Engineering Pseudomonas putida KT2440 for chain size tailor-made free fatty acid and oleochemical manufacturing

Chemical substances

All chemical substances have been bought from Sigma-Aldrich (United States) except in any other case described.

Plasmids and strains

Plasmids and strains used on this examine are listed in Supplementary Tables 10, 11. The plasmids and strains have been deposited within the public model of JBEI registry (http://public-registry.jbei.org) and are bodily obtainable from the corresponding writer upon request. All plasmids have been constructed through Gibson or Golden Gate Meeting utilizing normal protocols. All heterologous genes have been codon optimized for P. putida KT2440 and ordered as gBlocks from IDT.

Tradition circumstances and plasmid transformations

E. coli cultures used for cloning have been grown in LB medium at 37 °C at 200 RPM, whereas P. putida cultures used for pressure building have been grown in LB medium at 30 °C at 200 RPM. Choice markers have been used on the following ultimate concentrations: kanamycin (50 μg/mL), gentamicin (30 μg/mL), chloramphenicol (20 μg/mL), and sucrose (20% w/v). Episomal plasmids have been reworked into E. coli and P. putida through electroporation. Briefly, to organize competent cells 1 mL of in a single day tradition was centrifuged in 1.7 mL Eppendorf tubes at 21,300×g for 1 min. The supernatant was discarded, and pellets have been washed with nanopure water 3 times. After the ultimate wash the pellet was resuspended in 100 μL of nanopure water and 1 μL of purified plasmid was added to the competent cells. Cells have been electroporated in a 0.1 cm cuvette at 1.8 kV. After electroporation, cells have been resuspended in 500 μL LB and incubated at 37 °C at 200 RPM (E. coli) for one hour or 30 °C at 200 RPM (P. putida) for 2 hours earlier than plating. Except in any other case famous, cultures have been grown in 50 mL 25 mm × 150 mm round-bottom borosilicate tradition tubes.

Progress media

For FFA degradation assays EZ Wealthy medium (Teknova) supplemented with 10 mM glucose was used. For FFA and FAMEs manufacturing assays both EZ Wealthy medium supplemented with 100 mM glycerol, Terrific Broth medium (EMD Millipore), or diluted sorghum hydrolysate was used. Sorghum hydrolysate was diluted 4x with M9 minimal salts medium to a ultimate 1x M9 salts focus. After preparation, all media have been sterile filtered, not autoclaved, by way of a 0.22 μm bottle-top filter.

Sorghum hydrolysate manufacturing

Sorghum (30% w/w of complete weight), choline hydroxide (4% w/w of dry sorghum), lactic acid (6.3% of dry sorghum), acetic acid (5.8% of dry sorghum), and water have been added to a ten L Parr reactor with a complete working mass of three kg. Lactic and acetic acid was added to react with choline hydroxide and produce cholinium lactate and cholinium acetate in situ because the pretreatment catalysts. The fabric was pretreated for 3 hours at 140 °C at 80 RPM. Subsequently, the pretreated slurry was cooled to 25 °C and the pH was adjusted to five.0 with 6 N hydrochloric acid. A 9:1 ratio of cellulase CTec3 and hemicellulase HTec3 NS 22244 at 10 mg protein/g of biomass was added to the Parr reactor and enzymatic saccharification was performed at 50 °C for 72 h at 80 RPM. After 72 h, the hydrolysate was centrifuged, and the supernatant was filtered by way of a 0.45um bottle-top filter. Following filtration, the pH of the hydrolysate was adjusted to 7.0 with 5 N NaOH. Lastly, the hydrolysate was filtered as soon as extra by way of a 0.22μm bottle-top filter. The ultimate hydrolysate contained 47.1 g/L glucose, 25.6 g/L xylose, 16.9 g/L acetic acid, and 17.3 g/L lactic acid.

Technology of CoA ligase knockout mutants

Deletion mutants in P. putida have been constructed by allelic change as described beforehand⁴⁹. Briefly, 1 kb homology areas upstream and downstream of the goal gene, together with the beginning and cease codons, have been cloned into pMQ30. Plasmids have been then conjugated into P. putida utilizing E. coli S17 strains. Transconjugants have been chosen on gentamicin and chloramphenicol LB plates after which grown in a single day in LB with no antibiotics. In a single day cultures have been diluted 100x, 100 μL of which was plated on LB agar with no NaCl that was supplemented with 10% (wt/vol) sucrose. Putative deletions have been reproduction plated on LB agar with no NaCl supplemented with 10% (wt/vol) sucrose and LB agar with gentamicin. Colonies that grew on sucrose, however not gentamicin, have been screened through PCR with primers flanking the goal gene to substantiate gene deletion.

