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Bacteriocin production in lactic acid bacteria (LAB) has always been considered as a highly desirable trait as it enhances the strain's utility in different industrial applications. Bacteriocin producing LAB strains are considered to have higher bacterial fitness as they are able to easily establish themselves into target microbial niche and hence are more effective starter cultures in food fermentation and/or probiotic strains. The rapid advancement in genomic research revealed the true bacteriocin producing capacity of some select novel LAB strains capable of producing multiple bacteriocins which further improves their utility in different application systems. What is common to these novel strains is the remarkable sharing of some elements in the biosynthetic process enabling them to accomplish the extraordinary feat of producing multiple bacteriocins without exhausting its energy. Contrary to the common understanding that biosynthetic enzymes are specific to their cognate bacteriocins, multiple bacteriocin producing strains employ shared biosynthetic elements between their multiple bacteriocins. The quorum-sensing three-component regulatory system, bacteriocin maturation and transport mechanisms are shared among multiple bacteriocins in these strains. Nevertheless, although these novel strains possess enormous application potential, their safety with regards to their potential virulence and pathogenicity needs to be confirmed through comprehensive genotypic characterization. Here, we compile the occurrence of multiple bacteriocin production in some novel LAB strains and highlight specific examples of the unique sharing mechanism of its biosynthetic machinery because a good understanding how these novel strains synthesize their multiple bacteriocins can aid in maximizing their application potential., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5'-ATTTTAGGATA-3') were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We demonstrate here that exopolysaccharide (EPS) production, cell aggregation, and bile resistance in Lactobacillus brevis KB290 are conferred by three eps genes (gtf27, gtf28, and orf29) located on the 42.4-kb plasmid pKB290-1. The predicted products of gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family whereas the orf29 gene product showed homology with the ABC transporter. On in silico analysis, these genes were found to be widely distributed among lactobacilli from publicly available genomes and metagenomes, and their function is not yet elucidated. RT-PCR analysis showed that the eps genes were organised in an operon and their expression was markedly lower in arabinose- and xylose-containing media than in a glucose-containing medium. The three eps genes were cloned and expressed in homologous and heterologous strains. Considerably less EPS was produced by the plasmid-cured KB1802 strain than by the parental KB290 strain, whereas a similar amount was produced by the KB1802 strain expressing the three eps genes. The KB1802 strain expressing gtf27 and gtf28 but not orf29 did not produce EPS. Cell aggregation and bile resistance were also decreased in KB1802 strains but were complemented by eps genes. Moreover, the three eps genes conferred these phenotypes to a Lactobacillus plantarum strain. In conclusion, the three eps genes in pKB290-1 were sufficient for EPS biosynthesis with glucose and N-acetylglucosamine, and were responsible for cell aggregation and bile resistance. We consider these phenotypes to be at least partly responsible for KB290-specific properties., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (V e /V b ) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large V e /V b ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a V e /V b ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a V e /V b ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a V e /V b ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a V e /V b ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Lactococcin Z is a novel Lactococcus-specific bacteriocin produced by Lactococcus lactis QU 7 that shares 55.6% identity with lactococcin A. To identify the receptor targeted by lactococcin Z, several lactococcin Z-resistant mutants were generated from the sensitive strain, L. lactis IL1403. The resistant mutants showed difficulties in utilising mannose and glucose as sole carbon sources, contrary to their pattern of growth in the presence of galactose as a sole carbon source. Mutations were found in the ptnC and ptnD genes of lactococcin Z-resistant mutants, which encode the mannose phosphotransferase system (Man-PTS) components, IIC and IID, respectively; therefore, IIC and IID are proposed as potential receptors employed by lactococcin Z and are the same receptors targeted by lactococcin A. Both lactococcins A and Z share a high percentage identity in their N-termini regions in contrast to their C-termini that show less similarity; this may explain the difference in their action mechanisms as well as the lack of cross-immunity between them. Although lactococcin Z showed bactericidal activity, it neither dissipated membrane potential nor formed pores on the membranes of sensitive cells, in sharp contrast to the pore-forming lactococcin A., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH 4 ) 2 HPO 4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Binding to lipid II is an important step in the mode of action of most lantibiotics targeting the bacterial cell wall. We applied the Bacillus subtilis two-component system, LiaRS, that is known to respond to antibiotics interfering with lipid II cycle, in order to evaluate lipid II binding activity of known bacteriocins and also to identify lipid II binding moieties in lantibiotic nukacin ISK-1. Using this method, we confirmed that the methyllanthionine ring in nukacin ISK-1 is crucial for lipid II binding as previously indicated. In this study, we further identified that the three N-terminal lysine residues (K1, K2, and K3) and the glycine (G5) residue in nukacin ISK-1 are also important in lipid II binding., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Glycerol is a by-product in the biodiesel production process and considered as one of the prospective carbon sources for microbial fermentation including lactic acid fermentation, which has received considerable interest due to its potential application. Enterococcus faecalis isolated in our laboratory produced optically pure L-lactic acid from glycerol in the presence of acetic acid. Gas chromatography-mass spectrometry analysis using [1, 2-(13)C2] acetic acid proved that the E. faecalis strain QU 11 was capable of converting acetic acid to ethanol during lactic acid fermentation of glycerol. This indicated that strain QU 11 restored the redox balance by oxidizing excess NADH though acetic acid metabolism, during ethanol production, which resulted in lactic acid production from glycerol. The effects of pH control and substrate concentration on lactic acid fermentation were also investigated. Glycerol and acetic acid concentrations of 30 g/L and 10 g/L, respectively, were expected to be appropriate for lactic acid fermentation of glycerol by strain QU 11 at a pH of 6.5. Furthermore, fed-batch fermentation with 30 g/L glycerol and 10 g/L acetic acid wholly exhibited the best performance including lactic acid production (55.3 g/L), lactic acid yield (0.991 mol-lactic acid/mol-glycerol), total yield [1.08 mol-(lactic acid and ethanol)]/mol-(glycerol and acetic acid)], and total carbon yield [1.06 C-mol-(lactic acid and ethanol)/C-mol-(glycerol and acetic acid)] of lactic acid and ethanol. In summary, the strain QU 11 successfully produced lactic acid from glycerol with acetic acid metabolism, and an efficient fermentation system was established without carbon loss., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

