The branching ratio of enzymatically synthesized α-glucans impacts microbiome and metabolic outcomes of in vitro fecal fermentation.

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon.

Characterization and applications of biomacromolecule structurally similar to glycogen as a dispersion aid and skin protection agent.

Glycogen is a naturally occurring or metabolically synthesized biological macromolecule found in a wide range of living organisms, including animals, microorganisms, and even plants. However, naturally sourced glycogen poses challenges for industrial use. This study focused on a biological macromolecule referred to as glycogen-like particles (GLPs), detailing the production methods and biological properties of these particles. In vitro enzymatic production of GLPs was successfully achieved. GLPs synthesized through a simultaneous enzymatic reaction using sucrose had significant changes in their structure and functionality based on the branching enzyme (BE) to amylosucrase (ASase) ratio. As this ratio increased, the GLPs developed higher molecular weights and greater density, solubility, and branching degree while reducing size and turbidity. Structural changes in these enzymes were not observed beyond a critical BE/ASase ratio. Uniformly dispersed curcumin powder was generated in 50 % (w/v) aqueous GLP solution, and the GLPs were non-toxic to human skin keratinocytes at a concentration of 2.5 mg/mL. GLPs with lower branching inhibited tyrosinase activity and melanin synthesis, while those with more long chains displayed effective UV-blocking. By manipulating the BE/ASase ratio, GLPs were shown to display diverse chemical structures and physical characteristics, suggesting their potential application in the food and cosmetics industries.

GBSS mutations in an SBE mutated background restore the potato starch granule morphology and produce ordered granules despite differences to native molecular structure.

Potato starch with mutations in starch branching enzyme genes (SBEI, SBEII) and granule-bound starch synthase gene (GBSS) was characterized for molecular and thermal properties. Mutations in GBSS were here stacked to a previously developed SBEI and SBEII mutation line. Additionally, mutations in the GBSS gene alone were induced in the wild-type variety for comparison. The parental line with mutations in the SBE genes showed a âˆ¼ 40 % increase in amylose content compared with the wild-type. Mutations in GBSS-SBEI-SBEII produced non-waxy, low-amylose lines compared with the wild-type. An exception was a line with one remaining GBSS wild-type allele, which displayed ∼80 % higher amylose content than wild-type. Stacked mutations in GBSS in the SBEI-SBEII parental line caused alterations in amylopectin chain length distribution and building block size categories of whole starch. Correlations between size categories of building blocks and unit chains of amylopectin were observed. Starch in GBSS-SBEI-SBEII mutational lines had elevated peak temperature of gelatinization, which was positively correlated with large building blocks.

Mechanism of action of three different glycogen branching enzymes and their effect on bread quality.

Bread staling adversely affects the quality of bread, but starch modification by enzymes can counteract this phenomenon. Glycogen branching enzymes (GBEs) used in this study were isolated from Deinococcus geothermalis (DgGBE), Escherichia coli (EcGBE), and Vibrio vulnificus (VvGBE). These enzymes were characterized and applied for starch dough modification to determine their role in improving bread quality. First, the branching patterns, activity on amylose and amylopectin, and thermostability of the GBEs were determined and compared. EcGBE and DgGBE exhibited better thermostable characteristics than VvGBE, and all GBEs exhibited preferential catalysis of amylopectin over amylose but different degrees. VvGBE and DgGBE produced a large number of short branches. Three GBEs degraded the starch granules and generated soluble polysaccharides. Moreover, the maltose was increased in the starch slurry but most significantly in the DgGBE treatment. Degradation of the starch granules by GBEs enhanced the maltose generation of internal amylases. When used in the bread-making process, DgGBE and VvGBE increased the dough and bread volume by 9 % and 17 %, respectively. The crumb firmness and retrogradation of the bread were decreased and delayed significantly more in the DgGBE bread. Consequently, this study can contribute to understanding the detailed roles of GBEs in the baking process.

Improving the thermal stability and branching efficiency of Pyrococcus horikoshii OT3 glycogen branching enzyme.

