Sung-Hee Park

Expanding substrate specificity of GT‐B fold glycosyltransferase via domain swapping and high‐throughput screening

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT‐B superfamily, which is composed of separate N‐ and C‐domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N‐terminal fragments of kanamycin GT (kanF) and the C‐terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate‐based pH color assay was newly developed for the selection of functional domain‐swapped GTs, and a mutant (HMT31) with a crossover point (N‐kanF‐669 bp and 753 bp‐gtfE‐C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2‐DOS (aglycone substrate for KanF) glucosylation using dTDP‐glucose (glycone substrate for GtfE) with kcat/Km of 162.8 ± 0.1 mM−1 min−1. Moreover, HMT31 showed improved substrate specificity toward seven more NDP‐sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2‐DOS‐based aminoglycoside derivatives. Biotechnol. Bioeng. 2009;102: 988–994.

Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source.

In this study, a biosugar obtained from empty fruit bunch (EFB) of oil palm by hot water treatment and subsequent enzymatic saccharification was used for undecylprodigiosin production, using Streptomyces coelicolor. Furfural is a major inhibitor present in EFB hydrolysate (EFBH), having a minimum inhibitory concentration (MIC) of 1.9mM, and it reduces utilization of glucose (27%), xylose (59%), inhibits mycelium formation, and affects antibiotic production. Interestingly, furfural was found to be a good activator of undecylprodigiosin production in S. coelicolor, which enhanced undecylprodigiosin production by up to 52%. Optimization by mixture analysis resulted in a synthetic medium containing glucose:furfural:ACN:DMSO (1%, 2mM, 0.2% and 0.3%, respectively). Finally, S. coelicolor was cultured in a fermenter in minimal medium with EFBH as a carbon source and addition of the components described above. This yielded 4.2μg/mgdcw undecylprodigiosin, which was 3.2-fold higher compared to that in un-optimized medium.

A novel function of Streptomyces integration host factor (sIHF) in the control of antibiotic production and sporulation in Streptomyces coelicolor

Bacterial integration host factors (IHFs) play important roles in site-specific recombination, DNA replication, transcription, genome organization and bacterial pathogenesis. In Streptomyces coelicolor, there are three putative IHFs: SCO1480, SCO2950 and SCO5556. SCO1480 or Streptomyces IHF (sIHF) was previously identified as a transcription factor that binds to the promoter region of redD, the pathway-specific regulatory gene for the undecylprodigiosin biosynthetic gene cluster. Here we show that production of the pigmented antibiotics actinorhodin and undecylprodigiosin is strongly enhanced in sihf null mutants, while sporulation was strongly inhibited, with an on average 25% increase in spore size. Furthermore, the sihf mutant spores showed strongly reduced viability, with high sensitivity to heat and live/dead staining revealing a high proportion of empty spores, while enhanced expression of sIHF increased viability. This suggests a major role for sIHF in controlling viability, perhaps via the control of DNA replication and/or segregation. Proteomic analysis of the sihf null mutant identified several differentially expressed transcriptional regulators, indicating that sIHF may have an extensive response regulon. These data surprisingly reveal that a basic architectural element conserved in many actinobacteria such as mycobacteria, corynebacteria, streptomycetes and rhodococci may act as a global regulator of secondary metabolism and cell development.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

Abstractγ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

Low mass cutoff evasion with qz value optimization in ion trap

An ion trap is a powerful analyzer because of its high resolution, high sensitivity, and multistage mass analysis (MS(n)) capabilities. Multiple fragmentation analysis provides useful information regarding peptide sequence and biomolecular structure; however, this approach is limited by an inherent low mass cutoff (LMCO) derived from collision-induced dissociation (CID). To avoid the LMCO for application of an ion trap to iTRAQ, we optimized the q(z) value, which is a parameter that is proportional to the applied fundamental AC radio frequency voltage of a tandem mass spectrometry (MS/MS) event. Considering that many ion trap MS analyses employ CID as the MS/MS method, this method can be a practical one without any instrumental changes.

