Thioclava arenosa sp. nov., isolated from sea sand.

A Gram-staining-negative, non-spore-forming, non-motile, rod-shaped, facultatively anaerobe bacterial strain, designated CAU 1312T, was isolated from sea sand of Eurwangri beach, South Korea. The strain's taxonomic position was investigated using a polyphasic approach. CAU 1312T grew at temperatures from 20 to 40 °C, in the range of pH 6.0-9.0 and at salinities from 1-4 % (w/v). The results of phylogenetic analysis based on the 16S rRNA gene sequence revealed that CAU 1312T represented a member of the genus Thioclava and was most closely related to Thioclava atlantica 13D2W-2T (similarity 96.53 %). The strain contained Q-10 as the predominant menaquinone and summed feature 8 (C18 : 1ω7c/ω6c) as the major fatty acid. The polar lipids of CAU 1312T consisted of phosphatidylethanolamine, phosphatidylglycerol, two aminophospholipids, a phosphoglycolipid, and two unidentified phospholipids. The DNA G+C content was 64.7 mol%. On the basis of phenotypic and chemotaxonomic properties and phylogenetic inference, CAU 1312T is considered to represent a novel species of the genus Thioclava, for which the name Thioclava arenosa sp. nov. is proposed. The type strain is CAU 1312T(=KCTC 52190T=NBRC 111989T).

Tessaracoccus arenae sp. nov., isolated from sea sand.

A Gram-stain positive, non-spore-forming, non-motile, facultatively anaerobic bacterial strain, designated CAU 1319T, was isolated from sea sand and the strain's taxonomic position was investigated using a polyphasic approach. Strain CAU 1319T grew optimally at 30 °C and at pH 7.5 in the presence of 2 % (w/v) NaCl. Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain CAU 1319T belongs to the genus Tessaracoccus, and is closely related to Tessaracoccus lapidicaptus IPBSL-7T (similarity 97.69 %), Tessaracoccus bendigoensis Ben 106T (similarity 95.64 %) and Tessaracoccus flavescens SST-39T (similarity 95.84 %). Strain CAU 1319T had ll-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, MK-9 (H4) as the predominant menaquinone, and anteiso-C15 : 0 as the major fatty acid. The polar lipids consisted of phosphatidylglycerol, phosphatidylinositol, two unidentified aminolipids, three unidentified phospholipids and one unidentified glycolipid. Predominant polyamines were spermine and spermidine. The DNA-DNA hybridization value between strain CAU 1319T and T. lapidicaptus IPBSL-7T was 24 %±0.2. The DNA G+C content of the novel strain was 69.5 mol%. On the basis of phenotypic and chemotaxonomic properties, as well as phylogenetic relatedness, strain CAU 1319Tshould be classified as a novel species of the genus Tessaracoccus, for which the name Tessaracoccus arenae sp. nov. is proposed. The type strain is CAU 1319T(=KCTC 39760T=NBRC 111973T).

Nonlabens halophilus sp. nov., isolated from reclaimed land.

A Gram-stain-negative, orange-pigmented, non-spore-forming, non-motile, aerobic, rod-shaped bacterial strain, designated CAU 1131T, was isolated from reclaimed land. Strain CAU 1131T grew optimally at 30 °C and at pH 6.5 in the presence of 4 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1131T was grouped into the genus Nonlabens, and was most closely related to Nonlabens. marinus S1-08T (95.9 % 16S rRNA gene sequence similarity). The strain possessed+ MK-6 as the predominant menaquinone and iso-C15 : 0, iso-C15:0 3-OH, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) as the major cellular fatty acids. The polar lipid profile was determined to comprise phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid, an unidentified glycolipid and two unidentified lipids. The DNA G+C content was 38.7 mol%. On the basis of data from phenotypic, chemotaxonomic and phylogenetic inference, strain CAU 1131T represents a novel species of the genus Nonlabens, for which the name Nonlabens halophilus sp. nov. is proposed. The type strain is CAU 1131T (=KCTC 52177T=NBRC 111996T).

Marimonas arenosa gen. nov., sp. nov., isolated from sea sand.

