SPABBATS: A pathway-discovery method based on Boolean satisfiability that facilitates the characterization of suppressor mutants

Abstract

Background: Several computational methods exist to suggest rational genetic interventions that improve the productivity of industrial strains. Nonetheless, these methods are less effective to predict possible genetic responses of the strain after the intervention. This problem requires a better understanding of potential alternative metabolic and regulatory pathways able to counteract the targeted intervention.

Results: Here we present SPABBATS, an algorithm based on Boolean satisfiability (SAT) that computes alternative metabolic pathways between input and output species in a reconstructed network. The pathways can be constructed iteratively in order of increasing complexity. SPABBATS allows the accumulation of intermediates in the pathways, which permits discovering pathways missed by most traditional pathway analysis methods. In addition, we provide a proof of concept experiment for the validity of the algorithm. We deleted the genes for the glutamate dehydrogenases of the Gram-positive bacterium Bacillus subtilis and isolated suppressor mutant strains able to grow on glutamate as single carbon source. Our SAT approach proposed candidate alternative pathways which were decisive to pinpoint the exact mutation of the suppressor strain.

Conclusions: SPABBATS is the first application of SAT techniques to metabolic problems. It is particularly useful for the characterization of metabolic suppressor mutants and can be used in a synthetic biology setting to design new pathways with specific input-output requirements.

Figure 1

Key reactions for glutamate biosynthesis and degradation in Bacillus subtilis. Glutamate is the universal amino group donor in all living cells and in that way links the carbon and nitrogen metabolisms. In B. subtilis the synthesis of glutamate depends on the glutamate synthase GltAB. In addition, the genome encodes two glutamate dehydrogenases, RocG and GudB, although the latter is inactive in the laboratory B. subtilis strain 168 (see text). The synthesis and degradation of glutamate are tightly regulated in response to the availability of carbon and nitrogen sources.

Figure 2

Predictions of alternative pathways for glutamate utilization based on SAT techniques. A B. subtilis strain (GP28) was constructed that lacks the glutamate dehydrogenases. An evolutionary adaptation resulted in a strain (GP717) that acquired the capacity to grow on glutamate as single carbon source. Using a SAT based search algorithm (see Materials and Methods) we predicted four alternative pathways that could be activate in the GP717. The genes coding for the enzymes in orange were analyzed further (see text). 4-m-2-o-p-ate = 4-methyl-2-oxo-pentanoate; 3-m-2-o-b-ate = 3-methyl-2-oxo-butanoate.

Figure 3

Comparison of gene expression patterns between mutant and parental strains, based on the predictions of the SPABBATS algorithm for pathway analysis. The predictions of the SPABBATS algorithm (see Figure 2) were further characterised by transcription analysis. The expression of the ald and bcd genes remains constant between the mutant (GP717) and parental (GP28) strains, suggesting that these genes are not involved in the newly activated catabolic pathway. In contrast, the expression of the ansAB operon is strongly increased in the mutant. This hints to a gain of function in the mutant strain that was analyzed further.

Figure 4

Requirement of the aspartase gene in the alternative pathway for glutamate utilization. The SPABBATS algorithm (see Figure 2) and the transcription analysis (see Figure 3) suggested that the overexpression of the asparaginase and aspartase genes (ansAB) is the cause for the metabolic gain of function of the mutant strain GP717. To prove this, the ansAB operon was deleted in the GP717 strain. The resulting strain GP1154 lost the capacity to utilize glutamate as single carbon source. This strongly indicates that the induction of the aspartase gene is required and sufficient for the newly activated catabolic pathway. CE = Minimal medium containing 0.5% glutamate, CE-Glc = CE medium with an addition 0.5% glucose.