Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Abstract

Enantiopure L-glyceraldehyde-3-phosphate (L-GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L-glyceraldehyde (L-GA) by ATP is used for the synthesis of L-GAP. L-GAP has a half-life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca , but not by L-GAP and inorganic orthophosphate. Moreover, equal amount of Ca exerts a different degree of inhibition relative to the activity without the addition of Ca depending on the Mg to ATP molar ratio. If the Mg to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred-tank reactor is the most efficient process for the synthesis of L-GAP.

Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites

“…In addition, the biocatalytic methods of synthesis and their natural scope are of much interest for exploring and widening the capabilities of metabolic enzymes. (2 S )-glyceraldehyde 3-phosphate has been synthesized by biocatalytic phosphorylation of (2 S )-glyceraldehyde (see Figure 17 ) using glycerol kinase [ 116 , 117 ], while preparing (2 R )-glyceraldehyde 3-phosphate required the enantiocomplementary dihydroxyacetone kinase for catalyzing the phosphorylation of (2 R )-glyceraldehyde [ 118 ]. The synthesis of an unnatural analogue substituted with a triple bond at the C2-position, 2-ethynyl-(2 R )-glyceraldehyde 3-phosphate, a key islatravir intermediate, was achieved by phosphorylating 2-ethynyl-(2 R )-glyceraldehyde (see Figure 17 ), with complete conversion at 0.2 M concentration, using a kinase which after the initial discovery of a low activity pantothenate kinase from E. coli , directed evolution and further engineering of the enzyme was obtained as a kinase variant with 100-fold better activity [ 119 ].…”

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…[76] By optimizing reaction conditions for the phosphorylations, >90% conversions have been achieved for seven substrates. [76] While L-glyceraldehyde-3-phosphate can be synthesized by asymmetric phosphorylation of glyceraldehyde catalyzed by glycerol kinase, [77,78] the central metabolite D-glyceraldehyde-3-phosphate can be formed by an enantioselective dihydroxyacetone kinasecatalyzed 3-phosphorylation of D-glyceraldehyde. [79] An evolved pantothenate kinase, together with the acetylphosphate/acetate kinase system for ATP regeneration (Figure 5), enabled selective phosphorylation of unnatural (R)-2-ethynyl-glyceraldehyde to (R)-2ethynylglyceraldehyde 3-phosphate, which was a key intermediate in the biocatalytic synthesis of islatravir.…”

Horizons of Systems Biocatalysis and Renaissance of Metabolite Synthesis

“…The intensification of a biocatalytic process for a selected route and the level of integration depends on the limiting step in the process design and needs to consider its debottlenecking (Figure ) with the development of the optimum reaction conditions with high substrate concentrations and low enzyme concentrations, the scaling concept, the type of reactor and subsequent operations for the optimum performance of a robust and stable overall process with high space‐time yield . Analysing the reaction kinetics, enzyme inhibition, instable products and other process bottlenecks has been useful for selecting the most suitable reaction conditions and reactor type, as shown in the case of the synthesis of glyceraldehyde‐3‐phosphates . The common inhibition of enzymatic reactions by substrates, products or byproducts can be overcome by a number of different approaches, such as using solid adsorbents for substrate and product inhibition, protecting reactive aldehyde groups as dimethylacetals or utilizing an enzymatic reaction for removing an inhibiting byproduct .…”