There has been tremendous growth in the production of optically pure l-lactic acid from lignocellulose-derived sugars. In this study, Enterococcus mundtii QU 25 was used to ferment a glucose/xylose mixture to l-lactic acid. Maintenance of the xylose concentration at greater than 10 g/L achieved homo-lactic acid fermentation and reduced the formation of byproducts. Furthermore, carbon catabolite repression (CCR) was avoided by maintaining the glucose concentration below 25 g/L; therefore, initial concentrations of 25 g/L glucose and 50 g/L xylose were selected. Supplementation with 5 g/L yeast extract enhanced the maximum xylose consumption rate and consequently increased lactic acid production and productivity. Finally, a 129 g/L lactic acid without byproducts was obtained with a maximum lactic acid productivity of 5.60 g/(L·h) in fed-batch fermentation with feeding a glucose/xylose mixture using ammonium hydroxide as the neutralizing agent. These results indicate a potential for lactic acid production from glucose and xylose as the main components of lignocellulosic biomasses., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Butanol has recently attracted attention as an alternative biofuel because of its various advantages over other biofuels. Many researchers have focused on butanol fermentation with renewable and sustainable resources, especially lignocellulosic materials, which has provided significant progress in butanol fermentation. However, there are still some drawbacks in butanol fermentation in terms of low butanol concentration and productivity, high cost of feedstock and product inhibition, which makes butanol fermentation less competitive than the production of other biofuels. These hurdles are being resolved in several ways. Genetic engineering is now available for improving butanol yield and butanol ratio through overexpression, knock out/down, and insertion of genes encoding key enzymes in the metabolic pathway of butanol fermentation. In addition, there are also many strategies to improve fermentation technology, such as multi-stage continuous fermentation, continuous fermentation integrated with immobilization and cell recycling, and the inclusion of additional organic acids or electron carriers to change metabolic flux. This review focuses on the most recent advances in butanol fermentation especially from the perspectives of genetic engineering and fermentation technology., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The development and implementation of renewable materials for the production of versatile chemical resources have gained considerable attention recently, as this offers an alternative to the environmental problems caused by the petroleum industry and the limited supply of fossil resources. Therefore, the concept of utilizing biomass or wastes from agricultural and industrial residues to produce useful chemical products has been widely accepted. Lactic acid plays an important role due to its versatile application in the food, medical, and cosmetics industries and as a potential raw material for the manufacture of biodegradable plastics. Currently, the fermentative production of optically pure lactic acid has increased because of the prospects of environmental friendliness and cost-effectiveness. In order to produce lactic acid with high yield and optical purity, many studies focus on wild microorganisms and metabolically engineered strains. This article reviews the most recent advances in the biotechnological production of lactic acid mainly by lactic acid bacteria, and discusses the feasibility and potential of various processes., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Nukadoko is a fermented rice bran mash traditionally used for pickling vegetables in Japan. To date, the production of both homemade and commercial nukadoko depends on natural fermentation without using starter cultures. Here, we monitored chemical and microbiological changes in the initial batch fermentation of nukadoko. Nukadoko samples were prepared by spontaneous fermentation of four different brands of rice bran, and microbiome dynamics were analyzed for 2 months. In the first week, non-Lactobacillales lactic acid bacteria (LAB) species, which differed among the samples, grew proportionally to pH decrease and lactate increase. Thereafter, Lactobacillus plantarum started growing and consumed residual sugars, causing further lactate increase in nukadoko. Finally, microbial communities in all tested nukadoko samples were dominated by L. plantarum. Taken together, our results suggest that the mixture of the fast-growing LAB species and slow-growing L. plantarum may be used as a suitable starter culture to promote the initial fermentation of nukadoko., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Biological functions of a DUF95 superfamily protein in the biosynthesis gene cluster of a novel circular bacteriocin, leucocyclicin Q (LcyQ), were characterized in this paper. Sequence analysis and database search of the regions flanking the LcyQ structural gene lcyQ revealed four open reading frames (lcyR, lcyB, lcyC, and lcyD) related to bacteriocin biosynthesis. LcyD shares some similarity to the DUF95 superfamily proteins, often found in the biosynthetic gene clusters of circular bacteriocins. Mass spectrometry