Rapid and robust squashed spore/colony PCR of industrially important fungi.

Autor: Yuan G; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Czajka JJ; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Dai Z; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Hu D; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA., Pomraning KR; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Hofstad BA; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Kim J; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Robles AL; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA., Deng S; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Shuang.Deng@pnnl.gov.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA. Shuang.Deng@pnnl.gov., Magnuson JK; Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Jon.Magnuson@pnnl.gov.; DOE Agile BioFoundry, Emeryville, CA, 94608, USA. Jon.Magnuson@pnnl.gov.
Jazyk: angličtina
Zdroj: Fungal biology and biotechnology [Fungal Biol Biotechnol] 2023 Jul 08; Vol. 10 (1), pp. 15. Date of Electronic Publication: 2023 Jul 08.
DOI: 10.1186/s40694-023-00163-0
Abstrakt: Background: Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals.
Results: In this study we developed a rapid and robust technique called "Squash-PCR" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains.
Conclusion: The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.
(© 2023. The Author(s).)
Databáze: MEDLINE
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