Amy Gene Research Articles

High AMY1 copy number protects against obesity in Portuguese young adults

Background: The highly polymorphic copy number variation (CNV) in the salivary amylase gene (AMY1) has been associated with obesity in different populations. However, some authors have failed to reproduce these findings.Aim: To investigate the association between AMY1 CNV and obesity in young adults of Portuguese origin.Subjects and methods: This study evaluated AMY1 gene copy number (CN) in 262 individuals: 155 females and 107 males, aged 18–34 years-old (mean age = 21.08). The number of AMY1 copies was estimated in a QX100 droplet digital PCR (ddPCR) system (Bio-Rad Laboratories, Hercules, CA).Results: Defining a case group with obese and overweight individuals, logistic regression did not show a significant association between AMY1 CNV and risk of overweight/obesity in the whole population (p = 0.489). However, after testing case-control data in the sub-set of samples above the third quartile (CN ≥10), a significant association was found between lower AMY1 copy number and risk of obesity (OR = 0.532; p = 0.034), even when adjusted for age and sex (OR = 0.527; p = 0.039). In concordance, all participants with >10 AMY1 copies were normal weight controls (n = 20) or overweight (n = 6).Conclusion: The results suggest that high AMY1 gene copy number protects against obesity in Portuguese young adults.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Altered Salivary Alpha-Amylase Secretion in Patients with Ulcerative Colitis.

Purpose Patients with ulcerative colitis (UC) frequently present with psychological disturbances as well as dysfunctions of autonomic nervous system (ANS). Salivary alpha-amylase (sAA) secretion is predominantly controlled by sympathetic nervous activity, while salivary fluid secretion is by parasympathetic nervous activity. Thus, it is speculated that alterations of salivary secretion may be addressed in UC populations. Methods Thirty-five UC patients as well as 32 age- and sex-matched healthy controls were enrolled. Saliva samples before and after citric acid stimulation were collected from each participant, and salivary flow rate (SFR) was calculated accordingly. Western blotting and quantitative PCR were applied to measure the sAA level and sAA gene (AMY1) copy number, respectively. The psychological disorders, anxiety and depression, were evaluated by the scoring system of Hospital Anxiety and Depression Scale (HADS) for each participant. Results We observed robustly increased prevalence of anxiety (p < 0.001) as well as depression (p < 0.001) in UC patients relative to controls. Interestingly, we detected elevated basal (p = 0.015) and stimulated (p = 0.021) sAA levels in the UC populations compared to controls. However, no differences were found for basal (p = 0.643) or stimulated (p = 0.402) SFR between the two study groups. Besides, AMY1 gene copy number was comparable between UC patients and controls. Conclusions Our results reveal an overactivity of the sympathetic nervous system and a normal activity of the parasympathetic nervous system in the UC population.

The Amylases of Insects.

Alpha-amylases are major digestive enzymes that act in the first step of maltopolysaccharide digestion. In insects, these enzymes have long been studied for applied as well as purely scientific purposes. In many species, amylases are produced by multiple gene copies. Rare species are devoid of Amy gene. They are predominantly secreted in the midgut but salivary expression is also frequent, with extraoral activity. Enzymological parameters are quite variable among insects, with visible trends according to phylogeny: Coleopteran amylases have acidic optimum activity, whereas dipteran amylases have neutral preference and lepidopteran ones have clear alkaline preference. The enzyme structure shows interesting variations shaped by evolutionary convergences, such as the recurrent loss of a loop involved in substrate handling. Many works have focused on the action of plant amylase inhibitors on pest insect amylases, in the frame of crop protection by transgenesis. It appears that sensitivity or resistance to inhibitors is finely tuned and very specific and that amylases and their inhibitors have coevolved. The multicopy feature of insect amylases appears to allow tissue-specific or stage-specific regulation, but also to broaden enzymological abilities, such as pH range, and to overcome plant inhibitory defenses.

Comparisons of Copy Number, Genomic Structure, and Conserved Motifs for α-Amylase Genes from Barley, Rice, and Wheat.