FFA degradation assays

Examined strains have been inoculated in 5 mL of EZ Wealthy medium supplemented with 10 mM glucose and grown in a single day at 30 °C and 200 RPM. The cultures and a clean medium-only management have been then again diluted 10x in EZ Wealthy medium supplemented with 10 mM glucose and spiked with 250 μM of every even-chained FFA between C6 and C16. Following this, cultures have been grown at 30 °C for 48 h. The remaining FFAs have been derivatized and quantified utilizing GC/MS.

Progress assays

Carbon supply development assays have been carried out as beforehand described²⁷. Briefly, in a single day cultures have been inoculated into 5 mL of LB medium from single colonies, and grown at 30 °C. These cultures have been washed 3 times in carbon-free MOPS (morpholinepropanesulfonic acid) minimal medium, which is comprised of 32.5 μM CaCl₂, 0.29 mM Ok₂SO₄, 1.32 mM Ok₂HPO₄, 8 μM FeCl₂, 40 mM MOPS, 4 mM tricine, 0.01 mM FeSO₄, 9.52 mM NH₄Cl, 0.52 mM MgCl₂, 50 mM NaCl, 0.03 μM (NH₄)₆Mo₇O₂₄, 4 μM H₃BO₃, 0.3 μM CoCl₂, 0.1 μM CuSO₄, 0.8 μM MnCl₂, and 0.1 μM ZnSO₄. After washing, cultures have been diluted 100x into 500 μL of MOPS minimal medium with 10 mM glucose or 10 mM FFA (C6-C12) was spiked in as a sole carbon supply in 48-well plates (Falcon, 353072). Tergitol NP-40 was added to the medium to a ultimate focus of 1% (v/v) to assist solubilize the FFAs. Plates have been sealed with a gas-permeable microplate adhesive movie (Breathe-Straightforward®, Sigma-Aldrich, Z380059), after which optical density at 600 nm (OD600) was monitored with a Biotek Synergy H1M at 30 °C for 48 h with quick steady shaking (Agilent, Santa Clara, CA). To conduct development assays on strains containing an IPM overlay, in a single day cultures have been diluted 100x into 5 mL of LB medium containing a 1:10 isopropyl myristate overlay in 50 mL 25 mm × 150 mm round-bottom borosilicate tradition tubes and grown at 30 °C, 200 RPM, for 48 h. Periodically, 100 μL of the tradition’s aqueous part was transferred to a transparent backside 96-well plate (VWR, CORN3912) and the OD600 was measured with a SpectraMax M2 plate reader.

FFA and FAME manufacturing assays

Strains have been inoculated in 5 mL of medium containing kanamycin and grown in a single day at 30 °C and 200 RPM. The media used for in a single day cultures matched the media used for the manufacturing assay. Strains grown in sorghum hydrolysate needed to be slowly tailored to this medium. First, strains have been inoculated in a 20x dilution of hydrolysate in M9 minimal salts medium in a single day, and subsequently again diluted right into a 4x dilution of hydrolysate in M9 minimal salts and as soon as once more grown in a single day. The subsequent day, all in a single day cultures have been again diluted 10x into kanamycin-containing medium and grown at 30 °C for 4 h earlier than including arabinose to a ultimate focus of 0.2% v/v. A 1:10 isopropyl myristate overlay was added to FAME-producing cultures on the time of induction. Following induction with arabinose, cultures have been grown for a further 48 h earlier than quantifying FFA and FAME manufacturing through GC/MS.

FFA derivatization and quantification through GC/MS

To derivatize FFAs in liquid tradition, 300 μL of tradition was added to a 1.5 mL screw cap microcentrifuge tube (VWR 16466-064) containing 15 μL of 40% tetrabutylammonium hydroxide. Every pattern was spiked with a nonanoic acid inner normal to a ultimate focus of 250 μM. Then, 600 μL of dichloromethane containing 0.5% 2,3,4,5,6-pentafluorobenzyl bromide (v/v) was added to every pattern. The samples have been incubated in an Eppendorf thermomixer at 50 °C and 1400RPM for 20 min. Following incubation, the samples have been centrifuged at 21,300×g for 10 min and the natural layer was collected for GC/MS evaluation. In parallel, clean medium samples containing 0 μM, 50 μM, 100 μM, 250 μM, 500 μM, or 750 μM combination of every even-chained FFA between C6 and C16 have been equally spiked with a nonanoic acid inner normal and derivatized in triplicate.

Samples have been run on an Agilent 6890 GC and Agilent 5973 MS utilizing a 30 m × 0.250 mm × 0.25 μm HP-5MS column with a helium circulate of 1.3 mL/min. The GC was run in splitless mode with a 1 μL pattern injection and a continuing inlet temperature and switch line temperature of 250 °C. The oven beginning temperature was 80 °C and held for 1 min. Then, the oven temperature was elevated at a charge of 10 °C/min till 150 °C. Lastly, the temperature was elevated at a charge of 20 °C/min till 300 °C and held for five min. A solvent delay of 6.80 min was used and the MS was run in scan mode (50.0–350.0 amu). The extracted ion chromatograms at 181 m/z, a outstanding fragment ion for pentafluorobenzyl esters, have been analyzed through Chemstation Enhanced Knowledge Evaluation program. We recognized compounds of curiosity by comparability with derivatized genuine requirements.