analysis showed accumulation of active mature LcyQ inside lcyD knockout cells. Heterologous expression of lcyD demonstrated that it confers robust immunity against LcyQ. Peptide release/binding assay revealed that the immunity could be attributed to the secretion of LcyQ to the cell exterior. Thus, the DUF95 superfamily protein has a dual function in the biosynthesis of LcyQ, as an immunity-associated transporter and as a secretion-aiding agent. Accumulation of mature LcyQ inside the cell in lcyD knockout strains, further implied that cyclization occurs within the cell. To the best of our knowledge, this is the first report on LcyQ cyclization inside the cell and the dual role of a DUF95 superfamily protein in circular bacteriocin biosynthesis., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4 and mixed sugars containing cellobiose and xylose was studied to establish efficient butanol production process without carbon catabolite repression (CCR). Although batch culture with glucose and xylose exhibited apparent CCR, we achieved simultaneous consumption of cellobiose and xylose. Moreover, preculture of the N1-4 strain with xylose yielded maximum butanol and solvent concentrations (16 and 23 g/L, respectively). Thus, we succeeded in ABE fermentation with mixed sugars of hexose and pentose, without CCR, by using wild-type ABE-producing clostridia. We also investigated the effect of various ratios of cellobiose and xylose on the fermentation process and yield. Increasing initial xylose concentration improved butanol and solvent concentrations and maximum xylose consumption rate. Fed-batch culture with cellobiose and xylose showed rapid and simultaneous sugar consumption and improved maximum consumption rate of both sugars., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In a previous in vitro study, we reported that the potential mechanism of the cholesterol-lowering effect of Lactobacillus brevis 119-2 isolated from turnip Tsuda kabu was the incorporation of cholesterol to cell membrane. In this study, we analyzed serum cholesterol and hepatic gene expression of Sprague-Dawley (SD) rats kept on a cholesterol diet with or without L. brevis 119-2 for 2 weeks, to evaluate the cholesterol-lowering effect in vivo. Serum cholesterol levels were significantly reduced in SD rats kept on a diet including L. brevis 119-2 compared with that in SD rats kept on a diet without L. brevis 119-2, and both viable and dead L. brevis 119-2 induced this effect. Hepatic gene analysis by DNA microarray suggested that the potential mechanism of the cholesterol-lowering effect of L. brevis 119-2 in vivo was inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity by insulin induced gene (Insig) protein, and induction of catabolism of cholesterol to bile acid by Cyp7a1 (cytochrome P450 a1). In addition, we found that inclusion of L. brevis 119-2 in the diet decreased serum low density lipoprotein (LDL) cholesterol levels by inducing overexpression of the LDL receptor gene. In contrast, Lactobacillus acidophilus ATCC 43121 increased high density lipoprotein cholesterol levels by inducing overexpression of the ATP-binding cassette sub-family. A member 1 (Abca1) and Angiopoietin-like 3 (Angptl3) genes. These results suggest that L. brevis 119-2 decreases the risk of atherosclerosis by lowering serum cholesterol, ameliorating the effect of fatty liver., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Two chemically defined media, CDM-1G and CDM-1X, that use glucose and xylose as carbon sources, respectively, were prepared for Lactococcus lactis strain IO-1. The maximal cell density at 600 nm in CDM-1G exceeded 2. Omission growth experiments indicated that IO-1 is auxotrophic for 2 vitamins and 6 amino acids., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We previously reported a butanol production process with pH-stat continuous feeding of dl-lactic acid and glucose as the co-substrate (Oshiro et al., Appl. Microbiol. Biotechnol., 87, 1177-1185, 2010). To accomplish butanol production from completely inedible substrates, in this study, we investigated acetone-butanol-ethanol (ABE) fermentation of Clostridium saccharoperbutylacetonicum N1-4 with lactic acid by using pentose as the co-substrate. Examination for optimum co-substrate indicated that arabinose was superior to glucose and xylose for ABE fermentation. Actually batch culture with lactic acid and arabinose without pH control exhibited higher butanol production (7.11 g/l) and lactic acid consumption (2.02 g) than those (6.62 g/l and 1.45 g, respectively) with glucose. Fed-batch culture without pH control increased these values to 12.08 g/l and 15.60 g/l butanol production, and to 3.83 g and 5.91 g lactic acid consumption by feeding the substrate once and twice, respectively. Finally, the result of gas chromatography-mass spectroscopy analysis using [1,2,3-(13)C(3)]-lactic acid indicated that lactic acid was converted to butanol with the efficiency of 51.9%. Thus, we established a novel high butanol production from lactic acid using arabinose as the co-substrate in simple fed-batch culture., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20-30°C) than enterocins NKR-5-3A, C and D (25°C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR-5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter.

