Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry.

In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker's yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species' several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation.

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 µm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.

Diversity and Metabolism of Microbial Communities in a Hypersaline Lake along a Geochemical Gradient.

In the south of western Siberia (Russia), there are many unique and unexplored soda, saline, and freshwater lakes. In this study, the results are presented on microbial diversity, its metabolic potential, and their relation with a set of geochemical parameters for a hypersaline lake ecosystem in the Novosibirsk region (Oblast). The metagenomic approach used in this work allowed us to determine the composition and structure of a floating microbial community, the upper layer of silt, and the strata of bottom sediments in a natural saline lake via two bioinformatic approaches, whose results are in good agreement with each other. In the floating microbial community and in the upper layers of the bottom sediment, bacteria of the Proteobacteria (Gammaproteobacteria), Cyanobacteria, and Bacteroidetes phyla were found to predominate. The lower layers were dominated by Proteobacteria (mainly Deltaproteobacteria), Gemmatimonadetes, Firmicutes, and Archaea. Metabolic pathways were reconstructed to investigate the metabolic potential of the microbial communities and other hypothetical roles of the microbial communities in the biogeochemical cycle. Relations between different taxa of microorganisms were identified, as was their potential role in biogeochemical transformations of C, N, and S in a comparative structural analysis that included various ecological niches.

E. coli aggregation and impaired cell division after terahertz irradiation.

In this study we demonstrated that exposure of Escherichia coli (E. coli) to terahertz (THz) radiation resulted in a change in the activities of the tdcABCDEFGR and matA-F genes (signs of cell aggregation), gene yjjQ (signs of suppression of cell motility), dicABCF, FtsZ, and minCDE genes (signs of suppression of cell division), sfmACDHF genes (signs of adhesin synthesis), yjbEFGH and gfcA genes (signs of cell envelope stabilization). Moreover, THz radiation induced E. coli csg operon genes of amyloid biosynthesis. Electron microscopy revealed that the irradiated bacteria underwent increased aggregation; 20% of them formed bundle-like structures consisting of two to four pili clumped together. This could be the result of changes in the adhesive properties of the pili. We also found aberrations in cell wall structure in the middle part of the bacterial cell; these aberrations impaired the cell at the initial stages of division and resulted in accumulation of long rod-like cells. Overall, THz radiation was shown to have adverse effects on bacterial populations resulting in cells with abnormal morphology.

Metagenomics dataset used to characterize microbiome in water and sediments of the lake Solenoe (Novosibirsk region, Russia).

This is data on the microbial diversity in the floating cyanobacterial community and sediment samples from the lake Solenoe (Novosibirsk region, Russia) obtained by metagenomic methods. Such a detailed data of the microbial diversity of the Novosibirsk oblast lake ecosystem was carried out for the first time. The purpose of our work was to reveal microbial taxonomic diversity and abundance, metabolic pathways and new enzyme findings the studied lake ecosystem using the next-generation sequencing (NGS) technology and metagenomic analysis. The data was obtained using metagenomics DNA whole genome sequencing (WGS) on Illumina NextSeq and NovaSeq. The raw sequence data used for analysis is available in NCBI under the Sequence Read Archive (SRA) with the BioProjects and SRA accession numbers: PRJNA493912 (SRR7943696), PRJNA493952 (SRR7943839) and PRJNA661775 (SRR12601635, SRR12601634, SRR12601633) corresponding to floating cyanobacterial community and sediment layers samples, respectively.

Young «oil site¼ of the Uzon Caldera as a habitat for unique microbial life.

