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 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 a s 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~cumen ted .~ , ’~~ , ’~ 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 l i t e r a t ~ r e ~ - ~ , ’ ~ ? ~ ~ 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

secondary metabolic activity and intracellular protein and ATP levels.

MATERIALS AND METHODS

Cell Line

Lithosperrnurn erythrorhizon cell line transformed with Agrobacteriurn tumefaciens Ti plasmid was obtained from Dr. Chang Ryul Liu at the Genetic Research Institute, Taeduck, Korea.

Cell Cultivation

Cell suspensions were cultivated as described in our earlier paper.16 Thirteen-day-old cells were transferred (20% in- oculum v/v) into production medium (M-9l' with 5 wt % sucrose and without hormones). The culture volume was approximately 150 mL after inoculation. The medium was adjusted to pH 5.9 and overlaid with liquid paraffin 1:6 (v/v) before autoclaving. Liquid paraffin was used to fa- cilitate in situ extraction and measurement of product.' Cultivation was carried out in 500-mL shake flasks with foam-plug closures at 25°C in the dark at 125 rpm on a rotary shaker. In this article, control conditions refers to cul- tivation in hormone-free M-9 medium as described above.

Sudrose-rich (C-rich) and nitrate-rich (N-rich) media were prepared as follows. To prepare C-rich medium, hormone-free M-9 medium'' (20% concentration without sucrose or nitrate) was prepared as the basal medium and the concentrations of sucrose and nitrate were adjusted to give a final concentration of 30-40 g/L sucrose and 15-25 mg/L nitrogen (as nitrate) after addition to the culture. The volume of addition was adjusted to compen- sate for the cumulative sample volume withdrawn from the flask. Approximately 5 -6-mL samples were taken. Hormone-free 2.5-fold concentrated M-9 medium" was designated as N-rich. Upon addition of N-rich medium, the resultant concentration of nitrogen was typically between SO and 65 mg/L and sucrose was between 20 and 30 g/L. It must be mentioned that the C-rich and N-rich definitions are based upon the nitrogen content of the medium.

Measurement of Cell Growth, Product, and Nutrient Levels

Cell growth, sucrose, and nitrate in the medium were measured by previously published methods.16 Shikonin concentration was measured by the method of Heide et a1.8

Cellular Protein Content

The cell mass of known fresh weight was homogenized in 5 mL Tris-HC1 (0.2 M , pH 7.1) using a tissue dismember- ator (Fisher Scientific). The homogenate was centrifuged at 500g for 10 min and the protein content of the supernatant was measured by the method of Bradford' using bovine serum albumin as the standard.

Cellular Adenosine Triphosphate Content

Cells of known fresh weight were extracted in 3 mL of ice-cold 90% dimethylsulfoxide (DMSO) by vortexing for 30 s. Extracts were stored at -70°C until further analysis. Parallel controls revealed that the loss of adenosine triphos- phate (ATP) in cell extracts was in the range of 1-7% during this period. This difference has been ignored in the measurements. Prior to measurement, extracts were thawed at 4°C and centrifuged at 500g for 10 min. The supernatant was diluted 100-fold in N-(2-hydroxyethyl)piperazine-N'- 2-ethanesulfonic acid (HEPES) buffer (0.5 mM, pH 7.8) and 0.1-mL aliquots were used for ATP determination by luminometry. A Turner TD-20e luminometer (Turner De- signs, Mountain View, CA) was used to monitor the emitted luminescence of the ATP coupled luciferin-luciferase enzy- matic reaction. Lumit-QM luciferin-luciferase and diluent were obtained from Lumac BV (Lumac BV, Landgraaf, Netherlands). The ATP was quantified by comparing the relative luminescence units (RLUs) measured from cell extracts with those produced by known ATP standards (prepared in HEPES buffer) measured by the same assay.

Soluble Phenolics

Total soluble phenolics in the cell-free culture medium were measured spectrophotometrically by the method of Swain and Hillis as reported by Seitz et al.15 Caffeic acid was used as the standard.

