Susanne L. Jensen

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules

BackgroundStarch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.ResultsIn this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.ConclusionsThis is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Characterization of the Sulfolobus host-SSV2 virus interaction.

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus–host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines.

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO(2), ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO(2) in comparison to the other strains of Dekkera. These strains were inoculated (10(3)-10(4)cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 degrees C and 21 degrees C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10(5)cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 degrees C while slower growth was observed at 15 degrees C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 degrees C compared to 21 degrees C.

Future Cereal Starch Bioengineering: Cereal Ancestors Encounter Gene Technology and Designer Enzymes

ABSTRACT The importance of cereal starch production worldwide cannot be overrated. However, the qualities and resulting values of existing raw and processed starch do not fully meet future demands for environmentally friendly production of renewable, advanced biomaterials, functional foods, and biomedical additives. New approaches for starch bioengineering are needed. In this review, we discuss cereal starch from a combined universal bioresource point of view. The combination of new biotechniques and clean technology methods can be implemented to replace, for example, chemical modification. The recently released cereal genomes and the exploding advancement in whole genome sequencing now pave the road for identifying new genes to be exploited to generate a multitude of completely new starch functionalities directly in the cereal grain, converting cereal crops to production plants. Newly released genome data from cereal ancestors can potentially allow for the reintroduction of cereal traits including, for e...

Structural and physical properties of granule stabilized starch obtained by branching enzyme treatment

Chemical cross-linking of starch is an important modification used in the industry for granule stabilization. It has been demonstrated that treatment with branching enzyme (BE) can stabilize the granular structure of starch and such treatment thereby provides a potential clean alternative for chemical modification. This study demonstrates that such BE-assisted stabilization of starch granules led to partial protection from BE catalysis of both amylose (AM) and amylopectin (AP) in their native state as assessed by triiodide complexation, X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). The granule stabilizing effects were inversely linked to hydration of the starch granules, which was increased by the presence of starch-phosphate esters and suppressed by extreme substrate concentration. The data support that the granule stabilization is due to the intermolecular transglycosylation occurring in the initial stages of the reaction prior to AM-AP phase separation. The enzyme activity needed to obtain granule stabilization was therefore dependent on the hydration capability of the starch used.

The deposition and characterization of starch in Brachypodium distachyon

Highlight text A thorough study of starch biosynthesis and deposition in a non-domesticated wild grass was performed using Brachypodium distachyon as a model.

Levanase from Bacillus subtilis hydrolyses β-2,6 fructosyl bonds in bacterial levans and in grass fructans.

A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3 U mg(-1) using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (β-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.