Journal of Microbiology

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Development of a Novel D‑Lactic Acid Production Platform Based on Lactobacillus saerimneri TBRC 5746: Kitisak Sansatchanon , Pipat Sudying , Peerada Promdonkoy , Yutthana Kingcha , Wonnop Visessanguan , Sutipa Tanapongpipat , Weerawat Runguphan , Kanokarn Kocharin; J. Microbiol. 2023;61(9):853-863. Published online September 14, 2023; DOI: https://doi.org/10.1007/s12275-023-00077-x

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D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains, Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism. We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and observed a titer of 99.4 g/L and a yield of 0.90 g/g glucose (90% of the theoretical yield). However, we also observed L-lactic acid production, which reduced the product’s optical purity. We then used CRISPR/dCas9-assisted transcriptional repression to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.

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Industrial–scale production of various bio–commodities by engineered microbial cell factories: Strategies of engineering in microbial robustness
Ju-Hyeong Jung, Vinoth Kumar Ponnusamy, Gopalakrishnan Kumar, Bartłomiej Igliński, Vinod Kumar, Grzegorz Piechota
Chemical Engineering Journal.2024; 502: 157679. CrossRef
Microbial Cell Factories: Biodiversity, Pathway Construction, Robustness, and Industrial Applicability
Rida Chaudhary, Ali Nawaz, Mireille Fouillaud, Laurent Dufossé, Ikram ul Haq, Hamid Mukhtar
Microbiology Research.2024; 15(1): 247. CrossRef
Adaptive Evolution for the Efficient Production of High-Quality d-Lactic Acid Using Engineered Klebsiella pneumoniae
Bo Jiang, Jiezheng Liu, Jingnan Wang, Guang Zhao, Zhe Zhao
Microorganisms.2024; 12(6): 1167. CrossRef
Enhancing D-lactic acid production from non-detoxified corn stover hydrolysate via innovative F127-IEA hydrogel-mediated immobilization of Lactobacillus bulgaricus T15
Yuhan Zheng, Feiyang Sun, Siyi Liu, Gang Wang, Huan Chen, Yongxin Guo, Xiufeng Wang, Maia Lia Escobar Bonora, Sitong Zhang, Yanli Li, Guang Chen
Frontiers in Microbiology.2024;[Epub] CrossRef

Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis: Ozan Ertekin , Meltem Kutnu , Aslı Aras Ta&# , Mustafa Demir , Ayten Yazgan Karata&# , Gülay Özcengiz; J. Microbiol. 2020;58(4):297-313. Published online January 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9064-0

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Abstract

Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA􍿁::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLCMS/ MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment- specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoCregulated proteins.

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Biocontrol Ability of Strain Bacillus amyloliquefaciens SQ-2 against Table Grape Rot Caused by Aspergillus tubingensis
Suran Li, Shuangshuang Dai, Lei Huang, Yumeng Cui, Ming Ying
Journal of Agricultural and Food Chemistry.2024; 72(44): 24374. CrossRef
Isolation and identification of a novel Bacillus velezensis strain JIN4 and its potential for biocontrol of kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae
Xin Zhao, Yang Zhai, Lin Wei, Fei Xia, Yuanru Yang, Yongjian Yi, Hongying Wang, Caisheng Qiu, Feng Wang, Liangbin Zeng
Frontiers in Plant Science.2024;[Epub] CrossRef
Signatures of kin selection in a natural population of the bacteria Bacillus subtilis
Laurence J Belcher, Anna E Dewar, Chunhui Hao, Melanie Ghoul, Stuart A West
Evolution Letters.2023; 7(5): 315. CrossRef
Comparative biological network analysis for differentially expressed proteins as a function of bacilysin biosynthesis in Bacillus subtilis
Meltem Kutnu, Elif Tekin İşlerel, Nurcan Tunçbağ, Gülay Özcengiz
Integrative Biology.2022; 14(5): 99. CrossRef
Probiotic effects of the Bacillus velezensis GY65 strain in the mandarin fish, Siniperca chuatsi
Jiachuan Wang, Defeng Zhang, Yajun Wang, Zhijun Liu, Lijuan Liu, Cunbin Shi
Aquaculture Reports.2021; 21: 100902. CrossRef
Bacilysin within the Bacillus subtilis group: gene prevalence versus antagonistic activity against Gram-negative foodborne pathogens
Catherine Nannan, Huong Quynh Vu, Annika Gillis, Simon Caulier, Thuy Thanh Thi Nguyen, Jacques Mahillon
Journal of Biotechnology.2021; 327: 28. CrossRef
Impact of spatial proximity on territoriality among human skin bacteria
Jhonatan A. Hernandez-Valdes, Lu Zhou, Marcel P. de Vries, Oscar P. Kuipers
npj Biofilms and Microbiomes.2020;[Epub] CrossRef

