Si-Sun Choi

Detection of microorganisms using terahertz metamaterials

Microorganisms such as fungi and bacteria cause many human diseases and therefore rapid and accurate identification of these substances is essential for effective treatment and prevention of further infections. In particular, contemporary microbial detection technique is limited by the low detection speed which usually extends over a couple of days. Here we demonstrate that metamaterials operating in the terahertz frequency range shows promising potential for use in fabricating the highly sensitive and selective microbial sensors that are capable of high-speed on-site detection of microorganisms in both ambient and aqueous environments. We were able to detect extremely small amounts of the microorganisms, because their sizes are on the same scale as the micro-gaps of the terahertz metamaterials. The resonant frequency shift of the metamaterials was investigated in terms of the number density and the dielectric constants of the microorganisms, which was successfully interpreted by the change in the effective dielectric constant of a gap area.

Identification and characterization of wblA-dependent tmcT regulation during tautomycetin biosynthesis in Streptomyces sp. CK4412.

Tautomycetin (TMC) is an unusual linear polyketide compound esterified with a cyclic anhydride. It exhibits novel activated T cell-specific immunosuppressant as well as anti-cancer activities. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412, including a TMC pathway-specific gene, tmcN, the over-expression of which led to a significant increase in TMC productivity. In addition, we also reported that WblA acts as a global down-regulator of antibiotic biosynthesis through pathway-specific regulation in Streptomyces species. Here, we confirm that TmcT acts as another TMC pathway-specific regulator within the TMC biosynthetic cluster. Specifically, tmcT deletion resulted in the complete loss of TMC production, whereas complementation with a tmcT-carrying integrative plasmid significantly restored TMC biosynthesis. We also identified a 0.39kb wblA ortholog (named wblA(tmc)) from Streptomyces sp. CK4412 via genomic DNA library screening that showed 96% amino acid identity compared to a previously-known S. coelicolor wblA. Targeted gene disruption of wblA(tmc) in Streptomyces sp. CK4412 exhibited approximately 3-fold higher TMC productivity than that in the wild-type strain. Moreover, transcription analyses of the TMC biosynthetic and regulatory genes revealed that the expression of tmcT was strongly down-regulated by wblA(tmc). These results imply that the TMC biosynthetic regulation network is controlled by two pathway-specific positive regulator, WblA(tmc)-dependent TmcT as well as WblA(tmc)-independent TmcN in Streptomyces sp. CK4412.

Cloning and Heterologous Expression of a Large-sized Natural Product Biosynthetic Gene Cluster in Streptomyces Species

Actinomycetes family including Streptomyces species have been a major source for the discovery of novel natural products (NPs) in the last several decades thanks to their structural novelty, diversity and complexity. Moreover, recent genome mining approach has provided an attractive tool to screen potentially valuable NP biosynthetic gene clusters (BGCs) present in the actinomycetes genomes. Since many of these NP BGCs are silent or cryptic in the original actinomycetes, various techniques have been employed to activate these NP BGCs. Heterologous expression of BGCs has become a useful strategy to produce, reactivate, improve, and modify the pathways of NPs present at minute quantities in the original actinomycetes isolates. However, cloning and efficient overexpression of an entire NP BGC, often as large as over 100 kb, remain challenging due to the ineffectiveness of current genetic systems in manipulating large NP BGCs. This mini review describes examples of actinomycetes NP production through BGC heterologous expression systems as well as recent strategies specialized for the large-sized NP BGCs in Streptomyces heterologous hosts.

Post-PKS Tailoring Steps of a Disaccharide-Containing Polyene NPP in Pseudonocardia autotrophica

A novel polyene compound NPP identified in a rare actinomycetes, Pseudonocardia autotrophica KCTC9441, was shown to contain an aglycone identical to nystatin but to harbor a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic activity. Although the nppDI was proved to be responsible for the transfer of first polyene sugar, mycosamine in NPP biosynthesis, the gene responsible for the second sugar extending glycosyltransferase (GT) as well as NPP post-PKS tailoring mechanism remained unknown. Here, we identified a NPP-specific second sugar extending GT gene named nppY, located at the edge of the NPP biosynthetic gene cluster. Targeted nppY gene deletion and its complementation proved that nppY is indeed responsible for the transfer of second sugar, N-acetyl-glucosamine in NPP biosynthesis. Site-directed mutagenesis on nppY also revealed several amino acid residues critical for NppY GT function. Moreover, a combination of deletions and complementations of two GT genes (nppDI and nppY) and one P450 hydroxylase gene (nppL) involved in the NPP post-PKS biosynthesis revealed that NPP aglycone is sequentially modified by the two different GTs encoded by nppDI and nppY, respectively, followed by the nppL-driven regio-specific hydroxylation at the NPP C10 position. These results set the stage for the biotechnological application of sugar diversification for the biosynthesis of novel polyene compounds in actinomycetes.

