in Lithospermum erythrorhizon Cell Culture by Alternating Carbon and Nitrogen Feeding Strategy Venkatesh Srinivasan Department of Chemical Engineering,State University of New York, Buffalo,New York 13260 Dewey D.Y.Ryu* Department of Chemical Engineering,University of California,Davis, California 95616 Revised April 22,1993/Accepted April 27,1993 Stationary phase cell suspension cultures of Agrobac- was concluded in our earlier work16 that a low nitrogen terium tumefaciens transformed Lithospermum ery- level in the culture medium was essential for shikonin throrhizon respond to additions of sucrose-rich(C-rich) production by the cell line.In fact,the addition of excess medium with a 2-3-fold increase in the accumulation of shikonin derivatives and a 3-3.5-fold increase in nitrate to a production stage batch culture upon nitrate the accumulation of soluble phenolics while showing exhaustion reduced the product yield by 70-80%.How- a modest (10-30%)increase in cell concentration. ever,this nitrate effect was found to depend on the level Conversely,the addition of nitrate-rich(N-rich)medium of sucrose (carbon source)present at the time of nitrate resulted in 25-35%increase in biomass concentration but only 2-9%increase in shikonin production and addition,thereby suggesting that the inhibitory effect of ~3%increase in the yield of soluble phenolics.Repeated high nitrogen levels on shikonin yield was in fact mediated additions of C-rich medium resulted in only a modest by availability of the carbon source.A similar conclusion (less than 10%)improvement in shikonin production was also reached by Davies24 regarding the influence of over the levels obtained after the first application. nitrogen on polyphenol production by cell cultures of Paul's No obvious correlation could be discerned between scarlet rose.Our studies have also indicated a general intracellular ATP levels or protein synthesis patterns and the pattern of shikonin accumulation following the trend of increasing volumetric productivity of shikonin addition of C-rich medium,suggesting that the precursor derivatives in batch culture with increasing initial sucrose diversion mechanism is not generally applicable in concentrations in the range of 2-5 wt %Therefore a our cell line.It was found that alternating feeding logical strategy was to test the response of the culture of N-rich and C-rich media could be used as an effective strategy for enhancing the productivity of plant to additions of sucrose.Accordingly,when the sucrose secondary metabolite.1993 John Wiley Sons,Inc. level in the medium had dropped below 5 g/L,a known Key words:L.erythrorhizon.shikonin.carbon and quantity of sterile sucrose solution was added to the culture. nitrogen feeding However,it was found that the addition of sucrose alone (data not shown)resulted in negligible sucrose uptake and no enhancement of shikonin yield.Cell viability was INTRODUCTION also found to decrease.The inclusion of low levels of The infuence of various environmental parameters such nitrate and other salts alleviated this problem,suggesting as light intensity,medium composition,dissolved oxy- that the uptake of sucrose by stationary phase cultures gen,and temperature on the production of shikonin was limited by the depletion of other nutrients,possibly derivatives (naphthoquinone pigments)by cell cultures of phosphorus and nitrogen.Consequently,C-rich and N-rich Lithospermum erythrorhizon is well documented.6.7.9.17 Op- media(see Materials and Methods)were used in the ensuing timized media for growth and production of cell line experiments.Several reports in the literature2-5.12.14 have M-18 have been formulated.18 In an earlier article,16 indicated a stimulatory effect by elevated levels of carbon we reported the influence of the nitrogen source on source on natural product yields. shikonin production and the activities of the biosynthetic In this article we report on the continued stimulation enzymes phenylalanine ammonia lyase and 3-hydroxy- of shikonin production by stationary phase cultures of 3-methylglutaryl CoA reductase in an Agrobacterium L.erythrorhizon by additions of sucrose-rich media and tumefaciens transformed cell line of L.erythrorhizon.It we suggest a control strategy for improving the yield of secondary metabolites in this cell line.We have also To whom all correspondence should be addressed. examined the possibility of a general correlation between Biotechnology and Bioengineering,Vol.42,Pp.793-799(1993) 1993 John Wiley Sons,Inc. CCC0006-3592/93/070793-07Improvement of Shikonin Productivity in Lithospermum