Structural and functional modification of kudzu starch using α-amylase and transglucosidase.

The large agglomeration of starch paste in hot water, and fast retrogradation tendency and low transparency of starch gel restrict widespread application of kudzu starch. To improve the above defects, kudzu starch was modified with sequentially α-amylase (AA) and transglucosidase (TG), the latter for varying times. The results indicated that, compared to kudzu starch, amylose content and molecular weight of AA/TG-treated starches reduced by 20.07% and 69.50%, respectively. The proportion of A chain increased by 68.68%, whereas B1, B2 and B3 chains decreased by 14.28%, 48.29% and 23.44%, respectively. The degree of branching dramatically increased by 128.3%. After AA→TG treatment, the changes of starch structure enhanced the functional properties of kudzu starch. The solubility, paste clarity and gelatinization temperature increased, whereas the relative crystallinity, viscosity, storage and loss moduli decreased. Overall, the AA→TG modification would be desirable to improve the functional properties of kudzu starch to expand more large-scale application.

The effects of Pueraria mirifica extract, diadzein and genistein in testosterone-induced prostate hyperplasia in male Sprague Dawley rats.

Pueraria mirifica (PM) is a medicinal plant native to Thailand contained high amount of phytoestrogen and possesses anticancer activity. This study reports the effect of P. mirifica extract, phytoestrogen of diadzein and genistein for its benign prostate hyperplasia properties in testosterone-induced prostate hyperplasia in male Sprague Dawley rats. The P. mirifica extract was evaluated for its total phenols, flavonoid and antioxidant activity using DPPH, FRAP and metal chelating assay. The assessment of P. mirifica, diadzein and genistein against benign prostate hyperplasia was determined in testosterone-induced prostate hyperplasia in male Sprague Dawley rats. The total phenol was higher than flavonoid but showed low antioxidant activity of DPPH, FRAP and metal chelating. The aqueous PM extract at 1000 mg/kg significantly increased testosterone levels in testosterone-induced rats by 13% while diadzein and genistein increased it by 11% and 17% respectively. However, levels of FSH, LH, triglyceride and HDL are not affected by the oral administration of PM, diadzein and genistein to the rats. Similarly, total protein, albumin, globulin, total bilirubin, conjugated bilirubin, alkaline phosphatase, alanine aminotransferase, AST, and G-glutamyltransferase showed no significant difference as compared with negative control rats. The body weight of the rats, testis, kidney and liver showed no toxic effect. The zinc content increased significantly and the zinc transporter gen of ZnT4 and ZIP4 highly expressed suggesting that the PM, diadzein and genistein plays essential role in modulating prostate zinc homeostasis. Similarly, the expression of IL-6, AR and ER was significantly reduced indicating functioning in regulation of prostate growth and acts as anti-inflammatory role in preventing BPH. In conclusion, the results indicated that PM reduced BPH and contributed to the regulation in the zinc transport expression of the prostate cells in the benign prostate hyperplasia (BPH).

Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound.

Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.

Molecular cloning and functional characterization of two divergent 4-coumarate†:†coenzyme A ligases from Kudzu (Pueraria lobata).

As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.

Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase.

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).

Isoprene hydrocarbons production upon heterologous transformation of Saccharomyces cerevisiae.

