Protein design and engineering of a de novo pathway for microbial production of 1,3‐propanediol from glucose

Abstract

Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products.

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

“…The genes of SerC R42W/R77W and SerC WT were amplified from the constructed vector pET28a‐SerC R42W/R77W and pET28a‐SerC WT , respectively. Linearized vectors containing the genes of PDC and YqhD were achieved using PCR and the plasmid pZA‐GDH‐PDC‐YqhD as templates with primers having 15 bp extensions homologous at its ends. The PCR‐generated fragment and linearized vector were set up in an In‐Fusion cloning reaction using the In‐Fusion HD Cloning Kits (Clontech; TaKara Bio USA Inc., USA).…”

Discovery of a Potentially New Subfamily of ELFV Dehydrogenases Effective for l‐Arginine Deamination by Enzyme Mining

“…20 canonical amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine) and two non‐proteinogenic amino acids (ornithine and citrulline) were used as substrates to detect the oxidative deamination activities of the enzymes. Either NAD or NADP was used as electronic acceptor during the enzyme assay and the formation of NAD(P)H was measured by monitoring the absorbance change at 340 nm as described before …”

“…The advantage of in silico approach is that new enzymes can be discovered very quickly. This is especially important for constructing de novo pathways which often include several novel bioreactions without natural counterparts …”

“…Enzymes with new functions can be identified if they show different conservation pattern of specificity determined motifs (residues) with known enzyme subfamily. In this study, we use amino acid dehydrogenase as an example to discover new enzymes which can catalyze the deamination of positively charged amino acid l ‐arginine to its corresponding keto acid (Figure B) – a rare reaction in amino acid dehydrogenase family …”

“…They have been widely used for the synthesis of natural and non‐natural amino acids and also as diagnostic reagents. Recently, amino acid dehydrogenase has also been applied for the production of novel alcohols and diols . The ELFV dehydrogenase is one of the largest superfamily of amino acid dehydrogenase, including four known subfamilies, namely glutamate, leucine, phenylalanine, and valine dehydrogenases.…”