A potential modification of the production of the essential amino acid: L‐threonine

Abstract

L‐threonine is one of the 9 essential amino acids not produced by mammals. Relying on their diet to acquire it, the food industry as well as the pharmaceutical industry have been trying to efficiently mass produce L‐threonine. Currently, L‐threonine is produced through fermentation of E. coli, which also produces endotoxins that require additional costly purification steps to remove. C. glutamicum, a micro‐organism Generally Regarded As Safe (GRAS), could alternatively be used to produce L‐threonine since it is used to produce other essential amino acids. However, feedback inhibition of the L‐threonine biosynthetic pathway limits the yield of this amino acid from fermentation. One step of the L‐threonine biosynthetic pathway requires phosphorylation of Homoserine by the kinase ThrB. However, as the concentration of L‐threonine in the cell increases, it imparts competitive inhibition on the kinase activity of ThrB and thereby decreases the flux through this biosynthetic pathway. To avoid this negative feedback on threonine biosynthesis in C. glutamicum, we have engineered and characterized variants of ThrB. The studies focus on the conserved SSAN motif in ThrB that interacts with both enzyme substrates, Homoserine and L‐threonine, where the Alanine residue at position 20 was mutated to either Valine, Leucine, Serine or Glycine. Of those four mutants, only the A20G variant retained wild‐type levels of enzymatic activity. The ThrB‐A20G variant showed a 5.4‐fold decrease in feedback inhibition by L‐threonine. X‐ray crystal structures of CglThrB‐WT were obtained showing an overall conserved structure when compared to its homologues. The main differences residing in two loops: one interacting with the adenosine moiety of ATP, the other interacting with the magnesium necessary for stabilizing of the phosphate moieties of ATP. An apo structure solved at 1.8 Å resolution featured proteolysis of two seemingly highly dynamic α‐helices capping the active site. Inspecting the active site of the E. coli homologue in complex with phosphoaminophosphonic acid‐adenylate ester (AMP‐PNP) and Homoserine (1H72), two arginine side chains interact with the carboxylate group of Homoserine while a conserved threonine side chain interacts with the β‐phosphate of AMP‐PNP. Interestingly, these three residues are conserved in the sequence of most Homoserine kinases. The obtained apo structures of CglThrB‐WT shed light on the dynamics of the enzyme suggesting that the active site needs to be dynamic to promote binding of the substrates. Hence, the α‐helix featuring the Arg194 and Thr190 residues required for substrate interactions is highly disordered in apo structures resulting in Arg194 and Thr190 interacting with solvent. Further investigation of the role of Arg194 could potentially result in decrease dynamicity of that specific region promoting crystal formation of CglThrB‐WT in complex with Homoserine or L‐threonine and decreasing feedback inhibition by L‐threonine. Mutation of Thr190, the residue directly interacting with the hydroxyl on the side chain of L‐threonine, could also result in decreased feedback inhibition.