• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1257-1267
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• 2023

Although rational genetic engineering is nowadays the favored method for microbial strain improvement, building up mutant libraries based on directed evolution for improvement is still in many cases the better option. In this regard, the demand for precise and efficient screening methods for mutants with high performance has stimulated the development of biosensor-based high-throughput screening strategies. Genetically encoded biosensors provide powerful tools to couple the desired phenotype to a detectable signal, such as fluorescence and growth rate. Herein, we review recent advances in engineering several classes of biosensors and their applications in directed evolution. Furthermore, we compare and discuss the screening advantages and limitations of two-component biosensors, transcription-factor-based biosensors, and RNA-based biosensors. Engineering these biosensors has focused mainly on modifying the expression level or structure of the biosensor components to optimize the dynamic range, specificity, and detection range. Finally, the applications of biosensors in the evolution of proteins, metabolic pathways, and genome-scale metabolic networks are described. This review provides potential guidance in the design of biosensors and their applications in improving the bioproduction of microbial cell factories through directed evolution.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1362-1368
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• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.831-839
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• 2023

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 ㎍/ml) and UN-C137 (6889.72 ± 70.25 ㎍/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 ㎍/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.881-888
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• 2010

solvent-tolerant bacterium strain, MH6, was isolated by hydrophilic organic solvent DMSO enrichment in the medium and identified as Serratia marcescens. The extracellular protease with novel organic-solvent-stable properties from strain MH6 was purified and characterized. The molecular mass of the purified protease was estimated to be 52 kDa on SDS-PAGE. The open reading frame (ORF) of the MH6 protease encoded 504 amino acids with 471 amino acid residues in the mature protease. Based on the inhibitory effects of EDTA and 1,10-phenathroline, the MH6 protease was characterized as a metalloproteinase. The enzyme activity was increased in the presence of $Ni^{2+}$, $Mg^{2+}$, and $Ca^{2+}$. The protease could also be activated by the nonionic surfactants Tween 80 (1.0%) and Triton X-100 (1.0%). The protease showed remarkable solvent stability in the presence of 50% (v/v) solutions of long-chain alkanes and long-chain alcohols. It was also fairly stable in the presence of 25% solutions of hydrophilic organic solvents. Owing to its high stability in solvents and surfactants, the MH6 protease is an ideal candidate for applications in organic catalysis and other related fields.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.408-412
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• 2009

A study on the screening and isolation of microorganism was performed for the removal of main malodor, such as ammonia, produced from the livestock farm. The main malodor components in livestock farm are ammonia, volatile fatty acids, sulfur compounds and trimethylamine. Damages to man and livestock were originated from malodors mainly due to ammonia, and thus ammonia reduction experiments were performed. Sludge of sewage treatment plant was inoculated in the sesame dregs culture, from which ammonia gas was produced. An aerobically grown, pure cultured isolated from the 10th enrichment culture was analyzed by 16S rRNA sequencing and identified as Alcaligenes sp. NS-1. This strain NS-1 precultured in the sesame dregs was found to remove ammonia gas with an efficiency of approximately 99-100% at an average concentration of 40 ppmv of ammonia gas. When the strain NS-1 sprayed to pig excrements, the removal efficiency at an average concentration of 100 ppmv of ammonia was approximately 60% after 16 hr.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.322-328
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• 2005

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the $\beta$-galactosidase activity ($\beta$-GAL) of a recombinant Agrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing $\beta$-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration­dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of $\beta$-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.

• Journal of Microbiology and Biotechnology
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• v.28 no.3
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• pp.409-417
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• 2018

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis, which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

• Microbiology and Biotechnology Letters
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• v.6 no.2
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• pp.59-63
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• 1978

The Screening of fungi producing naringinase was done. A strain of Aspergillus midulans showed the highest naringinase activity among 447 strains those were isolated from soil, spoiled citrus friuts and stock cultures. The cultural Conditions of Asp. nidulans for production of naringinase were studied. A strain of Asp. nidulans showed higher activity when it was cultivated at 30$^{\circ}C$ for 3 days on wheat bran media supplemented with 2.0％ naringin, 0.2％ (NH$_4$)$_2$ SO$_4$ and 0.2％ CaCO$_3$.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.228-232
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• 2003

Lovastatin produced by Aspergillus terreus via polyketide pathway is a secondary metabolite with high anti-hypercholesterolemic activity. In this paper we are going to present effective strain development strategies for lovastatin production by comparing the productivity of the mutants obtained through traditional rational screening process and protoplast fusion method. Mutants resistant against various antibiotics and/or antimetabolites showed significantly higher lovastatin productivity than the corresponding mother strains, demonstrating that rational screening method was very efficient in selecting high yielding producers. Recombinant fusants obtained using protoplast fusion between high producers were observed to have very different morphology and physiology as represented by the production and secretion of lovastatin, as well as cell growth pattern. In parallel with the strain development, optimization process for the production medium was carried out in order to find optimal concentrations of the medium components using such a powerful statistical method as response surface method (RSM). It was concluded that not only the optimum production medium but also good morphological characteristics of the high-yielding producers led to higher lovastatin production.

• Archives of Pharmacal Research
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• v.10 no.2
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• pp.103-109
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• 1987

To find microorganisms producing esterase inhibitors, microbes were isolated from soil samples that were collected at different locations in Korea and screened for inhibitory activities. One of the inhibitor-producing strains was named strain DMC-498. This strain was found to be a new species of the genus Streptomyces by comparison with the characteristics of morphology and metabolisms of the other species of the genus.