scholarly journals In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan

Abstract Background: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway.Results: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologousβ-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11~8.51 folds and 3.0~9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg/g DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains.Conclusions: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan

Abstract Background : Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganisms is still limited by the poor efficiency of the heterologous pathway. Results: To address the bottleneck of astaxanthin yield in microbes, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modules of β-carotene ketolase ( crtW ) and hydroxylase ( crtZ ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05 mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination. Conclusions: Recombination of heterologous modules by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high-throughput screening of heterologous pathways by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW , but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathways.


2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan

Abstract Bacground: Astaxanthin is a kind of tetraterpene with strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganism is still limited by the poor efficiency of heterologous pathway.Results: To address the bottleneck of astaxanthin yield in microbe, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modular ofβ-carotene ketolase (crtW) and hydroxylation (crtZ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination.Conclusions: Recombination of heterologous modular by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high throughput screening of heterologous pathway by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW, but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathway.


Author(s):  
I. F. Gorlov ◽  
A. A. Mosolov ◽  
G. V. Komlatskiy ◽  
M. A. Nesterenko ◽  
K. D. Nimbona ◽  
...  

The article presents materials on the study of the possibility of reproduction and increase in the herd of highly productive cows through the use of embryo transplantation technology. The classical (in vivo) and more modern, developing (in vitro) methods of embryotransfer, their positive and negative sides are considered in detail. The possibility of accelerating the breeding process by using the method of transplantation, in which from one cow can be obtained from 10 to 100 calves, which will allow for 4-5 years, almost any herd (of any size and breed) with the help of biotechnology to turn into a cattle-breeding enterprise of the most modern level. At the same time, heifers obtained from unproductive cows can be used as "surrogate" mothers who are transplanted with the best donor embryos, which allows to obtain a full-fledged offspring adapted to local environmental conditions. A detailed scheme of obtaining, evaluation, storage, as well as the cost and economic effect of embryo transplantation was calculated, the market was evaluated, the required annual volume of transplants and the number of donor cows for large livestock farms were determined. As a positive example of "Scientific-production enterprise "Centre of biotechnology and embryo transfer" in 2014, implemented a project for accelerated replacement and genetic improvement of the dairy herd, engraftment averaged 57-69%, and the economic effect of the enterprise from getting a single animal by the method of embryo transfer, compared with imports of similar close in quality, ranged from 60 to 100 thousand rubles on his head. It is shown that it is necessary to organize at the state level a developed service for embryo transplantation to reduce the cost of embryo transfer and the possibility of creating in a short time in the country's own highly productive breeding nucleus of dairy and beef cattle, which will reduce, and in the future completely eliminate, import dependence on cattle products.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.


Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1428
Author(s):  
Ramachandran Chelliah ◽  
Eun-Ji Kim ◽  
Eric Banan-Mwine Daliri ◽  
Usha Antony ◽  
Deog-Hwan Oh

In the present study, we screened for potential probiotic yeast that could survive under extreme frozen conditions. The antimicrobial and heat-stable properties of the isolated yeast strains Saccharomyces boulardii (S. boulardii) (KT000032, KT000033, KT000034, KT000035, KT000036, and KT000037) was analyzed and compared with commercial probiotic strains. The results revealed that the tested S. boulardii KT000032 strain showed higher resistance to gastric enzymes (bile salts, pepsin, and pancreatic enzyme) at low pH, with broad antibiotic resistance. In addition, the strain also showed efficient auto-aggregation and co-aggregation abilities and efficient hydrophobicity in the in-vitro and in-vivo C. elegens gut model. Further, the KT000032 strain showed higher antimicrobial efficiency against 13 different enteropathogens and exhibited commensal relationships with five commercial probiotic strains. Besides, the bioactive compounds produced in the cell-free supernatant of probiotic yeast showed thermo-tolerance (95 °C for two hours). Furthermore, the thermo-stable property of the strains will facilitate their incorporation into ready-to-eat food products under extreme food processing conditions.


2020 ◽  
Author(s):  
Hua Sang ◽  
Jiali Liu ◽  
Fang Zhou ◽  
Xiaofang Zhang ◽  
Jingwei Zhang ◽  
...  

<p></p><p>Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by <i>in vitro</i> viability assays and <i>in vivo</i> tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly <i>in vivo</i> experiments.</p><br><p></p>


2021 ◽  
Vol 22 (22) ◽  
pp. 12502
Author(s):  
Shoji Kokubo ◽  
Shinobu Ohnuma ◽  
Megumi Murakami ◽  
Haruhisa Kikuchi ◽  
Shota Funayama ◽  
...  

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.


Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 702 ◽  
Author(s):  
Christian T. Stackhouse ◽  
James R. Rowland ◽  
Rachael S. Shevin ◽  
Raj Singh ◽  
G. Yancey Gillespie ◽  
...  

Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology and in gauging therapeutic response. It remains to be seen whether or not cancer model-specific genes may be directly or indirectly, through changes to tumor microenvironment, manipulated to improve the concordance of in vitro derivative models with in vivo models yielding better prediction of therapeutic response.


Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3784
Author(s):  
Anne M. Noonan ◽  
Amanda Cousins ◽  
David Anderson ◽  
Kristen P. Zeligs ◽  
Kristen Bunch ◽  
...  

Inhibitor of apoptosis (IAP) proteins are frequently upregulated in ovarian cancer, resulting in the evasion of apoptosis and enhanced cellular survival. Birinapant, a synthetic second mitochondrial activator of caspases (SMAC) mimetic, suppresses the functions of IAP proteins in order to enhance apoptotic pathways and facilitate tumor death. Despite on-target activity, however, pre-clinical trials of single-agent birinapant have exhibited minimal activity in the recurrent ovarian cancer setting. To augment the therapeutic potential of birinapant, we utilized a high-throughput screening matrix to identify synergistic drug combinations. Of those combinations identified, birinapant plus docetaxel was selected for further evaluation, given its remarkable synergy both in vitro and in vivo. We showed that this synergy results from multiple convergent pathways to include increased caspase activation, docetaxel-mediated TNF-α upregulation, alternative NF-kB signaling, and birinapant-induced microtubule stabilization. These findings provide a rationale for the integration of birinapant and docetaxel in a phase 2 clinical trial for recurrent ovarian cancer where treatment options are often limited and minimally effective.


Sensors ◽  
2019 ◽  
Vol 19 (16) ◽  
pp. 3502 ◽  
Author(s):  
Se-Hong Min ◽  
Alexander R. French ◽  
Keelan J. Trull ◽  
Kiet Tat ◽  
S. Ashley Varney ◽  
...  

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.


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