Short-chain ketone production by engineered polyketide synthases in Streptomyces albus

Yuzawa, Satoshi; Mirsiaghi, Mona; Jocic, Renee; Fujii, Tatsuya; Masson, Fabrice; Benites, Veronica T.; Baidoo, Edward E. K.; Sundström, Eric; Tanjore, Deepti; Pray, Todd; George, Anthe; Davis, Ryan W.; Gladden, John M.; Simmons, Blake A.; Katz, Leonard; Keasling, Jay D.

doi:10.1038/s41467-018-07040-0

Cited by 56 publications

(53 citation statements)

References 32 publications

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“…albus production runs Engineered S. albus spores were grown in 12 mL of TSB medium containing nalidixic acid (50 µg/mL), apramycin (50 µg/mL) and spectinomycin (200 µg/mL) for 4-5 days at 30°C. Three mL of the overnight culture was used to seed 30 mL of 10% media 042 and 90% plant hydrolysate (Yuzawa et al, 2018a), supplemented with 2.4 grams/liter of valine and nalidixic acid (50 µg/mL), which was grown for 10 days at 30°C. For production runs of lactones, an overlay of 4 mL of dodecane was added to retain the product.…”

Section: Production Runsmentioning

confidence: 99%

“…Previously, our group has engineered three major PKS elements in the first module of lipomycin: 1) an inserted TE to produce 3-hydroxy acids (Yuzawa et al, 2017a), 2) a KR knockout and AT domain swap to produce short chain ketones (Yuzawa et al, 2018a(Yuzawa et al, , 2017b, and 3) reductive loop (RL) exchanges to produce saturated, short chain carboxylic acids (Zargar et al, 2019). The design space expands considerably with multiple module systems, and in this work, we build on our single module platform by combining multiple PKS manipulations (KR knockouts, reductive loop (RL) swaps, AT swaps, fused TE) in a biomodular system to produce novel biomolecules, namely biofuels and specialty chemicals.…”

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confidence: 99%

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A bimodular PKS platform that expands the biological design space

Zargar

Valencia

Wang

et al. 2020

Preprint

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Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges.Collectively, our work expands the biological design space and moves the field closer to the production of "designer" biomolecules. Highlights• Engineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable bio-based chemicals • A reductive loop swap and ketoreductase knockout used to produce 20 mg/mL of a novel ethyl ketone, a gasoline replacement • An acyltransferase swap and reductive loop swap successfully produced δ-lactone, a potential fragrant compound • Incomplete reduction and premature hydrolysis observed in engineered modules

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Section: Production Runsmentioning

confidence: 99%

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confidence: 99%

A bimodular PKS platform that expands the biological design space

Zargar

Valencia

Wang

et al. 2020

Preprint

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“…Finally, it is important to consider if the target molecule can be produced in a more practical or economically favorable means through other biosynthetic pathways. While PKSs have recently been engineered to produce short-chain ketones, potential gasoline blending agents, from plant biomass at titers of 1 g/L [12], fatty acid and isoprenoid synthesis are currently more established production pathways with higher yields [13]. This holistic economic analysis of the target molecule can guide the extent of engineering efforts required for production.…”

Section: Pks Designmentioning

confidence: 99%

“…In this process, carbon source, nitrogen source, phosphate, and other nutrients are carefully adjusted via a one-factor-at-a-time method or statistical method [71]. In the case of short chain ketone production by an engineered PKS in S. albus, when the carbon source was supplemented by the plant-biomass hydrolysates the product titer increased more than four-fold and further titer increases were achieved by feeding specific acyl-CoA precursors [12]. Aside from traditional methods, recently developed machine learning algorithms have great potential to accelerate the media optimization process [72].…”

Section: Production and Analysismentioning

confidence: 99%

Technical Advances to Accelerate Modular Type I Polyketide Synthase Engineering towards a Retro-biosynthetic Platform

Pang

Valencia

Wang

et al. 2019

Biotechnol Bioproc E

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Modular type I polyketide synthases (PKSs) are multifunctional proteins that are comprised of individual domains organized into modules. These modules act together to assemble complex polyketides from acyl-CoA substrates in a linear fashion. This assembly-line enzymology makes engineered PKSs a potential retro-biosynthetic platform to produce fuels, commodity chemicals, speciality chemicals, and pharmaceuticals in various host microorganisms, including bacteria and fungi. However, the realization of this potential is restricted by practical difficulties in strain engineering, protein overexpression, and titer/yield optimization. These challenges are becoming more possible to overcome due to technical advances in PKS design, engineered heterologous hosts, DNA synthesis and assembly, PKS heterologous expression, and analytical methodology. In this review, we highlight these technical advances in PKS engineering and provide practical considerations thereof.

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“…Second, it is usually the method of choice for the synthesis of "unnatural natural products." In this case, it consists either in the design and assembly of new biosynthetic gene clusters (4) or in the engineering of biosyn-thetic enzymes such as the modular nonribosomal peptide synthetases (NRPS) (5)(6)(7) and polyketide synthases (PKS) (8,9). Such approaches are often referred to as combinatorial biosynthesis.…”

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Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp

Aubry

Pernodet

Lautru

2019

Appl Environ Microbiol

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With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain. Volume 85 Issue 16 e00485-19 aem.asm.org 2 on July 10, 2020 by guest http://aem.asm.org/ Downloaded from cloning methods in Streptomyces species, prolific producers of specialized metabolites. These vectors were designed on the model of the SEVA plasmids, although the exact architecture of these plasmids could not be used for our application. The 12 vectors were proven to be functional by the verified integration in the chromosome of three commonly used Streptomyces species. We also illustrate two possible uses of our vectors. We first refactored the albonoursin gene cluster using BioBrick assembly. Second, we genetically complemented our cgc22 mutant strain, CGCL030 (cgc22 is involved in congocidine biosynthesis [30]), by constructing a gene cassette constituted of a promoter, an RBS, cgc22, and a terminator using ligase chain reaction assembly. RESULTS AND DISCUSSIONDesign of the vectors. The vectors were designed to meet the following specifications. It should be possible to use several vectors in the same strain (orthogonality), so different antibiotic resistance cassettes and different systems of integration at specific sites in the chromosome of Streptomyces should be used for the construction of the vectors. The vectors should be E. coli/Streptomyces shuttle vectors so that genetic constructions can be prepared in E. coli before being introduced into Streptomyces strains; thus, an E. coli origin of replication has to be included. It should be possible to introduce the vectors into Streptomyces strains by E. coli/Streptomyces intergeneric conjugation, so the presence o...

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Short-chain ketone production by engineered polyketide synthases in Streptomyces albus

Cited by 56 publications

References 32 publications

A bimodular PKS platform that expands the biological design space

A bimodular PKS platform that expands the biological design space

Technical Advances to Accelerate Modular Type I Polyketide Synthase Engineering towards a Retro-biosynthetic Platform

Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp

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