Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L⁻¹. The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. cerevisiae.     

Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways

The potent antimalarial sesquiterpene lactone, artemisinin, is produced in low quantities by the plant Artemisia annua L. The source and regulation of the isopentenyl diphosphate (IPP) used in the biosynthesis of artemisinin has not been completely characterized. Terpenoid biosynthesis occurs in plants via two IPP-generating pathways: the mevalonate pathway in the cytosol, and the non-mevalonate pathway in plastids. Using inhibitors specific to each pathway, it is possible to resolve which supplies the IPP precursor to the end product. Here, we show the effects of inhibition on the two pathways leading to IPP for artemisinin production in plants. We grew young (7–14 days post cotyledon) plants in liquid culture, and added mevinolin to the medium to inhibit the mevalonate pathway, or fosmidomycin to inhibit the non-mevalonate pathway. Artemisinin levels were measured after 7–14 days incubation, and production was significantly reduced by each inhibitor compared to controls, thus, it appears that IPP from both pathways is used in artemisinin production. Also when grown in miconazole, an inhibitor of sterol biosynthesis, there was a significant increase in artemisinin compared to controls suggesting that carbon was shifted from sterols into sesquiterpenes. Collectively these results indicate that artemisinin is probably biosynthesized from IPP pools from both the plastid and the cytosol, and that carbon from competing pathways can be channeled toward sesquiterpenes. This information will help advance our understanding of the regulation of in planta production of artemisinin.     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

Regulation of copy number and stability of phage λ derived pTCλ1 plasmid in the light of the dimer/multimer catastrophe hypothesis

The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.     

Studies on maintenance of vector pBK2 in Corynebacterium acetoacidophilum by co-culture experiments in a continuous bioreactor

A plasmid vector pBK2 was tested for maintenance in Corynebacterium acetoacidophilum and found to be 100% stable for 90 generations. In co-culture experiments in absence of any selection pressure it was able to cause washout of plasmid free cells. Deletion mutagenesis of the plasmid was done to identify the regions of DNA responsible for its stable maintenance. This led to the identification of a region of DNA in pBB1, an endogenous plasmid used in the construction of pBK2, which is responsible for providing a growth rate advantage to the plasmid containing cell in co-culture with plasmid-free cells. To explain this observed stability we postulate the production of a “growth-inhibiting factor” by the plasmid-containing cells. Inclusion of a death rate term for the plasmid free cells in the computer simulation of the growth kinetics in a chemostat successfully predicts, depending upon the magnitude of the kinetic parameters, a stable coexistence of the two or washout of the plasmid-free cells, an observation that matches the experimental data. Because of its stable maintenance and its ability to select against plasmid-free cells this host-vector system is a natural candidate for cloning and use in large-scale fermentations.     

Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase

UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH₂p) yielding uridine 5′-(β,γ-methylenetriphosphate) (UppCH₂p). The following bisphosphonates were not acceptors of UMP: alendronate, pamidronate, clodronate and etidronate. UDP-glucose serves as uridylyl donor to triphosphate derivatives of the mevalonate pathway: farnesyl (far-PPP), geranyl (ger-PPP) and isopentenyl (iso-PPP), with formation of farnesyl-tetraphosphouridine (far-ppppU); geranyl-tetraphosphouridine (ger-ppppU) and isopentenyl-tetraphosphouridine (iso-ppppU). The K_m (mM) and V_max (mU/mg protein) values determined for these substrates were: 0.32 ± 0.07 and 4.9 ± 0.6; 0.21 ± 0.06 and 5.7 ± 0.8; 0.51 ± 0.14 and 2.0 ± 0.2, respectively. The K_m and V_max values for methylenebisphosphonate were 1.1 ± 0.2 mM and 4055 ± 96 mU/mg protein, respectively.     

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.     

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways

Carotenoids are isoprenoid pigments of industrial and nutritional interest. Although they are produced in non-carotenogenic Escherichia coli engineered with the appropriate biosynthetic genes, only a limited pool of their metabolic precursors is available in these bacteria. We have compared the production of carotenoids (lycopene) in strains in which the supply of precursors was enhanced either by upregulating the endogenous pathway via overexpression of deoxyxylulose 5-phosphate synthase (DXS) or by incorporating an exogenous MVA+ operon. In strains expressing DXS under the control of a leaky IPTG-inducible promoter, lycopene accumulation was increased up to 8-fold in the absence of inducer. Addition of IPTG, however, negatively affected lycopene production. Although induction of too high levels of the MVA+ operon enzymes also appeared to cause interference with cell metabolism, supplementation with mevalonate (to be metabolized into carotenoid precursors) resulted in a 10-fold increase in lycopene levels in cells with a near wild-type background. An additional 2-fold increase (up to 228 mg/l) was obtained using an engineered BL21 strain. These results confirm that the MVA+ pathway is most convenient to upregulate the production of carotenoids (lycopene) production in E. coli but that factors other than precursor supply should be considered for high pigment accumulation levels.     

Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotic-producing Streptomyces strains

We have isolated a mutant lacking 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity from a terpenoid antibiotic (terpentecin) producer, Streptomyces griseolosporeus MF730-N6, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgg) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6. The corresponding hmgs gene for HMG-CoA reductase was also cloned from Streptomyces sp. KO-3988, which produces the terpenoid antibiotic furaquinocin. Sequence analysis of hmgg and hmgs showed that both genes encode polypeptides of 353 amino acids which are 84% identical to each other. A search of protein sequence databases revealed that both gene products were also similar to HMG-CoA reductases from a variety of other organisms, including Streptomyces sp. CL190 (hmgg is 89% and hmgs 85% identical to its CL190 homolog), sea urchin (40.3 and 40.5%), German cockroach (37.6 and 38.4%), and Camptotheca acuminata (39.7 and 40.8%). Received: 17 May 1999 / Accepted: 10 September 1999     

Coenzyme Q - Biosynthesis and functions

In addition to its role as a component of the mitochondrial respiratory chain and our only lipid-soluble antioxidant synthesized endogenously, in recent years coenzyme Q (CoQ) has been found to have an increasing number of other important functions required for normal metabolic processes. A number of genetic mutations that reduce CoQ biosynthesis are associated with serious functional disturbances that can be eliminated by dietary administration of this lipid, making CoQ deficiencies the only mitochondrial diseases which can be successfully treated at present. In connection with certain other diseases associated with excessive oxidative stress, the level of CoQ is elevated as a protective response. Aging, certain experimental conditions and several human diseases reduce this level, resulting in serious metabolic disturbances. Since dietary uptake of this lipid is limited, up-regulation of its biosynthetic pathway is of considerable clinical interest. One approach for this purpose is administration of epoxidated all-trans polyisoprenoids, which enhance both CoQ biosynthesis and levels in experimental systems.     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Design and synthesis of non-hydrolyzable homoisoprenoid α-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases

An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure–activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.