Patent Application
20230124115
The present invention is in the field of polyketides.
Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated β-carbon through a reductive loop exchange.
In an aspect the invention provides methods of making delta-lactones comprising engineering lipomycin PKS to produce the delta-lactones. Engineered PKSs are used to construct dimethylated, single-methylated delta-lactones, and with facile manipulations, nonmethylated delta-lactones, which are valuable fragrances. In particular, we engineered the lipomycin PKS to produce triketide lactones. In the first module, we performed an acyltransferase swap with a BorAT and in the second module we performed a reductive loop swap with the NanA2 module. We performed two reductive loop swaps, from SpnB and NanA2, but only NanA2 worked. With another AT swap in the second module, we can programmably produce the non-methylated delta lactone. The methods employ a malonyl-CoA selecting analog in the first and second module, a KR only in the first module, and a full reductive loop in the second module. As the lipomycin PKS takes a variety of starters, the methods may be used to make delta lactones of varying size. Generation of lactones is of great industrial utility, and PKS engineering has been proposed (e.g. Kaus et al, Nat. Prod. Rep., 2018, 35, 1070) but successfully engineering lipomycin PKS to generate lactones was challenging, unexpected and unprecedented.
In embodiments:
In an aspect the invention provides related compositions including a composition comprising an engineered lipomycin PKS1 gene (or gene product) altered with an AT-swap from borreledin and a LipPKS2 altered with a donor reductive loop from NanA2, configured to produce a single-methylated lactone.
In embodiments the compositions comprise another AT swap on LipPKS2 from borreledin, configured to produce a non-methylated delta lactone.
The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited, such as recited herein.
Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.
The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
We sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that enables the engineering of PKSs to produce desired, unnatural products.
Rational reprogramming of PKS enzymes for the biosynthesis of new polyketides has been a major research thrust over the past three decades.1-3 PKSs load a malonyl-CoA analog onto the acyl carrier protein (ACP) using the acyltansferase (AT) domain and extend the growing chain from the ketosynthase (KS) domain through a decarboxylative Claisen condensation reaction. After chain extension, the β-carbonyl reduction state is determined by the module's reductive domains, namely the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER), which generate the β-hydroxyl, α-β alkene, or saturated β-carbons respectively, when progressively combined. Unlike fatty acid synthases, which faithfully produce saturated fatty acids, PKSs have this variability in β-carbonyl reduction. Consequently, multiple studies have reported PKS module engineering for various β-carbon oxidation states.4-8 However, design strategies for introduction of reductive loop exchanges (i.e. KR-DH-ER domains) into partially reductive modules remain elusive. Here we compare bioinformatic and chemoinformatic approaches to guide reductive loop (RL) exchanges and develop a new method for RL exchanges based on the chemical similarity of the RL substrate. Chemoinformatics, an interdisciplinary field blending computational chemistry, molecular modeling and statistics to analyze structure-activity relationships, was first established for drug discovery.9 A chemoinformatic approach to PKS engineering could be valuable, particularly in RL exchanges where the KR and DH domains are substrate-dependent1: acyl chain length has critically affected dehydration in stand-alone DH10 and full PKS module studies.7,13
Chemoinformatic methods such as atom pair (AP) similarity, which characterizes atom pairs (e.g. length of bond path, number of π electrons), and maximum common substructure (MCS) similarity, which identifies the largest common substructure between two molecules,14 could beneficially describe substrate profiles. While divergent in chemical characterization, both similarity methods translate to a Tanimoto coefficient with a range of 0 (least similar) to 1 (most similar).14 We hypothesized that chemosimilarity between the substrates of donor and acceptor modules in RL exchanges may correlate with production levels, thereby leading to engineered modules that better control the reductive state of the β carbon.