Barley is an important crop for the production of malt and beer. However, crops such as rice and wheat are rarely used for malting. α-amylase is the key enzyme that degrades starch during malting. In this study, we compared the genomic properties, gene copies, and conserved promoter motifs of α-amylase genes in barley, rice, and wheat. In all three crops, α-amylase consists of four subfamilies designated amy1, amy2, amy3, and amy4. In wheat and barley, members of amy1 and amy2 genes are localized on chromosomes 6 and 7, respectively. In rice, members of amy1 genes are found on chromosomes 1 and 2, and amy2 genes on chromosome 6. The barley genome has six amy1 members and three amy2 members. The wheat B genome contains four amy1 members and three amy2 members, while the rice genome has three amy1 members and one amy2 member. The B genome has mostly amy1 and amy2 members among the three wheat genomes. Amy1 promoters from all three crop genomes contain a GA-responsive complex consisting of a GA-responsive element (CAATAAA), pyrimidine box (CCTTTT) and TATCCAT/C box. This study has shown that amy1 and amy2 from both wheat and barley have similar genomic properties, including exon/intron structures and GA-responsive elements on promoters, but these differ in rice. Like barley, wheat should have sufficient amy activity to degrade starch completely during malting. Other factors, such as high protein with haze issues and the lack of husk causing Lauting difficulty, may limit the use of wheat for brewing.

Divergent taxonomic and functional responses of microbial communities to field simulation of aeolian soil erosion and deposition.

Aeolian soil erosion and deposition have worldwide impacts on agriculture, air quality and public health. However, ecosystem responses to soil erosion and deposition remain largely unclear in regard to microorganisms, which are the crucial drivers of biogeochemical cycles. Using integrated metagenomics technologies, we analysed microbial communities subjected to simulated soil erosion and deposition in a semiarid grassland of Inner Mongolia, China. As expected, soil total organic carbon and plant coverage were decreased by soil erosion, and soil dissolved organic carbon (DOC) was increased by soil deposition, demonstrating that field simulation was reliable. Soil microbial communities were altered (p<.039) by both soil erosion and deposition, with dramatic increase in Cyanobacteria related to increased stability in soil aggregates. amyA genes encoding α-amylases were specifically increased (p=.01) by soil deposition and positively correlated (p=.02) to DOC, which likely explained changes in DOC. Surprisingly, most of microbial functional genes associated with carbon, nitrogen, phosphorus and potassium cycling were decreased or unaltered by both erosion and deposition, probably arising from acceleration of organic matter mineralization. These divergent responses support the necessity to include microbial components in evaluating ecological consequences. Furthermore, Mantel tests showed strong, significant correlations between soil nutrients and functional structure but not taxonomic structure, demonstrating close relevance of microbial function traits to nutrient cycling.

Starch Digestion-Related Amylase Genetic Variant Affects 2-Year Changes in Adiposity in Response to Weight-Loss Diets: The POUNDS Lost Trial.

Salivary and pancreatic amylases (encoded by AMY1 and AMY2 genes, respectively) are responsible for digesting starchy foods. AMY1 and AMY2 show copy number variations that affect differences in amylase amount and activity, and AMY1 copies have been associated with adiposity. We investigated whether genetic variants determining amylase gene copies are associated with 2-year changes in adiposity among 692 overweight and obese individuals who were randomly assigned to diets varying in macronutrient content. We found that changes in body weight (BW) and waist circumference (WC) were significantly different according to the AMY1-AMY2 rs11185098 genotype. Individuals carrying the A allele (indicating higher amylase amount and activity) showed a greater reduction in BW and WC at 6, 12, 18, and 24 months than those without the A allele (P < 0.05 for all). The association was stronger for long-term changes compared with short-term changes of these outcomes. The genetic effects on these outcomes did not significantly differ across diet groups. In conclusion, the genetic variant determining starch metabolism influences the response to weight-loss dietary intervention. Overweight and obese individuals carrying the AMY1-AMY2 rs11185098 genotype associated with higher amylase activity may have greater loss of adiposity during low-calorie diet interventions.

