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.     

Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6,051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.     

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.     

利用Bio-BglBrick方法表达紫杉醇生物合成酶

目的:进行紫杉醇药物生物合成前五步催化酶二磷酸盐合酶(GGPPS)、紫杉二烯合酶(TS)、紫杉二烯5α羟化酶(THY5α)、紫杉二烯5α-O-乙酰转移酶(TAT)和紫杉烷10β羟化酶(TDH)基因在大肠杆菌异源生物合成途径的组建及串联,实现单个表达及串联表达,并试图通过连续生物催化获得紫杉醇中间体紫衫二烯。方法:依据合成生物学中Brick基因组装方法,通过对载体pET30a的酶切位点进行定向改造,设计独特的BglⅡ/BamHⅠ和XbaⅠ/SpeⅠ串联表达盒,在大肠杆菌BM Rosetta(DE3)中表达产物。结果:设计多基因表达盒,实现紫杉醇药物生物合成前五步催化酶的单个表达,GGPPS和TS以及THY5α、TAT和TDH的串联表达。结论:利用BglBrick/BioBrick基因组装方法,可以实现紫杉醇生物合成催化酶的快速组装及后续表达。     

Monoterpene biosynthesis pathway construction in Escherichia coli

Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions.     

代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。     

Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.

The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.     

Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens‐mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene‐producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene‐forming gene. Notably, by co‐overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well‐established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.     

Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when omega,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the alpha-isoprene unit in the Z configuration, indicating that Rv1086 (omega,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.     

Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human

E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45-50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22-31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene.     

Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.     

1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.     

Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

The structure of ent-copalyl diphosphate synthase reveals three α-helical domains (α, β and γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in ent-copalyl diphosphate synthase but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.