ABSTRACT
CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion, replacement and deletion due to its simplicity and high efficiency. The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast. In our previous research, GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast, in order to eliminate the requirement of galactose supplementation for induction. The maximum artemisinic acid production by engineered S. cerevisiae 1211-2 (740 mg/L) was comparable to that of the parental strain 1211 without galactose induction. Unfortunately, S. cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment. The artemisinic acid yield in the engineered S. cerevisiae 1211-2 was only 20%-25% compared with that of S. cerevisiae 1211. The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production. A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA, resulting in S. cerevisiae 1211-3. This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding, and the final artemisinic acid yield (> 20 g/L) was comparable to that of the parental strain S. cerevisiae 1211. In this study, an engineered yeast strain producing artemisinic acid without galactose induction was obtained. More importantly, it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding, which provides a reference for the production of other natural products in yeast chassis.
Subject(s)
Artemisinins , Fermentation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Dao-di herbs are preferred by long-term clinical application of Chinese medicine, they are produced in a specific area and affected by the specific production and processing methods. Dao-di herbs have a high reputation, compared with other regions produced by the same kind of herbs with good quality, good efficacy and stable quality characteristics. Geo-herbal is a unique concept that has been formed by the history of Chinese civilization for thousands of years and the history of Chinese medicine. According to the definition of geo-herbal, it can be seen that there are some differences in the quality and efficacy of the same kind of medicinal herbs in different regions, and there is some similarity in the specific area. In this study, based on the content of artemisinic acid in sampling points and its potential environmental factors, the effects of geographical environment factors on the spatial distribution of artemisinic acid content in Artemisia annua were studied by using the geophysical model. The results show that the spatial distribution of artemisinic acid content is the result of a combination of multiple factors. The effects of environmental factors on the spatial distribution of artemisinic acid were in the order of soil type (0.233)> radiation (0.208)> vegetation type (0.192)> elevation (0.171)> sunshine (0.170)> annual mean temperature (0.153) >annual precipitation (0.111)> slope (0.110)> relative humidity. Among them, the soil type and the amount of radiation are the main influencing factors, and the main influencing area is in the soil type as the initial soil and the average annual radiation of 1 200-1 400 kWh•m⁻². The main influencing factors selected in this study can be used to monitor the spatial distribution of artemisinic acid by remote sensing technology, so as to provide the theoretical basis for the cultivation of A. annua.
ABSTRACT
The antimalarial activity of artemisinin and its derivatives have been widely recognized.A deep scientific research had produced many patent documents.In order to formulate strategies on research and patent application rationally,it is necessary to carry an analysis existing patent application systematically.Necessary patent bibliographic information was searched and downloaded using professional patent database.And then,patent and industrial pattern within were extracted comprehensively.Main subjects included artemisinin,artemisinic acid and arteannuin B.The results showed that through a series of statistical analysis,information on life cycle of artemisinin patent applications,application efficiency preferences,excellent applications and high value patents was obtained.It was concluded that in this field,the patent technology life cycle is in mature stage.Among them,artemisinin has a relatively large space for development.The most popular applications are anti-parasite,anti-tumor and anti-infection.The core theme of internal reference is to treat skin diseases and to suppress cancer.
ABSTRACT
Objective: To investigate the effects of artemisinic acid on proliferation of K562 cells and its mechanism in vitro.Methods: MTT assay was used to detect the effects of artemisinic acid on proliferation of K562 cells.The flow cytometry was used to analyze the distribution of cell cycle.Morphological changes of cells were observed by electron microscopy.Results: The proliferation of K562 cells was inhibited by intervention of artemisinic acid significantly after 24 hours and 48 hours,and the effects were in concentration-dependent manner.The cell number in G0/G1 increased and apoptosis was induced after the treatment with artemisinic acid for 48 hours,the morphological characteristic of apoptosis was observed by electron microscopy.Conclusion: Artemisinic acid can inhibit the proliferation of K562 cells,which may be-due to induction of apoptosis and prohibit on of tumor cells from entering cell cycle.