Genetic variation in the AMY1 gene is associated with dietary carbohydrate and starch intake in a young adult population

BackgroundGenetic variation contributes to individual differences in energy and nutrient intake and may be a contributing factor to the selection of certain foods and macronutrient preference. Salivary amylase is encoded by the AMY1 gene, which displays high copy number variation (CNV) and plays an important role in starch digestion. Research has found that AMY1 CNV is positively correlated with both salivary amylase concentration and activity and higher AMY1 copy numbers have been found in populations with high starch diets. Individual single nucleotide polymorphisms (SNPs) near the amylase genes have been found to correlate with AMY1 copy number within populations. The objective of this study was to determine whether common variants in the AMY1 gene are associated with habitual dietary intake.MethodsFasting blood samples were drawn from a total of 1,600 ethno‐culturally diverse participants aged 20–29 years from the Toronto Nutrigenomics and Health Study for genotyping of 9 SNPs associated with AMY1 copy number: rs4244372 (T/A), rs11577390 (C/T), rs1566154 (A/G), rs10881197(G/C), rs2132957 (A/G), rs11185098(G/A), rs1999478(C/A), rs1330403(A/G) and rs6696797(A/G). Dietary intake was assessed using a one‐month, 196‐item Toronto‐modified Willett food frequency questionnaire. An analysis of covariance adjusted for age, sex, ethnicity, BMI, and physical activity was used to determine the association between AMY1 genotypes and dietary intake.ResultsAmong Caucasians (n = 620), carriers of the minor allele in rs10881197 had a significantly lower energy intake (1838 vs 2089 kcal/day, p = 0.005). Carriers of the minor allele in rs11185098 had significantly higher total carbohydrate (CHO) and starch intakes (273 vs 266 g CHO/day, p =0.03 and 122 vs 117g starch/day, p= 0.02 respectively). Among East Asians (n = 465), carriers of the minor allele in rs1999478 had significantly higher total energy and sugar intakes (1985 vs 1828 kcal/day, p = 0.02 and 113 vs 105 g sugar/day, p = 0.03 respectively). No other associations were observed between AMY1 variants and carbohydrate or starch intake.ConclusionPolymorphisms in the AMY1 gene are associated with dietary intake patterns. Identifying the genetic determinants of dietary intake and food preference may enhance our understanding of excessive caloric intake and obesity, a common risk factor of many chronic conditions and provide further evidence to support the use of personalized nutrition recommendations.Support or Funding InformationResearch support from Mitacs

High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system

BackgroundWe recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, PamyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536.ResultsUsing the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the PamyQ′ promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter PHpaII–PamyQ′ produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter PHpaII–PamyQ′ also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter PHpaII–PamyQ′ system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications.ConclusionsThe dual-promoter PHpaII–PamyQ′ system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

Copy number variation of human AMY1 is a minor contributor to variation in salivary amylase expression and activity

BackgroundSalivary amylase in humans is encoded by the copy variable gene AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes.ResultsAMY1 copy number was significantly correlated with the variation observed in salivary amylase production (11.7% of variance, P < 0.0005) and enzyme activity (13.6% of variance, P < 0.0005) but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052).ConclusionsProduction of salivary amylase is correlated with AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

IntroductionThe human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual.MethodsSeven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods.ResultsWe found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples.ConclusionsDespite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Lineage-Specific Evolutionary Histories and Regulation of Major Starch Metabolism Genes during Banana Ripening.