In practical applications, the gelatinisation temperature of starch is high. Most current glycogen branching enzymes (GBEs, EC 2.4.1.18) exhibit optimum activity at moderate or low temperatures and quickly lose their activity at higher temperatures, limiting the application of GBEs in starch modification. Therefore, we used the PROSS strategy combined with PDBePISA analysis of the dimer interface to further improve the heat resistance of hyperthermophilic bacteria Pyrococcus horikoshii OT3 GBE. The results showed that the melting temperature of mutant T508K increased by 3.1 °C compared to wild-type (WT), and the optimum reaction temperature increased by 10 °C for all mutants except V140I. WT almost completely lost its activity after incubation at 95 °C for 60 h, while all of the combined mutants maintained >40 % of their residual activity. Further, the content of the α-1,6 glycosidic bond of corn starch modified by H415W and V140I/H415W was approximately 2.68-fold and 1.92-fold higher than that of unmodified corn starch and corn starch modified by WT, respectively. Additionally, the glucan chains of DP < 13 were significantly increased in mutant modified corn starch. This method has potential for improving the thermal stability of GBE, which can be applied in starch branching in the food industry.

Designing a Specific Pretreatment on Corn Starch to Facilitate Enzymatic Rearrangement of Glycosidic Bonds for Efficiently Reducing Starch Digestibility.

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.

A model for the reproduction of amylopectin cluster by coordinated actions of starch branching enzyme isoforms.

Amylopectin is a highly branched glucan which accounts for approximately 65-85% of starch in most plant tissues. It is crucially important to understand the biosynthetic process of this glucan in regulating the structure and functional properties of starch granules. Currently, the most accepted ideas of structural feature and biosynthesis of amylopectin are that amylopectin is composed of a branched element called "cluster" and that the essential process of amylopectin biosynthesis is to reproduce a new cluster from the existing cluster. The present paper proposes a model explaining the whole process of amylopectin biosynthesis as to how the new cluster is reproduced by concerted actions of multiple isoforms of starch biosynthetic enzymes, particularly by combinations of distinct roles of starch branching enzyme (BE) isoforms. This model proposes for the first time the molecular mechanism as to how the formation of a new cluster is initiated, and the reason why BEI can play a major role in this step. This is because BEI has a rather broad chain-length preference compared to BEIIb, because a low preference of BEI for the substrate chain-length is advantageous for branching a couple of elongated chains that are not synchronously formed and thus these chains having varied lengths could be safely attacked by this isoform. On the contrary, it is unlikely that BEIIb is involved in this reaction because it can react to only short chains having degree of polymerization of 12-14. BEIIa is possibly able to complement the role of BEI to some extent, because BEIIa can attack basically short chains but its chain-length preference is lower compared with BEIIb. The model implies that the first branches mainly formed by BEI to construct the amorphous lamellae whereas the second branches predominantly formed by BEIIb are located mainly in the crystalline lamellae. This paper provides new insights into the roles of BEI, BEIIb, and BEIIa in amylopectin biosynthesis in cereal endosperm.

The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

Stochastic modelling of a three-dimensional glycogen granule synthesis and impact of the branching enzyme.

In humans, glycogen storage diseases result from metabolic inborn errors, and can lead to severe phenotypes and lethal conditions. Besides these rare diseases, glycogen is also associated to widely spread societal burdens such as diabetes. Glycogen is a branched glucose polymer synthesised and degraded by a complex set of enzymes. Over the past 50 years, the structure of glycogen has been intensively investigated. Yet, the interplay between the detailed three-dimensional glycogen structure and the related enzyme activity is only partially characterised and still to be fully understood. In this article, we develop a stochastic coarse-grained and spatially resolved model of branched polymer biosynthesis following a Gillespie algorithm. Our study largely focusses on the role of the branching enzyme, and first investigates the properties of the model with generic parameter values, before comparing it to in vivo experimental data in mice. It arises that the ratio of glycogen synthase over branching enzyme reaction rates drastically impacts the structure of the granule. We deeply investigate the mechanism of branching and parametrise it using distinct lengths. Not only do we consider various possible sets of values for these lengths, but also distinct rules to apply them. We show how combining various values for these lengths finely tunes glycogen macromolecular structure. Comparing the model with experimental data confirms that we can accurately reproduce glycogen chain length distributions in wild type mice. Additional granule properties obtained for this fit are also in good agreement with typically reported values in the experimental literature. Nonetheless, we find that the mechanism of branching must be more flexible than usually reported. Overall, our model provides a theoretical basis to quantify the effect that single enzymatic parameters, in particular of the branching enzyme, have on the chain length distribution. Our generic model and methods can be applied to any glycogen data set, and could in particular contribute to characterise the mechanisms responsible for glycogen storage disorders.

Genetic Diversification of Starch Branching Enzymes during Maize Domestication and Improvement.

