A búzaszem textúráját meghatározó fehérjék tanulmányozása in vitro rendszerekben = Study of proteins that determine the texture of wheat grain in in vitro systems

A pályázat keretében felszaporítottuk, és klónoztuk 3 búza friabilin fehérje génjét. A klónozott géneket E. coli-ban kifejeztettük. A puroindolinA génből létrehoztunk 3 mutáns változatot is, hogy a cisztein hidak és a triptofán gazdag hurok szerepét tudjuk vizsgálni. A termelt fehérjékkel funkcionális vizsgálatokat végeztünk. Bizonyítottuk, hogy mind a természetes eredetű, mind a heterológ rendszerben előállított friabilin fehérjék specifikusan kötődnek a tisztított búzakeményítőhöz. Igazoltuk, hogy a kenyérbúzából izolált keményítőhöz hasonlóan durum búzából származó keményítő is köti a friabilineket. Munkánk során meggyőzően bizonyítottuk, hogy egy kb. 30 kDa méretű gliadin típusú fehérjefrakció jelen van az érett keményítőszemcsék felszínén. Ezek a fehérjék képesek a keményítőhöz kötődni in vitro, puroindolinok jelenlétében és hiányában egyaránt. Az ?-gliadinok és a keményítő közötti erős kötés magyarázhatja azt a tényt, hogy ha nincs jelen mindkét vad típusú puroindolin, a búzaszem kemény. A puroindolinok és a szemkeménységet befolyásoló tartalékfehérjék vizsgálatával úttörő munkát végzünk, mely lehetővé teszi a keménység szélesebb látókörű szemlélését, megértését. | We have amplified and cloned 3 wheat friabilin genes. The friabilin genes were expressed in E. coli and the produced proteins were used in functional studies. We have also designed and expressed 3 puroindoline A mutant to determine the role of cistein bridges and the tryptophan rich loop. We were able to show that both natural and heterologously expressed frabilin binds starch, including durum starch. We have proved that an approx. 30 kDa sized gliadin type protein fraction is present on the surface of mature starch granules. These proteins are able to bind starch in vitro, regardless whether puroindolines are present or absent. The strong interaction between ?-gliadins and starch could be the cause of kernel hardness in the absence of wild type puroindolines. Investigating puroindolines and storage proteins effecting texture provides necessary information to understand wheat kernel hardness

A búza 1Bxz HMW glutenin alegység túltermelés molekuláris alapjainak vizsgálata = Study of the molecular bases of the overexpression of wheat 1Bx7 HMW glutenin subunit

Megklónoztuk és megszekvenáltuk a Glu-1Bx gén promóterét két ismert külföldi (egy normál- és egy túltermelő) és három hazai (egy Bx7-et átlag alatti szinten termelő, egy normál- valamint egy túltermelő) búza fajtából. A túltermelő búzafajták promóterében azonosítottunk egy 43 bp hosszú inszerciót, amely az alul- és normáltermelő fajtákban nincs jelen. Az inszerció kimutatására kodomináns PCR markert terveztünk és a markert ismert nemzetközi fajtákon validáltuk. Megklónoztuk és megszekvenáltuk a Glu-1Bx gén fehérjét kódoló szakaszát két hazai (egy normáltermelő, Bx7* allélt hordozó és egy túltermelő, Bx7 allélt hordozó) búza fajtából. A Bx7 allél 3? végén azonosítottunk egy 18 bp hosszú inszerciót, amely a Bx7* allélban nincs jelen. Az inszerció kimutatására kodomináns PCR markert terveztünk és a markert ismert nemzetközi fajtákon validáltuk. Kidolgoztunk egy real-time PCR módszert a Glu-1Bx géndózisának meghatározására és meghatároztuk a Glu-1Bx géndózisát 8 ismert külföldi (ebből 6 Bx7 túltermelő) búza fajtában és 3 régi magyar búza fajtában. | The promoter of the Glu-1Bx gene was cloned and sequenced from two international (a Bx normal- and a Bx over-expressing) whaet varieties and three Hungarian (Bx under-, normal- and over-expressing) wheat varieties. In the promoter of the overexpressing varieties a 43 bp insertion was found, which is absent from the promoters of the normal- and under-expressing varieties. We have developped a codominant PCR marker for the identification of this 43 bp insertion. The marker was validated on well known international wheat varieties. We have cloned and sequenced the coding region of the Glu-1Bx gene from two Hungarian varieties (normal-expressing Bx7* allel and over-expressing Bx7 allel). At the 3' end of the coding region of the Bx7 allel a 18 bp insertion was found, which is not present in the Bx7* allel. We have developped a codominant PCR marker for the identification of this 18 bp insertion and validated this marker on well known international varieties. We have developped a real-time PCR method for measuring the copy number of the Glu-1Bx gene and determined the Glu-1Bx gene dosage in 8 international 3 Hungarian wheat varieties

