scholarly journals Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 589
Author(s):  
Ricardo Correia ◽  
Bárbara Fernandes ◽  
Paula M. Alves ◽  
Manuel J.T. Carrondo ◽  
António Roldão
Keyword(s):  
Ph Value ◽  
Culture Conditions ◽  
Adaptation Process ◽  
Adaptive Laboratory Evolution ◽  
Constant Growth Rate ◽  
Laboratory Evolution ◽  
Culture Ph ◽  
Influenza Hemagglutinin ◽  
Standard Culture ◽  
High Five Cells

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

scholarly journals The genetics of catalase in Drosophila melanogaster: isolation and characterization of acatalasemic mutants.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 643-652 ◽  
Author(s):  
W J Mackay ◽  
G C Bewley
Keyword(s):  
Drosophila Melanogaster ◽  
Free Radical ◽  
Catalase Activity ◽  
Oxygen Toxicity ◽  
Threshold Effect ◽  
Culture Conditions ◽  
Free Radical Damage ◽  
Isolation And Characterization ◽  
Standard Culture ◽  
Hypomorphic Alleles

Abstract Activated oxygen species have been demonstrated to be the important agents in oxygen toxicity by disrupting the structural and functional integrity of cells through lipid peroxidation events, DNA damage and protein inactivation. The biological consequences of free radical damage have long been hypothesized to be a causal agent in many aging-related diseases. Catalase (H2O2:H2O2 oxidoreductase; EC 1.15.1.1) is one of several enzymes involved in the scavenging of oxygen free radicals and free radical derivatives. The structural gene for catalase in Drosophila melanogaster has been localized to region 75D1-76A on chromosome 3L by dosage responses to segmental aneuploidy. This study reports the isolation of a stable deficiency, Df(3L)CatDH104(75C1-2;75F1), that uncovers the catalase locus and the subsequent isolation of six acatalasemic mutants. All catalase mutants are viable under standard culture conditions and recessive lethal mutations within the 75Cl-F1 interval have been shown not to affect catalase activity. Two catalase mutations are amorphic while four are hypomorphic alleles of the Cat+ locus. The lack of intergenic complementation between the six catalase mutations strongly suggests that there is only one functional gene in Drosophila. One acatalesemic mutation was mapped to position 3-47.0 which resides within the catalase dosage sensitive region. While complete loss of catalase activity confers a severe viability effect, residual levels are sufficient to restore viability to wild type levels. These results suggest a threshold effect for viability and offer an explanation for the general lack of phenotypic effects associated with the known mammalian acatalasemics.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

2019 ◽  
Vol 20 (22) ◽  
pp. 5737 ◽  
Author(s):  
Miriam González-Villanueva ◽  
Hemanshi Galaiya ◽  
Paul Staniland ◽  
Jessica Staniland ◽  
Ian Savill ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Strain Engineering ◽  
Cupriavidus Necator ◽  
Superior Performance ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Cupriavidus Necator H16

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

Structure of vegetative organs in essential oil rose under standard culture conditions and long-term conservation in vitro

2021 ◽  
pp. 185-190
Author(s):  
I.V. Mitrofanova ◽  
V.A. Brailko ◽  
N.P. Lesnikova-Sedoshenko ◽  
N.N. Ivanova ◽  
O.V. Mitrofanova
Keyword(s):  
Essential Oil ◽  
Culture Conditions ◽  
Vegetative Organs ◽  
Standard Culture

Experimental studies on Lecanora rupicola (L.) Zahlbr.: chemical and microscopical investigations of the mycobiont and re-synthesis stages

2006 ◽  
Vol 38 (6) ◽  
pp. 577-585 ◽  
Author(s):  
Georg BRUNAUER ◽  
Armin HAGER ◽  
Wolf Dietrich KRAUTGARTNER ◽  
Roman TÜRK ◽  
Elfie STOCKER-WÖRGÖTTER
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Sequence Data ◽  
Experimental Studies ◽  
Culture Conditions ◽  
Voucher Specimen ◽  
Dna Sequence Data ◽  
Standard Culture ◽  
Voucher Specimens ◽  
Scanning Electron

Culture experiments that trigger the axenically grown mycobionts of Lecanora rupicola to produce the polyketide chemosyndrome typical of the naturally grown lichen are reported. This chemosyndrome comprises lecanoric, haematommic and orsellinic acids, sordidone, eugenitol and atranorin, all of which were hardly produced under standard culture conditions. The only exception was arthothelin that was only present in the voucher specimen. It has been shown that almost the complete acetyl-polymalonyl-pathway leading to depsides and chromones can be induced in culture, but apparently not the xanthones. The mycobiont was also successfully re-synthesized with its original photobiont, as confirmed by Scanning Electron Microscope studies (SEM). Cultures of the resynthesised lichen biosynthesized additional satellite substances, which were not detected either in the voucher specimens or in the aposymbiontically (without the photobiont) grown mycobiont cultures. The identity of cultured mycobionts of L. rupicola was confirmed by comparing ITS-DNA-sequence data from the original lichen with publicly available (GeneBank) sequences of that species.

scholarly journals ALEdb 1.0: a database of mutations from adaptive laboratory evolution experimentation

2018 ◽  
Vol 47 (D1) ◽  
pp. D1164-D1171 ◽  
Author(s):  
Patrick V Phaneuf ◽  
Dennis Gosting ◽  
Bernhard O Palsson ◽  
Adam M Feist
Keyword(s):  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals Application of Microalgal Stress Responses in Industrial Microalgal Production Systems

Marine Drugs ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 30
Author(s):  
Jia Wang ◽  
Yuxin Wang ◽  
Yijian Wu ◽  
Yuwei Fan ◽  
Changliang Zhu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Production Systems ◽  
Strain Improvement ◽  
Value Added ◽  
Product Yield ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Theoretical Support ◽  
Further Development ◽  
Value Added Products

Adaptive laboratory evolution (ALE) has been widely utilized as a tool for developing new biological and phenotypic functions to explore strain improvement for microalgal production. Specifically, ALE has been utilized to evolve strains to better adapt to defined conditions. It has become a new solution to improve the performance of strains in microalgae biotechnology. This review mainly summarizes the key results from recent microalgal ALE studies in industrial production. ALE designed for improving cell growth rate, product yield, environmental tolerance and wastewater treatment is discussed to exploit microalgae in various applications. Further development of ALE is proposed, to provide theoretical support for producing the high value-added products from microalgal production.

scholarly journals Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Thomas Perli ◽  
Dewi P.I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
B Vitamin

AbstractQuantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

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