A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor

Abstract

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15–20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.

Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK

“…With the exception of the tetracenomycins and tiancimycins, all members within this series contained the key β-hydroxy quinone signature of the native substrate CAR ( 1 ). The simplest among these were the substituted anthraquinones alizarin ( 2 , 13%), purpurin ( 3 , 13%), and 9,10-seco-7-deoxynogalamycinone ( 4 , 91%) . Anthraquinones represent a structurally and functionally diverse class of pharmacophores in which subtle structural changes can notably alter functional outcomes. − Comparative analysis of the anthraquinone set revealed CAR-like DnrK β-hydroxy regioselectivity in 4 .…”

“…The CAR-like tetracenes methylated by DnrK included aranciamycin ( 7 , 97%; Figure S1), 7-deoxyaranciamycin ( 8 , 96%; also known as SM 173B), steffimycin B ( 9 , 15%), and various nogalamycinones [nogalavinone ( 10 , 76%), auramycinone ( 11 , 96%), 7-deoxynogalamycinone ( 12 , 62%), and deoxyauramycinone ( 13 , 20%), respectively]. , Compared to CAR ( 1 ), the aranciamycin D ring has a C8 methoxy and differs slightly in the C7 glycosyl and C9 aliphatic substitutions. DnrK turnover of 7 and the C7-deoxy analogue of its aglycon ( 8 , lacking both C7-OH and its appended 2- O -methyl- l -rhamnose) was quantitative, and the corresponding DnrK aranciamycin product confirmed as 4- O -methoxyarancinamycin ( 7a ) via product isolation and structure elucidation (Figures S2 and S3).…”

Rethinking Biosynthesis of Aclacinomycin A

“…In an effort to advance aklavinone biosynthesis, a group from Stanford University leveraged bimodular PKSs to produce hexaketides and octaketides [ 130 ]. Recently, by optimizing promoters, enzymes, and chassis cells, a recombinant Streptomyces strain was engineered, which produced 15–20 mg/L aklavinone [ 131 ]. More importantly, a total of 37 genomes were shown to harbor the gene clusters for the biosynthesis of aclacinomycin, indicating the feasibility of screening strains for the high-level production of ACM-A [ 132 , 133 ].…”

Diverse Combinatorial Biosynthesis Strategies for C–H Functionalization of Anthracyclinones

“…3 Previously, we developed a BioBricks platform for the improved biosynthesis of anthracyclinones. 6 We developed vectors to produce four anthracyclinone scaffolds: aklavinone (1), 9-epiaklavinone (2), auramycinone (3), and nogalamycinone (4) (Figure 1A). In addition, endogenous activity from the host strain Streptomyces coelicolor M1152ΔmatAB led to the production of 7-deoxygenated versions of these compounds, including 7-deoxy-aklavinone (5), 7-deoxy-9-epi-aklavinone (6), 7-deoxy-auramycinone (7), and 7-deoxy-nogalamycinone (8) (Figure 1B).…”

“…Further reactions by methyltransferase (MET), fourth-ring cyclase (4-CYC), and ketoreductase (7-KR) enzymes furnish the core tetracyclic aromatic carbon skeletons (Figure A) . Previously, we developed a BioBricks platform for the improved biosynthesis of anthracyclinones . We developed vectors to produce four anthracyclinone scaffolds: aklavinone ( 1 ), 9- epi -aklavinone ( 2 ), auramycinone ( 3 ), and nogalamycinone ( 4 ) (Figure A).…”