Research Summary

It is exciting to expand the scope of our intellectual curiosity in biological systems, and to combine biological principles with the spirit of engineering. It has been our overall research interest to pursue interdisciplinary research encompassing topics from engineering problems in the production of value-added biomaterials to fundamental scientific problems in biology. Specifically, we are highly interested in the research areas of microbial systems and synthetic biology. Our research pursues a systems approach to decode microbial genomes and utilize the information-rich data types for synthetic biology applications. It is intensively based on the growing availability of high-resolution omics data types and diverse synthetic biology tools.

We have established five flourishing and successful research programs in our laboratory, focusing on several important problems in engineering microbial cells to exhibit better performance as a cell chassis for the production of biochemicals and pharmaceuticals. It includes (1) minimal genome construction, (2) conversion of CO2/CO into biochemicals using acetogenic bacteria, and (3) antibiotics production using Streptomyces. In addition, we elucidate (4) the cellular traits for screening drug targets in pathogenic Staphylococcus aureus and (5) the host-microbe interaction for better probiotic performance of microbes and treatment of pine wilt disease by fungi.

I. Minimal Genome Chassis

Recent advances in synthetic biology have changed our idea of a cell, from a unit of an organism to an assembly of thousands of genetic parts. Furthermore, systems biology approach has been used to collect data on multiple parts together to understand how they harmonize within cells. Despite the efforts, we are unable to fully understand a simple bacterial cell. In the case of the most intensively studied model organism, E. coli, there are over a thousand genes with functions that are yet unknown or only predicted. Furthermore, some genetic elements, such as insertion sequence elements (IS elements), induce genomic instability by randomly mutating the genome, thereby increasing genomic complexity and decreasing the predictability of cellular behavior.

To overcome the problems of complexity and predictability, the development of a genome with a reduced number of parts, called the minimal genome or the minimal-gene-set, composed of the smallest set of genes sufficient to maintain self-replicable life with no stresses was proposed. The cells equipped with the minimal genome, called minimal cells, are supposed to have reliable and predictable phenotypes, since a vast number of genomic parts that may act differently from our predictions have been removed. It has been hypothesized that the minimal cells conserve cellular energy, otherwise wasted due to transcription and translation of unnecessary genes. Thus, minimal genomes (or minimal cells) are expected to be potential platforms for the production of value-added biochemicals and proteins with unprecedented yield. With current technology, organisms having only the necessary parts of the genome are difficult to construct. Instead, cells with reduced or a simplified genome have been constructed.

These fascinating minimal genomes, however, often show unpredictable phenotypes such as growth retardation, although genes necessary for survival remain intact. For instance, all essential genes of M. mycoides (JCVI-syn3.0) were contained in the initial design for a minimal genome; however, a viable genome could only be constructed after “quasi-essential genes”, which are not strictly essential but are required for robust growth, were included in the minimal genome. In contrast to this ‘bottom-up’ approach to genome design, several E. coli strains harboring reduced genomes have been constructed by sequential genome reduction mostly without growth retardation in rich media. However, when genome-reduced strains are grown in minimal medium, their growth rate is often reduced. The decreased growth rate and other unpredictable phenotypes can be attributed to our limited understanding of bacterial genome processes, such as synthetic lethality and interactions between interconnected cellular components, making it difficult to construct minimal genomes. We have reviewed these scientific points in Biotechnol. J. (2015) and Essays Biochem. (2016).

Our laboratory has designed and constructed a minimal E. coli genome that lacks 1000 non-essential genes out of 4600 genes (MS56). Although the strain showed higher biochemical production capability under rich medium condition, it showed extremely slow growth in industrially relevant fermentation medium. To compensate, we deployed an iterative design-build-test-learn cycle of systems and synthetic biology to further optimize minimal genome system.

II. Next-generation CO2/CO Fixing System

Fossil fuel has been a valuable resource for humankind since the industrial revolution, by provid­ing power to heavy steam engines. The same engines, furthermore, enabled humans to drain more fuels from the ground, giving more power to the engines to extract even more fuel. The posi­tive feedback contributed to the exponential growth of technology in the last three hundred years and has led to an increase in the population. Concomitantly with the technological and population explosion, fossil fuel has caused environmental pollution by emitting 3 trillion tons of CO2 and CO (C1 compound). The accumulation of C1 compound has raised the global temperature.

Acetogenic bacteria is considered to be the most efficient microorganism for fixing C1 compound as they gain energy from operating the pathway, in contrast to the other C1 compound-fixing bacteria that spend energy during the uptake. The Wood-Ljungdahl pathway (WLP) in the microorganism converts C1 into acetyl-CoA, which is an important cellular precursor that is converted into biofuels, such as acetic acid, ethanol, and butanol. Despite the potential to reduce C1 compound in the atmosphere, lack of a systemic understanding, complex layers of regulation system, and inefficient electron delivery has limited the construction of a cellular factory optimized for producing the desired chemical. Instead, most of the studies were focused on understanding the biochemistry of WLP and physiological change of acetogen.

Our objective is to design and construct C1 compound recycling microorganisms for production of value-added chemicals. To meet the objective, molecular level insight has been obtained via Genome-Seq, RNA-Seq, Ribo-Seq, dRNA-Seq, and Term-Seq. The results revealed functional genes required for C1 compound fixation and their regulatory systems. Later, the obtained information-rich data types will be integrated with the CRISPR-Cas9 genetic modification tool to engineer regulatory systems for optimizing transcriptional and translational expression for C1 compound fixation. Using the modified organism, electron-transferring nanomaterials, which accepts electron from light, will be attached to microorganisms to donate electrons, utilizing an alternative energy source. Integration of the technologies will facilitate the construction of an optimal C1 fixing and biochemical-producing cellular factory.

III. Understanding Multi-drug Resistant Pathogens

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an epidemic health threat and a major worldwide healthcare burden. An important question is the genetic bases for community-acquired MRSA (CA-MRSA) to propagate in healthy people of a community, become rapidly epidemic, and display different antibiotic susceptibility, compared with healthcare-acquired MRSA (HA-MRSA). Expression and production of virulence factors are tightly regulated by a variety of elements such as transcriptional regulators, quorum-sensing, and regulatory RNAs in pathogenic bacteria

IV. Antibiotics-producing Microbial Cell Factory

Streptomyces, which are soil-dwelling, gram-positive bacteria with a high G+C content, are members of the largest genus of actinobacteria, with over 900 described species. They have been renowned for their ability to produce bioactive secondary metabolites including antiparasitics, antitumorals, antifungals, and mainly, antibiotics and immunosuppressants. Most secondary metabolites are typically synthesized by multi-enzyme complexes encoded in secondary metabolite biosynthetic gene clusters (BGCs). Production of secondary metabolites is also closely connected to the primary metabolism, which provides the precursor molecules controlled by subtle and precise transcriptional and translational regulatory networks. Recent genomic studies have indicated that individual Streptomyces species generally possess more than thirty BGCs, which have a vast potential to produce a diverse array of metabolites and were ‘silent’ in laboratory growth conditions. Discovering novel bioactive compounds by activating these silent clusters in these bacteria has attracted major attention due to the rapid rise in antibiotic-resistant pathogens.

Our objective is to reveal the transcriptional and translational control of the BGCs. The obtained information-rich data types are integrated with genetic modification tools to engineer the regulatory systems and the function of BGCs.

  • [2016] The Dynamic Transcriptional and Translational Landscape of the Model Antibiotic Producer Streptomyces coelicolor A3(2), Nat. commun., 7:11605