A rapid identification method for common astigmatid species based on multiplex polymerase chain reaction.

Astigmatid mites are economically significant pests of stored products and sources of inhalant allergens causing allergic rhinitis and asthma worldwide. The morphological identification of astigmatid mites at the species level is often a difficult task due to their small size, phenotypic similarity and lack of diagnostic characters. We used multiplex polymerase chain reaction (PCR) to identify astigmatid mite species, which could complement the morphological data for the species-specific identification of mites. Internal ribosomal transcribed spacer (ITS) sequences (i.e., partial 18S, the full length of ITS1-5.8S-ITS2 and partial 28S) from eight astigmatid species (Acarus siro, Tyrophagus putrescentiae, Suidasia nesbitti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Lepidoglyphus destructor, Chortoglyphus arcuatus and Gohieria fuscus) were obtained by DNA extraction and then sequenced after PCR amplification. Specific primers were designed in the ITS2 region manually. Results revealed that an identification method for eight common astigmatid species was established based on multiplex PCR, which should be effective for the identification of other species of mites by redesigning species-specific primers in future experiments.

The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae.

A challenge associated with the ethanol productivity under very-high-gravity (VHG) conditions, optimizing multi-traits (i.e. byproduct formation and stress tolerance) of industrial yeast strains, is overcome by a combination of metabolic engineering and genome shuffling. First, industrial strain Y12 was deleted with a glycerol exporter Fps1p and hetero-expressed with glyceraldehydes-3-phosphate dehydrogenase, resulting in the modified strain YFG12 with lower glycerol yield. Second, YFG12 was subjected to three rounds of drug resistance marker-aided genome shuffling to increase its ethanol tolerance, and the best shuffled strain TS5 was obtained. Compared with wild strain Y12, shuffled strain TS5 not only decreased glycerol formation by 14.8%, but also increased fermentation rate and ethanol yield by 3.7% and 7.6%, respectively. Moreover, the system of genetic modification and Cre/loxP in aid of three different drug-resistance markers presented in the study significantly improved breeding efficiency and will facilitate the application of breeding technologies in prototrophic industrial microorganisms.

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance.

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.

Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in Saccharomyces cerevisiae.

Acetic acid existing in a culture medium is one of the most limiting constraints in yeast growth and viability during ethanol fermentation. To improve acetic acid tolerance in Saccharomyces cerevisiae strains, a drug resistance marker-aided genome shuffling approach with higher screen efficiency of shuffled mutants was developed in this work. Through two rounds of genome shuffling of ultraviolet mutants derived from the original strain 308, we obtained a shuffled strain YZ2, which shows significantly faster growth and higher cell viability under acetic acid stress. Ethanol production of YZ2 (within 60 h) was 21.6% higher than that of 308 when 0.5% (v/v) acetic acid was added to fermentation medium. Membrane integrity, higher in vivo activity of the H+-ATPase, and lower oxidative damage after acetic acid treatment are the possible reasons for the acetic acid-tolerance phenotype of YZ2. These results indicated that this novel genome shuffling approach is powerful to rapidly improve the complex traits of industrial yeast strains.