AIMS: Isoprene (2-methyl-1,3-butadiene; C(5) H(8) ) is naturally produced by photosynthesis and emitted in the atmosphere by the leaves of many herbaceous, deciduous and woody plants. Fermentative yeast and fungi (Ascomycota) are not genetically endowed with the isoprene production process. The work investigated whether Ascomycota can be genetically modified and endowed with the property of constitutive isoprene production. METHODS AND RESULTS: Two different strategies for expression of the IspS gene in Saccharomyces cerevisiae were employed: (i) optimization of codon usage of the IspS gene for specific expression in S. cerevisiae and (ii) multiple independent integrations of the IspS gene in the rDNA loci of the yeast genome. Copy number analysis showed that IspS transgenes were on the average incorporated within about 25% of the endogenous rDNA. Codon use optimization of the Pueraria montana (kudzu vine) IspS gene (SckIspS) for S. cerevisiae showed fivefold greater expression of the IspS protein compared with that of nonoptimized IspS (kIspS). With the strategies mentioned earlier, heterologous expression of the kudzu isoprene synthase gene (kIspS) in S. cerevisiae was tested for stability and as a potential platform of fermentative isoprene production. The multi-copy IspS transgenes were stably integrated and expressed for over 100 generations of yeast cell growth and constitutively produced volatile isoprene hydrocarbons. Secondary chemical modification of isoprene to a number of hydroxylated isoprene derivatives in the sealed reactor was also observed. CONCLUSION: Transformation of S. cerevisiae with the Pueraria montana var. lobata (kudzu vine) isoprene synthase gene (IspS) conferred to the yeast cells constitutive isoprene hydrocarbons production in the absence of adverse or toxic effects. SIGNIFICANCE AND IMPACT OF THE STUDY: First-time demonstration of constitutive isoprene hydrocarbons production in a fermentative eukaryote operated through the mevalonic acid pathway. The work provides concept validation for the utilization of S. cerevisiae, as a platform for the production of volatile hydrocarbon biofuels and chemicals.

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques.

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA515) was evaluated following single-turnover saturating flashes. The decay of ΔA515 was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S2Q(B)⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S2Q(B)⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.

Optimization of a heterologous pathway for the production of flavonoids from glucose.

The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose.

A genomic approach to isoflavone biosynthesis in kudzu (Pueraria lobata).

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6'-deoxy- and 6'-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.

Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism.

The concept of "photosynthetic biofuels" envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO(2) and H(2)O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO(2), without a prior requirement for the harvesting, dewatering and processing of the respective biomass.

Mutational analysis of conserved outer sphere arginine residues of chalcone synthase.

Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the (372)GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity.

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells.

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.

An investigation on the germination of seeds of Kwao Kreu Kao (Pueraria candollei Grah. ex Benth) as affected by both water soakings and hot air oven treatments.

This experiment was carried out at the Faculty of Technology, Mahasarakham University, Mahasarakham 4400, Thailand to investigate effects of different water-soaking durations and hot air oven treatments on the germination of seeds of Kwao Kreu Kao (Pueraria candollei Grah. ex Benth) plants. The experiment was laid in a Randomised Complete Block Design with four replications. The results showed that after tested for Electrical Conductivity (EC) values for cracking of seeds, all seeds being used were at a normal condition (with an average EC value of 28.56 microS cm(-1) g(-1)) and all seeds were ready for germination. Strength on impermeability of seeds declined after soaking in water for 10 h and onwards then the treated seeds had increased in weights. However, after treated under hot air oven, dry weights of all seeds became similar. Germination percentage of all treatments was most rapid during the first three weeks of germination period and later slowly increased with time. At day 91 after sowing, T2 gave the highest percentage of germination (52%) and the lowest was found with T1 (control) with 31.25%. Again at day 91 after sowing, T2 gave the highest mean value of plant numbers (16.38) and the lowest was found with T1 (7.28). Numbers of abnormal seedlings determined at day 63 after sowing were lowest with T2 (6.25%) and worst with T4 (20.14%). Again at day 63 after sowing, plant height was significantly tallest with T2 (3.88 cm) and the lowest was found with T4 (2.71 cm). Numbers of leaves were not significantly different among the treated plants reaching a highest value of 11.25 leaves plant(-1) for T3. It may be concluded that T2 was the best treatment for use in germinating seeds of Pueraria candollei Grah. ex Benth plants. Further improvements on longevity and high percentage of germination of seeds were discussed and suggested.

Evolution of the isoprene biosynthetic pathway in kudzu.

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi.

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.

An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5'-deoxyflavonoids in transgenic tobacco.

We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5'-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.