Nagamani Bora

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.

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

BackgroundThe 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.ResultsWe 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.ConclusionThis 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.

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.

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

BackgroundPichia 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.ResultsAddition 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 (kLa) 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.ConclusionsWe 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.

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)).

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 industrys 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 proteins 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.

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.

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.

Smear Ripened Cheeses

Cheeses are produced at local and industrial scales generating over 1,000 varieties of cheese in Europe. Most cheeses have a complex successional microbial flora through milk fermentation, curd maturation and storage, to cheese maturation. Microorganisms are introduced with raw materials, by deliberate inoculation and from the environment. Microorganisms are responsible for most of the changes which produce cheese, give it its organoleptic properties and contribute to its preservation, but they may include pathogens, of which Listeria monocytogenes is the most common. The activity of aminotransferase, enzymatic degradation of L-methionine and the subsequent formation of volatile sulphur compounds leads to the development of the typical flavour in smear cheese. The characteristic features of cheese evolve from complex interaction of the metabolic activities of the smear cheese flora. So it is essential to define and identify surface microflora to enable the selection of strains that generate the colour, aroma and organoleptic qualities of specific cheeses, and to screen for anti-listerial activity.

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).