Bioinformatic studies of PKS evolution have guided engineering efforts in closely related biosynthetic gene clusters (BGCs).15,16 We therefore undertook a phylogenetic analysis of the reductive domain common to all RLs, the ketoreductase (KR). The KR not only reduces the β-keto group to a β-hydroxyl, but also sets the stereochemistry of the β-group and, if a branched extender is used, sets the α-carbon stereochemistry resulting in subtypes A1, A2, B 1, B2 (
To evaluate the importance of chemical similarity and phylogenetic distance in RL exchanges, we swapped diverse, full RLs into the first module of the lipomycin PKS (LipPKS1) using conserved residues as exchange sites (
We identified four donor RLs (IdmO, indanomycin, S. antibioticus; SpnB, spinosyn, S. spinosa; AurB, aureothin, S. aureofaciens; NanA2, nanchangamycin, S. nanchangensis) to swap into LipPKS1. A pairwise comparison of phylogenetic distance and amino acid sequence identity determined that IdmO, AurB, and SpnB have the highest KR similarities to LipPKS1 (
Consistent with our hypothesis, we found a perfect correlation between titers of the desired product and the AP/MCS chemosimilarities between donor and LipPKS1 module substrates (Rs=1.00 and p=0.00) (
Based on these results, we took a chemoinformatic approach to further test our hypothesis that chemosimilarity of RL substrates is critical to PKS engineering. Using the ClusterCAD17 database, we identified donor RLs from laidlomycin and monensin that use a KR substrate (identical to the NanA2 KR substrate) with the highest chemically similarity to LipPKS1 (
To better demonstrate the utility of this approach, we further evaluated RL exchanges where AP and MCS chemosimilarity diverge and tested this method in modules located at the center of assembly lines, thus requiring docking domain interactions and larger substrates. We therefore performed RL swaps on the second module of lipomycin, LipPKS2 (
Abbreviations: PKS polyketide synthase; KS ketosynthase; AT acyltransferase; ACP acyl carrier protein; KR ketoreductase; DH dehydratase; ER enoylreductase; RL reductive loop; AP atom pair; MCS maximum common substructure; BGC biosynthetic gene cluster
Traditionally engineered to produce novel pharmaceuticals, 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. With their assembly-line process and vast biological design space, PKSs could be rationally programmed to sustainably produce new biomolecules with superior properties. 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. Through reductive loop exchanges, ketoreductase knockouts, and acyltransferase swaps, we produced a novel ethyl ketone and δ-lactone as potential gasoline and fragrance specialty chemical replacements, respectively. Collectively, our work expands the biological design space to the production of “designer” biomolecules.
Engineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable biochemical
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.
Advances in biotechnology have only begun to capitalize on the biomolecular design space. Referred to as the parvome, ‘parv-’ meaning small and ‘-ome’ denoting group, the world of cell-based molecules is vastly larger than the known chemical design space (Davies, 2011). De novo biomolecular production efforts have sought to capitalize on this space to generate new biofuels, commodity chemicals, and specialty chemicals (King et al., 2016). Beyond developing molecules with superior properties, biosustainable production of these molecules could contribute to a substantial reduction in carbon emissions, which is needed to avoid potentially devastating climate change (Matthews et al., 2009). Generally, biosynthesis of unnatural molecules often relies on broad substrate ranges (Rodriguez et al., 2014) and promiscuous activity in enzymes (Khersonsky et al., 2006). While major advances have been made in protein engineering, redesigning proteins to generate novel bioactivity and achieve new products remains a major challenge (Kumar et al., 2018).
Polyketide synthases synthesize an astonishing diversity of natural products including, but not limited to, anticancer, antimicrobial, and immunomodulating compounds (reviewed by (Robbins et al., 2016)). Assembly-line-like, modular polyketide synthases (PKSs), a subset of Type I PKSs, are often linked in a collinear fashion, creating a design space that could be rationally reprogrammed to produce many valuable biomolecules (Cai and Zhang, 2018; Yuzawa et al., 2018b; Zargar et al., 2017, 2018). Each module's cycle begins with a Claisen condensation reaction between the growing chain on the ketosynthase (KS) domain and a malonyl-CoA analog on the acyl carrier protein (ACP) that was loaded by the acyltransferase domain (AT) (
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, 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, RL swaps, AT swaps, fused TE) in a biomodular system to produce novel biomolecules, namely biofuels and specialty chemicals.
Short chain ketones (C3-C7) have been noted for their potential as gasoline additives because of their high octane numbers (McCormick et al., 2017), and we recently tested their fuel combustion properties in a gasoline called CARBOB, a specially formulated Blendstock for Oxygenate Blending formula mandated by the state of California (Yuzawa et al., 2018a). While most ketones showed superior properties to the common biofuel butanol, methyl-branched C5 and C6 ketones even had comparable fuel properties to isooctane (octane numbers, energy density, boiling point, melting point, and flash point). Longer chain ketones (above C7) were too expensive to synthesize for testing, but it is likely they are candidates as gasoline blending agents as well, and possibly possess combustion properties comparable or superior to traditional gasoline molecules. We therefore sought to produce longer chain ketones as an example of de novo production of biomolecules in a bimodular system.