BACKGROUND: The Uzon Caldera is one of the places on our planet with unique geological, ecological, and microbiological characteristics. Uzon oil is the youngest on Earth. Uzon oil has unique composition, with low proportion of heavy fractions and relatively high content of saturated hydrocarbons. Microbial communities of the «oil site¼ have a diverse composition and live at high temperatures (up to 97 °C), significant oscillations of Eh and pH, and high content of sulfur, sulfides, arsenic, antimony, and mercury in water and rocks. RESULTS: The study analyzed the composition, structure and unique genetics characteristics of the microbial communities of the oil site, analyzed the metabolic pathways in the communities. Metabolic pathways of hydrocarbon degradation by microorganisms have been found. The study found statistically significant relationships between geochemical parameters, taxonomic composition and the completeness of metabolic pathways. It was demonstrated that geochemical parameters determine the structure and metabolic potential of microbial communities. CONCLUSIONS: There were statistically significant relationships between geochemical parameters, taxonomic composition, and the completeness of metabolic pathways. It was demonstrated that geochemical parameters define the structure and metabolic potential of microbial communities. Metabolic pathways of hydrocarbon oxidation was found to prevail in the studied communities, which corroborates the hypothesis on abiogenic synthesis of Uzon hydrothermal petroleum.

Creation of an Online Platform for Identification of Microorganisms: Peak Picking or Full-Spectrum Analysis.

Identification of microorganisms by MALDI-TOF mass spectrometry is a very efficient method with high throughput, speed, and accuracy. However, it is significantly limited by the absence of a universal database of reference mass spectra. This problem can be solved by creating an Internet platform for open databases of protein spectra of microorganisms. Choosing the optimal mathematical apparatus is the pivotal issue for this task. In our previous study we proposed the geometric approach for processing mass spectrometry data, which represented a mass spectrum as a vector in a multidimensional Euclidean space. This algorithm was implemented in a Jacob4 stand-alone package. We demonstrated its efficiency in delimiting two closely related species of the Bacillus pumilus group. In this study, the geometric approach was realized as R scripts which allowed us to design a Web-based application. We also studied the possibility of using full spectra analysis (FSA) without calculating mass peaks (PPA), which is the logical development of the method. We used 74 microbial strains from the collections of ICiG SB RAS, UNIQEM, IEGM, KMM, and VGM as the models. We demonstrated that the algorithms based on peak-picking and analysis of complete data have accuracy no less than that of Biotyper 3.1 software. We proposed a method for calculating cut-off thresholds based on averaged intraspecific distances. The resulting database, raw data, and the set of R scripts are available online at https://icg-test.mydisk.nsc.ru/s/qj6cfZg57g6qwzN.

Metagenome-Assembled Genome of Halomonas sp. Isolate SL48-SHIP-3 from the Microbial Mat of Salt Lake Number 48 (Novosibirsk Region, Russia).

The Halomonas sp. isolate SL48-SHIP-3 genome was obtained from metagenomics sequencing of the microbial mat of Salt Lake Number 48 (54.201806N, 78.179194E; Novosibirsk region, Russia). The sequenced and annotated genome is 2,575,909 bp and encodes 2,368 genes.

Metagenome-Assembled Genome Sequence of Phormidium sp. Strain SL48-SHIP, Isolated from the Microbial Mat of Salt Lake Number 48 (Novosibirsk Region, Russia).

The Phormidium sp. strain SL48-SHIP genome was obtained from metagenomics sequencing of the microbial mat of Salt Lake No. 48 (54.201806 N, 78.179194 E; Novosibirsk Region, Russia). The sequenced and annotated genome is 4,384,607 bp and encodes 3,807 genes.

Draft Genome Sequence of Halorubrum sp. Strain 48-1-W from a Saline Lake in the Novosibirsk Region, Russia.

Halorubrum sp. strain 48-1-W was isolated from a water sample from a saline lake (Novosibirsk Region, Russia, 54°14'N 78°13'E). The sequenced and annotated genome is 3,584,929 bp and contains 3,506 genes.

Draft Genome Sequence of Bacillus altitudinis Strain KL4, Isolated from Bottom Sediments in Lake Krotovaya Lyaga (Novosibirsk Region, Russia).

