Interplay between lipid metabolism and polyketide production in Streptomyces coelicolor.

COMBA S., A. ARABOLAZA AND GRAMAJO H.

Saint Felliu de Guixols

Conferencia; ESF Synthetic Biology of Antibiotics production; 2011

ESF-EMBO

Polyketides comprise one of the major families of natural bioactive products, which include a large number of medically important compounds. They are typically assembled by condensation of several units of short-chain acil-CoA molecules. Streptomyces are widely known as polyketides producers. In these organisms particularly, malonyl-CoA has a pivotal role: It is the precursor of fatty acids and phospholipids necessary for cell growth, but it is also involved in the biosynthesis of both secondary metabolites and triglyceride (TAG) storage lipid. According to this, reducing TAG content in the model bacteria Streptomyces coelicolor, by mutating key genes of its biosynthetic pathway does increase the production of the polyketide actinorhodin (Act), suggesting that TAG actually competes with Act for same substrates. In this sense, the carbon flux of malonyl-CoA from primary metabolism to secondary metabolism and the availability of this molecule is a key factor that determines the yield of polyketide antibiotics production. Here we describe the design and construction of a S. coelicolor host strain suitable for the production of malonyl-CoA based compounds. To achieve this end, we engineered the cell by deleting genes of competitive pathways and inserting novel enzymatic activities, thus redirecting the malonyl-CoA flux to the desired product. We employed the CH999 strain (act cluster deficient mutant), a widely used host for in vivo production of engineered natural products; and a mutant strain deficient in TAG biosynthesis. In these genetic background, we also overexpressed the S. coelicolor  Acetyl-CoA Carboxylase (ACC) complex. As a straight proof of concept we overproduce free fatty acid (a malonyl-CoA demanding product) in this recombinant microorganism by expressing the thioesterase tesA from Escherichia coli. These expressions were achieved using ad hoc customized vectors that consist in modular-combinable DNA features with BioBricks format. Using this approach, we constructed several plasmids carrying the genes coding for the target enzymes under different endogenous Streptomyces promoters, which are transcriptionally active in late exponential or stationary phase of growth. Thus, were able to exert a temporal control of genes expression in order to minimize the interference with cell chassis and optimize the timing of gene induction accordingly to the metabolic state of the cell. Then, we evaluate the production of fatty acid as an indicator of malonyl-CoA availability.