As illustrated in
To produce the ethyl ketones, we conjugated the engineered Lip1 and Lip2 with the phiC31 and VWB integrases, respectively, into the genome of Streptomyces albus J1074. After 10-day production runs, we harvested the samples and measured titers of the final product and side products. We observe production of the desired product after heating and acidification with a titer of 20.6 mg/L (
Over the past ten years, the market for genetically modified microbial production of fragrance aroma chemicals has grown, and an important fragrant compound class is δ-lactones (Gupta, 2015). While not commercially used, 3-isopropyl-6-methyltetrahydropyranone has been synthesized previously as a potentially fragrant δ-lactone (Plessis and Derrer, 2001). The engineered production of this compound requires a combination of an AT swap in LipPKS1 and a RL swap in LipPKS2, which has not been shown before. Previously, we analyzed AT domains and associated linkers to identify the boundaries for AT swaps while maintaining enzyme activity in LipPKS1 (Yuzawa et al., 2017b). An exchange of the native methylmalonyl-CoA of LipPKS1 with malonyl-CoA of the first module of the borreledin PKS (BorAT1) with a fused TE produced 3-hydroxy carboxylic acids, which could be used as organic building blocks (Yuzawa et al., 2017b). Here, we replaced the fused TE with the native Lip1 docking domains. For LipPKS2, we had previously introduced the NanA2 reducing loop, and when combined with the native LipPKS1, we engineered production of 3-isopropyl-4,6-dimethyltetrahydropyranone (Zargar et al., 2019). This combination of LipPKS1 with the AT swap and LipPKS2 with an RL exchange should produce 3-isopropyl-6-methyltetrahydropyranone (
As before, we integrated the genes encoding the engineered LipPKS1 and LipPKS2 into S. albus J1074 at the phiC31 and VWB phage attachment sites with phiC31 and VWB integrases, respectively. After 10 day production runs, we detected the programmed product with a titer of 40 μg/L (
While unaltered natural products and synthetic chemistry have been the basis of industrial molecules, de novo biomolecular designs contain an astonishing diversity of molecules that could have a transformational impact in many fields (Smanski et al., 2016). A biosynthetic platform based on PKSs represents a vast design space with an attractive programming basis. With the diversity of starter substrates (˜102), malonyl-CoA analogs (˜101), and stereochemistry arrangements (˜101), a unimodular system alone can feasibly produce over 10,000 molecules and each subsequent module increases the number by two orders of magnitude. Integrating other fields such as ‘click’ chemistry obtains new capabilities (Le Feuvre and Scrutton 2018; Kalkreuter et al. 2019; Zhu et al. 2015).
Previously, we engineered the first module of lipomycin to make an array of molecules through AT swaps, RL swaps, KR knockouts, and a fused TE. Here, we have shown that we can leverage the knowledge we gained in unimodular systems to combine engineering manipulations, and successfully generate novel biofuels and specialty chemicals in the host Streptomyces albus.
All clusters were expressed from the S. albus chromosome under control of the GapDH(E1) promoter from Eggerthella lenta. Junction sites for reductive loop exchanges were determined by those reported by Hagen et al. through multiple sequence alignment with MUSCLE (Hagen et al. 2016; Edgar 2004). The plasmids along with their associated information have been deposited in the public version of JBEI registry.
Cloning of LipPKS1 with Full Reductive Loop Modules and Native Docking Domain
The phiC31 Streptomyces integrase vectors were used as described by Phelan et al. to integrate the LipPKS1 reductive loop swap modules (Phelan et al., 2017). The native docking domain sequences of LipPKS1 were codon optimized for E. coli and synthesized by Gen9 (since acquired by Ginkgo Bioworks). They were cloned through Golden Gate assembly into the LipPKS1 module with an inserted RL from NanA2 from Zargar et al (Zargar et al., 2019).
Cloning of LipPKS1 with AT-Swap and Native Docking Domain
The phiC31 integrase vectors (Phelan et al., 2017) were used to integrate the AT-swapped LipPKS1 module into the phiC31 site in the S. albus chromosome. The Lip1 native docking domain sequences of LipPKS1 were cloned into the AT-swapped LipPKS1 module from Yuzawa et al. (Yuzawa et al., 2017b) through Golden Gate assembly.
Cloning of LipPKS2 with KR Knockout and Fused DEBS Thioesterase
The VWB Streptomyces integrase vectors were used to integrate the LipPKS2 KR− module (Phelan et al., 2017). The native LipPKS2 was codon optimized for E. coli with a single point mutation S1547A into the KR active site to mutate the catalytic serine to alanine, thereby abolishing KR activity, and synthesized by Gen9 (since acquired by Ginkgo Bioworks). The fused DEBS thioesterase domain was placed at the C-terminus of the ACP domain through Golden Gate assembly.
Cloning of LipPKS2 with Full Reductive Loop Modules and Fused DEBS Thioesterase
The VWB Streptomyces integrase vectors were used to integrate the LipPKS2 reductive loop modules cloned previously (Zargar et al., 2019).
Conjugation of phiC31 Integrase LipPKS1 Constructs into Streptomyces albus J1074
E. coli ET12567/pUZ8002 was transformed with LipPKS1 plasmids and selected for on LB agar containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and apramycin (50 μg/mL). A single colony was inoculated into 5 mL of LB containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and apramycin (50 μg/mL) at 37° C. The overnight culture was used to seed 10 mL of LB containing the same antibiotics, and the new culture was grown at 37° C. to an OD600 of 0.4-0.6. The E. coli cells were pelleted by centrifugation, washed twice with LB, and resuspended in 500 μL of LB. Fresh S. albus J1074 spores were collected from a mannitol soy agar plate with 5 mL of 2×YT and incubated at 50° C. for 10 min. The spores (500 μL) and the E. coli cells (500 μL) were mixed, spread onto mannitol soy agar, and incubated at 30° C. for 16 hours. 1 mL of both nalidixic acid (20 μg/mL) and apramycin (40 μg/mL) were added to the plate and allowed to dry. The plate was then incubated for 3-4 days at 30° C. A single colony was inoculated into TSB containing nalidixic acid (25 μg/mL) and apramycin (25 μg/mL). After 3-4 days, a 1 mL aliquot was taken for genomic isolation using the Maxwell kit (Promega, Cat# AS1490, Madison Wis.). Successful integration was verified using qPCR. The remainder of the culture was spread onto a MS plate and incubated at 30° C. for 2-3 days. The spores were collected from the plate with 3-4 mL of water and mixed with glycerol to prepare a 25% glycerol stock, which was stored at −80° C. for long-term storage.
