Meinhart H. Zenk

Immobilized plant cells for the production and transportation of natural products

There has been considerable interest in the past few years in the area of immobilized entities, such as enzymes and microbial cells [ 11, Preparations of this kind have proven to be of value not only for studies of a fundamental nature but also for a number of practical applications including the production and transfo~ation of food stuff additives and drugs. The advantages of using such preparations include, in particular, a lower cost in biotechnolo~cal applications because of the possibility of reusing the ‘biocatalyst’ and there is no need for separation of the product from the catalyst. Furthermore, immobilized preparations are ideally suited for flow-through processes; these advantages of immobilized systems have been amply reviewed [2,3]. In this study we report on an extension of the use of the immob~ization technique to include also, for the first time, plant cells. Isolated growing plant cells have been discussed as a potential source for the production and biotr~sfo~ation of natural products (e.g., [4]). The same advantages mentioned above should also apply to immobilized plant cells. In addition, it appears likely that the chemical potential of normally slow growing plant cells (under batch conditions) could be more efficiently exploited by immobilization. Three examples of the use of immobilized plant cells are given here. First the de novo synthesis of ~thraquinones by entrapped cells of Mo~ndu is demonstrated, second the formation of indole alkaloids from distant precursors by Cat~~Q~th~~ cells and third, the position and stereospecific hydroxyla-

Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata

Abstract A radioimmunoassay technique has been developed for the measurement of digoxigenin glycosides in crude extracts from both fresh and dried leaf material of Digitalis lanata , The antibody, obtained by immunizing rabbits against a conjugate of digoxin with human serum albumin, had a high affinity ( K a = 0.8 × 10 10 l/mol) for digoxin and permitted detection of as little as 60 fmol digoxin (45 pg) per 0.1 ml of sample. The antiserum was highly specific for digoxigenin and its glycosides, with only diginatin showing a substantial cross reactivity (30%). The use of [ 3 H]-labelled and [ 125 I]-labelled digoxin as tracer and of dextran-coated charcoal or ammonium sulfate for separation did not change the specificity of the assay nor the properties of the standard curve. This method has been found to correlate with the usual fluorimetric determination of digoxin, but is more sensitive by a factor of 10 4 . A correlation analysis of 8 and 30 different D. lanata plants (leaf discs and drugs analysed with both methods) gave correlation coefficients of r = 0.989 and r = 0.907 respectively. The analysis of a single leaf disc, 3 mm in diameter (obtained from a fresh leaf), gave an exact measure of the digoxin content found in the dried leaf drug ( r = 0.973). With a semi-automated technique, about 2000 quantitative analyses per week can be performed by one person, thus providing the potential to screen plants for use in breeding or tissue culture work. The distribution of digoxigenin equivalents in single seeds, seedlings and plants of different ages has also been investigated.

Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis

Abstract The purification and characterization of an NADP(H) specific cinnamyl alcohol oxidoreductase is reported. The enzyme, which has been purified 600 ×, shows an absolute specificity for the cinnamyl moiety. The reaction is readily reversible but is strongly inhibited by aldehyde substrates. The enzyme belongs to class A of NAD(P) specific oxidoreductases. The distribution of this activity throughout a wide variety of taxonomically different plant groups as well as plant parts has revealed a possible correlation with lignin biosynthesis.

Production of rosmarinic acid by cell-suspension cultures of Coleus blumei

larger amounts the pattern of elution Of radioactivity was clearly very, similar. Table 1 gives the results from a typical experiment of three determinations made. In all cases nearly all the radioactive label was found in the benzene fraction which contains the aromatic residues. We conclude that, under the conditions described, methyl palmitate is eluted by benzene from an alumina chromatographic column. It is likely that other esters of long-chain monocarboxylic acids behave in the same manner. This work was supported by National Science Foundation grant no. ENV 76-08310. Received August 8, 1977

Partial purification and properties of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase from higher plants

Abstract The partial purification and characterization of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) from two plant sources growing as cell cultures are reported. The enzymes have been purified 50-and 16-fold, respectively, and show an absolute specificity for p -coumaroyl-CoA and caffeoyl-CoA as well as for quinate, and are responsible for the synthesis of p -coumaroylquinate and caffeoylquinate (chlorogenic acid). The distribution of this transferase activity in a variety of plant cell cultures and differentiated plants is reported.

Three novel enzymes involved in the reduction of ferulic acid to coniferyl alcohol in higher plants: ferulate: Co a ligase, feruloyl-Co a reductase and coniferyl alcohol oxidoreductase

Using a cell-free system from cambial tissue of Sulix alba the reduction of ferulate to coniferyl alcohol has been shown for the first time, unequivocally, to occur in higher plants [l] . This conversion is dependent on ATP, CoA and reduced pyridine nucleotides. A reaction sequence has been postulated on the basis of these experiments involving the intermediate formation of feruloyl-CoA and coniferyl aldehyde which is in accordance with previous assumptions for this mechanism in lignin biosynthesis as recently reviewed [2]. In the present paper, a cell-free preparation of a lignifying higher plant which reduces ferulic acid to coniferyl alcohol is described. The overall reaction is composed of three individual enzymatic steps. Proof is presented that ferulate is first activated by a cinnamate: CoA ligase to feruloyl-CoA which is in turn reduced by NADPH to coniferyl aldehyde. This compound is further reduced to coniferyl alcohol, with NADPH again participating as the reductant.

