ABSTRACT
Glutathione (GSH), a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9-associated-system (CRISPR-Cas), in which gRNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1b/FGP. As a result, the prototrophic strain W303-1b/FGPPT showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1b/FGPPT that might have great interests in industrial fermentation.
ABSTRACT
Objective To investigate the effect of heterologous auxotrophy markers from non-Candida albicans (C. albicans) yeasts (C.dubliniensis HIS1\[C.d.HIS1\], C. maltosa LEU2\[C.m.LEU2\] and C.dubliniensis ARG4\[C.d.ARG4\]) on adaptation to various stresses of C. albicans knockout mutants (by His1-Leu2-Arg4 strategy). Methods Fusion PCR strategy was used to construct recombination fragment, which was then used to transform SN152 by lithium acetate method. Genomic PCR was used to confirm the integration of the three selection markers. Spot assays were employed to assess the sensitivities of different reintegrated strains to different stresses. Results The results of PCR showed that seven reintegrated strains containing C.m. LEU2, C.d. HIS1 and C.d. ARG4 were successfully constructed. The results of spot assay showed that there were no differences between the seven reintegration strains and SN152 in responses to the tested stresses, including pH stress, oxidative stress, antifungal agent stress, metal ion stress, cell wall stress, osmotic stress and DNA damage agent stress. We also found that only strains which integrated C.d. HIS1 marker could survive from the 0.02% SDS stress. Conclusion The three heterologous selection marker C.m. LEU2, C.d. HIS1 and C.d. ARG4 do not influence the sensitivity of C. albicans to most stresses; comparison can be made between C. albicans knockout mutants with different markers, and SN152 can be used as parental control. While assessing the sensitivity to SDS, the results vary with different selection markers, and SN152 can not be used as a unified control; therefore comparison should be made with C.d. HIS1 kept consistent.
ABSTRACT
We screened 349 isolates of P. aeruginosa from cystic fibrosis (CF+) and non-cystic fibrosis (CF-) patients for the auxotrophy. Fourteen (4.0 percent) were auxotrophic and among them only one was recovered from CF-patient showing that this characteristic is strongly associated with cystic fibrosis. In total, a requirement for 5 different compounds (or combination) was verified and, of these, methionine was the most common single amino acid required. Only one auxotrophic isolate was no able to produce biofilm in vitro.
Subject(s)
Humans , Airway Obstruction , Cystic Fibrosis , Drug Resistance, Microbial , Pseudomonas Infections , Pseudomonas aeruginosa/isolation & purification , Biofilms , Methods , MethodsABSTRACT
Aspergillus oryzae is an important microorganism,which was widely used in the fields of food,brewing,pharmacy and fermentation industry etc and was termed as a generally regarded as safe(GRAS) organism by the United States Food and Drug Administration(FDA).The strategies for promoting the expression of homologous and heterologous proteins in Aspergillus oryzae were reviewed.Which included the usage of strong promoters,multicopies of coding genes,optimization of culture medium and overexpression of the hemoglobin domains(HBD) etc.Heterologous proteins were usually exhibited low expression in Aspergillus oryzae due to the degradation by the proteinases from host.Therefore,development of proteinase auxotrophic stains of Aspergillus oryzae as host is neccessary.In addition,fusion of the heterologous protein with a highly secreted protein of Aspergillus oryzae was an alternative way to prompt the expression of heterologous protein in Aspergillus oryzae.