The Bacillus altitudinis strain KL4 was isolated from bottom sediments in Lake Krotovaya Lyaga (Novosibirsk Region, Russia, 53.7°N, 77.9°E). The sequenced and annotated genome is 3,738,419 bp long and carries 3,909 genes.

Draft Genome Sequence of Bacillus altitudinis Strain KU-skv2(2), Isolated from a Microbial Mat on an Anthropogenic Pipe from Caldera Uzon (Kamchatka, Russia).

Bacillus altitudinis strain KU-skv2(2) was isolated from a microbial mat on an anthropogenic pipe from Caldera Uzon (Kamchatka, Russia, 54°30'0.23″N, 160°0'15.18″E). The sequenced and annotated genome is 3,739,340 bp in size and encodes 3,929 genes.

Draft genome sequence of Anoxybacillus flavithermus KU2-6-11 isolated from hot-spring in Uzon caldera (Kamchatka, Russia).

The Anoxybacillus flavithermus KU2-6-11 was isolated from sediments of a nameless hot spring. The hot spring is located in Uzon caldera (Kamchatka, Russia). The sequenced and annotated genome is 2,646,305 bp and encodes 2787genes. The draft genome sequence of the Anoxybacillus flavithermus KU2-6-11 has been deposited at DDBJ/EMBL/GenBank under the accession PEDM01000000 and the sequences could be found at the site https://www.ncbi.nlm.nih.gov/nuccore/PEDM01000000.

Biodiversity of the microbial mat of the Garga hot spring.

BACKGROUND: Microbial mats are a good model system for ecological and evolutionary analysis of microbial communities. There are more than 20 alkaline hot springs on the banks of the Barguzin river inflows. Water temperature reaches 75 °C and pH is usually 8.0-9.0. The formation of microbial mats is observed in all hot springs. Microbial communities of hot springs of the Baikal rift zone are poorly studied. Garga is the biggest hot spring in this area. RESULTS: In this study, we investigated bacterial and archaeal diversity of the Garga hot spring (Baikal rift zone, Russia) using 16S rRNA metagenomic sequencing. We studied two types of microbial communities: (i) small white biofilms on rocks in the points with the highest temperature (75 °C) and (ii) continuous thick phototrophic microbial mats observed at temperatures below 70 °C. Archaea (mainly Crenarchaeota; 19.8% of the total sequences) were detected only in the small biofilms. The high abundance of Archaea in the sample from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. In the microbial mats, primary products were formed by cyanobacteria of the genus Leptolyngbya. Heterotrophic microorganisms were mostly represented by Actinobacteria and Proteobacteria in all studied samples of the microbial mats. Planctomycetes, Chloroflexi, and Chlorobi were abundant in the middle layer of the microbial mats, while heterotrophic microorganisms represented mostly by Firmicutes (Clostridia, strict anaerobes) dominated in the bottom part. Besides prokaryotes, we detect some species of Algae with help of detection their chloroplasts 16 s rRNA. CONCLUSIONS: High abundance of Archaea in samples from hot springs of the Baikal rift zone supplemented our knowledge of the distribution of Archaea. Most archaeal sequences had low similarity to known Archaea. Metagenomic analysis of microbial communities of the microbial mat of Garga hot spring showed that the three studied points sampled at 70 °C, 55 °C, and 45 °C had similar species composition. Cyanobacteria of the genus Leptolyngbya dominated in the upper layer of the microbial mat. Chloroflexi and Chlorobi were less abundant and were mostly observed in the middle part of the microbial mat. We detected domains of heterotrophic organisms in high abundance (Proteobacteria, Firmicutes, Verrucomicrobia, Planctomicetes, Bacteroidetes, Actinobacteria, Thermi), according to metabolic properties of known relatives, which can form complete cycles of carbon, sulphur, and nitrogen in the microbial mat. The studied microbial mats evolved in early stages of biosphere formation. They can live autonomously, providing full cycles of substances and preventing live activity products poisoning.

Draft genome sequence of Thermoactinomyces sp. Gus2-1 isolated from the hot-spring Gusikha in Bargusin Valley (Baikal Rift Zone, Russia).

