Publication

An optimized high throughput platform, from genetic transformation to fermentation, to screen for secreted laccases in Aspergillus niger

Abstract

Many challenges are faced in the conversion of biomass into advanced biofuels, one of which is finding the correct organism for the job. The filamentous fungus Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degradation because it can secrete numerous hydrolytic enzymes, such as lignin-degrading enzymes and, in particular, laccase enzymes (1). However, low transformation efficiency has hindered efforts to unlock the full potential of this organism.This thesis presents a novel method to efficiently edit the genome of A. niger that overcomes several of the current challenges encountered when using CRISPR/Cas9 (2). We designed a genetic construct that is efficient and precise concerning knockout efficiency and phenotype control and demonstrated its utility for genetic knockouts, integrations, and alterations. Using the new CRISPR/Cas9 toolbox, we developed a high throughput platform to transform A. niger using robotics. We then transformed a library of 81 laccases that could be potentially secreted by A. niger in two different strain backgrounds and obtained six new laccases secreted by A. niger. We engineered a strain that increased total protein secretion fourfold and extracellular laccase activity threefold. We are currently applying for a patent for this discovery. We characterized each secreted enzyme by downscaling in micro-culture to study their optimum efficiency (e.g., media, pH, and fermentation time). These discoveries bring us significantly closer to generating strains that can produce a cocktail of laccases to unlock recalcitrant biomass for downstream processing. Additionally, the methods we developed will enable the rapid building and testing of genetic variants in A. niger for metabolic engineering, synthetic biology, and many other applications.

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Related concepts (33)
Genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA.
Aspergillus niger
Aspergillus niger is a mold classified within the Nigri section of the Aspergillus genus. The Aspergillus genus consists of common molds found throughout the environment within soil and water, on vegetation, in fecal matter, on decomposing matter, and suspended in the air. Species within this genus often grow quickly and can sporulate within a few days of germination. A combination of characteristics unique to A. niger makes the microbe invaluable to the production of many acids, proteins and bioactive compounds.
Aspergillus
Aspergillus (ˌæspərˈdʒɪləs) is a genus consisting of several hundred mould species found in various climates worldwide. Aspergillus was first catalogued in 1729 by the Italian priest and biologist Pier Antonio Micheli. Viewing the fungi under a microscope, Micheli was reminded of the shape of an aspergillum (holy water sprinkler), from Latin spargere (to sprinkle), and named the genus accordingly. Aspergillum is an asexual spore-forming structure common to all Aspergillus species; around one-third of species are also known to have a sexual stage.
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