Comparison of Two Aspergillus oryzae Genomes From Different Clades Reveals Independent Evolution of Alpha-Amylase Duplication, Variation in Secondary Metabolism Genes, and Differences in Primary Metabolism

Microbes (bacteria, yeasts, molds), in addition to plants and animals, were domesticated for their roles in food preservation, nutrition and flavor. Aspergillus oryzae is a domesticated filamentous fungal species traditionally used during fermentation of Asian foods and beverage, such as sake, soy sauce, and miso. To date, little is known about the extent of genome and phenotypic variation of A. oryzae isolates from different clades. Here, we used long-read Oxford Nanopore and short-read Illumina sequencing to produce a highly accurate and contiguous genome assemble of A. oryzae 14160, an industrial strain from China. To understand the relationship of this isolate, we performed phylogenetic analysis with 90 A. oryzae isolates and 1 isolate of the A. oryzae progenitor, Aspergillus flavus. This analysis showed that A. oryzae 14160 is a member of clade A, in comparison to the RIB 40 type strain, which is a member of clade F. To explore genome variation between isolates from distinct A. oryzae clades, we compared the A. oryzae 14160 genome with the complete RIB 40 genome. Our results provide evidence of independent evolution of the alpha-amylase gene duplication, which is one of the major adaptive mutations resulting from domestication. Synteny analysis revealed that both genomes have three copies of the alpha-amylase gene, but only one copy on chromosome 2 was conserved. While the RIB 40 genome had additional copies of the alpha-amylase gene on chromosomes III, and V, 14160 had a second copy on chromosome II and an third copy on chromosome VI. Additionally, we identified hundreds of lineage specific genes, and putative high impact mutations in genes involved in secondary metabolism, including several of the core biosynthetic genes. Finally, to examine the functional effects of genome variation between strains, we measured amylase activity, proteolytic activity, and growth rate on several different substrates. RIB 40 produced significantly higher levels of amylase compared to 14160 when grown on rice and starch. Accordingly, RIB 40 grew faster on rice, while 14160 grew faster on soy. Taken together, our analyses reveal substantial genome and phenotypic variation within A. oryzae.

Crucial role of the intracellular α-glucosidase MalT in the activation of the transcription factor AmyR essential for amylolytic gene expression in Aspergillus oryzae

We examined the role of the intracellular α-glucosidase gene malT, which is part of the maltose-utilizing cluster (MAL cluster) together with malR and malP, in amylolytic gene expression in Aspergillus oryzae. malT disruption severely affected fungal growth on medium containing maltose or starch. Furthermore, the transcription level of the α-amylase gene was significantly reduced by malT disruption. Given that the transcription factor AmyR responsible for amylolytic gene expression is activated by isomaltose converted from maltose incorporated into the cells, MalT may have transglycosylation activity that converts maltose to isomaltose. Indeed, transglycosylated products such as isomaltose/maltotriose and panose were generated from the substrate maltose by MalT purified from a malT-overexpressing strain. The results of this study, taken together, suggests that MalT plays a pivotal role in AmyR activation via its transglycosylation activity that converts maltose to the physiological inducer isomaltose.

Ethnic-specific association of amylase gene copy number with adiposity traits in a large Middle Eastern biobank

Studies assessing the impact of amylase genes copy number (CN) on adiposity report conflicting findings in different global populations, likely reflecting the impact of ancestral and ethnic-specific environment and lifestyle on selection at the amylase loci. Here, we leverage population size and detailed adiposity measures from a large population biobank to resolve confounding effects and determine the relationship between salivary (AMY1) and pancreatic (AMY2A) amylase genes CN and adiposity in 2935 Qatari individuals who underwent whole-genome sequencing (WGS) as part of the Qatar Genome Programme. We observe a negative association between AMY1 CNs and trunk fat percentage in the Qatari population (P = 7.50 × 10−3) and show that Qataris of Arab descent have significantly lower CN at AMY1 (P = 1.32 × 10−10) as well as less favorable adiposity and metabolic profiles (P < 1.34 × 10−8) than Qataris with Persian ancestry. Indeed, lower AMY1 CN was associated with increased total and trunk fat percentages in Arabs (P < 4.60 × 10−3) but not in Persians. Notably, overweight and obese Persians reported a significant trend towards dietary restraint following weight gain compared to Arabs (P = 4.29 × 10−5), with AMY1 CN showing negative association with dietary self-restraint (P = 3.22 × 10−3). This study reports an association between amylase gene CN and adiposity traits in a large Middle Eastern population. Importantly, we leverage rich biobank data to demonstrate that the strength of this association varies with ethnicity, and may be influenced by population-specific behaviors that also contribute to adiposity traits.

Molecular characterization of gene encoding halo tolerant amylase of Bacillus paralicheniformis isolated from Jazan region

Objectives: This study aims to characterize the gene encoding halo tolerant amylase of bacteria isolated from Jazan region. Materials and Methods: Soil samples were collected from several area of Jazan region, KSA. The samples were serially diluted and plateted on starch agar plates. The amylase producing bacteria were detected by iodine test. To determine the halophilic amylase producing bacteria, several colonies were tested for their ability to grow at higher concentrations of NaCl ranging from 7 to 16%. The bacteria was identified by 16S rRNA and the full length amylase gene was fully identified by sequencing using specific primers. Results: One bacterial halophilic isolate was able to grow on starch agar medium up to 14% NaCl. The Gram stain of the isolate indicated that it is Gram-positive, bacilli. The 16S rRNA gene homology study showed that the bacterial isolate was identified as Bacillus paralicheniformis. Two specific primers were designed named S1F, S1R, to amplify the amylase gene (AMY) region using PCR and the PCR product was sequenced. The sequencing results showed that the full-length amy gene of B. paralicheniformis was of 1452 encoding 483 amino acids. The expected M.Wt. of the protein expressed is of 55 KDa. Conclusion: We report the isolation, identification, and characterization of an isolate of halophilic bacterium isolated from Jazan region. Based on molecular identification, this isolate was identified as Bacillus paralicheniformis. This bacterial strain has an α-amylase gene in its genome and is able to produce extracellular α-amylase. Based on the findings of this work we propose that Bacillus paralicheniformis amy gene could be cloned into expression vector for large scale production.