RESULTS AND DISCUSSION

Figure 1 illustrates the growth and production of shikonin derivatives under control conditions (hormone-free M-9 medium, 5 wt % sucrose). Typically nitrate was consumed by days 6-8 and sucrose by days 12-15. A two- to threefold increase in fresh weight was typically observed. Production of shikonin derivatives started between days 6 and 8 and the maximum concentration varied between 140 and 200 mg/L in various experiments. The data shown here represent the mean of duplicate experiments.

Figures 2A-D illustrate the effects of adding C-rich and N-rich media. Soluble phenolics were measured as an indication of general secondary metabolic activity. All experiments were conducted in duplicate. Days 1-13 in Figure 2A represent cultivation under conditions of the con- trol experiment. A maximum cell yield of about 140 g/L was obtained. Shikonin production was observed to start on day 5 and a concentration of -125 mg/L was measured on day 11. The soluble phenolic content of the medium increased from -20 mg/L on day 5 to -160 mg/L on day 12. Shikonin and phenolic concentrations did not increase further until day 13. On day 13, C-rich medium was added to the culture aseptically. Sucrose and nitrate concentrations following the addition are shown in the figure. Sharp increases in soluble phenolics and shikonin derivative yields were observed after a lag of 24-36 h. Shikonin concentration increased almost twofold over a

794 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 7, SEPTEMBER 20, 1993

80

150

100

50

0

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0

Time (days)

Figure 1. Cultivation under control conditions in regular production medium (M-9 medium with 5 wt % sucrose and without phytohormones): (m) biomass, (0) nitrate, (0) sucrose, and (0) shikonin. Data represent means of duplicate experiments.

period of 8 days following the addition of fresh C- rich medium. A maximum concentration of -220 mg/L was measured. Correspondingly, the concentration of phenolics increased almost 3.5-fold over a 6-day period following the addition of C-rich medium. A maximum of -550 mg/L was measured. Following dilution of the culture with fresh C-rich medium, the cell concentration dropped from 140 g/L to -1OO/L, and subsequently a modest increase in cell fresh weight (-15%) over a period of 8 days could be observed. The N-rich medium was added to the culture on day 24 (Fig. 2A) to further test the validity of earlier observations regarding the inhibitory effect of high nitrate levels. Almost all the added nitrate and -83% of the added sucrose was consumed over a period of 9 days following the addition of N-rich medium (Fig. 2B). There was a noticeable increase in cell fresh weight (-25%) over the fresh weight on day 24. Conspicuously, there was almost no further increase in the concentration of soluble phenolics. A meager 3-4% increase was recorded. Likewise, the shikonin concentration showed only a very slight increase (-9%) over the level on day 24. These results further confirmed previous observations regarding the influence of excess nitrogen on shikonin production. They also reveal that shikonin production and general secondary metabolic activity are strongly stimulated by the addition of C-rich medium. Also, N-rich medium supported growth of the culture while preventing further product accumulation.

Due to continuously changing conditions during batch culture, there is continuous variation in cellular physiology. It may be argued, therefore, that the diverse effects of C-rich and N-rich media on cell growth and shikonin production, when administered in alternating succession, are really effects caused by altered cellular physiology during this period rather than direct influences of the added media. To

study this issue, the reverse sequence of media additions was tried (Figs. 2C, D). Following cultivation under control conditions for about 13 days, N-rich medium was added to the culture on day 13 (Fig. 2C). While cell growth was strongly stimulated (-43% increase), the yield of shikonin derivatives remained the same (-140 mg/L) during the 8-9-day period following the addition of N-rich medium. Soluble phenolics were not measured in this experiment. On day 23, C-rich medium was added to the culture. After a lag of 24-48 h, fresh accumulation of shikonin was noticed. This increase continued for a period of 10 days. A maximum concentration of 200-220 mg/L was measured, reflecting 35% increase over levels on day 23.

Given the evidence of the stimulatory effect of C-rich medium on shikonin productivity, a natural question arises. Will successive additions of C-rich medium result in further product accumulation or is there a limit to the stimulatory effect? Might there be some limitation imposed by the cell's biosynthetic apparatus at some stage?

