Reduction of d-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene

Jin, Q.,Li, L.,Moon, J.S.,Cho, S.K.,Kim, Y.J.,Lee, S.J.,Han, N.S. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>The n-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of D-lactate from pyruvate to L-lactate, we expressed the L-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The IdhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of IdhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to IdhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both L-LDH activity and L-lactate productivity during fermentation, decreasing the D-/L-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased L-lactate concentration and decreased D-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high L-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of D-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system

Jang, Y.J.,Seo, S.O.,Kim, S.A.,Li, L.,Kim, T.J.,Kim, S.C.,Jin, Y.S.,Han, N.S. Elsevier Science Publishers 2017 Journal of biotechnology Vol.251 No.-

<P>Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the 'killer' plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers.</P>

Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293

Li, L.,Eom, H.J.,Park, J.M.,Seo, E.,Ahn, J.E.,Kim, T.J.,Kim, J.H.,Han, N.S. IPC Science and Technology Press ; Elsevier Scienc 2012 Enzyme and microbial technology Vol.51 No.5

Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30<SUP>o</SUP>C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20<SUP>o</SUP>C, respectively, for the lactate oxidation reaction. The K<SUB>m</SUB> kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k<SUB>cat</SUB> values for pyruvate and lactate were 2900s<SUP>-1</SUP> and 2280s<SUP>-1</SUP>, respectively. The enzyme was not inhibited by Ca<SUP>2+</SUP>, Co<SUP>2+</SUP>, Cu<SUP>2+</SUP>, Mg<SUP>2+</SUP>, Mn<SUP>2+</SUP>, Na<SUP>+</SUP>, or urea, but was inhibited by 1mM Zn<SUP>2+</SUP> and 1mM SDS.

Lateral pterygoid muscle volume and migraine in patients with temporomandibular disorders

Sérgio Lúcio Pereira de Castro Lopes,André Luiz Ferreira Costa,Thiago de Oliveira Gamba,Isadora Luana Flores,Adriana Dibo Cruz,Li Li Min 대한영상치의학회 2015 Imaging Science in Dentistry Vol.45 No.1

Purpose: Lateral pterygoid muscle (LPM) plays an important role in jaw movement and has been implicated in Temporomandibular disorders (TMDs). Migraine has been described as a common symptom in patients with TMDs and may be related to muscle hyperactivity. This study aimed to compare LPM volume in individuals with and without migraine, using segmentation of the LPM in magnetic resonance (MR) imaging of the TMJ. Materials and Methods: Twenty patients with migraine and 20 volunteers without migraine underwent a clinical examination of the TMJ, according to the Research Diagnostic Criteria for TMDs. MR imaging was performed and the LPM was segmented using the ITK-SNAP 1.4.1 software, which calculates the volume of each segmented structure in voxels per cubic millimeter. The chi-squared test and the Fisher’s exact test were used to relate the TMD variables obtained from the MR images and clinical examinations to the presence of migraine. Logistic binary regression was used to determine the importance of each factor for predicting the presence of a migraine headache. Results: Patients with TMDs and migraine tended to have hypertrophy of the LPM (58.7%). In addition, abnormal mandibular movements (61.2%) and disc displacement (70.0%) were found to be the most common signs in patients with TMDs and migraine. Conclusion: In patients with TMDs and simultaneous migraine, the LPM tends to be hypertrophic. LPM segmentation on MR imaging may be an alternative method to study this muscle in such patients because the hypertrophic LPM is not always palpable.

Numerical studies on the effects of the lateral boundary on soil-structure interaction in homogeneous soil foundations

Li, Z.N.,Li, Q.S.,Lou, M.L. Techno-Press 2005 Structural Engineering and Mechanics, An Int'l Jou Vol.20 No.4

In this paper, the finite element method is applied to investigate the effect of the lateral boundary in homogenous soil on the seismic response of a superstructure. Some influencing factors are presented and discussed, and several parameters are identified to be important for conducting soil-structure interaction experiments on shaking tables. Numerical results show that the cross-section width L, thickness H, wave propagation velocity and lateral boundaries of soil layer have certain influences on the computational accuracy. The dimensionless parameter L/H is the most significant one among the influencing factors. In other words, a greater depth of soil layer near the foundation should be considered in shaking table tests as the thickness of the soil layer increases, which can be regarded as a linear relationship approximately. It is also found that the wave propagation velocity in soil layer affects the numerical accuracy and it is suggested to consider a greater depth of the soil layer as the wave propagation velocity increases. A numerical study on a soil-structure experimental model with a rubber ring surrounding the soil on a shaking table is also conducted. It is found the rubber ring has great effect on the soil-structure interaction experiments on shaking table. The experimental precision can be improved by reasonably choosing the elastic parameter and width of the rubber ring.

