I. Genome design

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.

Seleted Publications for Genome Design

• Kang, M.*, Kim, K.*, Choe, D., Cho, S., Kim, S.C., Palsson, B., Cho, B.K. (2019) Inactivation of a Mismatch-Repair System Diversifies Genotypic Landscape of Escherichia coli during Adaptive Laboratory Evolution, Frontiers in Microbiology, 10, 1845
• Choe, D.*, Lee, J.H.*, Yoo, M., Hwang, S., Sung, B.H., Cho, S., Palsson, B., Kim, S.C., Cho, B.K. (2019) Adaptive laboratory evolution of a genome-reduced Escherichia coli, Nature Communications, 10, 935
• Cho, S.*, Choe, D.*, Lee, E., Kim, S.C., Palsson, B.Ø., Cho, B.K. (2018) High-level dCas9 expression induces abnormal cell morphology in Escherichia coli, ACS Synthetic Biology, 7(4), 1085–1094
• Hwang, S., Choe, D., Yoo, M., Cho, S., Kim, S.C., Cho, S., Cho, B.K. (2018) Peptide transporter CstA imports pyruvate in Escherichia coli K-12, e00771-17
• Cho, B.K., Zengler, K., Qiu, Y., Knight, E.M., Park, Y.S., Barrett, C.L., Gao, Y., and Palsson, B.O. (2009) The transcription unit architecture of the Escherichia coli K-12 MG1655 genome. Nature Biotechnol., 27(11): 1043-1049. (Featured on the cover article of this issue).

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.

Seleted Publications for Next-generation CO2/CO Fixing System

• Shin, J.*, Kang, S.*, Song, Y., Jin, S., Lee, J., Lee, J.K., Kim, D.R., Kim, S.C., Cho, S., Cho, B.K. (2019) Genome engineering of Eubacterium limosum using expanded genetic tools and CRISPR-Cas9 system, ACS Synthetic Biology, 8, 9, 2059-2068
• Song, Y., Shin, J., Jin, S., Lee, J.K., Kim, D.R., Kim, S.C., Cho, S., Cho, B.K. (2018) Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth, BMC genomics, 19:837
• Shin, J., Song, Y., Jin, S., Lee, J.K., Kim, D.R., Kim, S.C., Cho, S., Cho, B.K.(2018) Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation, RNA, 24:1839-1855
• Song, Y., Shin, J., Jeong, Y., Jin, S., Lee, J.K., Kim, D.R., Kim, S.C., Cho, S, Cho, B.K. (2017) Determination of the Genome and Primary Transcriptome of Syngas Fermenting Eubacterium limosum ATCC 8486 . Scientific Reports, 7:13694.
• Shin, J., Song, Y., Jeong, Y. and Cho, B.K. (2016). Analysis of the core genome and pan-genome of autotrophic acetogenic bacteria., Front. Microbiol. 7:1531.

III. 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.

Seleted Publications for Antibiotics-producing Microbial Cell Factory

• Lee, N., Kim, W., Chung, J., Lee, Y., Cho, S., Jang, K.S., Kim, S.C., Palsson, B., Cho, B.K. (2020) Iron competition triggers antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus, ISME J., https://doi.org/10.1038/s41396-020-0594-6
• Jeong, Y*., Kim, J.N*., Kim, M.W., Bucca, G., Cho, S., Yoon, Y.J., Kim, B.K., Roe, J.H., Kim, S.C., Smith, C.P., and Cho, B.K. (2016) The Dynamic Transcriptional and Translational Landscape of the Model Antibiotic Producer Streptomyces coelicolor A3(2), Nat. commun., 7:11605
• Hwang, S., Lee, N., Jeong, Y., Lee, Y., Kim, W., Cho, S., Palsson, B., Cho, B.K. (2019) Primary transcriptome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces genomes, Nucleic Acids Res., 47(12):6114-6129
• Yi, J.S., Kim, M.W., Kim, M., Jeong, Y., Kim, E.J., Cho, B.K.*, and Kim, B.G.* (2017) A Novel Approach for Gene Expression Optimization through Native Promoter and 5’ UTR Combinations Based on RNA-seq, Ribo-seq, and TSS-seq of Streptomyces coelicolor, ACS Synth. Biol., 2017, 6 (3):555-565
• Lee, Y., Lee, N., Jeong, Y., Hwang, S., Kim, W., Cho, S., Palsson, B., Cho, B.K. (2019) The transcription unit architecture of Streptomyces lividans TK24, Front. Microbiol., 10, 2074

IV. Eco-friendly environmental control

Under construction