A Gram-stain-negative, non-motile, non-spore-forming, aerobic and short rod-shaped bacterial strain, CAU 1311T, was isolated from sea sand in the Republic of Korea. Strain CAU 1311T grew at temperatures from 20-37 °C, in the range of pH 6.5-10.0, and under various NaCl concentrations from 0-6 % (w/v). Phylogenetic analysis based on the 16S rRNA gene sequence of CAU 1311T showed that it formed a distinct lineage within the family Rhodobacteraceae as a separate deep branch, with 96.2 % or lower sequence identity to representatives of the genera Marivita, Aestuariivita, Mameliella, Sulfitobacter and Maritimibacter. The major fatty acid was C18  : 1ω7c and the predominant respiratory quinone was Q-10. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid and an unidentified glycolipid. The DNA G+C content was 60.7 mol%. On the basis of genotypic, phenotypic and chemotaxanomic findings, strain CAU 1311T could be classified as representing a novel species of a new genus of the family Rhodobacteraceae, for which the name Marimonas arenosa gen. nov., sp. nov. is proposed. The type strain of the type species is CAU 1311T (=KCTC 52189T=NBRC 111988T).

Transcriptomic analysis of Lactococcus chungangensis sp. nov. and its potential in cheese making.

Lactococcus lactis has a played a prominent role in food industry from traditional milk fermentations to industrial scale processes. Extensive studies on the biochemical, physiological, and genetic aspects of L. lactis are evident from published literature. Recently, another novel species, Lactococcus chungangensis, was isolated from activated sludge as the sixth member of the genus to be discovered. To date, no study has been conducted to explore the functional aspects of L. chungangensis to identify features similar to those in L. lactis that are relevant to the dairy industry. Hence, the present study was undertaken to identify functional genes relevant to dairy application through comparative transcriptomic analysis of L. chungangensis with L. lactis. In expression microarray data, 415 genes were upregulated and 1,500 were downregulated of the total 1,915 probes analyzed. Interesting findings from this study were the identification of functional genes such as aminohydrolase and S-adenosylmethionine in L. chungangensis, which are useful in flavor production in cheese making. Probing these genes by PCR and analyzing the sequence confirmed the presence of these genes. Phenotypic analysis of these genes was also investigated by growing the strain in different concentrations of skim milk media to confirm the ability of L. chungangensis to degrade casein in milk, which is the major precursor for flavor enhancing compounds. Other adaptive and stress-response genes such as cold shock and heat shock proteins were also revealed. All experimental investigations at the functional level suggest that L. chungangensis possesses some interesting genes which are of commercial significance, especially in cheese production.

The role of inorganic-phosphate, potassium and magnesium in yeast-flavour formation.

Inorganic-phosphate, potassium, and magnesium are key-minerals required for yeast growth, metabolism, and survival, the present work investigated its impact in yeast-flavour formation using a multi-factor experimental design, which was used to generate a range of phosphorous-potassium-magnesium resulting in a 28-point D-optimal design. Samples were evaluated using HPLC (ethanol), GC-MS (aroma), and CountStar (total yeast cell). Results revealed that yeast requires a minimal amount of inorganic-phosphate, potassium, and magnesium (250, 500, and 70 mg/L, respectively) to support yeast-growth and ethanol/flavour formation. Inorganic-phosphate was important for fatty acid esters formation/short chain fatty acid (SCFA) reduction. Potassium was important in the formation of acetate esters/higher alcohols. Magnesium was the most important inorganic element for ester formation/SCFA reduction; furthermore, ethanol production is magnesium-dependent. In conclusion, inorganic phosphate, potassium and magnesium play an important role in yeast-growth, esters and higher alcohols formation; and SCFA reduction. Ethanol formation is Mg-dependent.

Understanding the yeast host cell response to recombinant membrane protein production.

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield.

BACKGROUND: Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast. RESULTS: Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (µg OD595⁻¹) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (k(L)a) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium. CONCLUSIONS: We show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.

Surface microbial consortia from Livarot, a French smear-ripened cheese.

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield.

BACKGROUND: The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. RESULTS: We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. CONCLUSION: This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Diversity and biogeography of marine actinobacteria.