Conjugation of VWB Integrase LipPKS2 Constructs into Streptomyces albus
E. coli ET12567/pUZ8002 was transformed with LipPKS2 plasmids and selected for on LB agar containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and spectinomycin (200 μg/mL). A single colony was inoculated into 5 mL of LB containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and spectinomycin (200 μg/mL) at 37° C. The overnight culture was used to seed 10 mL of LB containing the same antibiotics, and the new culture was grown at 37° C. to an OD600 of 0.4-0.6. The E. coli cells were pelleted by centrifugation, washed twice with LB, and resuspended in 500 μL of LB. S. albus J1074 spores with an integrated LipPKS1, which were collected from a mannitol soy agar plate with 5 mL of 2×YT and incubated at 50° C. for 10 min. The spores (500 μL) and the E. coli cells (500 μL) were mixed, spread onto mannitol soy agar, and incubated at 30° C. for 16 hours. 1 mL of each nalidixic acid (20 μg/mL), apramycin (40 μg/mL), and spectinomycin (400 μg/mL) was added to the plate and allowed to dry. The plate was then incubated for 3-4 days at 30° C. A single colony was inoculated into TSB containing nalidixic acid (25 μg/mL), apramycin (25 μg/mL) and spectinomycin (200 μg/mL). After 3-4 days, a 1 mL aliquot was taken for genomic isolation through the Maxwell kit (Promega, Cat# AS1490, Madison Wis.). Successful integration was verified through qPCR. The remainder of the culture was spread onto a MS plate and incubated at 30° C. for 2-3 days. The spores were collected from the plate with 3-4 mL of water and mixed with glycerol to prepare a 25% glycerol stock, which was stored at -80° C. for long-term storage.
S. albus Production Runs
Engineered S. albus spores were grown in 12 mL of TSB medium containing nalidixic acid (50 μL), 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.
To detect acid side products, 1 mL of each sample was centrifuged at 5000 g for 10 minutes and 200 μL of the supernatant was removed. The supernatant was mixed with 200 μl of 100 μM hexanoic acid dissolved in methanol and filtered using Amicon Ultra Centrifugal filters, 3 KDa Ultracel, 0.5 mL device (Millipore). β-hydroxy (3-hydroxy-2,4-dimethylpentanoic acid) and saturated acids (2,4-dimethylpentanoic acid) were synthesized by Enamine (Cincinnati, USA) to greater than 95% purity.
10 mL of each sample was mixed with 2 mL of diethyl ether in a 15-mL conical tube and vortexed for 5 minutes. Each conical tube was centrifuged at 5000 g for 10 minutes and 1 mL of ether was removed and placed into a 2-mL flat bottom microcentrifuge tube. Air was gently blown over each sample in a chemical fume hood until dry. The extract was resuspended in 200 μL of methanol. 5-methyl-6-(propan-2-yl)oxan-2-one was synthesized by Enamine (Cincinnati, USA) to greater than 95% purity.
One mL of each sample was harvested in a 1.7-mL microcentrifuge tube. To each tube, 300 μL of ethyl acetate and 50 μL uL of formic acid were added. All tubes were wrapped in paraffin and heated for 60 minutes at 80° C. Samples were then placed on ice for 5 minutes and vortexed for 5 minutes. Each sample was centrifuged for 2 minutes at 10,000 g. One hundred microliters of ethyl acetate was removed from each sample and placed in a GC MS vial.
Electron ionization GC/MS analysis was performed on a G3950A-9000 GC (Agilent) using a J&W HP-5 ms Ultra Inert Intuvo GC column module (15 m length, 0.25 mm inner diameter, 0.25 μm film thickness). The GC was coupled to a mass selective detector (Agilent 5977B MSD) and an autosampler (Model 7693 Agilent). The GC oven was programmed at 60° C. for 3 minutes, ramping at 10° C./min until 120° C., and then ramping at 200° C./min to 300° C.; the injection port temperature was 250° C. Using an authentic standard, we determined a single-ion method of detection collecting data at m/z=57.00, m/z=85.00, m/z=142.00.
