Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident.

AIM: To assess the role of lactate as a precursor for butyrate biosynthesis in human colonic microflora. METHODS AND RESULTS: Three human faecal microfloras were incubated in vitro with media supplemented with 30 mmol l(-1) unenriched or 13C-enriched lactate. Lactate metabolism and short-chain fatty acid (SCFA) production were quantified. Lactate conversion to butyrate was investigated by gas chromatography-mass spectrometry and the pathways involved were identified by 13C nuclear magnetic resonance spectroscopy. All human faecal microfloras rapidly and completely fermented lactate, yielding approx. 19 mmol l(-1) total SCFAs. However, the SCFA composition varied markedly between microfloras. Butyrate was the main end-product for two microfloras but not for the third (60 and 61%vs 27% of the net concentration of SCFA produced respectively). The latter was typified by its ability to produce propionate as a major product (37%), and valerate (3%). 13C-Labelling showed that butyrate was produced through the acetyl-CoA pathway and that the three microfloras possessed significant differences in their metabolic pathways for lactate consumption. CONCLUSIONS: In contrast to the ruminal microflora, the human intestinal microflora can utilize both d- and l-lactate as precursors for butyrate synthesis. Inter-individual variation is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the butyrogenic capability of colonic prebiotics could be related to lactate availability. These findings will direct the development of selection strategies for the isolation of new butyrate-producing bacteria among the lactate-utilizing bacteria present in the human intestinal microfloras.

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

The formation of 3 alpha- and 3 beta-acetoxytropanes by Datura stramonium transformed root cultures involves two acetyl-CoA-dependent acyltransferases.

Tropine (tropan-3 alpha-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in transformed root cultures of Datura stramonium. A further acyltransferase activity that acetylates pseudotropine (tropan-3 beta-ol) with acetyl-coenzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in forming the observed alkaloid spectrum of D. stramonium roots discussed.

Strategies for the genetic manipulation of alkaloid-producing pathways in plants.

Increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. In the pathways forming indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. Such knowledge contributes the basis upon which it has become feasible to design a strategy by which the flux in these pathways may be enhanced at the genomic level. This review presents a summary of the state-of-the-art pertaining to these pathways and discusses the strategy to be adopted for a molecular approach to their manipulation, together with some of the pitfalls that may arise when trying to alter their natural regulation.

Alkaloid Production by Transformed Root Cultures of Cinchona ledgeriana.

Transformed root cultures of CINCHONA LEDGERIANA have been generated by infecting shoots cultured IN VITRO with AGROBACTERIUM RHIZOGENES LBA9402. These root cultures grow axenically in the absence of antibiotics or exogenous plant growth regulators in media containing Gamborg B5 salts at half or full strength with 3% (w/v) sucrose as the carbon source. They show a 6- to 8-fold increase in fresh weight in 28 days. Transformation has been confirmed by Southern blotting using [ (32)P]-labelled fragments from both the T (L)- and T (R)-DNA of the Ri plasmid. The cultures are shown to synthesise a range of quinoline alkaloids, of which quinine, cinchonidine, and quinidine are the major components. The level of these alkaloids changes with the age of the cultures, reaching a maximum at about 50 microg/g fresh weight after 45 days. In addition, the roots contain quinamine and a number of other, unidentified, indole alkaloids, one of which is the major alkaloid present. Only about 1% of the total alkaloid present is released to the medium.

Production of Hyoscyamine by 'Hairy Root' Cultures of Datura stramonium.

'Hairy root' cultures of DATURA STRAMONIUM were established following infection of aseptic leaves with AGROBACTERIUM RHIZOGENES. Transformation was confirmed by Southern blotting using [ (32)P]-labelled fragments of the T-DNA as probes. The transformed cultures grew in the absence of added phytohormones and cell mass increased 55-fold during 28 days incubation. Hyoscyamine was a major component of the alkaloid fraction and accounted for at least 0.3% of the dry matter, comparable to pot-grown plants from which the cultures were initiated. Production of hyoscyamine followed growth during the first 15 days of incubation, but continued to increase during the early part of stationary phase. The alkaloid was retained almost entirely in the root tissue. The effects of medium composition and pH on growth and hyoscyamine production are reported.

Quinoline Alkaloid Production by Transformed Cultures of Cinchona ledgeriana.

A culture of CINCHONA LEDGERIANA, transformed with AGROBACTERLUM TUMEFACIENS A6 capable of growing and producing quinoline alkaloids in medium free of exogenous phytohormones has been obtained. Unlike the untransformed culture of this species, addition of ZR in combination with either IAA or IBA to medium did not affect alkaloid production. Growing the transformed culture in the dark, however, produced a marked enhancement of alkaloid accumulation, up to 50 times that of cultures grown in the light. This dark-dependent accumulation was not confined to any particular time in the growth cycle, although the extent of the stimulatory effect increased the longer cultures were kept in darkness. Blue light was detrimental to alkaloid accumulation but in red or green light the level of accumulation was equivalent to that in the dark. Alternating cultures for several 28-day periods between light and dark conditions resulted in alternate periods of low and higfralkaloid productivity, indicating this was not an adaption effect. These findings correlate to previously reported differences in key enzymes from the biosynthetic pathway. Increasing the phosphate or nitrate concentration above that of Gamborg B5, increasing sucrose above 2% (w/v), replacing sucrose with glucose or adding tryptophan or casamino acids to the medium resuited in lower alkaloid yields by dark-grown cultures of the transformed line.

A spectrophotometric assay for strictosidine synthase.

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.

Radioimmunoassay for the quantitative determination of quinine in cultured plant tissues.

The development of a radioimmunossay for the determination of picogram amounts of quinine is described. The antiserum, raised against a conjugate of quinine and bovine serum albumin, is highly specific to the CINCHONA quinoline alkaloids having the 8S configuration and will not cross-react to any detectable extent with the 8R series. Neither will it cross-react with quininone or cinchonidinone, the common 9-keto precursors. Using this assay a number of clones of C. LEDGERIANA have been screened for their quinine content.