Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems

AbstractTo harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.

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GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100751118 ◽

2021 ◽

Vol 118 (26) ◽

pp. e2100751118 ◽

Cited By ~ 1

Author(s):

Aleksandra Nivina ◽

Sur Herrera Paredes ◽

Hunter B. Fraser ◽

Chaitan Khosla

Keyword(s):

Molecular Mechanisms ◽

Assembly Line ◽

Gene Families ◽

Gene Clusters ◽

Nucleotide Composition ◽

Polyketide Synthases ◽

Bacterial Genomes ◽

Biosynthetic Gene Clusters ◽

Bacterial Phyla ◽

Genetic Elements

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces. While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

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Engineering strategies for rational polyketide synthase design

Natural Product Reports ◽

10.1039/c8np00030a ◽

2018 ◽

Vol 35 (10) ◽

pp. 1070-1081 ◽

Cited By ~ 47

Author(s):

Maja Klaus ◽

Martin Grininger

Keyword(s):

Protein Interactions ◽

Assembly Line ◽

Polyketide Synthase ◽

Polyketide Synthases ◽

Protein Protein Interactions ◽

Important Protein ◽

Line P

In this review, we highlight strategies in engineering polyketide synthases (PKSs). We focus on important protein–protein interactions that constitute an intact PKS assembly line.

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Caerulomycin and Collismycin Antibiotics Share a trans Flavin-Dependent Assembly Line for 2,2’-Bipyridine Formation and Sulfur Fate Differentiation

10.1101/2020.12.07.415471 ◽

2020 ◽

Author(s):

Bo Pang ◽

Rijing Liao ◽

Zhijun Tang ◽

Shengjie Guo ◽

Zhuhua Wu ◽

...

Keyword(s):

Amino Acid ◽

Assembly Line ◽

Bond Formation ◽

Sulfhydryl Group ◽

Polyketide Synthases ◽

Differential Treatment ◽

Nonribosomal Peptides ◽

Peptide Synthetases ◽

In Trans ◽

Hybrid Antibiotics

ABSTRACTLinear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids though assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges the understanding in the diverse polymerization processes of amino acid and carboxylate monomers programmed by various NRPSs and PKSs in nature. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an unusual assembly line that involves NRPS activity to recruit a flavoprotein acting in trans and catalyze C-C bond formation and heterocyclization during 2,2’-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2’-bipyridine intermediates through differential treatment of the sulfhydryl group arising from L-cysteine incorporation. Subsequent L-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2’-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.

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Strategies in megasynthase engineering – fatty acid synthases (FAS) as model proteins

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.13.119 ◽

2017 ◽

Vol 13 ◽

pp. 1204-1211 ◽

Cited By ~ 8

Author(s):

Manuel Fischer ◽

Martin Grininger

Keyword(s):

Fatty Acid ◽

Bioactive Compounds ◽

Assembly Line ◽

Structural Information ◽

Natural Compounds ◽

Polyketide Synthases ◽

Therapeutic Applications ◽

Rational Engineering ◽

Fatty Acid Synthases

Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the “assembly-line synthetic concept” of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.

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Faculty Opinions recommendation of Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718232065.793489554 ◽

2014 ◽

Author(s):

Ikuro Abe ◽

Lihan Zhang

Keyword(s):

Rational Design ◽

Assembly Line ◽

Polyketide Synthases

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Rational Design of Modular Polyketide Synthases: Morphing the Aureothin Pathway into a Luteoreticulin Assembly Line

Angewandte Chemie International Edition ◽

10.1002/anie.201308176 ◽

2014 ◽

Vol 53 (6) ◽

pp. 1560-1564 ◽

Cited By ~ 31

Author(s):

Yuki Sugimoto ◽

Ling Ding ◽

Keishi Ishida ◽

Christian Hertweck

Keyword(s):

Rational Design ◽

Assembly Line ◽

Polyketide Synthases

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Unraveling the iterative type I polyketide synthases hidden in Streptomyces

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917664117 ◽

2020 ◽

Vol 117 (15) ◽

pp. 8449-8454 ◽

Cited By ~ 3

Author(s):

Bin Wang ◽

Fang Guo ◽

Chunshuai Huang ◽

Huimin Zhao

Keyword(s):

Assembly Line ◽

Gene Clusters ◽

Polyketide Synthases ◽

Type I ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Computational Tools ◽

End Products ◽

Fungal Natural Products ◽

Biased Distribution

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces. Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs) were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides.

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