Glucosidases involved in indole alkaloid biosynthesis of Catharanthus cell cultures

Strictosidine, an alkaloidal glucoside [ I-31, is a firmly established intermediate in monoterpenoid indole alkaloid biosynthesis. The enzyme catalyzing the condensation of tryptamine with the monoterpenoid glucoside secologanin was discovered in several plant cell cultures [2,4] and has been characterized from cultured Catharunthus roseus cells [S]. The step subsequent to strictosidine formation involves the removal of the glucose moiety of strictosidine yielding an unstable aglycone which in turn opens to form a highly reactive dialdehyde [6]. This intermediate undergoes further rearrangements and in our system finally yields ajmalicine and its two isomers [7] (see scheme 1). One crucial step, the removal of the glucose molecule from strictosidine, is most likely catalyzed by a P-D-glucosidase(s). The involvement of non-specific glucosidases in this reaction has been claimed using C. roseus [8]. Non-specific glucosidases hydrolyzed p-NPG as well as catalyzed the formation of indole alkaloids [8]. Both of these enzymes had mol. wt 55 000 and optimum pH 5.0-5.5, and were activated by tryptamine. It was furthermore claimed that these glucosidases are part of an enzyme aggregate termed ‘ajmalicine synthetase’. Here the involvement of glucosidases in indole alkaloid biosynthesis is studied using a specific assay system with strictosidine as substrate. We report the discovery and characterization in C. roseus cell cultures of two highly glucoalkaloid-specific fi-glucosidases, which disprove the claims in [8].

Isovincoside (strictosidine), the key intermediate in the enzymatic formation of indole alkaloids

Indole alkaloids are among the most complex and diverse low molecular natural products. Their biosynthesis has been investigated by precursor feeding experiments in vivo [l-4] using differentiated Apocynaceae plants. As a result of these studies synthetic vincoside, which was originally assigned the 3 (Y (S) stereochemistry, was found to be incorporated into serpentine and other Corynanthe type alkaloids in good yield while the incorporation of isovincoside with opposite configuration at C-3 was lower by a factor of lo3 [5]. In subsequent experiments the crucial problem of the configuration at C-3 of the alkaloidal secoiridoid glucoside was solved and three independent groups reported vincoside to possess a C-3 (R) /3 hydrogen [6-81. The revision of the absolute C-3 hydrogen stereochemistry to 3 (R) 0 was secured recently by X-ray diffraction experiments [9]. The problem of inversion arose in that vincoside, the C-3 (R) fl epimer, is considered the biosynthetic precursor of the Corynanthe-type alkaloids with 3 a (5’) stereochemistry. Recently, the limitations of in vivo experiments for the elucidation of alkaloid biosynthesis were overcome when Scott and Lee [lo] obtained a crude cell-free system from callus tissue of Cutharunthus roseus which was capable of synthesizing geissoschizine and ajmalicine. These experiments have been extended by us and it was shown that a cell-free preparation from fermenter-grown [ 111 C. roseus cells synthesized ajmalicine, 19.epiajmalicine and tetrahydroalstonine from tryptamine and secologanin in the presence of either NADPH or NADH [ 121. Furthermore, an immediate precursor of these alkaloids was found to accumulate in the absence of reduced pyridine nucleotides which was identified as 20,21 -didehydroajmalicine (cathenamine) with 3 ~(5’) configuration [131* In the present communication we report that using the cell-free system from Cuthurunthus and also an enzyme preparation from Rhuziu strictu cells we were able to isolate the enzymatically catalyzed primary condensation product between tryptamine and secologanin which proved in both cases to be a single product with 3 cr Q configuration. This product is converted by a crude enzyme preparation from C roseus cells to cathenamine and in the presence of reduced nucleotides to ajmalicine, 19.epiajmalicine and tetrahydroalstonine. In contrast to previous assumptions [5,8], the key stone in the intricate pathway which leads to the structurally diverse indole alkaloids is isovincoside (strictosidine) (3 ~1 (s)) and not vincoside (3 p (R)).

The homogentisate ring-cleavage pathway in the biosynthesis of acetate-derived naphthoquinones of the droseraceae

Abstract Photosynthesis experiments with 14 CO 2 established that of 16 Droseraceae species tested Drosophylum lusitanicum incorporated the highest amount of label into plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone). Tyrosine-[β- 14 C] fed to Drosophyllum was shown to label plumbagin efficiently (20% incorporation). Extensive chemical degradation of the labeled naphthoquinone showed, however, that the incorporation of tyrosine was indirect, the label being distributed throughout the molecule. It was established that plumbagin and the closely related 7-methyljuglone are biosynthesized via the acetate-polymalonate pathway. Tyrosine is broken down to acetate in this tissue via the homogentisate pathway, which was demonstrated by feeding and incorporation of label into plumbagin of intermediates such as homogentisate-[ 14 C], maleyl- and fumarylacetoacetate-[ 14 C]. Simultaneous application of tyrosine-[β- 14 C] and α,α′-bipyridyl, an inhibitor of the homogentisate oxigenase, led to an accumulation of homogentisate-[ 14 C] within the tissue. The degradation of tyrosine to acetate by Drosophyllum is not due to epiphytic bacteria since ring cleavage of tyrosine and formation of plumbagin from breakdown products occurred both within sterile grown plants and sterile cell suspension cultures. In tissue kept in darkness, plumbagin undergoes a slow turnover with a half life of about 400 hr.

Partial purification and properties of p-hydroxycinnamoyl-CoA: Shikimate-p-hydroxycinnamoyl transferase from higher plants

Abstract The partial purification and characterization of p -hydroxycinnamoyl-CoA: shikimate- p -hydroxycinnamoyl transferase (CST) from cell suspension cultures of Cichorium endivia is reported. The enzyme has been purified 126-fold and shows an absolute specificity for p -coumaroyl-CoA and shikimic acid. The product of the enzyme reaction has been identified as 5- O - p -coumaroyl shikimic acid. A wide distribution of CST activity in a variety of plant cell suspension cultures and intact plants was also found.