Plant metabolic engineering: are we ready for phase two?
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Biotechnology↗
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Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol.
The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.
Acyltransferases↗
Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.
This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.
Basidiomycota↗
Research on the metabolic engineering of the direct oxidation pathway for extraction of phosphate from ore has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases.
The ability of some bacteria to dissolve poorly soluble calcium phosphates (CaPs) has been termed 'mineral phosphate solubilizing' (MPS). Since most microorganisms and plants must assimilate P via membrane transport, biotransformation of CaP into soluble phosphate is considered an essential component of the global P cycle. In many Gram-negative bacteria, strong organic acids produced in the periplasm via the direct oxidation pathway have been shown to dissolve CaP in the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may function in the ecophysiology of many soil bacteria. There is interest in using MPS bacteria for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a 'biofertilizer' analogous to nitrogen fixation. Our laboratory has spent 20 years studying superior MPS bacteria. Screening genomic libraries in the appropriate E. coli genetic background can 'trap' PQQ or GDH genes from these bacteria via functional complementation. In setting the 'trap' for PQQ genes, we have identified DNA fragments that apparently induce PQQGDH activity in E. coli with no sequence homology to known PQQ genes. These data suggest that E. coli may have an alternative, inducible PQQ biosynthesis pathway. Finally, a novel protein engineering strategy to increase the catalytic rate of PQQGDH has emerged and will be discussed.
Amino Acid Sequence↗
Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.
A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.
Cloning, Molecular↗
Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains.
Recombinant Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) [P(3HB-co-3HHx)] from dodecanoic acid. By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt %, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 (g/L)/h. The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol %. The 3HHx fraction in the copolymer could be increased by 3-fold when the Aeromonas hydrophila orf1 gene was coexpressed with the PHA biosynthesis genes.
3-Hydroxybutyric Acid↗
Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli.
1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.
Escherichia coli↗
Improving 1,3-propanediol production from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn-glycerol-3-phosphate.
High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli (E. coli) strains expressing genes from the Klebsiella pneumoniae dha (K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. coli can be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E. coli strains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. coli strains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E.coli Lin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coli K10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation.
Anaerobiosis↗
Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli.
We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.
Bioreactors↗
Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold.
Genetically engineered rice (Oryza sativa L.) with the ability to synthesize glycinebetaine was established by introducing the codA gene for choline oxidase from the soil bacterium Arthrobacter globiformis. Levels of glycinebetaine were as high as 1 and 5 micromol per gram fresh weight of leaves in two types of transgenic plant in which choline oxidase was targeted to the chloroplasts (ChlCOD plants) and to the cytosol (CytCOD plants), respectively. Although treatment with 0.15 M NaCl [corrected] inhibited the growth of both wild-type and transgenic plants, the transgenic plants began to grow again at the normal rate after a significantly less time than the wild-type plants after elimination of the salt stress. Inactivation of photosynthesis, used as a measure of cellular damage, indicated that ChlCOD plants were more tolerant than CytCOD plants to photoinhibition under salt stress and low-temperature stress. These results indicated that the subcellular compartmentalization of the biosynthesis of glycinebetaine was a critical element in the efficient enhancement of tolerance to stress in the engineered plants.
Acclimatization↗
Metabolic engineers in the Bavarian Alps.
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Biotechnology↗
Feedback regulation in metabolic engineering
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Journal Article↗
Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells.
The eukaryotic cell cycle is regulated by a complex network of many proteins. Effective reprogramming of this complex regulatory apparatus to achieve bioprocess goals, such as cessation of proliferation at high cell density to allow an extended period of high production, can require coordinated manipulation of multiple genes. Previous efforts to establish inducible cell-cycle arrest of Chinese hamster ovary (CHO) cells by regulated expression of the cyclin-dependent kinase inhibitor (CDI) p21 failed. By tetracycline-regulated coexpression of p21 and the differentiation factor CCAAT/enhancer-binding protein alpha (which both stabilizes and induces p21), we have achieved effective cell-cycle arrest. Production of a model heterologous protein (secreted alkaline phosphatase; SEAP) has been increased 10-15 times, on a per cell basis, relative to an isogenic control cell line. Because activation of apoptosis response is a possible complication in a proliferation-arrested culture, the survival gene bcl-xL was coexpressed with another CDI, p27, found to enable CHO cell-cycle arrest predominantly in G1 phase. CHO cells stably transfected with a tricistronic construct containing the genes for these proteins and for SEAP showed 30-fold higher SEAP expression than controls.
Alkaline Phosphatase↗
Metabolic engineering and human disease.
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Biotechnology↗
Metabolic engineering by genome shuffling.
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Catalysis↗
Metabolic engineering of Candida tropicalis for the production of long-chain dicarboxylic acids.
We have engineered an industrial strain of the yeast, Candida tropicalis, for the efficient production of long-chain dicarboxylic acids, which are important raw materials for the chemical industry. By sequential disruption of the four genes encoding both isozymes of the acyl-CoA oxidase which catalyzes the first reaction in the beta-oxidation pathway, alkane and fatty acid substrates have been successfully redirected to the omega-oxidation pathway. Consequently, the conversion efficiency and chemical selectivity of their terminal oxidation to the corresponding dicarboxylic acids has been improved to 100 percent. The specific productivity of the bioconversion has been increased further by amplification of the cytochrome P450 monooxygenase and NADPH-cytochrome reductase genes encoding the rate-limiting omega-hydroxylase in the omega-oxidation pathway. The amplified strains demonstrated increased omega-hydroxylase activity and a 30% increase in productivity compared to the beta-oxidation-blocked strain in fermentations. The bioconversion is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain-lengths ranging from 12 carbons to 22 carbons and also avoids the undesirable chain modifications associated with passage through the beta-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening. It is now possible to efficiently produce a wide range of previously unavailable saturated and unsaturated dicarboxylic acids with a high degree of purity.
Candida↗
Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway.
There is an increasing demand for peptide-mimicking molecules to modulate the interactions between proteins of pharmaceutical and agrochemical interest and their target polypeptides. Unnatural L-alpha-amino acids differing from the 20 naturally proteinogenic amino acids only in their side chain are ideal for this purpose, but their chemical synthesis is complex. Here we describe a fermentation-based approach for biosynthesis of unnatural amino acids after re-engineering the cysteine-biosynthetic pathway in Escherichia coli. O-acetylation of serine, the committed step of the pathway, was released from feedback inhibition by mutating the serine acetyltransferase gene. Next, the naturally broad substrate specificity of O-acetylserine sulfhydrylase was exploited for the direct in vivo incorporation of an unnatural side chain in a semisynthetic fermentation process comparable to the production of beta-lactams. O-acetyl-L-serine extruded from the cells by way of the O-acetylserine efflux protein was amenable to further biotransformations.
Amino Acids↗