We proposed a kinetic simulation model of xylose metabolism in Lactococcus lactis IO-1 that describes the dynamic behavior of metabolites using the simulator WinBEST-KIT. This model was developed by comparing the experimental time-course data of metabolites in batch cultures grown in media with initial xylose concentrations of 20.3-57.8 g/l with corresponding calculated data. By introducing the terms of substrate activation, substrate inhibition, and product inhibition, the revised model showed a squared correlation coefficient (r2) of 0.929 between the experimental time-course of metabolites and the calculated data. Thus, the revised model is assumed to be one of the best candidates for kinetic simulation describing the dynamic behavior of metabolites. Sensitivity analysis revealed that pyruvate flux distribution is important for higher lactate production. To confirm the validity of our kinetic model, the results of the sensitivity analysis were compared with enzyme activities observed during increasing lactate production by adding natural rubber serum powder to the xylose medium. The experimental results on pyruvate flux distribution were consistent with the prediction by sensitivity analysis.

Lantibiotics are one of the most promising alternative candidates for future antibiotics that maintain their antibacterial efficacy through many mechanisms. Of these mechanisms, some modes of activity have recently been reported, providing opportunities to show these peptides as potential candidates for forthcoming applications. Many findings providing new insight into the detailed molecular activities of numerous lantibiotics are constantly being uncovered. The combination of antibiotic mechanisms in one lantibiotic molecule shows its diverse antimicrobial usefulness as a future generation of antibiotic. Since lantibiotics do not have any known candidate resistance mechanisms, the discovered distinct modes of activity may revolutionize the design of anti-infective drugs through the knowledge provided by these super molecules. In this review, we discuss the rising assortment of lantibiotics, with special emphasis on their structure-function relationships, addressing the unique activities involved in their individual modes of action.