Improving initial xylose metabolism in recombinant Saccharomyces cerevisiae

The aim of the thesis is to improve the initial steps of xylose metabolism in recombinant Saccharomyces cerevisiae. S. cerevisiae takes up xylose of poor affinity by means of hexose transporters. Metabolic control analysis was used to investigate whether the low xylose utilisation rate is due to inefficient transport. Xylose transport was found to have little control over the aerobic xylose utilisation rate in strain TMB3001, due to its high steady-state intracellular xylose concentration. In strain TMB3260, due to the overexpression of xylose reductase (XR), the intracellular xylose concentration was lower than in TMB3001 and transport had stronger control over the xylose flux at low extracellular xylose concentrations. Candida intermedia PYCC4715 was identified as being a yeast very efficient in utilising xylose. Kinetic characterisation of the xylose transport revealed the presence of a low-affinity facilitated diffusion system and a high-affinity proton symport system. The former appeared to be constitutive, whereas the latter was repressed by glucose and xylose. Both systems were of high capacity compared with known transport systems from other xylose utilising yeasts, which may facilitate cloning in S. cerevisiae. The conversion of xylose to xylulose by XR and xylitol dehydrogenase (XDH) leads to an imbalance in cofactor regeneration. This, in turn, results in the excretion of xylitol by xylose-utilising recombinant S. cerevisiae. Xylitol production can be circumvented by substituting xylose isomerase (XI) for XR and XDH. XI from Streptomyces rubiginosus was expressed inactively in S. cerevisiae due to misfolding of the protein. XI from Thermus thermophilus is actively expressed in S. cerevisiae, but possesses insufficient activity at 30°C. Three mutants were isolated after error-prone PCR and selection in Escherichia coli. Although the catalytic rate constants were 5-9 fold as high for the mutants as for the wild type enzyme, but the catalytic efficiency was not improved significantly. The mutats have lower sensitivity to inhibition by xylitol, which makes them especially suitable for expression in S. cerevisiae

The Streptomyces rubiginosus xylose isomerase is missfolded when expressed in Saccharomyces cerevisiae.

The Streptomyces rubiginosus xylA gene was cloned and expressed in Saccharomyces cerevisiae. No xylose isomerase activity could be detected. The produced xylose isomerase protein was insoluble and could only be recovered from cell lysates by extraction with the detergent sodium dodecyl-sulfate. In contrast, expression of the xylA gene from Thermus thermophilus in the same host strain resulted in soluble xylose isomerase protein with activities of 1 U mg−1 protein. Comparison of available 3D models suggests that the higher number of intra-subunit ion-bridges in the Thermus thermophilus xylose isomerase may stabilise the protein structure and promote folding by Saccharomyces cerevisiae

Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization

For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important. As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed. After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost. Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Ga12 are important xylose-transporting proteins. Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose. Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions. The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures

High capacity xylose transport in Candida intermedia PYCC4715.

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (small mu, Greekmax=0.5 h−1, 30°C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km=0.2 mM, Vmax=7.5 mmol h−1 g−1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K=50 mM, Vmax=11 mmol h−1 g−1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization. We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C, for the xylose transport was calculated from the response and elasticity coefficients. The value of C for both strains was found to be 7.5 g L-1. However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L-1, while C stayed below 0.2 for strain TMB3001 irrespective of xylose concentration. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 818-824, 2003

Metabolic engineering of Saccharomyces cerevisiae for xylose utilization.

Metabolic engineering of Saccharomyces cerevisiae for ethanolic fermentation of xylose is summarized with emphasis on progress made during the last decade. Advances in xylose transport, initial xylose metabolism, selection of host strains, transformation and classical breeding techniques applied to industrial polyploid strains as well as modeling of xylose metabolism are discussed. The production and composition of the substrates--lignocellulosic hydrolysates--is briefly summarized. In a future outlook iterative strategies involving the techniques of classical breeding, quantitative physiology, proteomics, DNA micro arrays, and genetic engineering are proposed for the development of efficient xylose-fermenting recombinant strains of S. cerevisiae