Journal of Microbiology

Journal Article

[Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract: Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Research Support, Non-U.S. Gov't

Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli: Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee; J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0283-z

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Abstract: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.

Review

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates: Do Young Kim , Hyung Woo Kim , Moon Gyu Chung , Young Ha Rhee; J. Microbiol. 2007;45(2):87-97.; DOI: https://doi.org/2528 [pii]

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Abstract: Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

Research Support, Non-U.S. Gov't

Molecular Characterization of Extracellular Medium-chain-length Poly(3-hydroxyalkanoate) Depolymerase Genes from Pseudomonas alcaligenes Strains: Do Young Kim , Hyun Chul Kim , Sun Young Kim , Young Ha Rhee; J. Microbiol. 2005;43(3):285-294.; DOI: https://doi.org/2211 [pii]

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Abstract: A bacterial strain M4-7 capable of degrading various polyesters, such as poly(e-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ_PalM4-7) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ_PalM4-7 was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35^oC. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ_PalLB19) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M_r of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ_PalM4-7) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced M_r of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ_PalLB19 and the PhaZ_PalM4-7 commonly share the lipase box, GISSG, in their catalytic domains, and utilize ^111Asn and ^110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by Penicillium pinophilum: Han, Jeen Sun , Son, Young Jong , Chang, Chung Soon , Kim, Mal Nam; J. Microbiol. 1998;36(2):67-73.

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Abstract: The extracellular poly(3-hydroxybutyrate)(PHB) depolymerase of Penicilliyum pinophilum ATCC 9644 was purified and characterized. When Penicillum sp. was grown in basal salt medium with PHB as a sole carbon source, higher temperature favored fungal mycelial growth (37℃>30℃>25℃), but enzyme production was lower ar 37℃ than at any other temperatures. The PHB depolymerase was purified using Sepharose CL06B and Sephacryl S0100HR column chromatography. The isolated enzyme was found to be composed of a single polypeptide chain with a molecular weight of about 35 kDa. The optimum condition for the enzyme was pH 6.0 and 50℃, Enzyme activity decreased sharply at temperatures above 50℃. The enzyme was found to be stable in the pH range of 2.0~10.0.1mM Fe^2+ reduced the enzyme activity by 55% abd 4 mM Fe^2+ inhibited it almost completely. The PHB depolymerase (10U) degraded 47% of solvent cast PHB film, while commercial lipase (1000U) of Rhizopus arrhizus degraded 10% of the same specimen, over a period of 48 hours.

Isolation of a Medium Chain Length Polyhydroxyalkanoic Acids Degrading Bacterium, Janthinobacterium lividum: Jin-Seo Park , Jeong-Youl Choi , Pil-Mun Joung , Seong Joo Park , Young Ha Rhe e , Kwang-Soo Shin; J. Microbiol. 2001;39(2):139-141.

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Abstract: Medium-chain length polyhydroxyalkanoic acids (MCL-PHAs) degrading bacterium was isolated from the soil. The bacterium was identified as Janthinobacterium lividum by its biochemical properties, cell membrane fatty acids composition, and 16S rDNA sequence analysis. The bacterium showed a similarity of 0.911 with J. lividum according to the cell membrane fatty acids analysis and a similarity of 97% in the 16S rDNA sequence analysis. Culture supernatant of the bacterium showed the highest depolymerase activity toward polyhydroxynonanoic acid (PHN) that did not degrade the poly-[beta]-hydroxybutyric acid (PHB). The esterase activity was also detected with p-nitrophenyl (PNP) esters of fatty acids such as PNP-octanoic acid, PNP-dodecanoic acid, PNP-decanoic acid, and PNP-hexanoic acid.

Purification and Characterization of Extracellular Poly(3-hydroxybutyrate) Depolymerase from Penicillium simplicissimum LAR13: Jee-Sun Han , Mal-Nam Kim; J. Microbiol. 2002;40(1):20-25.

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Abstract: An extracellular PHB depolymerase was purified from P. simplicissimum LAR13 cultural medium by Sepharose CL-6B chromatography. When the fungus was grown in a basal salt medium with poly(3-hydroxybutyrate) (PHB) as the sole carbon source, PHB depolymerase production reached maximum at its stationary phase. The mycelial growth rate was higher at 37 C than at 30 C and even higher than at 25 C, However, the enzyme production was lower at 37 C than 30 C or 25 C. The isolated enzyme is composed of a single polypeptide chain with a molecular mass of about 36 kDa as determined by SDS-PAGE. The optimum conditions for the enzyme activity are pH 5.0 and 45 C. The enzyme was stable for 30 min at a temperature lower than 50 C, and stable at pH higher than 2.0 but it was unstable at pH 1.0. 1 mM Fe^2+ reduced the enzyme activity by 56% and the enzyme was inhibited almost completely by 4 mM Fe^2+ . The enzyme was partially inhibited by phenylmethylsulfonyl fluoride and was very sensitive to diazo-DL-norleucine methyl ester, dithiothreitol and mercuric ion. However, N-p-tosyl-L-lysinechloromethyl ketone, p-hydroxymercuricbenzoate and N-acetylimidazole had no influence upon its activity.

Purification and Characterization of Poly(3-hydroxybutyrate) Depolymerase from a Fungal Isolate, Emericellopsis minima W2: Do Young Kim , Ji Hye Yun , Hyung Woo Kim , Kyung Sook Bae , Young Ha Rhee; J. Microbiol. 2002;40(2):129-133.

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Abstract: The fungus, Emericellopsis minima W2, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from a waste water sample. Production of the PHB depolymerase from E. minima W2 (PhaZ_Emi ) was significantly repressed in the presence of glucose. PhaZ_Emi was purified by column chromatography on Octyl-Sepharose CL-4B and Sephadex G-100. The molecular mass of the PhaZ_Emi , which consisted of a single polypeptide chain, was estimated to be 48.0 kDa by SDS-PAGE and its pI value was 4.4. The maximum activity of the PhaZ_Emi was observed at pH 9.0 and 55 C. It was significantly inactivated by 1 mM dithiothreitol, 2 mM diisopropyl fluorophosphate, 0.1 mM Tween 80, and 0.1 mM Triton X-100, but insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. The PhaZ_Emi efficiently hydrolyzed PHB and its copolyester with 30 mol% 3-hydroxyvalerate, but did not act on poly(3-hydroxyoctanoate). It also hydrolyzed p-nitrophenylacetate and p-nitrophenylbutyrate but hardly affected the longer-chain forms. The main hydrolysis product of PHB was identified as a dimer of 3-hydroxybutyrate.

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