Recently, lactic acid bacteria (LAB) have attracted much attention because of their potential application to probiotics and industrial applications as starters for dairy products or lactic acid fermentation. Additional emphasis is also being paid to them as commensal bacteria in gastrointestinal tract. Since LAB exhibit a stress response, insight into the relationship between stress proteins such as molecular chaperones and stress tolerance or adaptation is increasing gradually along with current research examining these important bacteria. Similar to other bacteria, one of the major stress-response systems in LAB is the expression of molecular chaperones. The recently completed genome sequencing of various LAB strains, combined with the development of advanced molecular techniques, have enabled us to identify molecular chaperones and to understand their regulation systems in response to various stresses. Furthermore, recent biochemical studies provided novel insight into the molecular mechanisms of LAB chaperone systems. This review highlights the physiological consequences and biochemical properties of molecular chaperones (especially sHsps, Hsp70, and Hsp100) in LAB and their use in biotechnological applications.

Fermented barley extract (FBE) obtained from a barley shochu by-product (shochu kasu) and its ethanol fractions were evaluated as a medium and supplement, respectively, for nisin A production by Lactococcus lactis subsp. lactis ATCC 11454. A Brix 2.5 FBE medium supplemented with glucose provided a high level of nisin A production with a nisin yield comparable to that from a nutritionally rich laboratory medium (basal medium). By adding the ethanol-insoluble (EI) fraction of FBE to the basal medium, nisin A production was enhanced concomitant with an increase in bacterial cell growth, while the ethanol-soluble (ES) fraction had a negative effect on nisin A production. These findings indicate that FBE obtained from shochu kasu can be utilized as a preferable medium for nisin A production and could be converted into a value-added food product having preservative functions. The procedure developed in this study would promote recycling of shochu kasu.

Nukadoko is the fermented rice bran bed traditionally used for pickling vegetables in Japan. Here, we investigate the bacterial community structure of nukadoko using several culture-independent methods. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of V2-V3 16S rRNA gene (16S rDNA) fragments amplified from a long-aged nukadoko bacterial community indicated seven predominant operational taxonomic units (OTUs) closely related to known Lactobacillus species. Phylogenetic analysis of these OTUs indicated a major cluster consisting of six OTUs including a dominant OTU closely related to Lactobacillus acidifarinae and one distinct OTU corresponding to Lactobacillus acetotolerans. L. acetotolerans was commonly detected as a dominant species in samples from different seasons. The succession of microbial community structure in the fermentation and ripening processes was investigated using a laboratory model nukadoko. The L. acidifarinae-like bacteria grew rapidly with a pH decrease in the first few days after inoculation, whereas L. acetotolerans grew slowly and became dominant after one week. Real-time quantitative polymerase chain reaction (Q-PCR) showed that the doubling time of L. acetotolerans was 12 h, while that of total bacteria was 4 h. Real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) targeting 16S rRNA showed a low metabolic activity of L. acetotolerans throughout the fermentation and ripening processes. Fluorescence in situ hybridization (FISH) showed that L. acetotolerans was a dominant bacterium in the ripening period and had a low metabolic activity. These results indicate that the slow-growing L. acetotolerans stably dominated nukadoko microbiota after the L. acidifarinae-like bacteria mainly contributed to the lactic acid fermentation of the rice bran.

Non-growing Clostridium saccharoperbutylacetonicum N1-4 hardly produced butanol from only butyrate. As adding glucose to the medium, butyrate utilization and butanol production were stimulated. Addition of 0.1 mM methyl viologen as electron carrier resulted in the highest yield of butanol of 0.671 mol/mol to butyrate and glucose.

A circuit diagram is proposed on the basis of an analysis of metabolic pathways of lactic acid bacteria, namely, a phosphoketolase pathway and a pentose phosphate/glycolic pathway. An augmented matrix was derived from carbon balances and stoichiometries from the circuit diagram, and solved by Gaussian elimination. Results indicated that 70% of the amount of xylose was metabolized through the pentose phosphate/glycolic pathway and that the yield of lactic acid from xylose ranged from 0.34 to 0.52 (mol/mol), whereas that of pyruvic acid was more than 1.4 (mol/mol).

Lantibiotics are a unique type of antimicrobial peptide produced by a large number of gram-positive bacteria that contain unusual amino acids, such as lanthionine and dehydrated amino acids. Ribosomally synthesized lantibiotic prepeptide consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events to yield a biologically active lantibiotic. Research on lantibiotics has drawn much attention in recent years and has undergone extensive progress as a step forward to the next paradigm. Unusual amino acids in lantibiotics solely contribute to their biological activity and also enhance their structural stability. Thus, enzymes involved in lantibiotic biosynthesis would have a high potential for peptide engineering by introducing unusual amino acids into desired peptides, which may establish a universal approach to advance the structural design of novel peptides, termed lantibiotic engineering. In this review, we focus on recent development with contemporary innovations and perspective of lantibiotic research.

Strains of Leuconostoc lactis SHO-47 and Le. lactis SHO-54, producing the clinically useful enzyme NAD-specific 6-phosphoglucanate dehydrogenase, were cultivated with a hydrolyzed birch wood xylan as the unique carbon source to produce D-lactic acid for poly(D-lactic acid). In addition to the strains SHO-47 and SHO-54, Lactococcus lactis IO-1, well known as a good xylose-utilizing lactic acid bacterium, was used as a control to confirm the extent of hemicellulose hydrolysis. The fermentation time for lactic acid of strains SHO-47 and SHO-54 was 12 h, and produced respectively 2.3 and 2.2 g/l lactic acid from 8.5 g/l hydrolyzed xylan, whereas the fermentation time of strain IO-1 was 21 h, and produced 1.3 g/l lactic acid. Xylooligosaccharides from xylobiose to xylohexose were utilized more rapidly than xylose in the cultures of strains SHO-47 and SHO-54. However, xylose concentration increased temporarily and then decreased in the culture of strain IO-1. On the other hand, xylooligosaccharides larger than xyloheptaose were not utilized by these three strains. The xylosidase activities of SHO-47, SHO-54, and IO-1 were induced by xylose or a mixture of xylobiose and xylotriose. The xylosidases of these three strains were localized in their cytoplasm.

Clostridium saccharoperbutylacetonicum N1-4 could not grow or produce butanol in excess sludge medium. However, adding glucose to the excess sludge medium resulted in a specific growth rate and butanol productivity of 0.29 h(-1) and 0.55 g/l/h, respectively, and the final butanol production reached 9.3 g/l. Since the content of suspended solids in medium reduced to less than 50% of the initial content during acetone-butanol-ethanol (ABE) fermentation, the sludge was quantitatively decreased by this fermentation employing this strain.

Tetragenococcus halophila originally isolated from soy sauce is a halophilic lactic acid bacterium which can grow under 4 M sodium chloride. T. halophila chaperonin composed of a core moiety of chaperonin 60 (cpn60) and a lid moiety of chaperonin 10 (cpn10), is thought to contribute to host halotolerant capability. In this report, we reconstituted and characterized the core complex of T. halophila chaperonin by using a recombinant T. halophila cpn60 (Tcpn60) overexpressed in Escherichia coli. The reconstitution of Tcpn60 was performed in the presence of 10 mM MgCl2, 2 mM ATP and 0.8 M (NH4)2SO4 and the resultant oligomer was purified by gel filtration chromatography. Electron microscopy of the reconstituted Tcpn60 revealed a double toroidal tetradecameric structure that is characteristic of bacterial cpn60. The T. halophila tetradecamer cpn60 exhibited an ATPase activity and a refolding activity of both chemically and thermally denatured enolases under wide range of salt concentrations. Furthermore, we demonstrated that heterologous expression of Tcpn60 allowed the normal growth of host Escherichia coli cells under salt stress conditions and this effect was further enhanced by co-expression with Tcpn10. These results suggested that Tcpn60 contributes to the halotolerance property of T. halophila cell as a tetradecamer complex, probably associated with the Tcpn10 complex.

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. The gene cluster of nukacin ISK-1 consists of at least nukAMTFEG, ORF1 and ORF7. In this study, we demonstrated the heterologous production of nukacin ISK-1 in Lactococcus lactis by the artificial polycistronic expression of nukAMTFEG-ORF7 under the control of the nisin-controlled expression (NICE) system. Consequently, the recombinant L. lactis showed antimicrobial activity. Mass analysis clarified the presence of nukacin ISK-1 produced in the culture supernatant. These results suggested that the recombinant L. lactis produced nukacin ISK-1 heterologously. Inactivation of nukA, -M or -T resulted in the complete loss of the nukacin ISK-1 production phenotype. This finding suggested that nukAMT are indispensably associated with the biosynthesis of nukacin ISK-1. To our knowledge, this is the first report of the heterologous production of lantibiotic using the NICE system.

Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coli without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T, halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli.

We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region up-stream of hrcA. The T. halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T. halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T. halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3-4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.

An efficient bioreactor, termed a 'synchronized fresh cell bioreactor', was developed and consisted of a pH-dependent substrate feed system coupled with cross flow filtration and turbidity control. The effect of high dilution rate and high cell density coupled with high cell viability on the production of l-lactic acid in continuous culture by Lactococcus lactis IO-1 in enzyme-hydrolysed sago starch medium was investigated. For all changes in dilution rate, cells responded in a synchronized way to the addition of glucose by increasing the rate of biomass formation. Consequently, a glucose-free feed solution was required to maintain the cell concentration at a particular pre-set value. This set-up facilitated the maintenance of the cells in a permanent log phase. At a cell concentration of 15 gl(-1) and a feed glucose concentration of 53 gl(-1), volumetric LA productivities of 8.2, 19.3 and 33.1 gl(-1)h(-1) were obtained at dilution rates of 0.21, 0.50 and 1.1 h(-1), respectively. The respective residual glucose concentrations in the spent medium were 1.90, 0.24 and 3.80 gl(-1). By increasing the cell density, the volumetric productivity increased proportionally. At high cell density, higher dilution rates resulted in lower lactate concentrations in the culture medium resulting in higher productivity. This reactor facilitated efficient operation with high cell viability by maintaining the cells in continuous growth phase for long-term fermentation. Therefore, the growth rate (mu) was calculated according to the Monod equation. Using this system, high specific productivities can be obtained which guarantees high commercial productivity at economical cost with only a small investment for setting up the sago industry.