Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion

Abstract

Methanol is a low‐cost and abundantly available feedstock derived from natural gas and syngas. Although bioconversion holds promise for producing desired chemicals from methanol under economically viable operating conditions, the efficiency is limited by unfavorable kinetics of methanol oxidation and assimilation. Herein, artificial fusion proteins were engineered to enhance methanol bioconversion. Nicotinamide adenine dinucleotide (NAD)‐dependent methanol dehydrogenase (Mdh), 3‐hexulose‐6‐phosphate synthase (Hps) and 6‐phospho‐3‐hexuloisomerase (Phi) from different sources were first screened for catalytic activity. Next, we designed six fusion proteins using the best enzyme candidates and flexible linkers. Fusing Mdh with Hps or Hps‐Phi increased the Vmax of methanol oxidation up to 5.8‐fold, and enhanced methanol conversion to fructose‐6‐phosphate up to 1.3‐fold. Interestingly, fusion engineering changed the polymerization states of proteins and produced larger multimers, which may be responsible for the changed catalytic characteristics. This fusion engineering approach can be coupled with other metabolic engineering strategies for enhanced methanol bioconversion to valuable chemicals.

Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum

“…The PCR product was inserted between the NdeI and XhoI sites of pET-21a(+) and fused with a C-terminal His Tag using the Clo-nExpress II One Step Cloning Kit (Vazyme Biotech, China). Recombinant plasmids were transformed into E. coli BL21 (DE3) and heterogeneous expression and purification of protein were conducted according to the procedure described previously 22 . The resultant strains were cultivated in LB medium at 37°C with shaking at 220 rpm.…”

“…Discover and directed evolution of more active Mdh candidates have been conducted to address the first bottleneck [18][19][20] . The strategy of enzyme co-localization and metabolite channeling has also been applied to drive oxidation of methanol to formaldehyde by constructing Mdh-Hps-Phi complexes 21,22 . To regenerate Ru5P more efficiently, the non-oxidative pentose phosphate pathway (PPP) from native methylotroph Bacillus methanolicus or the sedoheptulose-bisphosphatase (SBPase) from E. coli was overexpressed in synthetic methylotrophs to activate the SBPase pathway variant of the RuMP cycle 23,24 .…”

Strategies for Making Multimeric and Polymeric Bifunctional Protein Conjugates and Their Applications as Bioanalytical Tools

“…14,15,21 The genes encoding the proteins of interest can be expressed in one expression cassette, and the proteins can be joined by adding flexible or rigid linkers. 16,[21][22][23][24][25] Although helical linkers can improve the expression and bioactivity of fusion proteins in several cases, this strategy only works if the proteins of interest can be folded appropriately. Furthermore, only a limited number of protein units can be fused or conjugated.…”

“…14,15 Genetically fusing proteins enables their stoichiometric coupling with precisely controlled conjugation sites. [14][15][16] Bifunctional proteins that contain multimeric enzyme units can be obtained by genetic modification strategies that utilize multimeric binding proteins-such as streptavidin-as fusion partners. Enzymatic reactions provide an alternative strategy for the creation of multimeric or polymeric probes.…”

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle

“…The enzymes were purified by AKTA Purifier 10 using a Ni-NTA column (GE Healthcare, USA). The purified enzymes were desalted and exchanged into storage buffer (50 mM KPB, 1.0 mM MgSO4, 2 mM TCEP, 10% glycerol, pH 8.0) (Fan et al, 2018). The purified enzymes were stored at -80 °C.…”