Fungal diversity notes 253–366: taxonomic and phylogenetic contributions to fungal taxa

Li, G. J.,Hyde, K. D.,Zhao, R. L.,Hongsanan, S.,Abdel-Aziz, F. A.,Abdel-Wahab, M. A.,Alvarado, P.,Alves-Silva, G.,Ammirati, J. F.,Ariyawansa, H. A. Springer Science and Business Media 2016 FUNGAL DIVERSITY Vol.78 No.1

<P>Notes on 113 fungal taxa are compiled in this paper, including 11 new genera, 89 new species, one new subspecies, three new combinations and seven reference specimens. A wide geographic and taxonomic range of fungal taxa are detailed. In the Ascomycota the new genera Angustospora (Testudinaceae), Camporesia (Xylariaceae), Clematidis, Crassiparies (Pleosporales genera incertae sedis), Farasanispora, Longiostiolum (Pleosporales genera incertae sedis), Multilocularia (Parabambusicolaceae), Neophaeocryptopus (Dothideaceae), Parameliola (Pleosporales genera incertae sedis), and Towyspora (Lentitheciaceae) are introduced. Newly introduced species are Angustospora nilensis, Aniptodera aquibella, Annulohypoxylon albidiscum, Astrocystis thailandica, Camporesia sambuci, Clematidis italica, Colletotrichum menispermi, C. quinquefoliae, Comoclathris pimpinellae, Crassiparies quadrisporus, Cytospora salicicola, Diatrype thailandica, Dothiorella rhamni, Durotheca macrostroma, Farasanispora avicenniae, Halorosellinia rhizophorae, Humicola koreana, Hypoxylon lilloi, Kirschsteiniothelia tectonae, Lindgomyces okinawaensis, Longiostiolum tectonae, Lophiostoma pseudoarmatisporum, Moelleriella phukhiaoensis, M. pongdueatensis, Mucoharknessia anthoxanthi, Multilocularia bambusae, Multiseptospora thysanolaenae, Neophaeocryptopus cytisi, Ocellularia arachchigei, O. ratnapurensis, Ochronectria thailandica, Ophiocordyceps karstii, Parameliola acaciae, P. dimocarpi, Parastagonospora cumpignensis, Pseudodidymosphaeria phlei, Polyplosphaeria thailandica, Pseudolachnella brevifusiformis, Psiloglonium macrosporum, Rhabdodiscus albodenticulatus, Rosellinia chiangmaiensis, Saccothecium rubi, Seimatosporium pseudocornii, S. pseudorosae, Sigarispora ononidis and Towyspora aestuari. New combinations are provided for Eutiarosporella dactylidis (sexual morph described and illustrated) and Pseudocamarosporium pini. Descriptions, illustrations and/or reference specimens are designated for Aposphaeria corallinolutea, Cryptovalsa ampelina, Dothiorella vidmadera, Ophiocordyceps formosana, Petrakia echinata, Phragmoporthe conformis and Pseudocamarosporium pini. The new species of Basidiomycota are Agaricus coccyginus, A. luteofibrillosus, Amanita atrobrunnea, A. digitosa, A. gleocystidiosa, A. pyriformis, A. strobilipes, Bondarzewia tibetica, Cortinarius albosericeus, C. badioflavidus, C. dentigratus, C. duboisensis, C. fragrantissimus, C. roseobasilis, C. vinaceobrunneus, C. vinaceogrisescens, C. wahkiacus, Cyanoboletus hymenoglutinosus, Fomitiporia atlantica, F. subtilissima, Ganoderma wuzhishanensis, Inonotus shoreicola, Lactifluus armeniacus, L. ramipilosus, Leccinum indoaurantiacum, Musumecia alpina, M. sardoa, Russula amethystina subp. tengii and R. wangii are introduced. Descriptions, illustrations, notes and / or reference specimens are designated for Clarkeinda trachodes, Dentocorticium ussuricum, Galzinia longibasidia, Lentinus stuppeus and Leptocorticium tenellum. The other new genera, species new combinations are Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis from Neocallimastigomycota, Phytophthora estuarina, P. rhizophorae, Salispina, S. intermedia, S. lobata and S. spinosa from Oomycota, and Absidia stercoraria, Gongronella orasabula, Mortierella calciphila, Mucor caatinguensis, M. koreanus, M. merdicola and Rhizopus koreanus in Zygomycota.</P>

