Figure 1.
The Reverse Pathway Engineering (RPE) approach combines chemoinformatics and bioinformatics analyses.
The approach enables a flexible input of target compounds and reaction databases, and can result in an output for various analyses or applications. Using 3-methylbutanoic acid as an input compound, two of the proposed synthetic reactions are shown as an example. The complete retrosynthesis trees can be found in Figure 4.
Figure 2.
Leucine catabolism network in LAB, including the inter-conversion pathways between leucine degradation and valine catabolism (framed by blue box), and between leucine degradation and isoleucine catabolism (framed by green box).
Three branches of the subsequent degradation of alpha-keto isocaproate (KICA) are indicated: i) conversion to the corresponding aldehyde, alcohol or carboxylic acid via alpha-keto acid decarboxylation (depicted in black) or ii) the oxidative decarboxylation (depicted in blue) or iii) an alternative route resulting in α-hydroxy-isocaproate (HICA), as depicted in gold. The flavor compounds used as input for RPE approach are indicated in italics. The novel predicted reactions are indicated by red dashed arrows. Enzymes names are: BcAT, branched-chain aminotransferase; GDH, glutamate dehydrogenase; HycDH, hydroxyacid dehydrogenase; KdcA, alpha-ketoacid decarboxylase; AlcDH, alcohol dehydrogenase; AldDH, aldehyde dehydrogenase; EstA, esterase A; KaDH, alpha-ketoacid dehydrogenase complex; PTA, phosphotransacylase; ACK, acyl kinase.
Figure 3.
Leucine to 3-methylbutanol route as proposed by RPE.
The retrosynthesis tree was obtained from BioPath.Design using 3-methylbutanol as input.
Figure 4.
3-methylbutanoic acid synthesis routes from alpha-keto isocaproate as proposed by RPE.
The retrosynthesis trees correspond to the pathways shown in Figure 2. (a) Proposed route for 3-methylbutanal to 3-methylbutanoic acid. (b) Proposed oxidative decarboxylation route converting alpha-keto isocaproate (KICA) into 3-methylbutanoic acid. (c) Proposed route converting KICA into 3-methylbutanoic acid via alpha-hydroxy-isocaproate (HICA). A novel reaction converting HICA into 3-methylbutanoic acid by lactate 2-monooxygenase was proposed (step 2).
Figure 5.
The suggested reaction converting alpha-hydroxy-isocaproate (HICA) to 3-methylbutanoic acid (adapted from BioPath.Design).
The suggested reaction is shown in the upper part. One of the reference reactions is indicated together with the information on the enzyme which catalyzes it.
Figure 6.
Bootstrapped (n = 1000) neighbor-joining tree of the LOX homologs from LAB.
The functional equivalents (orthologs) of LOX from Aerococcus viridans are highlighted by the pink frame. Genome abbreviations and GI codes (NCBI accession codes) of the homologs are in parentheses. Different colors represent different orthologous groups. Diverse annotations have been assigned to the protein members in the lower groups, such as lactate dehydrogenase, isopentenyl pyrophosphate isomerase, and inosine-5-monophosphate dehydrogenase. Red squares indicate duplication events, and green dots represent speciation events.
Figure 7.
The predicted chemical reaction converts alpha-keto isocaproate (KICA) to 2-methylpropanal, connecting the leucine degradation and the valine degradation pathways.
The reference reaction converting alpha-keto-γ-methylthiobutyrate to methylsulfanyl-acetaldehyde was derived from the additional part of the BioPath.Database containing the reactions of the flavor-forming pathways from sulfur-containing amino acid degradation.