erythrorhizon Cell Culture by Alternating Carbon and Nitrogen Feeding Strategy Venkatesh Srinivasan Department of Chemical Engineering, State University of New York, Buffalo, New York 13260 Dewey D.Y. Ryu* Department of Chemical Engineering, University of California, Davis, California 956 16 Revised April 22, 1993/Accepted April 27, 1993 Stationary phase cell suspension cultures of Agrobac￾teriurn turnefaciens transformed Lithospermum ery￾throrhizon respond to additions of sucrose-rich (C-rich) medium with a 2-3-fold increase in the accumulation of shikonin derivatives and a 3-3.5-fold increase in the accumulation of soluble phenolics while showing a modest (10-30%) increase in cell concentration. Conversely, the addition of nitrate-rich (N-rich) medium resulted in 25-35% increase in biomass concentration but only 2-9% increase in shikonin production and - 3% increase in the yield of soluble phenolics. Repeated additions of C-rich medium resulted in only a modest (less than 10%) improvement in shikonin production over the levels obtained after the first application. No obvious correlation could be discerned between intracellular ATP levels or protein synthesis patterns and the pattern of shikonin accumulation following the addition of C-rich medium, suggesting that the precursor diversion mechanism is not generally applicable in our cell line. It was found that alternating feeding of N-rich and C-rich media could be used as an effective strategy for enhancing the productivity of plant secondary metabolite. 0 1993 John Wiley & Sons, Inc. Key words: L. erythrorhizon shikonin carbon and nitrogen feeding INTRODUCTION The influence of various environmental parameters such as light intensity, medium composition, dissolved oxy￾gen, and temperature on the production of shikonin derivatives (naphthoquinone pigments) by cell cultures of timized media for growth and production of cell line M-18 have been formulated.18 In an earlier article,16 we reported the influence of the nitrogen source on shikonin production and the activities of the biosynthetic enzymes phenylalanine ammonia lyase and 3-hydroxy- 3-methylglutaryl CoA reductase in an Agrobacterium tumefaciens transformed cell line of L. erythrorhizon. It Lithospermum erythrorhizon is well d~cumented.~,’~~,’~ O P- * To whom all correspondence should be addressed. was concluded in our earlier work16 that a low nitrogen level in the culture medium was essential for shikonin production by the cell line. In fact, the addition of excess nitrate to a production stage batch culture upon nitrate exhaustion reduced the product yield by 70430%. How￾ever, this nitrate effect was found to depend on the level of sucrose (carbon source) present at the time of nitrate addition, thereby suggesting that the inhibitory effect of high nitrogen levels on shikonin yield was in fact mediated by availability of the carbon source. A similar conclusion was also reached by Da~ies’,~ regarding the influence of nitrogen on polyphenol production by cell cultures of Paul’s scarlet rose. Our studies have also indicated a general trend of increasing volumetric productivity of shikonin derivatives in batch culture with increasing initial sucrose concentrations in the range of 2-5 wt %. Therefore a logical strategy was to test the response of the culture to additions of sucrose. Accordingly, when the sucrose level in the medium had dropped below 5 g/L, a known quantity of sterile sucrose solution was added to the culture. However, it was found that the addition of sucrose alone (data not shown) resulted in negligible sucrose uptake and no enhancement of shikonin yield. Cell viability was also found to decrease. The inclusion of low levels of nitrate and other salts alleviated this problem, suggesting that the uptake of sucrose by stationary phase cultures was limited by the depletion of other nutrients, possibly phosphorus and nitrogen. Consequently, C-rich and N-rich media (see Materials and Methods) were used in the ensuing experiments. Several reports in the literat~re~-~,’~?~~ have indicated a stimulatory effect by elevated levels of carbon source on natural product yields. In this article we report on the continued stimulation of shikonin production by stationary phase cultures of L. erythrorhizon by additions of sucrose-rich media and we suggest a control strategy for improving the yield of secondary metabolites in this cell line. We have also examined the possibility of a general correlation between Biotechnology and Bioengineering, Vol. 42, Pp. 793-799 (1993) 0 1993 John Wiley & Sons, Inc. CCC QQ06-3592/93/070793-Q7