Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Metabolic engineering in strawberry fruit uncovers a dormant biosynthetic pathway

Wild strawberry (Fragaria vesca) fruit contains several important phenylpropene aroma compounds such as eugenol, but cultivated varieties are mostly devoid of them. We have redirected the carbon flux in cultivated strawberry (Fragaria×ananassa) fruit from anthocyanin pigment biosynthesis to the production of acetates of hydroxycinnamyl alcohols, which serve as the precursors of the phenylpropenes, by downregulating the strawberry chalcone synthase (CHS) via RNAi-mediated gene silencing and, alternatively, by an antisense CHS construct. Simultaneous heterologous overexpression of a eugenol (EGS) and isoeugenol synthase (IGS) gene in the same cultivated strawberry fruits boosted the formation of eugenol, isoeugenol, and the related phenylpropenes chavicol and anol to concentrations orders of magnitude greater than their odor thresholds. The results show that Fragaria×ananassa still bears a phenylpropene biosynthetic pathway but the carbon flux is primarily directed to the formation of pigments. Thus, partial restoration of wild strawberry flavor in cultivated varieties is feasible by diverting the flavonoid pathway to phenylpropene synthesis through metabolic engineering.     

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza.     

Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the lld-ACV:bisACV ratio on the flux control

An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis--(l--aminoadipyl)-l-cysteinyl-d-valine (bisACV) from the first pathway intermediate lld-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both lld-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of lld-ACV, K_ACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of lld-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the lld-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of lld-ACV:bisACV to be in the range of 1–2 moles per moles emphasises the importance of a fast conversion of lld-ACV to IPN, and accumulation of lld-ACV above the K_m-value of the IPNS should therefore be avoided.     

Metabolic modeling of Rosmarinic acid biosynthetic pathway

Rosmarinic acid (RA) is an ester of caffeic acid and 3, 4-dihydroxyphenyllacticacid. It is commonly found in Coleus blumei, Salvia officinalis, Melissa officinalis and Rosmarinus officinalis. The biosynthesis of RA starts with precursor molecules L-phenylalanine and L-tyrosine. Simulation of RA biosynthetic pathway was done using Gepasi Software, includes the reaction kinetics of each step of the pathway and different integration methods such as Euler's method. Optimization of the significant parameters responsible for RA biosynthesis was carried out. As the goal of the work was to increase the productivity of i.e. to maximize the concentration of the RA, the final concentration of RA ([RA]t) was selected as an objective function and selected initial concentration of the Caffeoyl-3'-4'hydroxyphenyllactic acid (3'C4HPLA) as parameter constraint and varied its initial concentration as: 0≤ [3'C4HPLA]i ≤ 0.025. Several optimization methods such as Simulated annealing, Evolutionary algorithms and Genetic algorithms were used to optimize the objective function. After optimization the final concentration of RA was slightly higher (4.566132e-002 mM) than before optimization (4.047119e- 002 mM). On the basis of results obtained, it is clear that 4-hydroxyphenyllactic acid and 3'C4HPLA play major role in the high productivity of the RA.     

Metabolic engineering under uncertainty--II: analysis of yeast metabolism

Yeast metabolism has been used extensively in scientific investigations and industrial applications. Understanding the properties of the yeast metabolic network is crucial, yet unaccomplished due to its high complexity, the different culture conditions, and the uncertainty associated with kinetic parameters. We recently developed a computational and mathematical framework using Monte Carlo method in which parameter uncertainty can be addressed through large-scale sampling procedure. This framework was applied on the compartmentalized central carbon pathways of Saccharomyces cerevisiae metabolism considering the growth environment of batch and chemostat reactor and integrating information from metabolic flux analysis. Statistical analysis of the results indicates that yeast cells growing in batch culture condition exhibit dramatically different control schemes from those growing in a chemostat. The difference is mainly due to the feedback introduced by the constraints of the chemostat. The control of the enzymes on the rates of the substrate uptake, product excretion, and cell growth and its practical implication are discussed. Clustering of the reaction steps according to the similarity of their responses to enzyme activity perturbations reveals functional coupling of metabolic reactions.     

Metabolic engineering Corynebacterium glutamicum for the l-lysine production by increasing the flux into l-lysine biosynthetic pathway

The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l^?1 h^?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum.     

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.     