Exopolysaccharide from psychrotrophic Arctic glacier soil bacterium Flavobacterium sp. ASB 3-3 and its potential applications

A novel exopolysaccharide (EPS) producing psychrotrophic bacterium Flavobacterium sp. ASB 3-3 was isolated from Arctic glacier soil and identified. The optimum fermentation conditions for EPS production were an initial medium pH of 7.2 and an initial inoculum size of 5% (v/v). The maximum yield of EPS (7.25 ± 0.26 g L−1) was obtained after cultivation at 25 °C for 120 h with glycerol as the sole carbon source. The EPS was purified and its structural characteristics were analyzed by 1H and 13C NMR. The predominant repeating units of this EPS are (α, β) D-glucose and D-galactose and it is different from the structure of EPSs produced by other Arctic and Antarctic bacteria, which have mannose units. In addition, EPS has demonstrated a comparable emulsifying property than SDS and flocculating properties with kaolinite, suggesting their potential applications in various industries. The EPS also significantly improved the tolerance of Flavobacterium sp. and Escherichia coli from freeze–thaw cycles, suggesting that it might be used to survive in polar regions and it can have possible usage as microbial cryoprotectants.

Cell-free Escherichia coli-based system to screen for quorum-sensing molecules interacting with quorum receptor proteins of Streptomyces coelicolor.

ABSTRACT Quorum sensing (QS) is mediated by small molecules and involved in diverse cellular functions, such as virulence, biofilm formation, secondary metabolism, and cell differentiation. In this study, we developed a rapid and effective screening tool based on a cell-free Escherichia coli-based expression system to identify QS molecules of Streptomyces. The binding of QS molecules to γ-butyrolactone receptor ScbR was monitored by changes in the expression levels of the green fluorescent protein reporter in E. coli cell extract. Using this assay system, we could successfully confirm SCB1, a γ-butyrolactone molecule in Streptomyces coelicolor, binding to its known receptor, ScbR. In addition, we have shown that N-hexanoyl-dl-homoserine lactone, one of the QS molecules in many gram-negative bacteria, can regulate ScbR and trigger precocious antibiotic production in S. coelicolor. Our new method can be applied to other strains for which a screening tool for QS molecules has not yet been developed.

Metal removal and reduction potential of an exopolysaccharide produced by Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620

An exopolysaccharide (EPS) was produced from psychrotrophic Arctic glacier fore-field soil bacterium Pseudomonas sp. PAMC 28620 using glycerol enriched medium and the maximum productivity 7.24 ± 0.31 g L−1 of EPS was obtained after 168 h of fermentation. The EPS was purified and analysed by HPLC, GC-MS, FT-IR, 1H and 13C NMR. The EPS obtained from Arctic strain PAMC 28620 exhibits a distinctive structural composition and the constituent sugar monomers are rhamnose, galactose, glucose, fucose, mannose and ribose. The purified EPS has shown excellent flocculating and emulsification capacities with promising biotechnological and ecological implications. From the metal removal experiments, the EPS exhibited remarkable metal adsorption (99%) potential adopting the order Fe2+ > Cu2+ > Mg2+ > Zn2+ > Mn2+ > Ca2+. FE-SEM combined with EDX analysis has shown that the metal ions were complexed or immobilized onto the EPS matrix and further reduced to nanoparticles (150–950 nm). This study is significant in terms of metal removal and reduction potential of Arctic bacterial EPS and the possible ecological roles of the EPS in Arctic environment.

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by 13C-NMR.

Rapid Functional Screening of Streptomyces coelicolor Regulators by Use of a pH Indicator and Application to the MarR-Like Regulator AbsC

ABSTRACT To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production between a deletion mutant and a wild-type strain (WT) were compared. These differences are easily hidden by complex media. To determine the specific nutrient conditions that reveal such differences, we used a multiwell method containing different nutrients along with bromothymol blue. We found several nutrients that provide key information on characterization conditions. By comparing the growth of wild-type and mutant strains on screened nutrients, we were able to measure growth, organic acid production, and antibiotic production for the elucidation of regulator function. As a result of this method, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and α-ketoglutarate and increased extracellular actinorhodin (ACT) production in minimal medium containing glucose and alanine (MMGA). This method could therefore be a high-throughput method for the characterization of unknown regulators.