LC separation of short chain acids was conducted on an InfinityLab Poroshell HPHB-C18 reversed phase column (100 mm length, 3.0 mm internal diameter, 2.7 μm particle size; Agilent, United States) using a Waters Acquity Autopurification system, prep UHPLC-MS (ESI) (Waters, United States). The mobile phase for separating 2,4-dimethylpentanoic acid and 2,4-dimethylpent-2-enoic acid was composed of 10 mM ammonium acetate and 0.05% ammonium hydroxide in water (solvent A) and 10 mM ammonium acetate and 0.05% ammonium hydroxide in methanol (solvent B). The mobile phase for separating 3-hydroxy-2,4-dimethylpentanoic acid and 2,3-dimethyl-3-oxopentanoic acid was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). All acids were each separated via the following gradient: increased from 5 to 97.1% B in 6.5 min, held at 97.1% B for 1.3 min, decreased from 97.1 to 5% B in 0.4 min, and held at 5% B for an additional 2 min. The flow rate was held at 0.42 ml·min−1 for 8.2 min, and then increased from 0.42 to 0.65 ml·min−1 for an additional 2 min. The total LC run time was 10.8 min. Samples of 3 μl were injected into the LC column. Acids were detected via [M−H]-ions. The Agilent 1200 Rapid Resolution LC system was coupled to an Agilent 6210 TOF (Agilent Technologies, United States). Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. The drying and nebulizing gases were set to 111·min−1 and 301·bin−2, respectively, and a drying gas temperature of 330° C. was used throughout. Atmospheric pressure chemical ionization was conducted in the positive-ion mode with capillary and fragmentor voltages of 3.5 kV and 100 V, respectively. The skimmer, OCT1 RF, and corona needle were set to 50 V, 170 V, and 4 μA, respectively. The vaporizer was set to 350° C. The analysis was performed using an m/z range of 70 to 1100. Data acquisition and processing were performed using MassHunter software (Agilent Technologies, United States).
LC separation of triketide lactones was conducted on a Kinetex XB-C18 reversed phase column (100 mm length, 3 mm internal diameter, 2.6 μm particle size; Phenomenex, United States) using an Agilent 1200 Rapid Resolution LC system (Agilent Technologies, United States). The mobile phase was composed of water (solvent A) and methanol (solvent B). Lactones were each separated via the following gradient: increased from 30 to 90% B in 3.7 min, held at 94% B for 5.2 min, decreased from 90 to 30% B in 0.33 min, and held at 30% B for an additional 2.0 min. The flow rate was held at 0.42 ml·min−1 for 8.67 min, increased from 0.42 to 0.60 ml·min−1 in 0.33 min, and held at 0.60 ml·min−1 for an additional 2.0 min. The total LC run time was 11.0 min. The column compartment and autosampler temperatures were set to 50° C. and 6° C., respectively. Samples of 3 μl were injected into the LC column. The Agilent 1200 Rapid Resolution LC system was coupled to an Agilent 6210 TOF (Agilent Technologies, United States). Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. The drying and nebulizing gases were set to 101·min−1 and 251·bin−2, respectively, and a drying gas temperature of 325° C. was used throughout. Atmospheric pressure chemical ionization was conducted in the positive-ion mode with capillary and fragmentor voltages of 3.5 kV and 100 V, respectively. The skimmer, OCT1 RF, and corona needle were set to 50 V, 170 V, and 4 μA, respectively. The vaporizer was set to 350° C. Lactones were detected via [M+H]+ ions: m/z=157.1223. The analysis was performed using an m/z range of 70 to 1100. Data acquisition and processing were performed using MassHunter software (Agilent Technologies, United States).
E. coli ET12567
S. venezuelae
The phiC31 Streptomyces integrase vectors were used as described by Phelan et al to integrate the LipPKS1 reductive loop swap modules27. The native lipomycin module 1 with a fused DEBS thioesterase was used from Yuzawa et al30. Reductive loop sequences of IdmO, AurB, NanA2, and SpnB sequences were codon optimized for E. coli and amplified from Hagen et al7. Reductive loop sequences from MAS, MonA2, and LaidS2 were codon optimized for E. coli and synthesized by Gen9 (since acquired by Ginkgo Bioworks). Cloning was performed through Golden Gate assembly. All clusters were expressed under the GapDH(El) promoter from Eggerthella lenta. Junction sites for reductive loop sites were determined by those reported by Hagen et al through multiple sequence alignment with Muscle31. The plasmids along with their associated information have been deposited in the public version of JBEI registry.
The phiC31 integrase vectors were used to integrate the native LipPKS1 module. The cloning of the native docking domain to replace the DEBS thioesterase was performed through Golden Gate assembly. The VWB Streptomyces integrase vectors were used as described by Phelan et al1 to integrate the LipPKS2 reductive loop swap modules. The native LipPKS2 was codon optimized for E. coli and the native sequences synthesized with an attached DEBS thioesterase. Junction A reductive loop sites were determined through Muscle as in LipPKS1, and SpnB and NanA2 reductive loops were cloned through Golden Gate assembly. The plasmids along with their associated information have been deposited in the public version of JBEI registry.
