In heterologous protein productions by P. pastoris, methanol induction is generally initiated when cell concentration reaches very high density. The alternative strategy by initiating methanol induction at lower cells concentration was also reported to be effective in easing DO control, reducing toxic by-metabolites accumulation and increasing targeted proteins titers. However, the methanol/energy regulation mechanisms are seldom reported. We theoretically analyzed the methanol/energy metabolisms in protein expression process with the strategies of initiating induction at higher or lower cells concentrations, using monellin production as a prototype. When initiating induction at lower cells concentration and controlling induction temperature at 30°C, monellin concentration reached the highest levels of 2.62~2.71 g/L, which was 2.5~4.9 fold of those obtained with the strategy of initiating induction at higher cells concentration. With the desired induction strategy, 1) carbon metabolism ratio directing into the precursors synthesis route for monellin production reached the highest level of 65%, carbon metabolism ratios towards to precursors synthesis and ATP regeneration routes were regulated at relatively balanced levels; 2) monellin synthesis was completely cell growth associated, with the largest associated coefficient and higher specific growth rate; 3) theoretical NADH (energy) utilization efficiency η was the highest, and η stayed high levels (≥0.8) during most period (89%) within induction phase to supply sufficient energy in supporting monellin synthesis.

In this study, the alcoholysis of furfuryl alcohol (FA) into ethyl levulinate (EL) using a deep eutectic solvent (DES) composed of choline chloride (ChCl) and ethanol was investigated by experiments and calculations. Experimental results reveal that the addition of 5-sulfonic acid salicylic acid (5-SSA) can catalyze the alcoholysis of FA to produce EL. The combined presence of ChCl and 5-SSA significantly improved the selectivity for EL. The mechanism of the alcoholysis of FA to EL in acidic DES was investigated by density functional theory (DFT) calculations in Gaussian 03. It was found that hydrogen-bond acceptor ChCl is coupled with hydrogen-bond donor ethanol to form a structure similar to HCl and ethoxy, which facilitates the alcoholysis of FA into EL.