Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon.

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Two BAHD Acyltransferases Catalyze the Last Step in the Shikonin/Alkannin Biosynthetic Pathway.

Several Boraginaceae plants produce biologically active red naphthoquinone pigments, derivatives of the enantiomers shikonin and alkannin, which vary in acyl groups on their side chains. Compositions of shikonin/alkannin derivatives vary in plant species, but the mechanisms generating the diversity of shikonin/alkannin derivatives are largely unknown. This study describes the identification and characterization of two BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1), from Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae that primarily produces the shikonin/alkannin derivatives acetylshikonin and ß-hydroxyisovalerylshikonin. Enzyme assays using Escherichia coli showed that the acylation activity of LeSAT1 was specific to shikonin, whereas the acylation activity of LeAAT1 was specific to alkannin. Both enzymes recognized acetyl-CoA, isobutyryl-CoA, and isovaleryl-CoA as acyl donors to produce their corresponding shikonin/alkannin derivatives, with both enzymes showing the highest activity for acetyl-CoA. These findings were consistent with the composition of shikonin/alkannin derivatives in intact L erythrorhizon plants and cell cultures. Genes encoding both enzymes were preferentially expressed in the roots and cell cultures in the dark in pigment production medium M9, conditions associated with shikonin/alkannin production. These results indicated that LeSAT1 and LeAAT1 are enantiomer-specific acyltransferases that generate various shikonin/alkannin derivatives.

A Trp-574-Leu mutation in the acetolactate synthase (ALS) gene of Lithospermum arvense L. confers broad-spectrum resistance to ALS inhibitors.

Lithospermum arvense is a troublesome dicotyledonous winter annual weed of wheat in China. A L. arvense population (HN01) suspected of being resistant to acetolactate synthase (ALS) inhibitors was found in Henan Province, China. This study aimed to testify the sensitivity of this HN01 population to eight herbicides from 3 different modes of action, and to explore the potential target-site-resistance mechanism to tribenuron-methyl. The whole-plant bioassays indicated that the population was highly resistant to tribenuron-methyl (SU, 350-fold), pyrithiobac sodium (PTB, 151-fold), pyroxsulam (TP, 62.7-fold), florasulam (TP, 80.6-fold), and imazethapyr (IMI, 136-fold), but was sensitive to carfentrazone-ethyl and fluroxypyr-meptyl. ALS gene sequencing revealed that the Trp (TGG) was substituted by Leu (TTG) at codon 574 in resistant plants. In in vitro ALS assays, the concentration of tribenuron-methyl required to inhibit 50% ALS activity (I50) for HN01 was 117-fold greater than that required to inhibit a susceptible population (HN05), indicating that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron-methyl. To the best of our knowledge, this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L. arvense.

Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway.

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.

Metabolic control of respiratory levels in coenzyme Q biosynthesis-deficient Escherichia coli strains leading to fine-tune aerobic lactate fermentation.

A novel strategy to finely control the electron transfer chain (ETC) activity of Escherichia coli was established. In this study, the fine-tuning of the ubiquinone biosynthesis pathway was applied to further controlling ETC function in coenzyme Q8 biosynthesis-deficient E. coli strains, HW108 and HW109, which contain mutations in ubiE and ubiG, respectively. A competing pathway on the intermediate substrates of the Q8 synthesis pathway, catalyzed by diphosphate:4-hydroxybenzoate geranyltransferase (PGT-1) of Lithospermum erythrorhizon, was introduced into these mutant strains. A nearly theoretical yield of lactate production can be achieved under fully aerobic conditions via an in vivo, genetically fine-tunable means to further control the activity of the ETC of the Q8 biosynthesis-deficient E. coli strains.

Homogeneous purification and characterization of LePGT1--a membrane-bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon.

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 µg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.

Production of dehydrogingerdione derivatives in Escherichia coli by exploiting a curcuminoid synthase from Oryza sativa and a ß-oxidation pathway from Saccharomyces cerevisiae.

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an "artificial" biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4-coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a ß-oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway.

Curcuminoids, which are produced specifically by plants of the order Zingiberales, have long been used as food additives because of their aromatic, stimulant and colouring properties and as traditional Asian medicines because of their anti-tumour, antioxidant and hepatoprotective activities. Curcuminoids are therefore attractive targets for metabolic engineering. An artificial curcuminoid biosynthetic pathway, including reactions of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate : CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa), a type III polyketide synthase, was constructed in Escherichia coli for the production of curcuminoids. Cultivation of the recombinant E. coli cells in the presence of tyrosine or phenylalanine, or both, led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane. Another E. coli system carrying 4CL and CUS genes was also used for high-yield production of curcuminoids from exogenously supplemented phenylpropanoid acids: p-coumaric acid, cinnamic acid and ferulic acid. The yields of curucminoids were up to approximately 100 mg l(-1). Furthermore, this system gave approximately 60 mg curcumin l(-1) from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.

High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon.

Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.

Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon. Cloning and characterization of a ket enzyme in shikonin biosynthesis.

Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.