Starch is the most widespread and abundant storage carbohydrate in plants. It is also a major feature of cultivated bananas as it accumulates to large amounts during banana fruit development before almost complete conversion to soluble sugars during ripening. Little is known about the structure of major gene families involved in banana starch metabolism and their evolution compared to other species. To identify genes involved in banana starch metabolism and investigate their evolutionary history, we analyzed six gene families playing a crucial role in plant starch biosynthesis and degradation: the ADP-glucose pyrophosphorylases (AGPases), starch synthases (SS), starch branching enzymes (SBE), debranching enzymes (DBE), α-amylases (AMY) and β-amylases (BAM). Using comparative genomics and phylogenetic approaches, these genes were classified into families and sub-families and orthology relationships with functional genes in Eudicots and in grasses were identified. In addition to known ancestral duplications shaping starch metabolism gene families, independent evolution in banana and grasses also occurred through lineage-specific whole genome duplications for specific sub-families of AGPase, SS, SBE, and BAM genes; and through gene-scale duplications for AMY genes. In particular, banana lineage duplications yielded a set of AGPase, SBE and BAM genes that were highly or specifically expressed in banana fruits. Gene expression analysis highlighted a complex transcriptional reprogramming of starch metabolism genes during ripening of banana fruits. A differential regulation of expression between banana gene duplicates was identified for SBE and BAM genes, suggesting that part of starch metabolism regulation in the fruit evolved in the banana lineage.

PheS * , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens.

Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS * was constructed through a point mutation of the gene pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5mM p-chloro-phenylalanine, 100% of B. amyloliquefaciens strain SQR9 cells carrying pheS * were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS * and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS * as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS * in other Bacillus species was preliminarily assessed. The expression of PheS* in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS * could be used as a counter-selectable marker (CSM) in these strains.

Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering.

Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera.

A Phosphate Starvation-Inducible Ribonuclease of Bacillus licheniformis.

The BLi03719 protein of Bacillus licheniformis DSM13 belongs to the most abundant extracellular proteins under phosphate starvation conditions. In this study, the function of this phosphate starvation inducible protein was determined. An amino-acid sequence analysis of the BLi03719-encoding gene showed a high similarity with genes encoding the barnase of Bacillus amyloliquefaciens FZB42 and binase-like RNase of Bacillus pumilus SARF-032. The comparison of the control strain and a BLi03719-deficient strain revealed a strongly reduced extracellular ribonuclease activity of the mutant. Furthermore, this knockout mutant exhibited delayed growth with yeast RNA as an alternative phosphate and carbon source. These results suggest that BLi03719 is an extracellular ribonuclease expressed in B. licheniformis under phosphate starvation conditions. Finally, a BLi03719 mutant showed an advantageous effect on the overexpression of the heterologous amyE gene under phosphate-limited growth conditions.

Metabolomic Profile of Low-Copy Number Carriers at the Salivary α-Amylase Gene Suggests a Metabolic Shift Toward Lipid-Based Energy Production.

Low serum salivary amylase levels have been associated with a range of metabolic abnormalities, including obesity and insulin resistance. We recently suggested that a low copy number at the AMY1 gene, associated with lower enzyme levels, also increases susceptibility to obesity. To advance our understanding of the effect of AMY1 copy number variation on metabolism, we compared the metabolomic signatures of high- and low-copy number carriers. We analyzed, using mass spectrometry and nuclear magnetic resonance (NMR), the sera of healthy normal-weight women carrying either low-AMY1 copies (LAs: four or fewer copies; n = 50) or high-AMY1 copies (HAs: eight or more copies; n = 50). Best-fitting multivariate models (empirical P < 1 × 10-3) of mass spectrometry and NMR data were concordant in showing differences in lipid metabolism between the two groups. In particular, LA carriers showed lower levels of long- and medium-chain fatty acids, and higher levels of dicarboxylic fatty acids and 2-hydroxybutyrate (a known marker of glucose malabsorption). Taken together, these observations suggest increased metabolic reliance on fatty acids in LA carriers through β- and ω-oxidation and reduced cellular glucose uptake with consequent diversion of acetyl-CoA into ketogenesis. Our observations are in line with previously reported delayed glucose uptake in LA carriers after starch consumption. Further functional studies are needed to extrapolate from our findings to implications for biochemical pathways.