To address these questions, C-rich medium was added to cultures after 12 days of cultivation under control condi- tions (Fig. 2E). The anticipated increase in shikonin yield was observed. Shikonin levels increased -2.2-fold from 150 to 325 mg/L over 9-10 days following addition of C-rich medium. Cell growth increased by approximately 25% in this experiment. However, further addition of C-rich medium on day 24 resulted in very little con- sumption of the added sucrose (Fig. 2F) and showed only 12% increase in shikonin yield. A 20% increase in cell fresh weight was observed. A medium preparation without sucrose (C-rich salts) was also added on day 24 to some flasks in a parallel experiment (data not shown). While cell concentration increased by about 11%, product levels remained unchanged from levels on day 24. These results suggest the existence of some intrinsic limitation to the

SRlNlVASAN AND RYU: ALTERNATING CARBON AND NITROGEN FEEDING 795

' 4 2 0

2 A C-Rlch N-Alch

1 1 1 360 240 - 160' +

0 - - '0 120 ' 180

5 g 8 0 120 'D

- - 0 40 60 0

0 0 0 5 1 0 15 2 0 2 5 30 3 5

Time (days)

I Z B

C-Rch

1

Time (days) 0 5 10 15 20 25 30 3 5 4 0

Time (days)

t 2c

Time (days) Time (days)

Figure 2. Additions of C-rich or N-rich media at various stages during cultivation. Additions are indicated by arrows and appropriate labels. Typically, days 1-13 represent cultivation under control conditions and subsequent data are for cultivation in specific environments as indicated: (0) nitrate, (0) sucrose, (U) biomass fresh weight, and (0) shikonin, and (A) soluble phenolics. Data represent means of duplicate experiments.

796 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 7 , SEPTEMBER 20, 1993

100 . , . , . , . , . , . , . , .

fi 2 F

0 5 10 15 20 2 5 30 35 4 0

Time (days)

Figure 2. (continued)

continued stimulation of shikonin production by C-rich medium. It may be noted (Fig. 2C) that it was possible to stimulate production by C-rich medium by first cultivating in N-rich medium. These observations indicate that there may in fact be a biosynthetic potential for secondary metabolite synthesis that is exhausted by repeated cultiva- tion in a C-rich environment but which can be successfully replenished by brief periods of cultivation in N-rich or growth-supporting conditions. This biosynthetic potential could potentially involve energy reserves, levels of key biosynthetic enzymes, and other metabolic precursors. As a result, a control strategy, based on cycling the culture between C-rich and N-rich conditions, may make it possible to obtain higher culture productivity for extended periods of time.

It is well established that the start of secondary metabolic activity in many microorganisms corresponds closely to the depletion of cellular ATP.'o," While the cessation of cellular protein synthesis is usually a good indication of the onset of secondary metabolism, it fails to be so in those cases where limited cell growth is observed. Besides, the early biosynthetic pathway of shikonin derivatives consists of two branches. These are the phenylpropanoid branch leading to the synthesis of p-hydroxybenzoic acid from L-phenylalanine and the terpenoid pathway leading to the synthesis of the high-energy intermediate geranyl pyrophos- phate (GPP) from mevalonic acid (MVA) via isopentynyl pyrophosphate and dimethylallyl pyrophosphate. Many of the enzymes in the latter pathway such as GPP synthase and MVA pyrophosphate decarboxylase are ATP-requiring enzymes suggesting that the energy status of the cell may influence the production of shikonin derivatives. In an attempt to gain some understanding of the intracellular parameters that the proposed biosynthetic potential might correlate to, we measured the cellular protein and ATP concentrations under nonproducing and producing condi- tions. The results are shown in Figures 3A and B. Cells

