The bacterial actin-like cell division protein FtsA forms curved antiparallel double filaments upon binding of FtsN

Cell growth and division of walled bacteria depend on the synthesis and remodelling of peptidoglycan (PG). These activities are carried out by two multiprotein complexes, the elongasome and the divisome during cell elongation and division, respectively. Filaments of tubulin-like FtsZ form the cytoplasmic scaffold for divisome assembly, the Z-ring. In E. coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits downstream divisome components, including bitopic FtsN. FtsN is recruited late and activates the periplasmic PG synthase FtsWI. To start unravelling the activation mechanism involving FtsA and FtsN, we showed that E. coli FtsA forms antiparallel double filaments on lipid monolayers when also binding FtsN's cytoplasmic tail, and that Vibrio maritimus FtsA crystallised as an equivalent double filament. We structurally located the FtsA-FtsN interaction site in FtsA's IA-IC interdomain cleft and confirmed FtsA double filament formation in vivo using site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted on and in liposomes preferred negative Gaussian curvature, as was previously shown for the elongasome's actin, MreB. MreB filaments serve as curvature-sensing "rudders", orienting insertion of PG around the cell's circumference. We propose that curved antiparallel FtsA double filaments function similarly in the divisome: FtsA filaments, together with dynamic FtsZ filaments orient and concentrate cell-constricting septal PG synthesis in the division plane.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.

An integrative approach for high level production of glucuronoyl esterase in Pichia pastoris.

Objective: The commercial availability of lignin-modifying and accessory enzymes is limited, which delays the investigation of their functionality and development of industrial applications. Glucuronoyl esterase (GE) has been shown to improve fractionation of lignin-carbohydrate complexes, yet no pure commercial enzyme preparations are available. To improve accessibility to this enzyme for emerging research, this study reports a simple and effective heterologous expression strategy, coupled with a methanol-free production protocol in a bioreactor for high-level production of GE. This strategy was further validated by production of cellobiose dehydrogenase, a lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate, and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, in a two-step filtration process. This study describes a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research.