Multiple pathways for acetate assimilation in Streptomyces cinnamonensis

Abstract

In most bacteria acetate assimilation is accomplished via the glyoxylate pathway. Isocitrate lyase (ICL) and malate synthase (MS) are two key enzymes of this pathway, which results in the net generation of one molecule of succinyl-CoA from two acetyl-CoA molecules. Genetic and biochemical data have shown that genes encoding these key enzymes are present in streptomycetes, yet there has been no clear demonstration of the importance of these genes to acetate assimilation. In fact, for Streptomyces collinus an alternative butyryl-CoA pathway has been shown to be critical for growth on acetate as a sole carbon source. Crotonyl-CoA reductase (CCR) is a key enzyme in this pathway and catalyzes the last step of the conversion of 2-acetyl-CoA molecules to butyryl-CoA. In Streptomyces cinnamonensis C730.1, it has been shown that CCR and this butyryl-CoA pathway provide the majority of methylmalonyl-CoA and ethylmalonyl-CoA for monensin A biosynthesis in an oil-based fermentation medium. We have cloned a MS homologue gene from this strain. Reverse transcription and direct enzyme assays demonstrated that neither this nor other MS genes were expressed during fermentation in an oil-based fermentation of either the C730.1 or L1 strain (a ccr mutant). Similarly, no ICL activity could be detected. The C730.1 but not the L1 strain was able to grow on acetate as a sole carbon source. The Streptomyces coelicolor aceA and aceB2 genes encoding ICL and MS were cloned into a Streptomyces expression plasmid (a derivative of pSET152) to create pExIM1. Enzyme assays and transcript analyses demonstrated expression of both of these proteins in C730.1/pExIM1 and L1/pExIM1 grown in an oil-based fermentation and tryptic soy broth media. Nonetheless, L1/pExIM1, like L1, was unable to grow on acetate as a sole carbon source, and was unable to efficiently generate precursors for monensin A biosynthesis in an oil-based fermentation, indicating that the additional presence of these two enzyme activities does not permit a functional glyoxylate cycle to occur. UV mutagenesis of S. cinnamonensis L1 and L1/pExIM1 led to mutants which were able to grow efficiently on acetate despite a block in the butyryl-CoA pathway. Analysis of enzyme activity and monensin production from these mutants in an oil-based fermentation demonstrated that neither the glyoxylate cycle nor the butyryl-CoA pathway function, suggesting the possibility of alternative pathways of acetate assimilation.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S -adenosyl- l -methionine

“…The residual biosynthesis of salinosporamide B from ethylmalonyl-CoA in the housekeeping CCR (Strop_3612) deletion mutant may derive from SalG-catalyzed reductive carboxylation of crotonyl-CoA. Alternatively, an orthogonal pathway for acetate assimilation may function in S. tropica (7,20) as suspected in Streptomyces cinnamonensis in which genetic inactivation of its CCR likewise resulted in the reduced production of the ethylmalonyl-CoA-derived polyketide monensin A rather than abolishing it completely (32).…”

“…Biosynthesis of ethylmalonylCoA as a precursor of salinosporamide B is shown for comparison ( Fig. 3B) and is not encoded in the sal locus but constitutes rather a primary metabolic pathway for acetate assimilation and a source of building blocks for secondary metabolite production (19,20).…”

IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

“…Butyrivibrio fibrisolvens and Streptomyces collinus), acetyl-CoA is converted to butyryl-CoA via four reactions, involving acetyl-CoA acetyltransferase (thiolase), 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase (crotonase), and butyryl-CoA dehydrogenase (33)(34)(35).…”

“…Fuchs and co-workers (17) noted that in some organisms the enzymes from the first half of the cycle are missing and proposed that in this situation reversal of the second half of the pathway might be important for acetyl-CoA assimilation into succinyl-CoA. Interestingly, the first three reactions of this reverse sequence (acetoacetyl-CoA ␤-ketothiolase, 3-hydroxybutyryl-CoA dehydrogenase, and crotonyl-CoA hydratase) are identical to those in the acetyl-CoA assimilation pathway described for Streptomyces collinus, which converts acetyl-CoA to crotonyl-CoA (33). The latter, via the action of crotonyl-CoA reductase, is converted to butyryl-CoA, which is isomerized to isobutyrylCoA by the action of a stand-alone ICM.…”

AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes

“…In previous work we used a proteomic and genetic approach to identify and characterize the enzymes involved in allantoin metabolism in S. coelicolor (12). Upstream of allB and alc genes, which encode the first two enzymes of the pathway, allantoinase and allantoicase, respectively, we found the gene sco6246, which had previously been predicted to be the transcriptional regulator of the glyoxylate shunt (IcIR) (27) (Fig. 1A).…”