Conjugation of phiC31 Integrase LipPKS1 Constructs into Streptomyces albus
E. coli ET12567/pUZ8002 was transformed with LipPKS1 plasmids and selected for on LB agar containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and apramycin (50 μg/mL). A single colony was used to inoculate a 5 mL of LB containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and apramycin (50 μg/mL) at 37° C. The overnight culture was used to seed 10 mL of LB containing the same antibiotics, which was grown at 37° C. to an OD600 of 0.4-0.6. The E. coli cells were pelleted by centrifugation, washed twice with LB, and resuspended in 500 μL of LB. Fresh S. albus J1074 spores were collected from a mannitol soy agar plate with 5 mL of 2×YT and incubated at 50° C. for 10 min. The spores (500 μL) and the E. coli cells (500 μL) were mixed, spread onto mannitol soy agar, and incubated at 30° C. for 16 hours. After 1 mL addition of nalidixic acid (20 μg/mL) and apramycin (40 μg/mL) was added and allowed to dry, the plate was further incubated for 3-4 days at 30° C. A single colony was used to inoculate into TSB containing nalidixic acid (25 μg/mL) and apramycin (25 μg/mL). After 3-4 days, a 1 mL aliquot was taken for genomic isolation through the Maxwell kit (Promega, Cat#AS1490, Madison Wis.). Successful integration was verified through qPCR. The remainder of the culture was spread onto a MS plate and incubated at 30° C. for 2-3 days. The spores were collected from the plate with 3-4 mL of water and mixed with glycerol to prepare 25% glycerol stock. The glycerol stock was stored at −80° C. for long-term storage.
Conjugation of VWB Integrase LipPKS2 Constructs into Streptomyces albus
E. coli ET12567/pUZ8002 was transformed with LipPKS2 plasmids and selected for on LB agar containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and spectinomycin (100 μg/mL). A single colony was used to inoculate a 5 mL of LB containing kanamycin (25 μg/mL), chloramphenicol (15 μg/mL), and apramycin (100 μg/mL) at 37° C. The overnight culture was used to seed 10 mL of LB containing the same antibiotics, which was grown at 37° C. to an OD600 of 0.4-0.6. The E. coli cells were pelleted by centrifugation, washed twice with LB, and resuspended in 500 μL of LB. S. albus J1074 spores with an integrated LipPKS1 were collected from a mannitol soy agar plate with 5 mL of 2×YT and incubated at 50° C. for 10 min. The spores (500 μL) and the E. coli cells (500 μL) were mixed, spread onto mannitol soy agar, and incubated at 30° C. for 16 hours. After a 1-mL addition of nalidixic acid (20 μg/mL), apramycin (40 μg/mL), and spectinomycin (400 μg/mL) was added and allowed to dry, the plate was further incubated for 3-4 days at 30° C. A single colony was used to inoculate into TSB containing nalidixic acid (25 μg/mL), apramycin (25 μg/mL) and spectinomycin (100 μg/mL). After 3-4 days, a 1-mL aliquot was taken for genomic isolation through the Maxwell kit (Promega, Cat#AS1490, Madison Wis.). Successful integration was verified through qPCR. The remainder of the culture was spread onto a MS plate and incubated at 30° C. for 2-3 days. The spores were collected from the plate with 3-4 mL of water and mixed with glycerol to prepare 25% glycerol stock. The glycerol stock was stored at -80° C. for long-term storage.
S. albus Production Runs
Engineered S. albus spores were grown in 12 mL of TSB medium containing nalidixic acid (50 μg/mL) and apramycin (50 μg/mL) for 4-5 days at 30° C. for single module LipPKS1 studies. Bimodular studies also included spectinomycin (200 μg/mL). 3 mL of the overnight culture was used to seed 30 mL of 10% media 042 and 90% plant hydrolysate5, supplemented with 2.4 grams/liter of valine and nalidixic acid (50 μg/mL), which was grown for 10 days at 30° C. For bimodular production runs, an overlay of 4 mL of dodecane was added to retain the triketide lactone.
To detect β-keto, β-hydroxy, α-β alkene, and saturated acids, 1 mL of each sample was centrifuged at 5000×g for 10 minutes and 200 μL of the supernatant was removed. The supernatant was mixed with 200 μl of 100 μM hexanoic acid dissolved in methanol and filtered using Amicon Ultra Centrifugal filters, 3 kDa Ultracel, 0.5 mL device (Millipore). β-hydroxy (3-hydroxy-2,4-dimethylpentanoic acid) and saturated acids (2,4-dimethylpentanoic acid) were synthesized by Enamine (Cincinnati, USA) to greater than 95% purity.
Ten mL of each sample was mixed with 2 mL of diethyl ether in a 15 mL conical tube and vortexed for 5 minutes. Each conical tube was centrifuged at 5000 g for 10 minutes and 1 mL of ether was removed and placed into a 2 mL flat bottom microcentrifuge tube. Air was gently blown over each sample in a chemical fume hood until dry. The extract was resuspended in 200 μL of methanol with an internal standard of 100 μM δ-nonalactone (Sigma). 5-methyl-6-(propan-2-yl)oxan-2-one was synthesized by Enamine (Cincinnati, USA) to greater than 95% purity used as a standard to approximate 3,5-dimethyl-6-(propan-2-yl)oxan-2-one.
