URA3 affects artemisinic acid production by an engineered Saccharomyces cerevisiae in pilot-scale fermentation / 生物工程学报

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.

Disruption of URA-3 gene of a paclitaxel-producing fungus N8 / 国际生物医学工程杂志

Objective In order to get genetic markers,an auxotrophic paclitaxel-producing fungus named Pestalotiopsis malicola N8 strain was isolated by genetic modification.Methods Based on the homologous recombination,URA-3 which is the key gene for uracil synthetic route of Pestalotiopsis malicola N8 strain was knocked out.The transformants were screened by minimal medium with the combination of 5-fluoroorotic acid (5-FOA) and uracil.Results The results showed that the uracil auxotrophic strain was able to grow in the minimal medium containing 5-FOA and uracil while the wild type strain was not.Conclusions The uracil auxotrophic strain can be used as a new selection marker for future gene function studies of N8 strain.

Construction and identification of the double auxotrophic Candida parapolymorpha / 中华微生物学和免疫学杂志

Objective To construct a genetically-stable double auxotrophic,in which the uracil and leucine were mutated,using the Candida parapolymorpha ATCC26012 as materials.Methods Based on the physical and genetic engineering methods,the chromosome of the C.parapolymorpha strain was modified,where the ura3 and leu2 genes were directly mutated,to obtain the uracil and leucine double auxotrophic strain.Then the constructed strain was identified by the analysis of its biological properties,such as genetic stability,the change of the genes,and the physiologic and biochemical characteristics.Results The uracil and leucine double auxotrophic strain is obtained by screening.The biological identification results show that the obtained strain is genetically stable and the targeted genes are directly altered.In addition,the physiologic and biochemical analyses indicate that the auxotrophic can utilize various kinds of carbon and nitrogen nutrient sources,and its growth is good.Conclusion The successful construction of double auxotrophic mutant strains facilitated the genetic studies on C.parapolymorpha to meet various investigational purposes.Moreover,the constructed auxotrophic strains can be applied as advantageous host cells to express multiple proteins/antigens simultaneously,which is of great significance in the development of vaccines.