A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.)

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Genetic Similarity Analysis Based on Positive and Negative Sequence Patterns of DNA

Similarity analysis of DNA sequences can clarify the homology between sequences and predict the structure of, and relationship between, them. At the same time, the frequent patterns of biological sequences explain not only the genetic characteristics of the organism, but they also serve as relevant markers for certain events of biological sequences. However, most of the aforementioned biological sequence similarity analysis methods are targeted at the entire sequential pattern, which ignores the missing gene fragment that may induce potential disease. The similarity analysis of such sequences containing a missing gene item is a blank. Consequently, some sequences with missing bases are ignored or not effectively analyzed. Thus, this paper presents a new method for DNA sequence similarity analysis. Using this method, we first mined not only positive sequential patterns, but also sequential patterns that were missing some of the base terms (collectively referred to as negative sequential patterns). Subsequently, we used these frequent patterns for similarity analysis on a two-dimensional plane. Several experiments were conducted in order to verify the effectiveness of this algorithm. The experimental results demonstrated that the algorithm can obtain various results through the selection of frequent sequential patterns and that accuracy and time efficiency was improved.

Exploring the role of gene therapy for neurological disorders

Gene therapy is one the frontier fields of medical breakthroughs that poses as an effective solution to previously incurable diseases. The delivery of the corrective genetic material or a therapeutic gene into the cell restores the missing gene function and cures a plethora of diseases, incurable by the conventional medical approaches. This discovery holds the potential to treat many neurodegenerative disorders such as muscular atrophy, multiple sclerosis, Parkinson's disease (PD) and Alzheimer's disease (AD) among others. Gene therapy proves as a humane, cost effective alternative to the exhaustive often arduous and timely impossible process of finding matched donors and extensive surgery. It also overcomes the shortcoming of conventional methods to cross the blood brain barrier. However, the use of gene therapy is only possible after procuring the in-depth knowledge of the immuno-pathogenesis and molecular mechanism of the disease. The process of gene therapy can be broadly categorized into three main steps: elucidating the target gene, culling the appropriate vector, and determining the best mode of transfer; each step mandating pervasive research. This review aims to dissertate and summarize the role, various vectors and methods of delivery employed in gene therapy with special emphasis on therapy directed at the central nervous system (CNS) associated with neurodegenerative diseases.

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

What is in a lichen? A metagenomic approach to reconstruct the holo-genome of Umbilicaria pustulata

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are generally poor candidates for experimentation. Obtaining contiguous, high quality genomes for such symbiotic communities is technically challenging. Here we present the first assembly of a lichen holo-genome from metagenomic whole genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis – the fungus Umbilicaria pustulata (33 Mbp) and the green alga Trebouxia sp. (53 Mbp) – were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative cellular abundance of approximately 20:1 (fungus:alga). Gap-free, circular sequences for all organellar genomes were obtained. The community of lichen-associated bacteria is dominated by Acidobacteriaceae, and the two largest bacterial contigs belong to the genus Acidobacterium. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that G/C-rich inverted repeats paired with non-random sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

Understanding and eliminating the detrimental effect of endogenous thiamine auxotrophy on metabolism of the oleaginous yeast Yarrowia lipolytica

ABSTRACTThiamine is an essential vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this study, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing gene scTHI13, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase for the de novo thiamine synthesis in Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene, we engineered the first thiamine-prototrophic Y. lipolytica reported to date. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica.IMPORTANCEThiamine plays a crucial role as an essential cofactor for enzymes in carbon and energy metabolism of all living cells. Thiamine deficiency has detrimental consequences on cellular health. Yarrowia lipolytica, a non-conventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph, whose effects on cellular metabolism are not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host to study thiamine metabolism, especially as mammalian cells are also thiamine-auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamentals of thiamine deficiency on cellular metabolism of Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, discovery of thiamine-regulated elements enables development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.