Two milliliters of each production culture were harvested at 10 days, spun down at 10000 g for 10 minutes and resuspended in 0.75 mL of aqueous solution of 50 mM potassium phosphate, 300 mM NaCl, 10% glycerol and 2 mg/mL of lysozyme at pH 7.5. Samples were incubated for 1 hour at 30 degrees Celsius. Samples were then lysed with the ZR Fungal/Bacterial DNA Microprep kit (Zymo Research, Catalog No: D6007, Irvine, Calif.). Samples were loaded into the ZR Bashing/Bead Lysis tube and secured in a bead beater. Samples were shaken at a frequency of 30 per second for 5 minutes. The cell lysates were centrifuged at maximum speed in a Eppendorf benchtop centrifuge and the resulting supernatants were collected for proteomic analysis. The protein concentration of protein samples was determined by Bio-Rad DC Protein Assay (Bio-Rad #5000121) according to manufacture instruction. 20 ug protein of each sample was reduced and alkylated, followed by overnight trypsin digestion. The resulting tryptic peptide samples were subjected to LCMS analysis.
LC separation of short-chain acids was conducted on an InfinityLab Poroshell HPH-C18 reversed phase column (100 mm length, 3.0 mm internal diameter, 2.7 pm particle size; Agilent, United States) using a Waters Acquity Autopurification system, prep UHPLC-MS (ESI) (Waters, United States). The mobile phase was composed of 10 mM ammonium acetate and 0.05% ammonium hydroxide in water (solvent A) and 10 mM ammonium acetate and 0.05% ammonium hydroxide in methanol (solvent B) to separate 2,4-dimethylpentanoic acid and 2,4-dimethylpent-2-enoic acid. The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1%formic acid in methanol (solvent B) to separate 3-hydroxy-2,4-dimethylpentanoic acid and 2,3-dimethyl-3-oxopentanoic acid. All acids were each separated via the following gradient: increased from 5 to 97.1% B in 2.0 min, held at 97.1% B for 2.8 min, decreased from 97.1 to 5% B in 0.4 min, and held at 5% B for an additional 5.8 min. The flow rate was held at 0.42 ml·min−1 for 5.2 min, and then increased from 0.42 to 0.65 ml·min−1 for an additional 5.8 min. The total LC run time was 11 min. Samples were injected into the LC column at a volume of 15 μl. Acids were detected via [M−H]−ions: m/z=129.092; m/z=127.076; m/z=145.087; m/z=143.071. The analysis was performed using an m/z range of 70 to 500.
LC separation of triketide lactones was conducted on a Kinetex XB-C18 reversed phase column (100 mm length, 3 mm internal diameter, 2.6 μm particle size; Phenomenex, United States) using an Agilent 1200 Rapid Resolution LC system (Agilent Technologies, United States). The mobile phase was composed of water (solvent A) and methanol (solvent B). Lactones were each separated via the following gradient: increased from 30 to 90% B in 3.7 min, held at 94% B for 5.2 min, decreased from 90 to 30% B in 0.33 min, and held at 30% B for an additional 2.0 min. The flow rate was held at 0.42 ml·min−1 for 8.67 min, increased from 0.42 to 0.60 ml·min−1 in 0.33 min, and held at 0.60 ml·min−1 for an additional 2.0 min. The total LC run time was 11.0 min. The column compartment and autosampler temperatures were set to 50° C. and 6° C., respectively. Samples were injected into the LC column at a volume of 3 μl. The Agilent 1200 Rapid Resolution LC system was coupled to an Agilent 6210 TOF (Agilent Technologies, United States). Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. The drying and nebulizing gases were set to 101·min−1 and 251·bin−2, respectively, and a drying gas temperature of 325° C. was used throughout. Atmospheric pressure chemical ionization was conducted in the positive-ion mode with capillary and fragmentor voltages of 3.5 kV and 100 V, respectively. The skimmer, OCT1 RF, and corona needle were set to 50 V, 170 V, and 4 μA, respectively. The vaporizer was set to 350° C. Lactones were detected via [M+H]+ ions: m/z=157.1223; m/z=171.1379. The analysis was performed using an m/z range of 70 to 1100. Data acquisition and processing were performed using MassHunter software (Agilent Technologies, US).
Synthesis of 2,4-dimethyl-3-oxopentanoic acid. A solution of ethyl 4-methyl-3-oxopentanoate (1.5 g, 9.5 mmol), potassium carbonate (3.93 g, 28.5 mmol) and methyl iodide (1.62 g, 11.4 mmol) in THF (20 mL) was refluxed overnight under a N2 environment. The reaction mixture was allowed to cool to room temperature before subsequent filtration and concentration was carried out, resulting in the synthesis of ethyl 2,4-dimethyl-3-oxopentanoate as clear yellow oil. Without further purification, the oil was dissolved in 10 mL 1N NaOH aqueous solution and stirred overnight at room temperature. The solution was acidified to pH 1-2 using conc. HCl and the resulting mixture was extracted with diethyl ether (30 mL×3). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give the crude product, which was purified by flash column chromatography (silica, DCM: EA 1:1) to yield 2,4-dimethyl-3-oxopentanoic acid a pale-yellow oil (985 mg, 72% over two steps). 1H NMR (400 MHz, Chloroform-d) δ 4.06 (q, J=7.1 Hz, 1H), 2.74 (hept, J=6.9 Hz, 1H), 1.21 (d, J=7.1 Hz, 3H), 1.01 (dt, J=6.9, 2.5 Hz, 6H).
