High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea

Abstract

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or acetate as carbon and energy sources. This method provides an avenue for the first step in identifying new gene functions: associating a genetic mutation with a reproducible phenotype. Mutations in bona fide methanogenesis genes such as corrinoid methyltransferases and proton-translocating FH:methanophenazine oxidoreductase (Fpo) were also generated, opening the door to in vivo functional complementation experiments. Irradiation-based mutagenesis such as from ultraviolet (UV) light, combined with modern genome sequencing, is a useful procedure to discern systems-level gene function in prokaryote taxa that can be axenically cultured but which may be resistant to chemical mutagens.

Towards a Miniaturized Culture Screening for Cellulolytic Fungi and Their Agricultural Lignocellulosic Degradation

“…This method may not only quantitatively determine the amount of certain enzyme activities but also eliminate fungal isolates that do not grow well under submerged fermentation conditions. For instance, the microplate-based cultivation method has been implemented successfully, as previously reported in other applications, such as for enzymatic hydrolysis of lignocellulosic biomass according to [ 43 ], for mutant archaea and fungal strains using mutagenesis [ 25 , 44 ] and for fermentation production of citric acid, ethanol and glycerol by filamentous fungi [ 26 ].…”

Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

“…Such traditional screening methods generated large workloads, were time-consuming, and had low efficiency and high costs, and were gradually eliminated over time. Currently, high-throughput screening technologies based on microplate (24/48/96/384-deep well plates) technologies are widely used for screening mutant strains, biologically active substances, and candidate drugs [25][26][27] due to their similar characteristics to automatic parallel microreactors. In addition, spectrophotometry based microplate readers have huge advantages when compared with cylinder plate and high-performance liquid chromatography (HPLC) methods in terms of antibiotic identification, as they process large numbers of simultaneous samples and reduce reagent costs and use.…”