In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus

2005 ◽  
Vol 100 (2) ◽  
pp. 158-163 ◽  
Author(s):  
Akira Nakamura ◽  
Yasuaki Takakura ◽  
Hideo Kobayashi ◽  
Takayuki Hoshino
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Thermus Thermophilus ◽  
Vector System ◽  
Selection Marker ◽  
Hygromycin B

scholarly journals In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression

FEBS Letters ◽  
1995 ◽  
Vol 369 (2-3) ◽  
pp. 158-160 ◽  
Author(s):  
Andrey V. Karginov ◽  
Olga A. Karginova ◽  
Vera A. Spiridonova ◽  
Alexei M. Kopylov
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
Thermus Thermophilus

scholarly journals A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB

2020 ◽  
Vol 8 (11) ◽  
pp. 1738
Author(s):  
Abu Bakar Salleh ◽  
Siti Marha Baharuddin ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Mahiran Basri ◽  
...  
Keyword(s):  
Expression System ◽  
Vector System ◽  
Selection Marker ◽  
Hygromycin B ◽  
Gene Encoding ◽  
Methanol Induction ◽  
New Host ◽  
Yeast Expression System ◽  
Meyerozyma Guilliermondii ◽  
New Yeast

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter ‘ragi’ from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

scholarly journals Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhi-Bo Yan ◽  
Jing-Long Liang ◽  
Fu-Xing Niu ◽  
Yu-Ping Shen ◽  
Jian-Zhong Liu
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Genome Shuffling ◽  
Stilbene Synthase ◽  
Strain Engineering ◽  
Host Strain ◽  
E Coli ◽  
Enhanced Production

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

Безопасность соединений из группы терпенов ментанового ряда in vitro и in vivo

2020 ◽  
Vol 83 (4) ◽  
pp. 24-30
Author(s):  
Ирина Владимировна Акулина ◽  
Светлана Ивановна Павлова ◽  
Ирина Семеновна Степаненко ◽  
Назира Сунагатовна Карамова ◽  
Александр Владиславович Сергеев ◽  
...  
Keyword(s):  
Escherichia Coli

Проведено токсикологическое исследование соединений с антибактериальными свойствами из группы терпенов ментанового ряда в условиях in vitro и in vivo: лимонена (B34), его производного (+)-1,2-оксида лимонена (B60) и серосодержащего монотерпенового соединения 2-(1’-гидрокси-4’-изопренил-1’-метилциклогексил-2’-тио)метилэтаноата (B65). В условиях in vitro (культура опухолевых клеток HeLa) изучаемые монотерпены в диапазоне концентраций 2 – 200 мкг/мл обладали цитотоксичностью. Ингибирующая концентрация (ИК50) для B34 составила 231 (167 – 295) мкг/мл, для B60 – 181 (105 – 257) мкг/мл, ИК50 B65 – 229 (150 – 308) мкг/мл. Исследование генотоксичности показало, что B34 и B65 в диапазоне концентраций 50 – 1000 мкг/мл не индуцируют SOS мутагенез в клетках Escherichia coli PQ37, тогда как B60 в концентрациях 500 и 1000 мкг/мл проявляет генотоксичность. In vivo в остром эксперименте на беспородных мышах установлена низкая токсичность B34 и его производных при различных путях введения. Наименьший показатель острой токсичности имеет B65, в связи с чем дополнительно на крысах проведено изучение его хронической токсичности. Ежедневное внутрижелудочное введение B65 в разовых дозах, составляющих 1/10 и 1/20 ЛД50 (1000 мг/кг и 500 мг/кг), в течение 1 мес не вызывало гибели животных, значимых нарушений общего состояния, изменения динамики массы тела, морфопатологических изменений. Внутрижелудочное введение B65 крысам в высокой токсической дозе 2000 мг/кг (1/5 ЛД50) в течение месяца вызывает патоморфологические изменения структуры печени.

Garlic Extract (Allium sativum L.) Effectively Inhibits Staphylococcus aureus and Escherichia coli by Invitro Test

2020 ◽  
Vol 2 (2) ◽  
pp. 61-68
Author(s):  
Agnina Listya Anggraini ◽  
Ratih Dewi Dwiyanti ◽  
Anny Thuraidah
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Allium Sativum ◽  
Antibacterial Properties ◽  
Herbal Medicines ◽  
Garlic Extract ◽  
Dilution Method ◽  
Initial Stage

Infection is a disease caused by the presence of pathogenic microbes, including Staphylococcus aureus and Escherichia coli. Garlic (Allium sativum L.) has chemical contents such as allicin, alkaloids, flavonoids, saponins, tannins, and steroids, which can function as an antibacterial against Staphylococcus aureus and Escherichia coli. This study aims to determine the antibacterial properties of garlic extract powder against Staphylococcus aureus and Escherichia coli. This research is the initial stage of the development of herbal medicines to treat Staphylococcus aureus and Escherichia coli infections. The antibacterial activity test was carried out by the liquid dilution method. The concentrations used were 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL and 70 mg/mL. The results showed that the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus and Escherichia coli was 40 mg/mL and 50 mg / mL. Minimum Bactericidal Concentration (MBC) results for Staphylococcus aureus and Escherichia coli are 50 mg/mL and 70 mg/mL. Based on the Simple Linear Regression test, the R2 value of Staphylococcus aureus and Escherichia coli is 0.545 and 0.785, so it can be concluded that there is an effect of garlic extract powder on the growth of Staphylococcus aureus and Escherichia coli by 54.5% and 78.5%. Garlic (Allium sativum L.) extract powder has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

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