Shotgun proteomic was analyzed on an Agilent 6550 iFunnel Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, Calif.) coupled to an Agilent 1290 UHPLC system as described previously32. Peptides were separated on a Sigma—Aldrich Ascentis Peptides ES-C18 column (2.1 mm×100 mm, 2.7 μm particle size, operated at 60° C.) at a 0.400 mL/min flow rate and eluted with the following gradient: initial condition was 95% solvent A (0.1% formic acid) and 5% solvent B (99.9% acetonitrile, 0.1% formic acid). Solvent B was increased to 35% over 120 min, and then increased to 50% over 5 min, then up to 90% over 1 min, and held for 7 min at a flow rate of 0.6 mL/min, followed by a ramp back down to 5% B over 1 min where it was held for 6 min to re-equilibrate the column to original conditions. Peptides were introduced to the mass spectrometer from the LC by using a Jet Stream source (Agilent Technologies) operating in positive-ion mode (3,500 V). Source parameters employed gas temp (250° C.), drying gas (14 L/min), nebulizer (35 psig), sheath gas temp (250° C.), sheath gas flow (11 L/min), VCap (3,500 V), fragmentor (180 V), OCT 1 RF Vpp (750 V). The data were acquired with Agilent MassHunter Workstation Software, LC/MS Data Acquisition B.06.01 operating in Auto MS/MS mode whereby the 20 most intense ions (charge states, 2-5) within 300-1,400 m/z mass range above a threshold of 1,500 counts were selected for MS/MS analysis. MS/MS spectra (100-1,700 m/z) were collected with the quadrupole set to “Medium” resolution and were acquired until 45,000 total counts were collected or for a maximum accumulation time of 333 ms. Former parent ions were excluded for 0.1 min following MS/MS acquisition. The successfully identified peptides of engineered LipPKS1 were targeted in a SRM method developed on an Agilent 6460 QQQ mass spectrometer system coupled with an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, Calif.). Peptides were separated on an Ascentis Express Peptide C18 column [2.7-mm particle size, 160-Å pore size, 5-cm length×2.1-mm inside diameter (ID), coupled to a 5-mm×2.1-mm ID guard column with the same particle and pore size, operating at 60° C.; Sigma-Aldrich] operating at a flow rate of 0.4 ml/min via the following gradient: initial conditions were 98% solvent A (0.1% formic acid), 2% solvent B (99.9% acetonitrile, 0.1% formic acid). Solvent B was increased to 40% over 11 min, and was then increased to 80% over 1 min, and held for 1.5 min at a flow rate of 0.6 mL/min, followed by a ramp back down to 2% B over 0.5 min where it was held for 1 min to re-equilibrate the column to original conditions. The eluted peptides were ionized via an Agilent Jet Stream ESI source operating in positive ion mode with the following source parameters: gas temperature=250° C., gas flow=13 liters/min, nebulizer pressure=35 psi, sheath gas temperature=250° C., sheath gas flow=11 liters/min, capillary voltage=3500 V, nozzle voltage=0 V. The data were acquired using Agilent MassHunter version B.08.02. Acquired SRM data were analyzed by Skyline software version 20.1 (MacCoss Lab Software).
Amino acid sequences of KR and KS domains from 72 BGCs were extracted from ClusterCAD17. The sequences were aligned using Muscle v3.833. The alignments were manually curated using JalView34. The best amino acid substitution model for both the ketosynthases and ketoreductases phylogenies was LG+F+I+G4, and it was selected using the ModelFinder tool implemented in IQ-tree35. Finally the phylogeny was constructed using IQ-tree36, using the partitioned models with 10,000 bootstrap replicates and visualized with FigTree.
Spearman rank correlations were used to compare the significance of reductive loop exchanges. Production titers were normalized to the highest producer in the production of short chain acids in LipPKS1 swaps and triketide lactone production in LipPKS2 swaps. The datasets were combined and production titer and chemosimilarity were normalized to the highest production titer and most chemically similar reductive loop. The datasets were then tested for statistical significance through a Spearman correlation.
This application claims priority as a continuation application of PCT International Patent Application No. PCT/US2021/31213, filed May 7, 2021, which claims priority to U.S. Provisional Patent Application Ser. No. 63/021,554, filed May 7, 2020, both of which are hereby incorporated by reference.
The invention described and claimed herein was made utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63021554 | May 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/031213 | May 2021 | US |
| Child | 18053288 | US |
