Low temperature-induced systems failure inEscherichia coli: Insights from rescue by cold-adapted chaperones

Abstract Background Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. Results We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difficult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. Conclusions Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

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High rate domestic wastewater treatment at 15 °C using anaerobic reactors inoculated with cold-adapted sediments/soils – shaping robust methanogenic communities

Environmental Science Water Research & Technology ◽

10.1039/c8ew00410b ◽

2019 ◽

Vol 5 (1) ◽

pp. 70-82 ◽

Cited By ~ 6

Author(s):

Evangelos Petropoulos ◽

Yongjie Yu ◽

Shamas Tabraiz ◽

Aminu Yakubu ◽

Thomas P. Curtis ◽

...

Keyword(s):

Wastewater Treatment ◽

Low Temperature ◽

Domestic Wastewater ◽

High Rate ◽

Cold Adapted ◽

Anaerobic Reactors ◽

Domestic Wastewater Treatment

To choose the reactor format in which to employ a low temperature adapted seed for wastewater treatment, we compared a UASB and an AnMBRUASB (UF)reactor at low HRT and temperature (15 °C).

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Structure analysis of a glycosides hydrolase family 42 cold-adapted β-galactosidase from Rahnella sp. R3

RSC Advances ◽

10.1039/c6ra04529d ◽

2016 ◽

Vol 6 (44) ◽

pp. 37362-37369 ◽

Cited By ~ 4

Author(s):

Yuting Fan ◽

Jiang Yi ◽

Xiao Hua ◽

Yinghui Feng ◽

Ruijin Yang ◽

...

Keyword(s):

Low Temperature ◽

High Activity ◽

Structure Analysis ◽

Dairy Industry ◽

Cold Adapted ◽

Psychrotrophic Bacterium ◽

Hydrolase Family

The β-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-β-Gal), exhibits high activity at low temperature and has potential in the dairy industry.

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Biotechnological perspectives of microorganisms isolated from the Polar Regions

Microbiology Australia ◽

10.1071/ma18042 ◽

2018 ◽

Vol 39 (3) ◽

pp. 137

Author(s):

Viktoria Shcherbakova ◽

Olga Troshina

Keyword(s):

Wastewater Treatment ◽

Low Temperature ◽

Liquid Water ◽

Polar Regions ◽

Biotechnological Applications ◽

Cold Adapted ◽

Culture Independent ◽

Biotechnological Processes ◽

Improved Methods ◽

Culture Dependent

Polar permanently frozen grounds cover more than 20% of the earth's surface, and about 60% of the Russian territories are permafrost. In the permafrost environments, the combination of low temperature and poor availability of liquid water make these habitats extremely inhospitable for life. To date, both culture-dependent and culture-independent methods have shown that permafrost is a habitat for microorganisms of all three domains: Bacteria, Archaea and Eukarya. An overview of applying psychrophilic and psychrotolerant bacteria and archaea isolated from Arctic and Antarctic permafrost ecosystems in biotechnological processes of wastewater treatment, production of cold-adapted enzymes, etc. is discussed here. The study of existing collections of microorganisms isolated from permanently cold habitats, improved methods of sampling and enrichment will increase the potential biotechnological applications of permafrost bacteria and archaea producing unique biomolecules.

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Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

Applied and Environmental Microbiology ◽

10.1128/aem.00824-07 ◽

2007 ◽

Vol 73 (15) ◽

pp. 4849-4856 ◽

Cited By ~ 28

Author(s):

Ryoma Miyake ◽

Jun Kawamoto ◽

Yun-Lin Wei ◽

Masanari Kitagawa ◽

Ikunoshin Kato ◽

...

