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The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to byproduct generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Metal corrosion occurs anytime and anywhere in nature and the corrosion prevention has a great significance everywhere in national economic development and daily life. Here, we demonstrate a flexible hybrid nanogenerator (NG) that is capable of simultaneously or individually harvesting ambient thermal and mechanical energies and used for a self-powered cathodic protection (CP) system without using an external power source. Because of its double peculiarities of both pyroelectric and piezoelectric properties, a polarized poly(vinylidene fluoride) (PVDF) film-based NG was constructed to scavenge both thermal and mechanical energies. As a supplementary, a triboelectric NG was constructed below the pyro/piezoelectric NG to grab ambient mechanical energy. The output power of the fabricated hybrid NG can be directly used to protect the metal surface from the chemical corrosion. Our results not only verify the feasibility of self-powered CP-based NGs, but also expand potential self-powered applications.