Metabolic control of the tetrapyrrole biosynthetic pathway for porphyrin distribution in the barley mutant albostrians

The barley line albostrians exhibits a severe block in chloroplast development as a result of a mutationally induced lack of plastid ribosomes. White leaves of this mutant contain undifferentiated plastids, possess only traces of chlorophyll (Chl), and are photosynthetically inactive. Chl deficiency, combined with a continuous heme requirement, should lead to drastic changes in the tetrapyrrole metabolism in white versus green leaves. We analyzed the extent to which the synthesis rate of the pathway and the porphyrin distribution toward the Chl- and heme-synthesizing bifurcation is altered in the white tissue of albostrians. Expression and activity of several distinctively regulated enzymes, such as glutamyl-tRNAglu reductase, glutamate 1-semialdehyde aminotransferase, Mg- and Fe-chelatase, and Chl synthetase, were altered in white mutant leaves in comparison to control leaves. A drastic loss in the rate-limiting formation of 5-aminolevulinate and in the Mg-chelatase and Mg-protoporphyrin IX methyltransferase activity, as well as an increase in Fe-chelatase activity, accounts for a decrease in the metabolic flux and the re-direction of metabolites. It is proposed that the tightly balanced control of activities in the pathway functions by different metabolic feedback loops and in response to developmental state and physiological requirements. This data supports the idea that the initial steps of Mg-porphyrin synthesis contribute to plastid-derived signaling toward the nucleus. The barley mutant albostrians proved to be a valuable system for studying regulation of tetrapyrrole biosynthesis and their involvement in the bi-directional communication between plastids and nucleus.     

A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system

Sterol biosynthesis occurs in the ER and most sterol biosynthetic enzymes have transmembrane domains. However, due to difficulties in characterizing membrane protein-protein interactions, the nature of the sterol biosynthetic complex as well as in vivo interactions between various enzymes have not been described. We employed a split-ubiquitin membrane protein yeast two-hybrid system to characterize interactions between sterol biosynthetic proteins. Fourteen bait constructs were co-transformed into a reporter yeast strain with 14 prey constructs representing all sterol enzymatic reactions beginning with the synthesis of squalene. Our results not only confirmed several previous interactions, but also allowed us to identify novel interactions. Based on these results, ergosterol biosynthetic enzymes display specific protein-protein interactions forming a functional complex we designate, the ergosome. In this complex, Erg11p, Erg25p, Erg27p, and Erg28p appear to form a core center that can interact with other enzymes in the pathway. Also Erg24p and Erg2p, two enzymes that are sensitive to morpholine antifungals, appear to interact with one another; however, the profile of protein interaction partners appears to be unique. Erg2p and Erg3p, two enzymes catalyzing sequential reactions also appear to have different interaction partners. Our results provide a working model as to how sterol biosynthetic enzymes are topologically organized not only in yeast but in plant and animal systems that share many of these biosynthetic reactions.     

Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway

Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1 g/L) with a yield of 0.47 mol/mol glucose.     

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.     

Metabolic pathway analysis of a recombinant yeast for rational strain development

Elementary mode analysis has been used to study a metabolic pathway model of a recombinant Saccharomyces cerevisiae system that was genetically engineered to produce the bacterial storage compound poly-beta-hydroxybutyrate (PHB). The model includes biochemical reactions from the intermediary metabolism and takes into account cellular compartmentalization as well as the reversibility/irreversibility of the reactions. The reaction network connects the production and/or consumption of eight external metabolites including glucose, acetate, glycerol, ethanol, PHB, CO(2), succinate, and adenosine triphosphate (ATP). Elementary mode analysis of the wild-type S. cerevisiae system reveals 241 unique reaction combinations that balance the eight external metabolites. When the recombinant PHB pathway is included, and when the reaction model is altered to simulate the experimental conditions when PHB accumulates, the analysis reveals 20 unique elementary modes. Of these 20 modes, 7 produce PHB with the optimal mode having a theoretical PHB carbon yield of 0.67. Elementary mode analysis was also used to analyze the possible effects of biochemical network modifications and altered culturing conditions. When the natively absent ATP citrate-lyase activity is added to the recombinant reaction network, the number of unique modes increases from 20 to 496, with 314 of these modes producing PHB. With this topological modification, the maximum theoretical PHB carbon yield increases from 0.67 to 0.83. Adding a transhydrogenase reaction to the model also improves the theoretical conversion of substrate into PHB. The recombinant system with the transhydrogenase reaction but without the ATP citrate-lyase reaction has an increase in PHB carbon yield from 0.67 to 0.71. When the model includes both the ATP citrate-lyase reaction and the transhydrogenase reaction, the maximum theoretical carbon yield increases to 0.84. The reaction model was also used to explore the possibility of producing PHB under anaerobic conditions. In the absence of oxygen, the recombinant reaction network possesses two elementary modes capable of producing PHB. Interestingly, both modes also produce ethanol. Elementary mode analysis provides a means of deconstructing complex metabolic networks into their basic functional units. This information can be used for analyzing existing pathways and for the rational design of further modifications that could improve the system's conversion of substrate into product.     

Metabolic engineering of monoterpene synthesis in yeast

Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol-producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 μg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range.     

Metabolic engineering of sesquiterpene metabolism in yeast

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.     

Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast

We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described.