Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures

The activities of the biosynthetic enzmes 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.     

Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1-6) and three shikonofurans (7-9) from Lithospermum erythrorhizon. The nine isolated compounds 1-9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7-9) were greater than shikonin derivatives (1-6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC(50)=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1-6) were of almost equal efficiency with IC(50) value of 40μM and shikonofurans (7-9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.     

Regulation of shikonin production by glutamine in Lithospermum erythrorhizon cell cultures

Amino acid analysis has shown that Lithosperum erythrorhizon cell suspension cultures which are unable to produce shikonin derivatives in LS medium containing ammonium accumulate a large quantity of glutamine, as compared with shikonin-producing cells cultured in the production medium M9 containing nitrate as the sole nitrogen source. The addition of glutamine to M9 medium proved to be strongly inhibitory to shikonin production. Furthermore, culture experiments using an inhibitor of glutaminase suggested that shikonin synthesis is not inhibited by ammonium released from glutamine but by glutamine itself. These findings indicate that the repression of shikonin synthesis occurs in close association with an accumulation of glutamine in cultured cells grown in a medium containing ammonium.     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Induction of shikonin biosynthesis by endogenous polysaccharides in Lithospermum erythrorhizon cell suspension cultures

Endogenous polysaccharides capable of inducing shikonin biosynthesis in a growth medium were isolated from shikonin-producing Lithospermum cells cultured in a production medium. Chemical analysis showed that these active polysaccharides consist of galacturonic acid, galactose, arabinose and glucose. Their activity, however, was lost by a treatment with pectinlyase. Addition of a small amount of pectinase to cell cultures in the growth medium induced shikonin formation. This is the first report that endogenous polysaccharides participate in inducing normal secondary metabolism of higher plants.     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.     

Increased shikonin production by hairy roots of Lithospermum erythrorhizon in two phase bubble column reactor

Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period.     

Exploring the evolutionary process of alkannin/shikonin O-acyltransferases by a reliable Lithospermum erythrorhizon genome

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contig_N50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon’s roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT’s functional differentiation in evolution.     

紫草的制剂学研究进展

本文对国内有关紫草制剂的研究概况进行了综述,为研究和开发紫草制剂提供依据.     

Improvement of shikonin productivity in Lithospermum erythrorhizon cell culture by alternating carbon and nitrogen feeding strategy

Stationary phase cell suspension cultures of Agrobacterium tumefaciens transformed Lithospermum erythrorhizon 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 approximately 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. (c) 1993 John Wiley & Sons, Inc.     

Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound

This work demonstrates the use of low-energy ultrasound (US) to enhance secondary metabolite production in plant cell cultures. Suspension culture of Lithospermum erythrorhizon cells was exposed to low-power US (power density < or = 113.9 mW/cm(3)) for short periods (1-8 min). The US exposure significantly stimulated the shikonin biosynthesis of the cells, and at certain US doses, increased the volumetric shikonin yield by about 60%-70%. Meanwhile, the shikonin excreted from the cells was increased from 20% to 65%-70%, due partially to an increase in the cell membrane permeability by sonication. With combined use of US treatment and in situ product extraction by an organic solvent, or the two-phase culture, the volumetric shikonin yield was increased more than two- to threefold. Increasing in the number of US exposures during the culture process usually resulted in negative effects on shikonin yield but slight stimulation of shikonin excretion. US at relatively high energy levels caused slight cell growth depression (maximum 9% decrease in dry cell weight). Two key enzymes for the secondary metabolite biosynthesis of cells, phenylalanine ammonia lyase and p-hydroxybenzoic acid geranyltransferase, were found to be stimulated by the US. The US stimulation of secondary metabolite biosynthesis was attributed to the metabolic activity of cells activated by US, and more specifically, the defense responses of plant cells to the mechanical stress of US irradiation.     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.