Research Support, Non-U.S. Gov'ts

Packaging of Porcine Reproductive and Respiratory Syndrome Virus Replicon RNA by a Stable Cell Line Expressing Its Nucleocapsid Protein: Byung-Hak Song , Jeong-Min Kim , Jin-Kyoung Kim , Han-Saem Jang , Gil-Nam Yun , Eun-Jin Choi , Jae-Young Song , Sang-Im Yun , Young-Min Lee; J. Microbiol. 2011;49(3):516-523. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1280-1

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Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrep19/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-ΔN) with a 177-nucleotide deletion, removing the 3′-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4×102 to 1.1×102 infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

Complementation System for Helicobacter pylori: Jinmoon Kim , Sung-Whan Kim , Sungil Jang , D. Scott Merrell , Jeong-Heon Cha; J. Microbiol. 2011;49(3):481-486. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1196-9

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Abstract: Previously Langford et al. (2006) developed the pIR203C04 complementation system for Helicobacter pylori, which can be used to complement and restore phenotypic effects in H. pylori mutant, and furthermore they used the complementation system in vivo experiments to animals without altering the ability of strain SS1 to colonize mice. In their previous study, the pIR203C04 was able to transform 26695, SS1, J99, and 43504 H. pylori strains by an electroporation method. However, in the present study using a natural transformation the pIR203C04 transformed only 26695 H. pylori but not SS1, J99, 7.13, and G27 H. pylori strains. Since the useful complementation system has a limitation of narrow selection among H. pylori strains, we redesigned the complementation system for the improvement. The same intergenic chromosomal site between hp0203 and hp0204 was utilized for the new complementation system because the insertion at the intergenic site didn’t show any polar effects and disruption of other H. pylori genes. The genome sequence analysis showed that the intergenic regions among H. pylori strains may have too low homology to each others to do a homologous recombination. Thus, in addition to the short intergenic region, the fragments of the new complementation system included 3′ conserved parts of hp0203 and hp0204 coding regions. Between the fragments there are a chloramphenicol acetyltransferase cassette and multicloning sites, resulting in pKJMSH. DNA fragment of the interest can be cloned into the multicloning sites of pKJMSH and the fragment can be integrated at the intergenic region of H. pylori chromosome by the homologous recombination. Indeed, by the natural transformation, pKJMSH was able to transform all five H. pylori strains of 26695, SS1, J99, 7.13, and G27, which are common for the investigation of molecular pathogenesis. Thus, the new pKJMSH complementation system is applicable to most H. pylori wild-type stains.

cloning of Gene Encoding for Siderophore biosynthesis in Fluorescent Pseudomonas sp.: Koh, Han Cheol , Ha, Sung Cheol , Na, Jung A , Kim, Ho Sang , Yeo, Myeong Gu , Lee, Jung Sup , Kim, Sung Jun , Park, yeal; J. Microbiol. 1995;33(1):28-33.

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Abstract: Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

Molecular cloning of the arginine biosynthetic genes from corynebacterium glutamicum: Chun, Jae Yeon , Jung, Sam Il , Ko, Soon Young , Park, Mee Young , Kim, Soo Young , Lee, Heung Shickc , Cheon, Choong Ill , Min, Kyung Hee , Lee, Myeong Sok; J. Microbiol. 1996;34(4):355-362.

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Abstract: Complementation cloning of the argC, E, B D, F, and G genes in Corynebacterium glutamicum was done by transforming the genomic DNA library into the corresponding arginine auxotrophs fo Escherichia coli. Recombinant plasmids containing 6.7 kb and 4.8kb fragments complementing the E. coli arg B mutant were also able to complement the E. coli argC, E, A, D, and F mutants, indicating the clustered organization of the arginine biosynthetic genes within the cloned DNA fragments. The insert DNA fragments in the recombinant plasmids, named pRB1 AND pRB2, were physically mapped with several restriction enzymes. By further subcloning the entire DNA fragment containing the functions and by complementation analysis, we located the arg genes in the order of ACEBDF on the restriction map. We also determined the DNA nucleotide sequence of the fragment and report here the sequence of the argB gene. When compared to that with the nutant strain, higherh enzyme activity of N-acetylglutamate kinase was detacted in the extract of the mutant carrying the plasmid containing the putative argB gene, indicating that the plasmid contains a functional argB gene. Deduced amino acid sequence of the argB gene shows45%, 38%, and 25% identity to that from Bacillus strearothermophilus, Bacillus substilus, and E. coli respectively. Our long term goal is genetically engineering C. glutamicum which produces more arginine than a wild type strain does.

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