In vitro metabolic engineering for the production of α-ketoglutarate

Abstract

α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10gL glucuronate with 92% yield of aKG within 5h. The maximum productivity of 2.8gL h is the second highest reported in the biotechnological synthesis of aKG.

Efficient Bioconversion of Sucrose to High‐Value‐Added Glucaric Acid by In Vitro Metabolic Engineering

“…[24] Improvements to our system were achievedb yo ptimization of the reactionc onditions and enzyme concentrations, and by fed-batch addition of the ratelimiting enzyme (MIOX)at2 0a nd 40 h. This in vitro cascade reaction, which did not require ATP, is driven by exergonic reactions such as the CÀCc ycloisomerization catalyzed by MIPS and the dephosphorylation catalyzed by IMPase, which are accompanied by changes in DG 0 of À50.24 and À20.7 kJ mol À1 ,r espectively.S imilarly,i nt he last two steps of the cascade, catalyzed by MIOX and UDH, there are DG 0 values of À86.8 and À28.9 kJ mol À1 ,r espectively. The success of this ATP-free in vitro enzyme cascade enzyme system suggests that as imilara pproachm ight be used as an alternative to the biomanufacture of value-added products such as fructose 1,6-bisphosphate, [24] myo-inositol, [9a] a-ketoglutarate, [11] raffinose, and stachyose [16] from less costly and more abundant substrates than those used currently. The success of this ATP-free in vitro enzyme cascade enzyme system suggests that as imilara pproachm ight be used as an alternative to the biomanufacture of value-added products such as fructose 1,6-bisphosphate, [24] myo-inositol, [9a] a-ketoglutarate, [11] raffinose, and stachyose [16] from less costly and more abundant substrates than those used currently.…”

“…The effects of different pH values (5.5-8.5) were estimated at 30 8Cf or 12 h. The highest yield of GA was obtained at pH 7.5, so we chose this as the optimal pH value for subsequent work (Figure 3b). [11] Therefore, we used 2mm Mg 2 + and Fe 2 + in the optimized in vitro multi-enzyme system (Figure 3d). For reasonso fc ost, we chose 3mm NAD + (Figure 3c).…”

“…Futures tudies will investigate enzymei mmobilization techniques, [11] protein engineering strategies, [26] and database mining [27] to attempt to improve the stability,expression level, and activity of MIOX. To address this, we employed af ed-batch strategy of MIOX supplementation, which led to a1 .5-fold improvementi nt he yield of GA from sucrose (34.0 mm).…”

“…d-glucuronate, [11] and myo-inositol from xylose, starch, raw tomato, and amylose. [9,12] However, there are currently no reports on the production of GA through this approach.…”

Facile synthesis of (−)‐vibo‐quercitol from maltodextrin via an in vitro synthetic enzymatic biosystem

“…This type of biosystem can implement complicated biochemical reactions for manufacturing desired value-added products (Hodgman & Jewett, 2012;. To date, a variety of products, including organic acids (Ye et al, 2013), carbohydrates (You et al, 2013), electricity (Zhu & Zhang, 2017;Zhu et al, 2014), rare sugars (Wang et al, 2017), and biochemicals such as inositol (Beer et al, 2017;Jaturapaktrarak et al, 2014;Rieckenberg et al, 2014;, have been synthesized by several in vitro biosystems. To date, a variety of products, including organic acids (Ye et al, 2013), carbohydrates (You et al, 2013), electricity (Zhu & Zhang, 2017;Zhu et al, 2014), rare sugars (Wang et al, 2017), and biochemicals such as inositol (Beer et al, 2017;Jaturapaktrarak et al, 2014;Rieckenberg et al, 2014;, have been synthesized by several in vitro biosystems.…”