The Thermoactinomyces sp. strain Gus2-1 was isolated from hot-spring sediments sample from the hot-spring Gusikha in Bargusin Valley (Baikal Rift Zone, Russia). The sequenced and annotated genome is 2,623,309 bp and encodes 2513 genes. The draft genome sequence of the Thermoactinomyces sp. strain Gus2-1 has been deposited at DDBJ/EMBL/GenBank under the accession JPZM01000000 and the sequences could be found at the site https://www.ncbi.nlm.nih.gov/nuccore/JPZM01000000.

2.3THz radiation: Absence of genotoxicity/mutagenicity in Escherichia coli and Salmonella typhimurium.

The mutagenicity and genotoxicity in bacteria of 2.3THz radiation (THz) produced by a free-electron laser (NovoFEL) were evaluated; exposures were 5, 10, or 15min at average power 1.4W/cm(2). Two Ames mutagenicity test strains of Salmonella typhimurium, TA98 and TA102, were used. For the genotoxicity test, we measured SOS induction in Escherichia coli PQ37. No significant differences were found between exposed and control cells, indicating that THz radiation is neither mutagenic nor genotoxic under these conditions. Nevertheless, a small increase in total cell number of S. typhimurium after 15min exposure, and an increase in ß-galactosidase and alkaline phosphatase activities in E.coli PQ37, were observed, indicating some effect of THz radiation on cell metabolism. We also examined the combined effect of 4-NQO (8µM; positive control) and THz exposure (5min) on genotoxicity in E.coli PQ37. Unexpectedly, THz radiation decreased 4-NQO genotoxicity.

The role of environmental factors for the composition of microbial communities of saline lakes in the Novosibirsk region (Russia).

BACKGROUND: Nothing is currently known about microbial composition of saline lakes of the Novosibirsk region and its dependence on physical-chemical parameters of waters. We studied the structure of microbial communities of saline lakes of the Novosibirsk region and the effect of physical-chemical parameters of waters on microbial communities of these lakes. RESULTS: According to the ion content, the lakes were classified either as chloride or chloride-sulfate types. Water salinity ranges from 4.3 to 290 g L(-1). Many diverse microbial communities were found. Filamentous and colonial Cyanobacteria of the genera Scytonema, Aphanocapsa, and/or filamentous Algae dominated in littoral communities. Spatial and temporal organization of planktonic microbial communities and the quantities of Archaea and Bacteria were investigated using fluorescent in situ hybridization. We have found that the dominant planktonic component is represented by Archaea, or, less frequently, by Bacteria. Various phylogenetic groups (Bacteria, Archaea, Algae, and Cyanobacteria) are nonuniformly distributed. The principal component analysis was used to detect environmental factors that affect microorganism abundance. We found the principal components responsible for 71.1 % of the observed variation. It was demonstrated that two-block partial least squares was a better method than principal component analysis for analysis of the data. We observed general relationships between microbial abundance and water salinity. CONCLUSIONS: We have performed the first-ever study of the structure of the microbial communities of eleven saline lakes in the Novosibirsk region along with their physical-chemical parameters of waters. Our study demonstrates that saline lakes in the Novosibirsk region contain a unique microbial communities that may become a prolific source of microorganisms for fundamental and applied studies in various fields of ecology, microbiology, geochemistry, and biotechnology, and deserve further metagenomic investigation.

Identification of Bacillus strains by MALDI TOF MS using geometric approach.

Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html.

Draft genome sequence of a halorubrum h3 strain isolated from the burlinskoye salt lake (altai krai, Russia).

A Halorubrum H3 strain was isolated from a water and silt sample from Burlinskoye Lake (Altai Krai, Russia, 53°8'19″N 78°24'27″E). According to 16S rRNA sequences, this strain is most closely related to Halorubrum saccharovorum. The completely sequenced and annotated genome is 3,282,373 bp and contains 3,237 genes.