RESUMO
Pikromycin is an important precursor of drugs, for example, erythromycin. Hence, systems metabolic engineering for the enhanced pikromycin production can contribute to the development of pikromycin-related drugs. In this study, metabolic genes in Streptomyces venezuelae were systematically engineered for enhanced pikromycin production. For this, a genome-scale metabolic model of S. venezuelae was reconstructed and simulated, which led to the selection of 11 metabolic gene targets. These metabolic genes, including four overexpression targets and seven knockdown targets, were individually engineered first. Next, two overexpression targets and two knockdown targets were selected based on the 11 strains' production performances to engineer two to four of these genes together for the potential synergistic effects on the pikromycin production. As a result, the NM1 strain with AQF52_RS24510 (methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase) overexpression and AQF52_RS30320 (sulfite reductase) knockdown showed the best production performance among all the 22 strains constructed in this study. Fed-batch fermentation of the NM1 strain produced 295.25 mg/L of pikromycin, by far the best production titer using the native producer S. venezuelae, to the best of our knowledge. The systems metabolic engineering strategy demonstrated herein can also be applied to the overproduction of other secondary metabolites using S. venezuelae.
Assuntos
Engenharia Metabólica , Streptomyces , Macrolídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Branched chain amino acids (BCAA) are catabolized into various acyl-CoA compounds, which are key precursors used in polyketide productions. Because of that, BCAA catabolism needs fine tuning of flux balances for enhancing the production of polyketide antibiotics. To enhance BCAA catabolism for pikromycin production in Streptomyces venezuelae ATCC 15439, three key enzymes of BCAA catabolism, 3-ketoacyl acyl carrier protein synthase III, acyl-CoA dehydrogenase, and branched chain α-keto acid dehydrogenase (BCDH) were manipulated. BCDH overexpression in the wild type strain resulted in 1.3 fold increase in pikromycin production compared to that of WT, resulting in total 25 mg/L of pikromycin. To further increase pikromycin production, methylmalonyl-CoA mutase linked to succinyl-CoA production was overexpressed along with BCDH. Overexpression of the two enzymes resulted in the highest titer of total macrolide production of 43 mg/L, which was about 2.2 fold increase compared to that of the WT. However, it accumulated and produced dehydroxylated forms of pikromycin and methymycin, including their derivatives as well. It indicated that activities of pikC, P450 monooxygenase, newly became a bottleneck in pikromycin synthesis.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Antibacterianos/biossíntese , Macrolídeos/metabolismo , Streptomyces/metabolismo , Hidroxilação , Metabolismo SecundárioRESUMO
Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds.