Sequence analysis of <Emphasis Type="Italic">KmXYL1</Emphasis> genes and verification of thermotolerant enzymatic activities of xylose reductase from four <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.     

Model-based biotechnological potential analysis of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> central metabolism

The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here, we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modelling and optimisation methods. Our model reveals the organism’s potential for industrial application and metabolic engineering. Modelling results imply that the aeration regime can be used as a tool to optimise product yield and flux distribution in K. marxianus. Also rebalancing NADH and NADPH utilisation can be used to improve the efficiency of substrate conversion. Xylose is identified as a biotechnologically promising substrate for K. marxianus.     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

<Superscript>13</Superscript>C metabolite profiling to compare the central metabolic flux in two yeast strains

¹³C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms.     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Detection of virulence-related genes in <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> and their expression in response to different conditions

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.     

Functional Properties of <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis> Strains Isolated from Brazilian Goat Milk

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Biofilm production and other virulence factors in <Emphasis Type="Italic">Streptococcus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> isolated from clinical cases of bovine mastitis in Poland

Background

Mastitis is a common disease in dairy cattle throughout the world and causes considerable economic losses each year. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among bacteria of the genus Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland.

Results

Most of the isolates analysed demonstrated an ability to produce biofilm (over 70%). Virulence genes were searched for in the three most common streptococci in our experiment: S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33%), cylE (78%), bca (37%), and cfb (100%). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes of the species were Ia (n?=?8) and II (n?=?8), in addition to some strains that were not classified in any of the groups (n?=?6). Out of the eight selected genes for S. uberis (sua, pauA/skc, gapC, cfu, lbp, hasA, hasB, hasC), only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76% and 86% of the strains, respectively.

Conclusions

The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

Dairy propionibacteria as direct-fed microbials: in vitro effect on acid metabolism of <Emphasis Type="Italic">Streptococcus bovis</Emphasis> and <Emphasis Type="Italic">Megasphaera elsdenii</Emphasis>

Ruminal acidosis caused by accumulation of lactic acid, a decrease of pH in the rumen and subsequent imbalance of the rumen fermentation process, affects the health and productivity of dairy cows and beef cattle. Direct-fed microbials have potential for use in the control and prevention of ruminal acidosis. This study investigated the interaction between five strains of dairy propionibacteria, Megasphaera elsdenii and Streptococcus bovis in various co-culture combinations in a simulated rumen environment comprising unmodified rumen digesta supplemented with excess glucose. While suppression of lactic acid accumulation by both the dairy propionibacteria and M. elsdenii in the presence of S. bovis in the simulated rumen conditions was evident, propionibacteria were found to be more effective than M. elsdenii in controlling lactic acid levels.     

The growth kinetics and metabolic and antioxidant activities of the functional synbiotic combination of <Emphasis Type="Italic">Lactobacillus gasseri</Emphasis> 505 and <Emphasis Type="Italic">Cudrania tricuspidata</Emphasis> leaf extract

In a previous study, the synbiotic combination of selected Lactobacillus gasseri strains and Cudrania tricuspidata leaf extract (CT) was shown to significantly improve the functionality of fermented milk, and the greatest synbiotic effect was exhibited with L. gasseri 505. The aim of the present study was to investigate the growth kinetics and fermentation metabolism of this specific synbiotic combination. Fermentation was carried out in synthetic media and milk with or without CT supplementation using L. gasseri 505. Whole genome sequencing and comparative genomics analyses were conducted to verify the novelty of strain. Titratable acidity, pH, microbial population, and organic acid production were measured during the fermentation period. The addition of CT accelerated the acidification rate, supporting the growth of L. gasseri 505, and the production of fermentation metabolites such as lactic acid and pyruvic acid also significantly increased during fermentation of both of CT-supplemented synthetic media and milk. In particular, the formic acid and propionic acid in CT were significantly utilized during fermentation of milk by L. gasseri 505. Moreover, the antioxidant capacity of CT-supplemented fermented milk increased due to the release of bioactive compounds until the exponential growth phase, after which the antioxidant activity declined due to degradation and loss of potency. Therefore, this study established that L. gasseri 505 efficiently utilized the CT-related nutrients during fermentation producing resulting metabolites with health-promoting effects, although it is necessary to control the fermentation time to obtain dairy products with optimum functionality.     

Effect of <Emphasis Type="Italic">UCP2</Emphasis> and <Emphasis Type="Italic">UCP3</Emphasis> Genes Polymorphisms on Functional Traits in Dairy Cattle

The aim of this study was to investigate associations between genotypes of UCP2 and UCP3 genes, milk, and reproduction traits in dairy cattle. The study included two herds: Jersey cows and Polish Holstein-Friesian (Red and White strain) cows. All cows were genotyped using the PCR-RFLP method and allele frequencies were determined. Statistical analysis showed a significant association between polymorphism in the UCP3 gene and the milk yield and fat content of milk (P ≤ 0.05, P ≤ 0.01) and between the UCP2 gene and the calving interval (P ≤ 0.05). Information contained in this study may be useful in further analysis to define the role of analysed genes in relation to functional traits in dairy cattle, nevertheless, the obtained results should be verified by conducting research on a larger group of animals and various cattle breeds.     

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> IR-2 for high-temperature ethanol production

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 °C and over-expression of all PPP genes at 36 °C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 °C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Comparative genomic analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> GB-LP4 and identification of evolutionarily divergent genes in high-osmolarity environment

Lactobacillus plantarum is one of the widely-used probiotics and there have been a large number of advanced researches on the effectiveness of this species. However, the difference between previously reported plantarum strains, and the source of genomic variation among the strains were not clearly specified. In order to understand further on the molecular basis of L. plantarum on Korean traditional fermentation, we isolated the L. plantarum GB-LP4 from Korean fermented vegetable and conducted whole genome assembly. With comparative genomics approach, we identified the candidate genes that are expected to have undergone evolutionary acceleration. These genes have been reported to associate with the maintaining homeostasis, which are generally known to overcome instability in external environment including low pH or high osmotic pressure. Here, our results provide an evolutionary relationship between L. plantarum species and elucidate the candidate genes that play a pivotal role in evolutionary acceleration of GB-LP4 in high osmolarity environment. This study may provide guidance for further studies on L. plantarum.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.