Elucidating the genetic basis of starch pasting and gelatinization properties is crucial for enhancing the quality of maize and its utility as feed and industrial raw material. In maize, ZmSBE genes encode important starch branching enzymes in the starch biosynthesis pathway. In this study, we re-sequenced the genomic sequences of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII in three lines called 335 inbred lines, 68 landrace lines, and 32 teosinte lines. Analyses of nucleotide polymorphisms and haplotype diversity revealed differences in the selection patterns of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII during maize domestication and improvement. A marker-trait association analysis of inbred lines detected 22 significant loci, including 18 SNPs and 4 indels significantly associated with three maize starch physicochemical properties. The allele frequencies of two variants (SNP17249C and SNP5055G) were examined in three lines. The frequency of SNP17249C in ZmSBEIIb was highest in teosinte lines, followed by landrace lines, and inbred lines, whereas there were no significant differences in the frequency of SNP5055G in ZmSBEIII among the three lines. These results suggest that ZmSBE genes play an important role in the phenotypic variations in the starch physicochemical properties in maize. The genetic variants detected in this study may be used to develop functional markers for improving maize starch quality.

Biosynthesis of maltodextrin-derived glucan dendrimer using microbial branching enzyme.

Glucan dendrimers were developed with microbial branching enzyme (BE) treated maltodextrin. The molecular weight (Mw) of recombinant BE was 79.0 kDa, and its optimum activity was observed at pH 7.0 and 70 °C. BE converted different maltodextrins with dextrose equivalent value of 6 (MD6), 12 (MD12), or 19 (MD19) into the given glucan dendrimers, along with the marked increment of the molecular density (approximately 30-60 folds) and α-1,6 linkage percentage (up to 7.3-9.7%). Among three glucan dendrimers, the enzyme-treated MD12 showed a more homogeneous Mw distribution with the maximum Mw of 5.5 × 106 g/mol, indicating that higher substrate catalytic specificity of BE for MD12 substrate. During transglycosylation with MD12 for 24 h, the shorter chains (degree of polymerization, DP < 13) increased from 73.9% to 83.0%, accompanying by a reduction of medium chains (DP13-24) and long chains (DP > 24). Moreover, the slowly digestible and resistant nutritional fractions were increased by 6.2% and 12.5%, respectively. The results suggested that the potentiality of BE structuring glucan dendrimer with tailor-made structure and functionality for industrial application.

Effect of modification by maltogenic amylase and branching enzyme on the structural and physicochemical properties of sweet potato starch.

Sweet potato starch (SPSt) was treated sequentially with the combination of maltogenic amylase (MA) and branching enzyme (BE) (MA â†’ BE) or BE and MA (BE→MA) to modify its structural and physicochemical properties. Following the MA â†’ BE and BE→MA modifications, the degree of branching was increased from 12.02 % to 44.06 %; whereas, the average chain length (ACL) decreased from 18.02 to 12.32. Fourier-transform infrared spectroscopy and digestive performance analysis indicated that the modifications reduced hydrogen bonds and increased resistant starch in SPSt. Rheological analysis revealed that the storage and loss moduli of the modified samples were lower than those of the control samples, except for starch treated with MA alone. X-ray diffraction measurements suggested that the re-crystallisation peak intensities of the enzyme-modified starches were lower than those of the untreated sample. The retrogradation resistance ability of the analysed samples followed the order: BE→MA-starches > MA â†’ BE-starches > untreated starch. The relationship between the crystallisation rate constant and short branched chains (DP6-9) was well described by linear regression. This study provides a theoretical foundation for retarding the retrogradation of starch, which can improve food quality and extend the shelf-life of enzymatically modified starchy foods.

Biomimetic synthesis of maltodextrin-derived dendritic nanoparticle and its structural characterizations.

The maltodextrin-derived dendritic nanoparticle was fabricated using microbial branching enzyme and its structural characterizations were investigated. During biomimetic synthesis, molecular weight distribution of maltodextrin substrate with 6.8 × 104 g/mol shifted to the narrower and uniform distribution region with the larger molecular weight up to 6.3 × 106 g/mol (MD12). The enzyme-catalyzed product had the larger size, higher molecular density as well as higher percentage of α-1,6 linkage, accompanying by more chain accumulations of DP 6-12 and disappearance of DP > 24, suggesting the biosynthesized glucan dendrimer had a compact tighter branched structure. The interaction of molecular rotor CCVJ and local structure of dendrimer was monitored, displaying there was a higher intensity related with the numerous nano-pockets at the branch points of MD12. The maltodextrin-derived dendrimers had the single spherical particulate shape with the size range of 10-90 nm. The mathematical models were also established to reveal the chain structuring during enzymatic reaction. The above results showed that the biomimetic strategy for novel dendritic nanoparticle with controllable structure arising from branching enzyme treated maltodextrin, which would help to enlarge the panel of available dendrimer.

