Salvador Peirú

Production of the Potent Antibacterial Polyketide Erythromycin C in Escherichia coli

ABSTRACT An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-l-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-α-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-d-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis.

Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.

TDP-L-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

Chemobiosynthesis of new antimalarial macrolides.

ABSTRACT We have synthesized new derivatives of the macrolide antibiotics erythromycin and azithromycin. Novel deoxysugar moieties were attached to these standard antibiotics by biotransformation using a heterologous host. The resulting compounds were tested against several standard laboratory and clinically isolated bacterial strains. In addition, they were also tested in vitro against standard and drug-resistant strains of human malaria parasites (Plasmodium falciparum) and the liver stages of the rodent malaria parasite (Plasmodium berghei). Antibacterial activity of modified erythromycin and azithromycin showed no improvement over the unmodified macrolides, but the modified compounds showed a 10-fold increase in effectiveness after a short-term exposure against blood stages of malaria. The new compounds also remained active against azithromycin-resistant strains of P. falciparum and inhibited growth of liver-stage parasites at concentrations similar to those used for primaquine. Our findings show that malaria parasites have two distinct responses to macrolide antibiotics, one reflecting the prokaryotic origin of the apicoplast and a second, as-yet uncharacterized response that we attribute to the eukaryotic nature of the parasite. This is the first report for macrolides that target two different functions in the Plasmodium parasites.

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.