Keyword(s):

Low Temperature ◽

Protein Expression ◽

Recombinant Protein Expression ◽

Expression System ◽

Maximum Yield ◽

Broad Host Range ◽

Psychrophilic Bacterium ◽

T7 Promoter ◽

Cold Adapted ◽

Protein Expression System

ABSTRACT A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

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Methylovulum psychrotolerans sp. nov., a cold-adapted methanotroph from low-temperature terrestrial environments, and emended description of the genus Methylovulum

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.001046 ◽

2016 ◽

Vol 66 (6) ◽

pp. 2417-2423 ◽

Cited By ~ 19

Author(s):

Igor Y. Oshkin ◽

Svetlana E. Belova ◽

Olga V. Danilova ◽

Kirill K. Miroshnikov ◽

W. Irene C. Rijpstra ◽

...

Keyword(s):

Low Temperature ◽

Cold Adapted ◽

Emended Description ◽

Terrestrial Environments

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A Novel Subfamily of Endo-β-1,4-Glucanases in Glycoside Hydrolase Family 10

Applied and Environmental Microbiology ◽

10.1128/aem.01029-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 2

Author(s):

Fang Zhao ◽

Hai-Yan Cao ◽

Long-Sheng Zhao ◽

Yi Zhang ◽

Chun-Yang Li ◽

...

Keyword(s):

Low Temperature ◽

Substrate Specificity ◽

Glycoside Hydrolase ◽

Marine Bacterium ◽

High Salinity ◽

Binding Capacity ◽

Glycoside Hydrolase Family ◽

Salt Tolerant ◽

Content Type ◽

Cold Adapted

ABSTRACTAs classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identifiedArcticibacterium luteifluviistationiscarboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase.AlCMCase originated from an Arctic marine bacterium,Arcticibacterium luteifluviistationisSM1504T. It shows low identity (<35%) with other GH10 xylanases. The gene encodingAlCMCase was overexpressed inEscherichia coli. Biochemical characterization showed that recombinantAlCMCase is a cold-adapted and salt-tolerant enzyme.AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity ofAlCMCase is negligible. Correspondingly,AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides.AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity asAlCMCase. These results suggest thatAlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters ofAlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCECellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase,AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover,AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.

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Terrestrial models for extraterrestrial life: methanogens and halophiles at Martian temperatures

International Journal of Astrobiology ◽

10.1017/s1473550406002916 ◽

2006 ◽

Vol 5 (2) ◽

pp. 89-97 ◽

Cited By ~ 47

Author(s):

I.N. Reid ◽

W.B. Sparks ◽

S. Lubow ◽

M. McGrath ◽

M. Livio ◽

...

Keyword(s):

Low Temperature ◽

Extracellular Polymeric Substances ◽

Halophilic Archaea ◽

Methanosarcina Acetivorans ◽

Extraterrestrial Life ◽

Antarctic Species ◽

Cold Temperatures ◽

Cold Adapted ◽

Cold Environments ◽

The Galaxy

Cold environments are common throughout the Galaxy. We are conducting a series of experiments designed to probe the low-temperature limits for growth in selected methanogenic and halophilic Archaea. This paper presents initial results for two mesophiles, a methanogen, Methanosarcina acetivorans, and a halophile, Halobacterium sp. NRC-1, and for two Antarctic cold-adapted Archaea, a methanogen, Methanococcoides burtonii, and a halophile, Halorubrum lacusprofundi. Neither mesophile is active at temperatures below 5 °C, but both cold-adapted microorganisms show significant growth at sub-zero temperatures (−2 °C and −1 °C, respectively), extending previous low-temperature limits for both species by 4–5 °C. At low temperatures, both H. lacusprofundi and M. burtonii form multicellular aggregates, which appear to be embedded in extracellular polymeric substances. This is the first detection of this phenomenon in Antarctic species of Archaea at cold temperatures. The low-temperature limits for both psychrophilic species fall within the temperature range experienced on present-day Mars and could permit survival and growth, particularly in sub-surface environments. We also discuss the results of our experiments in the context of known exoplanet systems, several of which include planets that intersect the Habitable Zone. In most cases, those planets follow orbits with significant eccentricity, leading to substantial temperature excursions. However, a handful of the known gas giant exoplanets could potentially harbour habitable terrestrial moons.

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