Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes byZymomonas mobilis

Abstract

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.The biological conversion of lignocellulosic biomass to ethanol represents a potential major source of future domestic transportation fuels, but the current cost of converting biomass to fermentable sugars still needs to be reduced further (12). Most current strategies for ethanol production via biochemical conversion of lignocellulosic feedstocks utilize simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and cofermentation (SSCF) processes (8,21,22). The process configuration known as consolidated bioprocessing (CBP) (20) would alleviate the financial strain of producing saccharolytic enzyme cocktails by combining the necessary steps for ethanol production as the action of one microorganism.A particularly attractive microbial candidate for the development of a CBP microorganism is the Gram-negative fermentative bacterium Zymomonas mobilis. Z. mobilis has been studied for its exceptionally high ethanol production rate, yield, and tolerance to the toxicity of the final product (15-17, 20, 31-33, 35, 43). In addition, Z. mobilis has the ability to ferment sugars at low pH and has a naturally high tolerance to many of the inhibitory compounds found in hydrolysates derived from lignocellulosic biomass (45, 46) Furthermore, the use of the Entner-Doudoroff...

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

“…Considerable effort has been made to develop single microbes capable of both saccharification and fermentation to avoid the substantial expense of using saccharolytic enzyme mixtures (13). Heterologous expression of saccharolytic enzymes has been demonstrated in a number of organisms, including Saccharomyces cerevisiae, Zymomonas mobilis, Escherichia coli, and Bacillus subtilis to ferment various model cellulosic and hemicellulosic substrates (13)(14)(15). Although these approaches have resulted in progress in cellulose utilization, the overall enzyme activity is still very low compared with that of naturally cellulolytic organisms and the rates of hydrolysis are not sufficient for an industrial process (13).…”

Evaluation of Cellulase Production by Zymomonas mobilis

“…This finding correlated to our result that native endoglucanases from Z. mobilis ZM4 and TISTR551 represented the intracellular activity with 6-8 folds higher than extracellular activity or highly presented inside the cell rather than secreted out. Future development on the cellulase secretions can be processed by engineering the effective secretion system SecB-dependent and twin arginine translocation (TAT) to the native cellulases (Linger et al 2010).…”

“…The colonies were removed from the agar, and plates were flooded with 0.2% Congo red solution for 10 min then washed with 1 M NaCl. A clear zone surrounding colonies indicates cellulose hydrolysis (Wood 1980;Linger et al 2010). The diameters of the cleared zones in three replicates were measured.…”

“…This is considered as a promising strategy for reducing the production costs (Lynd et al 2005;Cardona and Sanchez 2007;Xu et al 2009;He et al 2014). CBP microbes for ethanol production from lignocellulosic materials can be developed using native cellulolytic microbes that are engineered to improve ethanol production, e.g., Trichoderma reesei, Aspergillus, and Fusarium (Ruiz et al 2007;Xu et al 2009, Piriya et al 2012, or engineered noncellulolytic organisms that exhibit high native ethanol production, e.g., Z. mobilis and Saccharomyces cerevisiae (Van Zyl et al 2007;Linger et al 2010;Apiwatanapiwat et al 2011;Vasan et al 2011;Den et al 2015;Gonçalves et al 2016).…”

“…For example, two cellulolytic enzymes E1 and GH12 from Acidothermus cellulolyticus were successfully expressed in Z. mobilis via its native signal secretion protein. Z. mobilis produces endogeneous extracellular cellulose activity against carboxymethyl cellulose (CMC) via both native and engineered cellulolytic expressing genes (Linger et al 2010). Cellulolytic enzymes from Cellulomonas fumi and Ruminococcus albus have been introduced, coexpressed, and secreted in Zymobacter palmae, which is then able to successfully ferment cellulosic polysaccharide (barley -glucan) to ethanol with 79.5% theoretical yield (Kojima et al 2013).…”