Production of Bacterial-Derived Enzymes in Aspergillus Niger with Applications in Lignocellulosic Biofuel Production

The development biofuels from Lignocellulosic (LC) biomass, which includes a variety of plant feedstocks such as agricultural and forestry residues and dedicated energy crops, is one route to clean and renewable alternatives to petroleum-base fuel. The first step in converting plant biomass to fuels is breaking down cellulose, the most abundant plant cell wall polymer, into fermentable sugars. Pretreament of LC biomass with ionic liquids (ILs) greatly reduces the difficulty in breaking down the plant cell wall, allowing more efficient enzymatic hydrolysis of cellulose to sugar. Unfortunately, currently available commercial cellulase cocktails are not active in the presence of ILs, an incompatibility that required the addition of expensive and wasteful washing steps. Due to the efficacy of IL pretreatment, finding IL-tolerant cellulolytic enzymes is necessary. An ideal place to look for such enzymes is high salt and high temperature environments that harbor halophilic and thermophilic bacteria, which tend to produce highly stable enzymes that may be IL-tolerant. Researchers at JBEI have identified 17 novel bacterialderived cellulase enzymes which have been isolated from enriched compost material. However, enzyme production at a scale large enough to be commercially viable is limited due to the inability of common laboratory heterologous expression systems (E. coli and Saccharomyces cerevisiae) to produce sufficient titers of these enzymes. A frequently used commercial host, Aspergillus niger (A. niger), which is known for large scale heterologous/endogenous protein production, is a promising solution to this limitation. The Fungal Biotechnology Group at JBEI is focused on understanding and engineering enzyme hyper production phenotypes into A. niger for use as a high-titer expression platform for cellulase enzymes discovered at JBEI. The Fungal Biotechnology group has created DNA constructs for expression of heterologous enzymes in A. niger and has begun screening these enzymes for expression. To determine how well these bacterial-derived enzymes express in A. niger, bacterial genes were transformed into A. niger and the enzymes were assayed for activity using the model cellulose substrates Azo-CM-cellulose (CMC), p-Nitrophenyl β-D-cellobioside (pNPC) or p-Nitrophenyl β-D-glucaopyranoside (pNPG) assays. Here we show that these enzymes can be functionally expressed in A. niger with varying degrees of success. Based on our initial results, we will perform further assays on these enzymes and apply forward and reverse genetic approaches to identify genetic features responsible for high protein secretion and engineer these features into a master high production strain.