Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7±0.85 mg/l as well as significant amounts of geranylgeraniol (33.1±5.6 mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.     

Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato

The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation. Phytoene and beta-carotene exhibited the greatest increases (2.4- and 2.2-fold, respectively). Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.     

牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes

1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC–MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.     

Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis

The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.¹ revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.² This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.³ Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.     

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis

The biomechanical functions of articular cartilage are governed largely by the composition and density of its specialized extracellular matrix. Relationships between matrix density and functional indices such as mechanical properties or interstitial solute diffusivities have been previously explored. However, direct correlations between mechanical properties and solute transport parameters have received less attention, despite potential application of this information for cartilage functional assessment both in vivo and in vitro. The objective of this study was therefore to examine relationships among solute diffusivities, mechanical properties, and matrix density of compressed articular cartilage. Matrix density varied due to natural variation among explants and due to applied static compression. Matrix density of statically compressed cartilage explants was characterized by glycoaminoglycan (GAG) weight fraction and fluid volume fraction, while diffusion coefficients of a wide range of solutes were measured to characterize the transport environment. Explant mechanical properties were characterized by a non-linear Young's modulus (axial stress-strain ratio) and a non-linear Poisson's ratio (radial-to-axial strain ratio). Solute diffusivities were consistently correlated with Young's modulus, as well as with explant GAG weight and fluid volume fractions. Therefore, in vitro mechanical tests may provide a means of assessing transport environments in cartilage-like materials, while in vivo measurements of solute transport (for example with magnetic resonance imaging) may be a useful complement in identifying localized differences in matrix density and mechanical properties.     

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.     

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.     

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.     

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

As a precursor of bioengineered heparin, heparosan is currently produced from Escherichia coli K5, which is pathogenic bacteria potentially causing urinary tract infection. Thus, it would be advantageous to develop an alternative source of heparosan from a non-pathogeneic strain. In this work we reported the biosynthesis of heparosan via the metabolic engineering of non-pathogenic E. coli BL21 as a production host. Four genes, KfiA, KfiB, KfiC and KfiD, encoding enzymes for the biosynthesis of heparosan in E. coli K5, were cloned into inducible plasmids pETDuet-1 and pRSFDuet-1 and further transformed into E. coli BL21, yielding six recombinant strains as follows: sA, sC, sAC, sABC, sACD and sABCD. The single expression of KfiA (sA) or KfiC (sC) in E. coli BL21 did not produce heparosan, while the co-expression of KfiA and KfiC (sAC) could produce 63mg/L heparosan in shake flask. The strain sABC and sACD could produce 100 and 120mg/L heparosan, respectively, indicating that the expression of KfiB or KfiD was beneficial for heparosan production. The strain sABCD could produce 334mg/L heparosan in shake flask and 652mg/L heparosan in 3-L batch bioreactor. The heparosan yield was further increased to 1.88g/L in a dissolved oxygen-stat fed-batch culture in 3-L bioreactor. As revealed by the nuclear magnetic resonance analysis, the chemical structure of heparosan from recombinant E. coli BL21 and E. coli K5 was identical. The weight average molecular weight of heparosan from E. coli K5, sAC, sABC, sACD, and sABCD was 51.67, 39.63, 91.47, 64.51, and 118.30kDa, respectively. This work provides a viable process for the production of heparosan as a precursor of bioengineered heparin from a safer bacteria strain.     

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.     

Distribution of mevalonate and glyceraldehyde 3-phosphate/pyruvate routes for isoprenoid biosynthesis in some Gram-negative bacteria and mycobacteria

Labeling experiments using [1-¹³C]acetate or [1-¹³C]glucose were performed with opportunistic pathogenic bacteria, with innocuous bacteria related to pathogenic species or with phytopathogenic species. The labeling pattern was determined in the isoprenic moiety of ubiquinone or menaquinone derivatives. These experiments showed that Acinetobacter, Citrobacter, Erwinia, Pseudomonas, Burkholderia, Ralstonia and Mycobacterium synthesize their isoprenoids via the mevalonate-independent glyceraldehyde 3-phosphate/pyruvate route. Enzymes of this novel bacterial metabolic route, which is apparently absent in vertebrates and man, therefore represent potential targets for a novel type of antibacterial drugs.     

Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),11(12)-diene] in transgenic tomato fruit

The taxanes are a group of polycyclic diterpenes produced by various species of yew. The potent anticancer drug paclitaxel (marketed as Taxol™) is the commercially most important taxane with annual sales in 2000 exceeding $1.6 billion. Paclitaxel is currently obtained either by direct extraction from yew trees or by the extraction of the precursor 10-deactilbaccatin III, which is then converted to paclitaxel by semi-synthesis. Apart from cost, one of the main draw backs to taxol in cancer treatment is the development of resistance by tumours, commonly due to the expression of ABC transporter efflux pumps which remove the drug from the target cell. A number of natural taxanes and semisynthetic derivates, have recently been shown to act as potent inhibitors of ABC transport proteins. These compounds have no effect upon microtubule polymerization (the normal target of paclitaxel), but have the ability to restore drug sensitivity when given in combination with paclitaxel to resistant cell lines. In work to be described elsewhere, we sort to carry out a structure function analysis of the ability of novel oxidised taxanes to act as ABC transporter inhibitors. For this study 100 mg or more of taxadiene [taxa-4(5),11(12)-diene], the first taxane in the paclitaxel pathway, was required as starting material from which to synthesize these compounds. Taxadiene is synthesised directly from geranylgeranyl diphosphate (GGPP), which is found in most plant tissues where it serves as a common precursor for many metabolites. The synthesis and use of GGDP are tightly regulated in most vegetative organs, however, in tomato fruit it is used almost exclusively for the production of coloured carotenoids which accumulate to high levels in the plastid as lycopene crystals. Expressing taxadiene synthase in a yellow-fruited tomato line that lacks the ability to utilise GGPP for carotenoid synthesis allowed GGPP normally utilised for making carotenoids to be re-routed for the production of taxadiene, allowing the facile extraction of 160 mg of highly pure taxadiene from 1 kg of freeze dried fruit.     

Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane

In higher plants, two independent pathways are responsible for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central five-carbon precursors of all isoprenoids. The cytosolic pathway, which involves mevalonate (MVA) as a key intermediate, provides the precursor molecules for sterols, ubiquinone, and certain sesquiterpenes, whereas the plastidial MVA-independent pathway is involved in the formation of precursors for the biosynthesis of isoprene, monoterpenes, diterpenes, carotenoids, abscisic acid, and the side chains of chlorophylls, tocopherols, and plastoquinone. Recent experiments provided indirect evidence for the presence of an export system for isoprenoid intermediates from the plastids to the cytosol in Arabidopsis thaliana. Here we report that isolated chloroplasts (from spinach, kale, and Indian mustard), envelope membrane vesicles, and proteoliposomes prepared from the solubilized proteins of envelope membranes (from spinach) are capable of the efficient transport of isopentenyl diphosphate and geranyl diphosphate. Lower rates of transport were observed with the substrates farnesyl diphosphate and dimethylallyl diphosphate, whereas geranylgeranyl diphosphate and mevalonate were not transported with appreciable efficiency. Our data suggest that plastid membranes possess a unidirectional proton symport system for the export of specific isoprenoid intermediates involved in the metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis.     

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.     

Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase

The recently discovered non-mevalonate pathway to isoprenoids, which uses glycolytic intermediates, has been modulated by overexpression of Escherichia coli d-1-deoxyxylulose 5-phosphate synthase (DXS) to increase deoxyxylulose 5-phosphate and, consequently, increase the isoprenoid precursor pool in E. coli. Carotenoids are a large class of biologically important compounds synthesized from isoprenoid precursors and of interest for metabolic engineering. However, carotenoids are not ordinarily present in E. coli. Co-overexpression of E. coli dxs with Erwinia uredovora gene clusters encoding carotenoid biosynthetic enzymes led to an increased accumulation of the carotenoids lycopene or zeaxanthin over controls not expressing DXS. Thus, rate-controlling enzymes encoded by the carotenogenic gene clusters are responsive to an increase in isoprenoid precursor pools. Levels of accumulated carotenoids were increased up to 10.8 times the levels of controls not overexpressing DXS. Lycopene accumulated to a level as high as 1333 μg/g dw and zeaxanthin accumulated to a level as high as 592 μg/g dw, when pigments were extracted from colonies. Zeaxanthin-producing colonies grew about twice as fast as lycopene-producing colonies throughout a time course of 11 days. Metabolic engineering of carbon flow from simple glucose metabolites to representatives of the largest class of natural products was demonstrated in this model system. Received: 6 August 1999 / Received revision: 25 October 1999 / Accepted: 5 November 1999