Changes of Key Enzymes in Sulfate Assimilation in Response to the S-deficiency in Rape (Brassicanapus L.)

L. S. Li,B. R. LEE,D. H. Kim,T. H. Kim 한국초지조사료학회 2008 한국초지조사료학회 학술대회논문집 Vol.2008 No.09

Numerical studies on the effects of the lateral boundary on soil-structure interaction in homogeneous soil foundations

Z. N. Li,Q. S. Li,M. L. Lou 국제구조공학회 2005 Structural Engineering and Mechanics, An Int'l Jou Vol.20 No.4

In this paper, the finite element method is applied to investigate the effect of the lateral boundary in homogenous soil on the seismic response of a superstructure. Some influencing factors are presented and discussed, and several parameters are identified to be important for conducting soil-structure interaction experiments on shaking tables. Numerical results show that the cross-section width L, thickness H, wave propagation velocity and lateral boundaries of soil layer have certain influences on the computational accuracy. The dimensionless parameter L/H is the most significant one among the influencing factors. In other words, a greater depth of soil layer near the foundation should be considered in shaking table tests as the thickness of the soil layer increases, which can be regarded as a linear relationship approximately. It is also found that the wave propagation velocity in soil layer affects the numerical accuracy and it is suggested to consider a greater depth of the soil layer as the wave propagation velocity increases. A numerical study on a soil-structure experimental model with a rubber ring surrounding the soil on a shaking table is also conducted. It is found the rubber ring has great effect on the soil-structure interaction experiments on shaking table. The experimental precision can be improved by reasonably choosing the elastic parameter and width of the rubber ring.

Activity of Enzymes Related to Nitrogen Metabolism Under S-deficiency in Leaves of Rape (Brassica napus L.)

B. R. LEE,L. S. Li,Y. L. Jin,D. H. Kim,T. H. Kim 한국초지조사료학회 2008 한국초지조사료학회 학술대회논문집 Vol.2008 No.09

Growth hormone and receptor gene mutations in Chinese Banna miniature pig

Deng, J.Z.,Hao, L.L.,Li, M.T.,Lang, S.,Zeng, Y.Z.,Liu, S.C.,Zhang, Y.L. The Korean Society for Integrative Biology 2011 Animal cells and systems Vol.15 No.4

The Banna miniature pig (BNMP) is a representative miniature pig breed in China. Even though BNMP dwarfism is obvious, its underlying causative mutations remain unknown. In this study, the BNMP and Large White pig (LWP) serum growth hormone (GH) and insulin-like growth factor (IGF-1) levels were detected by ELISA and compared. BNMP serum IGF-1 levels were significantly lower than LWP levels (P<0.05). The miniature condition may arise from mutations in the GH and GH receptor (GHR) genes. Therefore, GH and GHR cDNA from the BNMP were cloned into a pMD18-T vector by RT-PCR using the total RNA obtained from the BNMP's pituitary and liver tissues. Sequencing results indicated that the open reading frame of the BNMP GH gene is composed of a 26-residue signal peptide and a 191-residue mature peptide. The coding sequence of the BNMP GHR gene contained 639 amino acids, including a signal peptide that is 18 amino acids long. Two amino acid substitutions, A09V and R22Q, were found in the signal peptide of the GH gene. Additionally, the S104P mutation was found in the BNMP's mature GH protein. Four mutations in the cytoplasmic domain of GHR may influence the downstream signal transduction of GHR, which needs further experimental evidence.