were cultivated in either the growth medium (hormone-free Schenck and Hildebrandt medium with 5 wt % sucrose) or the normal production medium. No shikonin is produced in the growth stage cultures. A classic pattern of variation in cellular ATP content was observed. The ATP content increased from low levels during the lag phase and attained a maximum value in the mid- to late-exponential growth phase (Fig. 3A). Following this, a steady decrease was observed in the stationary phase and lag phase levels were reached again. Cellular protein, however, varied in different ways in the growth and production stages. In the growth medium, three distinct regions were observed (Fig. 3A). Protein content per fresh weight of cell mass increased during the lag and early exponential growth phases. It dropped to -33% of peak value and remained constant at this value during the midexponential phase, probably reflecting a brief period of balanced growth, and dropped an additional -60% during the late-exponential phase and remained essentially constant through the stationary phase. In the production medium, however, cellular protein content appeared to follow the growth curve (Fig. 3B). A pattern similar to the control culture (production stage, days 1 - 12, Fig. 3B) was also observed in the two stages of cultiva- tion following additions of C-rich medium (days 13-35, Fig. 3B). Cellular protein content appeared to be a function of culture dilution, and increases in protein content occurred over the same durations as increases in biomass. Rapid increases were also observed in cellular ATP content. We had hoped to see some characteristic changes in the patterns of cellular protein and ATP levels that might correlate to secondary metabolic activity, but this was not observed. The pattern of variation in ATP content was qualitatively similar in C-rich or N-rich medium. Clearly, a simple criterion such as cellular ATP or protein content is not a sufficient indicator of the proposed biosynthetic potential. Keeping in mind the complex regulatory mechanisms and the myriad of environmental stimuli (light, nutrient, and physical stresses, temperature, and infection by pathogens) that are known to affect secondary metabolite synthesis in culture plant cells, these processes are most likely mediated by complex intracellular signals in addition to those considered here. The detailed intracellular responses of our cell line to nutrient cycling need to be studied further.

The results described above therefore suggest that the cellular response, namely, cell growth in N-rich medium and enhanced production in C-rich medium, is not sig- nificantly affected by the alternating C-rich and N-rich environments during the period of time in which these ex- periments were conducted. The yield coefficients of cell and product were estimated to understand the nutrient re- quirement under different cultivation conditions (Table I). The product yield, Y p l x , is approximately 2-fold better in C-rich condition but is only one 16th in N-rich condition. This is probably so since a substantial portion of the prod- uct was produced in the late exponential and the stationary phases, a period when cell growth was not significant, a

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3 A

. 3 B

0 5 10 15 20 25

Tlme (days)

J

- F

E" 5 . -

640

9.9 1 120

t

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/ C-Rich ' t C-Rich / I

0 10 20 3 0 4 0 "

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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

Table I. Nutrient partitioning in different culture conditions.

Yield coefficient Control N rich C rich

YXIN 0.671 0.12 0.374 f 0.06 0.53 z 0.26 YXlC 0.16 * 0.05 0.06 * 0.006 0.017 * 0.003 YPlX 0.067 2 0.02 0.004 f 0.001 0.12 -c 0.02

Individual yield coefficients represent the averages from five sets of data for control conditions and four sets of N-rich or C-rich data. Y X I N = g dry weight cells/mmol NO3-. YX/C = g dry weight cells/mmol sucrose. Y p l x = mg shikonin derivatives/g dry weight cells.

exclusiveness of growth and shikonin production. Thus, the stimulation of shikonin production by C-rich medium cannot be explained entirely by diversion of precursors for growth into the shikonin biosynthetic pathways as suggested by Mizukami et al.13. The lack of repeated stimu- lation by C-rich medium indicated a saturation of metabolic activity. We also observed an 8-11% drop in cell viability between days 24 and 36 from the value on day 24 (71% measured by phenosafranin dye exclusion). It is also pos- sible that successive additions of C-rich medium resulted in altered cellular physiology. Interim cultivation in N-rich medium alleviated this problem.

Although the literature is rich with instances of hor- monal regulation and regulation by specific precursors of secondary metabolites, the effect of carbon and nitrogen source is still significant. As the relatively small changes in cell and product yields following switching between media demonstrates, it is possible to significantly improve overall shikonin production by cycling the environment of L. erythrorhizon cell cultures between N-rich and C-rich states. It is expected that by cultivation under semicontin- uous or continuous conditions, better control over product and cell yields can be achieved. Such studies are being actively considered.

We thank Dr. Liu Chang Ryul at the Genetic Research Institute in Daeduck, Korea for providing the Lithospermum erythrorhizon cell line and the National Science Foundation for its partial support of this research.

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