Deciphering the structural and functional characteristics of an innovative small cluster branched α-glucan produced by sequential enzymatic synthesis.

Highly branched α-glucan (HBAG) proved to be a promising material as an osmotic agent in peritoneal dialysis solutions. However, high resistance of HBAG to amylolytic enzymes might be a potential drawback for peritoneal dialysis due to its high degree of branching (20-30 %). To address this issue, we designed a small-clustered α-glucan (SCAG) with a relatively low molecular weight (Mw) and limited branching. Structural characteristics revealed that SCAG was successfully synthesized by modifying waxy rice starch (WRS) using sequential maltogenic α-amylase (MA) and starch branching enzyme (BE). The Mw of SCAG was 1.40 × 105 Da, and its (α1 â†’ 6) bonds ratio was 8.93 %, which was below that of HBAG. A relatively short branch distribution was observed in SCAG (CL = 6.27). Short-range orderliness of WRS was reduced from 0.749 to 0.322 with the MABE incubation. Additionally, SCAG had an extremely low viscosity (~12 cP) and nearly no retrogradation. Although the resistance of SCAG to amylolytic enzymes was enhanced by 15.22 % compared with native WRS, the extent was significantly lower than that of HBAG in previous studies. These new findings demonstrate the potential of SCAG as a novel functional α-glucan in food and pharmaceutical applications.

Production of highly branched α-limit dextrins with enhanced slow digestibility by various glycogen-branching enzymes.

α-Limit dextrins (α-LDx) are slowly digestible carbohydrates that attenuate postprandial glycemic response and trigger the secretion of satiety-related hormones. In this study, more highly branched α-LDx were enzymatically synthesized to enhance the slowly digestible property by various origins of glycogen branching enzyme (GBE), which catalyzes the transglycosylation to form α-1,6 branching points after cleaving α-1,4 linkages. Results showed that the proportion of branched α-LDx in starch molecules increased around 2.2-8.1 % compared to α-LDx from starch without GBE treatment as the ratio of α-1,6 linkages increased after different types of GBE treatments. Furthermore, the enzymatic increment of branching points enhanced the slowly digestible properties of α-LDx at the mammalian α-glucosidase level by 17.3-28.5 %, although the rates of glucose generation were different depending on the source of GBE treatment. Thus, the highly branched α-LDx with a higher amount of α-1,6 linkages and a higher molecular weight can be applied as a functional ingredient to deliver glucose throughout the entire small intestine without a glycemic spike which has the potential to control metabolic diseases such as obesity and type 2 diabetes.

Production of branched glucan polymer by a novel thermostable branching enzyme of Bifidobacterium thermophilum via one-pot biosynthesis containing a dual enzyme system.

Glycogen-like particles (GLPs) are applied in food, pharmaceutical, and cosmetics. The large-scale production of GLPs is limited by their complicated multi-step enzymic processes. In this study, GLPs were produced in a one-pot dual-enzyme system using Bifidobacterium thermophilum branching enzyme (BtBE) and Neisseria polysaccharea amylosucrase (NpAS). BtBE showed excellent thermal stability (half-life of 1732.9 h at 50 °C). Substrate concentration was the most influential factor during GLPs production in this system: GLPs yield and [sucrose]ini decreased from 42.4 % to 17.4 % and 0.3 to 1.0 M, respectively. Molecular weight and apparent density of GLPs decreased significantly with increasing [sucrose]ini. Regardless of the [sucrose]ini, the DP 6 of branch chain length was predominantly occupied. GLP digestibility increased with increasing [sucrose]ini, indicating that the degree of GLP hydrolysis may be negatively related to its apparent density. This one-pot biosynthesis of GLPs using a dual-enzyme system could be useful for the development of industrial processes.

Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product.

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).

Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria.

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

Efficient Accumulation of Amylopectin and Its Molecular Mechanism in the Submerged Duckweed Mutant.

Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant (sub-1) with abundant starch accumulation was obtained, whose content of amylopectin accounts for 84.04% of the starch granules. Compared with the wild type (Lemna aequinoctialis), the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6-12, DP 25-36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13-24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated ß-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.

Increasing the level of resistant starch in 'Presidio' rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes.

Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.