The actinomycetes, although not all the Actinobacteria, are easy to isolate from the marine environment. However, their ecological role in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate strains for search and discovery of new drugs. However, the marine environment has become a prime resource in search and discovery for novel natural products and biological diversity, and marine actinomycetes turn out to be important contributors. Similarly, striking advances have been made in marine microbial ecology using molecular techniques and metagenomics, and actinobacteria emerge as an often significant, sometimes even dominant, environmental clade. Both approaches - cultivation methods and molecular techniques - are leading to new insights into marine actinobacterial biodiversity and biogeography. Very different views of actinobacterial diversity emerge from these, however, and the true extent and biogeography of this are still not clear. These are important for developing natural product search and discovery strategies, and biogeography is a hot topic for microbial ecologists.

Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses.

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Large-scale production of secreted proteins in Pichia pastoris.

The production of recombinant therapeutic proteins is an active area of research in drug development. These bio-therapeutic drugs target nearly 150 disease states and promise to bring better treatments to patients. However, if new bio-therapeutics are to be made more accessible and affordable, improvements in production performance and optimization of processes are necessary. A major challenge lies in controlling the effect of process conditions on production of intact functional proteins. To achieve this, improved tools are needed for bio-processing. For example, implementation of process modeling and high-throughput technologies can be used to achieve quality by design, leading to improvements in productivity. Commercially, the most sought after targets are secreted proteins due to the ease of handling in downstream procedures. This chapter outlines different approaches for production and optimization of secreted proteins in the host Pichia pastoris.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review).

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

Deinococcus aquaticus sp. nov., isolated from fresh water, and Deinococcus caeni sp. nov., isolated from activated sludge.

The taxonomic positions of two environmental isolates from South Korea were established using a combination of genotypic and phenotypic data. The organisms, designated PB314(T) and Ho-08(T), were Gram-negative, rod-shaped and non-spore-forming and had chemotaxonomic properties consistent with their classification in the genus Deinococcus 16S rRNA gene tree, the highest sequence similarities being shown to the type strains of Deinococcus grandis (96.3-96.7 %) and Deinococcus indicus (96.3-96.4 %). The isolates shared relatively high 16S rRNA gene sequence similarity (98.1 %) but had a DNA-DNA relatedness value of only 22 %. Chemotaxonomic data revealed that both strains possess quinone system MK-8 as the predominant compound, C(16 : 1)omega7c and C(16 : 0) as major fatty acids and ornithine as a diamino acid in the peptidoglycan structure, corroborating our assignment of the strains to the genus Deinococcus. The results of phylogenetic analyses based on 16S rRNA gene sequences, DNA-DNA relatedness values and physiological and biochemical tests clearly demonstrated that the two strains represent distinct species. On the basis of these data, two novel species, Deinococcus aquaticus sp. nov. (type strain PB314(T) =KCTC 12552(T) =NBRC 101311(T)) and Deinococcus caeni sp. nov. (type strain Ho-08(T) =KCTC 12553(T) =NBRC 101312(T)), are proposed.

Mycetocola reblochoni sp. nov., isolated from the surface microbial flora of Reblochon cheese.

Four Gram-positive, aerobic, non-sporulating, rod-shaped bacteria isolated from the surface microflora of Reblochon cheese at the late stage of ripening had chemotaxonomic properties characteristic of members of the family Microbacteriaceae. The isolates had virtually identical SDS-PAGE whole-organism protein patterns, shared many chemical and phenotypic characteristics and formed an independent branch in the Microbacteriaceae 16S rRNA gene tree that was most closely related to the type strains of Mycetocola species. The new isolates had chemotaxonomic properties consistent with their classification in the genus Mycetocola but were readily distinguished from recognized members of this taxon based on DNA-DNA relatedness, whole-organism protein and phenotypic data. The combined genotypic and phenotypic data indicate that the isolates should be classified in the genus Mycetocola as members of a novel species, for which the name Mycetocola reblochoni sp. nov. is proposed. The type strain is LMG 22367(T) (=R-20377(T) =BRB-1L41(T) =DSM 18580(T)).

Agrococcus casei sp. nov., isolated from the surfaces of smear-ripened cheeses.

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2(T) (=DSM 18061(T)=LMG 22410(T)) is the type strain of Agrococcus casei sp. nov.