Investigating the initial steps of auricin biosynthesis using synthetic biology

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Abstract

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with d-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and β (KSα, KSβ). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-023-01591-2.

Key points

A new synthetic biology-based system for investigating antibiotic biosynthesis.
Efficient rabelomycin production using the initial landomycin biosynthetic genes.
Linking of two BGCs in the initial steps of auricin biosynthesis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-023-01591-2.

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Most cited references 43

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A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.

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Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Dae-Kyun Ro, Eric M Paradise, Mario Ouellet … (2006)

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

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P. K. Ajikumar, W-H Xiao, K. Tyo … (2010)

Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene--the first committed Taxol intermediate--approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid-forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.

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Author and article information

Contributors

Jan Kormanec:

ORCID: http://orcid.org/0000-0003-0377-6413

jan.kormanec@savba.sk

Journal

Journal ID (nlm-ta): AMB Express

Journal ID (iso-abbrev): AMB Express

Title: AMB Express

Publisher: Springer Berlin Heidelberg (Berlin/Heidelberg )

ISSN (Electronic): 2191-0855

Publication date (Electronic): 8 August 2023

Publication date PMC-release: 8 August 2023

Publication date Collection: 2023

Volume: 13

Electronic Location Identifier: 83

Affiliations

[1 ]GRID grid.419303.c, ISNI 0000 0001 2180 9405, Institute of Molecular Biology, , Slovak Academy of Sciences, ; Dubravska Cesta 21, 845 51 Bratislava, Slovak Republic

[2 ]GRID grid.419303.c, ISNI 0000 0001 2180 9405, Institute of Chemistry, , Slovak Academy of Sciences, ; 845 38 Bratislava, Slovak Republic

Author information

Jan Kormanec http://orcid.org/0000-0003-0377-6413

Article

Publisher ID: 1591

DOI: 10.1186/s13568-023-01591-2

PMC ID: 10409956

PubMed ID: 37552435

SO-VID: f1180ea5-6c19-4dcb-b2a2-57e8a0341021

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 25 July 2023

Date accepted : 2 August 2023

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100005357, Agentúra na Podporu Výskumu a Vývoja;

Award ID: APVV-19-0009

Award Recipient : Jan Kormanec

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: antibiotics,auricin,biosynthetic gene cluster,griseusin,synthetic biology,streptomyces

Data availability:

ScienceOpen disciplines: Biotechnology

Keywords: antibiotics, auricin, biosynthetic gene cluster, griseusin, synthetic biology, streptomyces

Investigating the initial steps of auricin biosynthesis using synthetic biology

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Most cited references 43

The neighbor-joining method: a new method for reconstructing phylogenetic trees.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli.

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