RESUMO
Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.
Assuntos
Melhoramento Genético/métodos , Macrolídeos/metabolismo , Malonil Coenzima A/biossíntese , Metaboloma/fisiologia , Engenharia de Proteínas/métodos , Streptomyces/fisiologia , Malonil Coenzima A/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Bacteria stringently regulate the synthesis of their membrane phospholipids, but the responsible regulatory mechanisms are incompletely understood. Bacillus subtilis FabF, the target of the mycotoxin cerulenin, catalyses the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. Here we show that B. subtilis strains containing the fabF1 allele, which codes for the cerulenin-insensitive protein FabF[I108F], overexpressed several genes involved in fatty acid and phospholipid biosynthesis (the fap regulon) and had significantly elevated levels of malonyl-CoA. These results pinpointed FabF[I108F] as responsible for the increased malonyl-CoA production, which in turn acts as an inducer of the fap regulon by impairing the binding of the FapR repressor to its DNA targets. Synthesis of acyl-ACPs by a cell-free fatty acid system prepared from fabF1 cells showed the accumulation of short- and medium-chain acyl-ACPs. These results indicate that the acyl-ACP chain length acceptance of FabF[I108F] is biased towards shorter acyl-ACPs. We also provide evidence that upregulation of FabF[I108F] is essential for survival and for resistance to cerulenin of fabF1 cells. These findings indicate that malonyl-CoA is a key molecule to monitor lipid metabolism functioning and trigger appropriate genetic and biochemical adjustments to relieve dysfunctions of this essential metabolic pathway.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Bacillus subtilis/enzimologia , Regulação Bacteriana da Expressão Gênica , Metabolismo dos Lipídeos/genética , Malonil Coenzima A/genética , Proteínas Repressoras/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/efeitos dos fármacos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Bacillus subtilis/genética , Cerulenina/farmacologia , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Malonil Coenzima A/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Regulon , Proteínas Repressoras/genéticaRESUMO
Malonyl-CoA is an essential intermediate in fatty acid synthesis in all living cells. Here we demonstrate a new role for this molecule as a global regulator of lipid homeostasis in Gram-positive bacteria. Using in vitro transcription and binding studies, we demonstrate that malonyl-CoA is a direct and specific inducer of Bacillus subtilis FapR, a conserved transcriptional repressor that regulates the expression of several genes involved in bacterial fatty acid and phospholipid synthesis. The crystal structure of the effector-binding domain of FapR reveals a homodimeric protein with a thioesterase-like 'hot-dog' fold. Binding of malonyl-CoA promotes a disorder-to-order transition, which transforms an open ligand-binding groove into a long tunnel occupied by the effector molecule in the complex. This ligand-induced modification propagates to the helix-turn-helix motifs, impairing their productive association for DNA binding. Structure-based mutations that disrupt the FapR-malonyl-CoA interaction prevent DNA-binding regulation and result in a lethal phenotype in B. subtilis, suggesting this homeostatic signaling pathway as a promising target for novel chemotherapeutic agents against Gram-positive pathogens.