Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature

Abstract

To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

Upgraded Bioelectrocatalytic N2 Fixation: From N2 to Chiral Amine Intermediates

“…8 For example, (R)-1b is a precursor of the antihypertensive dilevalol, 36,37 (R)-2b can be used to synthesize the bronchodilator formoterol, 38 and (R)-3b has the same backbone of the antiarrhythmic, antimyotonic, analgesic drug (R)-mexiletin 39,40 and antimyotonic, analgesic drug (R)-mexiletin. 39,40 In the bioelectrocatalytic upgraded N 2 fixation system, AlaDH, 41 DI, 42 HN-ωTA, 8 and nitrogenase (Figure S2) are stable enough and work steadily at 30 °C. The generation of NH 3 catalyzed by nitrogenase is the initial step in the reaction pathway.…”

“…In the bioelectrocatalytic upgraded N 2 fixation system, AlaDH, DI, HN-ωTA, and nitrogenase (Figure S2) are stable enough and work steadily at 30 °C. The generation of NH 3 catalyzed by nitrogenase is the initial step in the reaction pathway.…”

“…The activity of DI was determined at 30 °C by measuring the absorbance change at 578 nm in 1 min. 42 The enzyme reactions were carried out in an anaerobic screw-cap IR quartz cuvette (Reflex Analytical Co., Ridgewood, NJ, USA) with a degassed 100 mM MOPS buffer (pH 7.0) containing 2 mM NADH. Reduced methyl viologen (MV •+ , 100 μM), prepared with a rotating disc electrode by bulk electrolysis at −0.85 V vs SCE for overnight, was used as an electron donor.…”

“…The activity of DI was determined at 30 °C by measuring the absorbance change at 578 nm in 1 min . The enzyme reactions were carried out in an anaerobic screw-cap IR quartz cuvette (Reflex Analytical Co., Ridgewood, NJ, USA) with a degassed 100 mM MOPS buffer (pH 7.0) containing 2 mM NADH.…”

Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

“…The interaction between cohesins and dockerins is species and type-specific and allows controlled binding of several different enzymes next to each other (Carvalho et al, 2003;Cameron et al, 2015). Several synthetic protein scaffolds or designer cellulosomes have been expressed and tested for viability (You et al, 2012;You and Zhang, 2013;Chen et al, 2018;Kahn et al, 2019). Natural and designer cellulosomes can be thermostable (Kahn et al, 2019), allow self-assembly and co-immobilization (You and Zhang, 2013), and show remarkable flexibility in regards to possible activity combinations (Artzi et al, 2017).…”