Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli.

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.

Investigation of anticapsin biosynthesis reveals a four-enzyme pathway to tetrahydrotyrosine in Bacillus subtilis.

Bacillus subtilis produces the antibiotic anticapsin as an L-Ala-L-anticapsin dipeptide precursor known as bacilysin, whose synthesis is encoded by the bacA-D genes and the adjacent ywfGH genes. To evaluate the biosynthesis of the epoxycyclohexanone amino acid anticapsin from the primary metabolite prephenate, we have overproduced, purified, and characterized the activity of the BacA, BacB, YwfH, and YwfG proteins. BacA is an unusual prephenate decarboxylase that avoids the typical aromatization of the cyclohexadienol ring by protonating C(8) to produce an isomerized structure. BacB then catalyzes an allylic isomerization, generating a conjugated dienone with a 295 nm chromophore. Both the BacA and BacB products are regioisomers of H(2)HPP (dihydro-4-hydroxyphenylpyruvate). The BacB product is then a substrate for the short chain reductase YwfH which catalyzes the conjugate addition of hydride at the C(4) olefinic terminus using NADH to yield the cyclohexenol-containing tetrahydro-4-hydroxyphenylpyruvate H(4)HPP. In turn, this keto acid is a substrate for YwfG, which promotes transamination (with L-Phe as amino donor), to form tetrahydrotyrosine (H(4)Tyr). Thus BacA, BacB, YwfH, and YwfG act in sequence in a four enzyme pathway to make H(4)Tyr, which has not previously been identified in B. subtilis but is a recognized building block in cyanobacterial nonribosomal peptides such as micropeptins and aeruginopeptins.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense.

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of beta-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.

A new isopentenyl diphosphate isomerase gene from Camptotheca acuminata: cloning, characterization and functional expression in Escherichia coli.

Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. Here we report cloning, characterization and functional expression of a new cDNA encoding IPI from Camptotheca acuminata. The full-length cDNA was 1143 bp long designated as CaIPI (GenBank Accession Number: DQ839416), containing an open reading frame (ORF) of 930bp which encodes a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CaIPI was highly homologous with other IPI gene and the deduced amino acid sequence of CaIPI was similar to known plant IPIs and contained Cys-149 and Glu-212 active sites. Phylogenic analysis indicated that all IPIs could be divided into five groups and CaIPI belonged to plant IPIs' family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues. The result showed that CaIPI expression could be detected in roots, stems and tender leaves but could not in mature leaves and fruits, and the expression levels was much higher in stems than in roots and tender leaves. Finally, CaIPI was functionally expressed in engineered Escherichia coli in which the carotenoid pathway was reconstructed. In engineered E. coli, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange beta-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil the biosynthetic mechanism of camptothecin precursors.

Improving lycopene production in Escherichia coli by engineering metabolic control.

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.

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.

Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme.

The crtB gene encoding phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn2+ or Mg2+. The Km value for GGPP was determined as 41 microM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I50 value for the latter inhibitor was 15 microM. Lineweaver-Burk plots showed constant Km values in the presence or absence of squalestatin.

Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco.

Two distinct cDNA clones (IPI1 and IPI2) encoding IPI were isolated from Nicotiana tabacum. In situ expression of isopentenyl diphosphate isomerase-1 (IPI1)- and IPI2-green fluorescent protein fusion constructs revealed that IPI1 and IPI2 were localized in chloroplast and cytosol, respectively. The level of IPI1 mRNA was increased under high-salt and high-light stress conditions, while that of IPI2 mRNA was increased under high-salt and cold stress conditions. Both IPI transcripts were increased in an abscisic acid-independent manner. This is the first report of a cytosolic IPI. The results indicated that two distinct IPIs were differentially induced in response to stress.

The role of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in the control of cardiac lipid metabolism.

The postnatal mammalian heart uses mitochondrial fatty acid oxidation (FAO) as the chief source of energy to meet the high energy demands necessary for pump function. Flux through the cardiac FAO pathway is tightly controlled in accordance with energy demands dictated by diverse physiologic and dietary conditions. In this report, we demonstrate that the lipid-activated nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), regulates the expression of several key enzymes involved in cardiac mitochondrial FAO. In response to the metabolic stress imposed by pharmacologic inhibition of mitochondrial long-chain fatty acid import with etomoxir, PPARa serves as a molecular 'lipostat' factor by inducing the expression of target genes involved in fatty acid utilization including enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. In mice lacking PPARalpha (PPARalpha-/- mice), etomoxir precipitates a cardiac phenotype characterized by myocyte lipid accumulation. Surprisingly, this metabolic regulatory response is influenced by gender as demonstrated by the observation that male PPARalpha-/- mice are more susceptible to the metabolic stress compared to female animals. These results identify an important role for PPARalpha in the control of cardiac lipid metabolism.

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana.

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Cloning, expression, and purification of the functional Delta(3)-Delta(2)-enoyl-CoA isomerase fusion protein.

Delta(3)-Delta(2)-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length rat liver Delta(3)-Delta(2)-enoyl-CoA isomerase was previously cloned as pAG847. PCR methodologies were used to subclone the gene encoding the functional Delta(3)-Delta(2)-enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5(') primer. The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli. The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE and the molecular weight of the protein subunit was 30 kDa. The purified protein had a dimeric structure composed of identical subunits, and the molecular weight of the enzyme determined by gel chromatography was 60 kDa. Kinetic studies have been carried out and K(M) of 81 microM and V(max) of 292 micromol/min/mg were determined. The specific activity of the protein is 201 U/mg, which is significantly higher than that reported before for the same protein isolated from a natural source. The one-step purification of the highly active Delta(3)-Delta(2)-enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues.

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.