Linear regression was used to generate a normal curve for every derivatized FFA utilizing requirements between 0 and 750 μM. Extra particularly, the extracted ion chromatogram peak space of the FFA of curiosity was normalized by the extracted ion chromatogram peak space of the derivatized nonanoic acid inner normal inside every pattern to generate an space ratio for every FFA current in the usual. Equally, the common space ratios from organic replicates (n = 3) have been calculated for every FFA current within the samples and in comparison with a normal curve generated in the identical media to find out the focus of the FFA. If any calculated FFA space ratio inside a pattern fell outdoors the linear vary of the usual curve, then a contemporary aliquot of the pattern was diluted 5x in media, spiked with inner normal, and re-analyzed to extra precisely calculate the focus of the FFA.

FAME quantification through GC/MS

All medium-chain FAMEs have been discovered to evaporate from liquid media when maintained at 30 °C and 200RPM for 48 h. Subsequently, a 1:10 isopropyl myristate (IPM) overlay was utilized in all samples from which we quantified FAME titers. As soon as prepared for evaluation, 1 mL from the floor of every pattern was centrifuged at 21,300×g for 10 min. After centrifugation, 20 μL of the IPM layer was collected and diluted 10x with ethyl acetate spiked with 500 μM methyl nonanoate as an inner normal. In parallel, FAME requirements have been ready by making a 0 μM, 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, 1 mM, 1.5 mM, and a couple of mM combination of every even-chained FAME between C6 and C16 in a 1:10 resolution of IPM in ethyl acetate spiked with 500 μM methyl nonanoate as an inner normal in triplicate.

Samples have been run on an Agilent 6890 GC and Agilent 5973 MS utilizing a 30 m × 0.250 mm × 0.25 μm HP-5MS column with a helium circulate of 1.3 mL/min. The GC was run in splitless mode with a 1 μL pattern injection and a continuing inlet temperature and switch line temperature of 280 °C. The oven beginning temperature was 50 °C and held for 1 min. Then, the oven temperature was elevated at a charge of 10 °C/min till 160 °C. Lastly, the temperature was elevated at a charge of 30 °C/min till 250 °C and held for five min. A solvent delay of 4.8 min was used and the MS was run in scan mode (50.0–350.0 amu). The extracted ion chromatograms at 74 m/z, a outstanding fragment ion for FAMEs, have been analyzed through Chemstation Enhanced Knowledge Evaluation program. We recognized compounds of curiosity by comparability with genuine requirements.

Linear regression was used to generate a normal curve for every FAME utilizing requirements between 0 and a couple of mM. Extra particularly, the extracted ion chromatogram peak space of the FAME of curiosity was normalized by the extracted ion chromatogram peak space of the methyl nonanoate inner normal inside every pattern to generate an space ratio for every FAME current in the usual. Equally, the common space ratios from organic replicates (n = 3) have been calculated for every FAME current in samples and in comparison with the usual curve to find out the focus of the FAME.

Regardless of the usage of an IPM overlay, we discovered that medium-chain FAMEs have been nonetheless partially evaporating. To account for this, each manufacturing run had clean media controls (n = 3) that have been spiked with a 500 μM FAME combination earlier than the addition of the IPM overlay. These controls have been incubated alongside all organic samples and have been equally processed for FAME quantification. The typical space ratios of every FAME within the spiked FAME controls have been in comparison with the world ratios akin to 500 μM FAME in the usual curves and used to calculate an evaporation coefficient that accounts for the evaporation of FAMEs following the formulation beneath (Eq. 1):

the place A_normal is the anticipated space ratio of a specific 500 μM FAME as calculated from the usual curve and A_spiked is the noticed space ratio of a specific 500 μM FAME that was spiked right into a media management and incubated alongside organic samples. After calculating this coefficient for every FAME, this worth was multiplied by the unique calculated FAME focus to find out a FAME focus adjusted for evaporation.

Theoretical yield calculations

Utilizing the COBRApy metabolic modeling package deal in Python⁵⁰, the utmost theoretical yield was calculated with the P. putida KT2440 mannequin IJN1463⁵¹ assuming no carbon flux to biomass. Particular person thioesterase reactions have been added for every free fatty acid analyte and methyl transferase reactions for the methyl ester analytes.

Statistics and reproducibility

All experiments involving the quantification of FFAs or FAMEs from bacterial liquid cultures have been performed with three biologically unbiased samples (n = 3) the place every replicate was began from a unique colony. The reported values have been the imply of those biologically unbiased samples and error bars present the usual error of the imply.

Reporting abstract

Additional data on analysis design is on the market within the Nature Portfolio Reporting Abstract linked to this text.