Identification and Biotechnological Application of Novel Regulatory Genes Involved in Streptomyces Polyketide Overproduction through Reverse Engineering Strategy

Polyketide belongs to a family of abundant natural products typically produced by the filamentous soil bacteria Streptomyces. Similar to the biosynthesis of most secondary metabolites produced in the Streptomyces species, polyketide compounds are synthesized through tight regulatory networks in the cell, and thus extremely low levels of polyketides are typically observed in wild-type strains. Although many Streptomyces polyketides and their derivatives have potential to be used as clinically important pharmaceutical drugs, traditional strain improvement strategies such as random recursive mutagenesis have long been practiced with little understanding of the molecular basis underlying enhanced polyketide production. Recently, identifying, understanding, and applying a novel polyketide regulatory system identified from various Omics approaches, has become an important tool for rational Streptomyces strain improvement. In this paper, DNA microarray-driven reverse engineering efforts for improving titers of polyketides are briefly summarized, primarily focusing on our recent results of identification and application of novel global regulatory genes such as wblA, SCO1712, and SCO5426 in Streptomyces species. Sequential targeted gene manipulation involved in polyketide biosynthetic reguation synergistically provided an efficient and rational strategy for Streptomyces strain improvement. Moreover, the engineered regulation-optimized Streptomyces mutant strain was further used as a surrogate host for heterologous expression of polyketide pathway.

Rheological Study on Biodegradable Poly(3-Hydroxybutyrate) and Its Copolymer

Some aspects of rheological properties of microbial biodegradable copolymers containing 3-hydroxybutyrate and 3-hydroxyvalerate have been studied in this work, using both rotational rheometer with parallel plate geometry and capillary rheometer. Dynamic viscoelasticity as functions of time and temperature was also measured by Rheovibron.

Characterization of a non-phosphotransferase system for cis,cis -muconic acid production in Corynebacterium glutamicum

Cis,cis-muconic acid (CCM) is a biochemical material that can be used for the production of various plastics and polymers and is particularly gaining attention as an adipic acid precursor for the synthesis of nylon-6,6. In the current study, the production of CCM was first attempted by introducing a newly developed protocatechuate (PCA) decarboxylase from Corynebacterium glutamicum 13032 to inha103, which completed the biosynthetic pathway therein. To improve CCM productivity, a phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that consumed the existing glucose was developed, in the form of a strain with a non-PTS that did not consume PEP. To improve glucose uptake, we developed P25 strain, in which iolR (a transcriptional regulator gene) was additionally deleted. Strain P28, a P25 derivative expressing PCA decarboxylase, produced 4.01 g/L of CCM, which was 14% more than that produced by the parental strain. Moreover, strains P29 and P30, with an active pentose phosphate pathway and overexpressing important genes (qsuB) in the metabolic pathway, produced 4.36 and 4.5 g/L of CCM, respectively. Particularly, the yield per glucose in strain P30 was similar to that of the fed-batch culture of Escherichia coli, which has the highest reported yield of 22% (mol/mol). These results are underpinned by the characteristics of the non-PTS with increased PEP availability and a strain with deletion of the iolR gene, which greatly increased glucose uptake.

Pikromycin Production Stimulation through Antibiotic Down-regulatory Gene Disruption in Streptomyces venezuelae

The wblA gene, which encodes a whiB-like putative transcription factor, has been widely reported as an antibiotic biosynthesis down-regulator in Streptomyces coelicolor. The wblA ortholog from Streptomyces venezuelae was identified by sequence alignment with wblA from S. coelicolor. To determine the biological significance of wblA from S. venezuelae, chromosomal disruption of the wblAsve gene was performed by homologous recombination using the pKC1139 vector. The phenotypic difference between S. venezuelae and S. venezuelaeΔwblAsve was shown, and pikromycin production was quantified by HPLC analysis. Production of pikromycin by S. venezuelaeΔwblAsve was approximately 3.5-fold higher compared to that by S. venezuelae. To further show that the wblAsve gene was implicated in pikromycin production, wblAsve was cloned into Streptomyces integrative expression vector, which was independently introduced into both S. venezuelae and S. venezuelaeΔwblAsve by conjugation. HPLC analysis of complementation and overexpression strains showed an approximately 2.5-fold reduction in pikromycin production by the wblAsve overexpression mutant.

Redesign of antifungal polyene glycosylation: engineered biosynthesis of disaccharide-modified NPP

Polyene macrolides such as nystatin A1 and amphotericin B have been known to be potent antifungal antibiotics for several decades. Because the therapeutic application of polyenes is restricted by severe side effects such as nephrotoxicity, various chemical and biological studies to modify the polyene structure have been conducted to develop less-toxic polyene antifungals. A newly discovered nystatin-like polyene compound NPP was shown to contain an aglycone that was identical to nystatin but harbored a unique di-sugar moiety, mycosaminyl-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic toxicity. Additionally, a NPP-specific second sugar extending gene, nppY, was recently identified to be responsible for the transfer of a second sugar, N-acetyl-glucosamine, in NPP biosynthesis. In this study, we investigated biosynthesis of the glycoengineered NPP analog through genetic manipulation of the NPP A1 producer, Pseudonocardia autotrophica KCTC9441. NypY is another second sugar glycosyltransferase produced by Pseudonocardia sp. P1 that is responsible for the transfer of a mannose to the mycosaminyl sugar residue of nystatin. We blocked the transfer of a second sugar through nppY disruption, then expressed nypY in P. autotrophica △nppY mutant strain. When compared with nystain A1 and NPP A1, the newly engineered mannosylated NPP analog showed reduced in vitro antifungal activity, while exhibiting higher nephrotoxical activities against human hepatocytes. These results suggest for the first time that not only the number of sugar residues but also the type of extended second sugar moiety could affect biological activities of polyene macrolides.

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in Pseudonocardia autotrophica.

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.