유전자 형질전환을 통한 쌀 전분 분해효소 재조합 효모균주의 개발과 발효특성조사

Department of Cosmetology, Dongkang College, Gwangju 61200, Republic of Korea(Received April 4, 2016; Revised May 9, 2016; Accepted May 10, 2016)ABSTRACT: To improve antioxidant glutathione (GSH) content and saccharification ability in sake yeasts of Saccharomyces cerevisiae, the γ-glutamylcysteine synthetase gene (GSH1 ) from S. cerevisiae, glucoamylase gene (GAM1 ) and α-amylase gene (AMY ) from Debaryomyces occidentalis were co-expressed in sake yeasts for manufacturing a refreshing alcoholic beverage abundant in GSH from rice starch. The extracellular GSH content of the recombinant sake yeasts increased 1.5-fold relative to the parental wide-type strain. The saccharification ability by glucoamylase of the new yeast strain expressing both GAM1 and AMY genes was 2-fold higher than that of the yeast strain expressing only GAM1 gene when grown in the culture medium containing 2% (w/v) rice starch. It generated 11% (v/v) ethanol from 20% (w/v) rice starch and consumed up to 90% of the starch content after 7 days of fermentation.Key words: Saccharomyces cerevisiae, fermentation, glutathione, rice starch, saccharification*For correspondence.

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system.

Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

Low AMY1 Gene Copy Number Is Associated with Increased Body Mass Index in Prepubertal Boys.

BackgroundGenome-wide association studies have identified more than 60 single nucleotide polymorphisms associated with Body Mass Index (BMI). Additional genetic variants, such as copy number variations (CNV), have also been investigated in relation to BMI. Recently, the highly polymorphic CNV in the salivary amylase (AMY1) gene, encoding an enzyme implicated in the first step of starch digestion, has been associated with obesity in adults and children. We assessed the potential association between AMY1 copy number and a wide range of BMI in a population of Italian school-children.Methods744 children (354 boys, 390 girls, mean age (±SD): 8.4±1.4years) underwent anthropometric assessments (height, weight) and collection of saliva samples for DNA extraction. AMY1 copies were evaluated by quantitative PCR.ResultsA significant increase of BMI z-score by decreasing AMY1 copy number was observed in boys (β: -0.117, p = 0.033), but not in girls. Similarly, waist circumference (β: -0.155, p = 0.003, adjusted for age) was negatively influenced by AMY1 copy number in boys. Boys with 8 or more AMY1 copy numbers presented a significant lower BMI z-score (p = 0.04) and waist circumference (p = 0.01) when compared to boys with less than 8 copy numbers.ConclusionsIn this pediatric-only, population-based study, a lower AMY1 copy number emerged to be associated with increased BMI in boys. These data confirm previous findings from adult studies and support a potential role of a higher copy number of the salivary AMY1 gene in protecting from excess weight gain.

Complex Copy Number Variation of AMY1 does not Associate with Obesity in two East Asian Cohorts.

The human amylase gene locus at chromosome 1p21.1 is structurally complex. This region contains two pancreatic amylase genes, AMY2B, AMY2A, and a salivary gene AMY1. The AMY1 gene harbors extensive copy number variation (CNV), and recent studies have implicated this variation in adaptation to starch-rich diets and in association to obesity for European and Asian populations. In this study, we showed that by combining quantitative PCR and digital PCR, coupled with careful experimental design and calibration, we can improve the resolution of genotyping CNV with high copy numbers (CNs). In two East Asian populations of Chinese and Malay ethnicity studied, we observed a unique non-normal distribution of AMY1 diploid CN genotypes with even:odd CNs ratio of 4.5 (3.3-4.7), and an association between the common AMY2A CN=2 genotype and odd CNs of AMY1, that could be explained by the underlying haplotypic structure. In two further case-control cohorts (n=932 and 145, for Chinese and Malays, respectively), we did not observe the previously reported association between AMY1 and obesity or body mass index. Improved methods for accurately genotyping multiallelic CNV loci and understanding the haplotype complexity at the AMY1 locus are necessary for population genetics and association studies.