HyeonSeok Shin

Elucidation of the growth delimitation of Dunaliella tertiolecta under nitrogen stress by integrating transcriptome and peptidome analysis

Dunaliella tertiolecta LB 999 is an oleaginous microalgae species that produces large quantities of lipid and starch during nitrogen starvation; however, nitrogen starvation also limits the cell growth. In order to understand the underlying mechanisms of this phenomenon, the transcriptome and peptidome of D. tertiolecta LB 999 grown under different nitrogen and light conditions were analyzed. Integration of the de novo assembly of transcriptome sequencing reads with peptidome analysis revealed 13,861 protein-coding transcripts, including 33 transcripts whose expression patterns were significantly altered along with the growth phenotypes. Interestingly, 21 of these genes, which were highly enriched in the plastid region, were associated with chlorophyll synthesis and tetrahydrofolate-mediated C1 metabolism. Furthermore, intracellular glutamate levels are predicted to be the main factor that acts as a switch for the regulation of cell growth and carbon accumulation. These data provide the genetic information of D. tertiolecta for its future applications.

Rational Protein Engineering Guided by Deep Mutational Scanning

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design.

Exploring the Functional Residues in a Flavin-Binding Fluorescent Protein Using Deep Mutational Scanning

Flavin mononucleotide (FMN)-based fluorescent proteins are versatile reporters that can monitor various cellular processes in both aerobic and anaerobic conditions. However, the understanding of the role of individual amino acid residues on the protein function has been limited and has restricted the development of better functional variants. Here we examine the functional amino acid residues of Escherichia coli flavin mononucleotide binding fluorescent protein (EcFbFP) using the application of high-throughput sequencing of functional variants, termed deep mutational scanning. The variants were classified into 329 function-retained (FR) and 259 function-loss (FL) mutations, and further the mutational enrichment in each amino acid residues was weighed to find the functionally important residues of EcFbFP. We show that the crucial amino acid residues of EcFbFP lie among the FMN-binding pocket, turns and loops of the protein where conformation changes occur, and spatially clustered residues near the E56-K97 salt bridges. In addition, the mutational sensitivity of the critical residues was confirmed by site-directed mutagenesis. The deep mutational scanning of EcFbFP has demonstrated important implications for constructing better functioning protein variants.

Elucidation of Bacterial Genome Complexity Using Next-generation Sequencing

Next-generation sequencing (NGS) technologies generate higher resolution and less noise data that can allow the assembly of bacterial genome sequences. It also enables the characterization and quantification of transcriptomes, and the genome-wide profiling of DNA-protein interactions. With decreasing cost of NGS, such revolutionary advances in technology has become a powerful tool for studying bacterial genome complexity, which in turn will be used to design synthetic genome. This review describes the NGS approaches, the challenges associated with their application and the advances made so far in characterizing bacterial genomes, transcriptomes, and interactomes. We anticipate these high-throughput data to be a resourceful and broadly used for elucidating bacterial cells at the system level and furthermore, for the synthesis of intelligent biological systems for biotechnological purposes.

Elucidation of the bacterial communities associated with the harmful microalgae Alexandrium tamarense and Cochlodinium polykrikoides using nanopore sequencing

Interactions between microalgae and bacteria are often obligatory for harmful algal blooms (HABs). Here, we investigated the specific bacterial communities associated with Alexandrium tamarense and Cochlodinium polykrikoides, which cause ecological and economic damage during their blooms. To this end, the bacterial metagenome was selectively isolated from the two dinoflagellates and subsequently used for 16S rRNA analysis via the Nanopore MinION and Illumina sequencing platforms. Although the full-length 16S rRNA reads from the MinION platform showed high correlation in higher taxonomic ranks to the partial-length 16S rRNA reads from the Illumina platform, there was less correlation at the genus and species levels. MinION reads that are similar in the V3-V4 hypervariable regions with Illumina reads are classified to different taxonomies due to the extra information encoded in the full-length 16S rRNA reads. This indicates that bias arising from the short length Illumina reads can be supplemented by MinION reads. Furthermore, integrated analysis of the Illumina and MinION data showed that A. tamarense was predominantly enriched in the Roseobacter clade and C. polykrikoides was enriched in Gammaproteobacteria and Alphaproteobacteria. These results suggest that the association of different bacterial communities with A. tamarense and C. polykrikoides may be required for HABs.