Patent Application
20220356495
The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy as filed herewith was originally created on 15 Jul. 2022. The ASCII copy as filed herewith is named NREL 21-21_ST25.txt, is 14,467 bytes in size and is submitted with the instant application.
The biological conversion of gaseous waste streams (CO2, stranded natural gas, flue gas, biogas, landfill gas, etc.) to a platform chemicals such as 3-hydroxybutyrate, which can in turn be upgraded to fuels and polymers (e.g. polypropylene and polymers), is, hitherto fore, an unmet need for the generation of renewable commodity chemicals.
In an aspect, disclosed herein are a non-naturally occurring organism capable of converting gases selected from the group consisting essentially of CO2, stranded natural gas, flue gas, biogas, and landfill gas to 3-hydroxybutyrate. In an embodiment, the non-naturally occurring organism is a methanotrophic bacteria. In an embodiment, the organism is genetically engineered to overexpress PHB depolymerase and also lacks an acetoacetyl-CoA synthetase (AACS) pathway.
In an aspect, disclosed herein is a method for making 3-hydroxybutyrate comprising the step of contacting a non-naturally occurring organism with gases selected from the group consisting essentially of CO2, stranded natural gas, flue gas, biogas, and landfill gas. In an embodiment, the non-naturally occurring organism is a methanotrophic bacteria. In another embodiment, the non-naturally occurring organism is genetically engineered to overexpress PHB depolymerase and also lacks a AACS pathway.
Other objects, advantages, and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.
Using biological systems to produce chiral compounds like 3-hydroxybutyrate (3-HB) requires lower temperatures and produces less toxic waste, fewer emissions and by-products than conventional production routes, and thus represents a sustainable alternative to conventional chemical synthesis. Hydroxycarboxylic acids, like 3-HB, can be used widely as chiral precursor for antibiotics, pharmaceuticals and fungicides, while oligomerized 3-HB has the potential to serve as a drug or antioxidant delivery vector. As an abundantly produced physiological ketone, 3-hydroxybutyrate is also thought to have an array of potential medical applications—for example as treatment for patients with tear-deficient dry eye disease. In addition, monomeric 3-HB has the potential to serve as a substrate for high-value pure polyhydroxybutyrate (PHB) or its co-polymers with superior properties compared to microbially produced PHB. Another advantage of 3-HB compared to PHB is that as an extracellular product, 3-HB can be collected straight from the medium enabling cell reuse, which is useful for relatively slow growing biomass.
Various efforts are underway to produce microbial 3-HB. The halophilic bacteria Halomonas sp. KM-1, which stores intracellular PHB under aerobic conditions and secretes the monomer under microaerobic conditions, achieved secretion of one of the highest 3-HB titers to date. In optimized culture conditions this organism produced up to 40.3 g/L (R)-3-HB in a nitrate fed-batch cultivation with 20% (w/v) glucose. Most of the efforts to produce 3-HB in metabolically engineered strains has so far been focused on the expression of heterologous pathways in model organisms such as Escherichia coli or Saccharomyces cerevisiae. A recombinant E. coli strain, overexpressing both its native thioesterase yciA and its glucose-6-phosphate dehydrogenase zwf alongside a heterologous thiolase and reductase from Halomonas boliviensis yielded the maximum titer to date in E. coli (14.3 g/L). Maximum titers in the non-conventional yeast Arxula adeninivorans were achieved by overexpressing the bacterial thiolase thl and the enantiospecific reductase (phaB) from Cupriavidus necator H16. Being able to thrive on CO2 and sunlight, cyanobacteria have become an attractive host for the production secondary metabolites from cheap, renewable feedstock. However, with a maximum titer to date of 533.4 mg/L from a genetically engineered Synechocystis, cyanobacterial titers lack far behind those of their heterotrophic counterparts.
With CH4 and CO2 being the two most abundant anthropogenic greenhouse gases and Type II methanotrophs metabolizing both of those gases, these biocatalysts not only offer a promising greenhouse gas sink, but might also be an auspicious alternative production chassis for 3-HB. Various biochemical pathways can be envisioned to generate 3-HB levels in Type II methanotrophs. In this study, four different pathways were overexpressed, one of which yielded the highest titer of CH4-derived acid to our knowledge to date (
In an embodiment, disclosed herein are compositions of matter comprising non-naturally occurring organisms that generate 3HB.
Generation of 3HB-Production Strains and their 3HB Production Capacity
The 3HB production capability of five different M. trichosporium OB3b strains harboring one of four different overexpression plasmids was assessed. The same basic overexpression backbone was used for all four plasmids: a promoter and terminator sequence flanking the gene of interest (GOI) arranged as an operon on an IncP broad-host-range plasmid. Since the native mxaF gene showed high expression levels in transcriptomic studies and the upstream genomic region of this gene was found to drive strong gene expression of reporter proteins, this region was used as a promoter sequence in this study. Ribosome binding sites (RBS) linking the GOI were either selected from the native genome, designed with the RBS calculator or derived from the consensus RBS AGGAGG+6 or 9 base pair downstream. All four plasmids were introduced in M. trichosporium OB3b WT background. Considering that a simple bdh null mutant in R. eutropha H16 led to a 1.67 fold increase for the mutant compared to the wild type (18 mM (=1.87 g/L) to 30 mM (=3.12 g/L)) for the mutant 3-HB in the medium, the complete PHB operon+depolymerase was also introduced into backgrounds where the flux between acetyl-CoA and 3-HB would be interrupted (aacS and bdh null mutants).
Initial analysis for 3HB production capacity in batch culture in serum vials revealed three out of five overexpression strains capable of producing measurable amounts of 3HB in the supernatant while the other two strains did not exceed background 3-HB levels. If the native PHB depolymerase (pIPRJ016) was overexpressed by itself, 3-HB levels in the supernatant only increased significantly after cells reached a stage in their growth phase when they would naturally produce PHB. Interestingly, cells overexpressing the complete PHB pathway plus the PHB depolymerase, but lacking the native acetoacetate-CoA ligase gene aacS or butyrate dehydrogenase bdh, outperformed the respective WT background in 3-HB production—indicating 3-HB reassembly capacity in the WT background. Given enough time, a similar effect was observed in the KO background indicating a switch in cellular metabolite or reductant balance (
Further increase in 3-HB titers might be accomplished by treating the culture supernatant with a 3-HB oligomer hydrolase. It was shown that 3-HB in the culture supernatant of an E. coli co-expressing a Ralstonia eutropha PHB operon and an extracellular Paucimonas lemoignei PHB depolymerase gene could be increased about 10 fold in the presence of a 3HB oligomer hydrolase from R. pickettii T1. In the same study a R. eutropha bdh null mutant produced up to 3.12 g/L 3-HB under anaerobic conditions, indicating potential superior 3-HB production in M. trichosporium overexpressing the phaABCZ1Z2 operon in a bdh KO or a aacS/bdh double-KO background.
Cultivation and Growth Parameters.
M. trichosporium OB3b were routinely cultured at 30° C. in a modified nitrate mineral salts (NMS) medium. Selective plates for M. trichosporium OB3b plates were supplemented with 25 μg/ml kanamycin, 50 μg/ml spectinomycin, 20 μg/ml gentamicin, whereas only 10 μg/ml kanamycin and 10 μg/ml gentamicin was used for cultivation in liquid NMS in sealed glass serum bottles with orbital shaking at 200-250 rpm. Liquid cultures were inoculated at OD600=0.1 (batch cultures) or 0.4 (continuous reactor) with plate-harvested biomass. Batch cultures were cultured as 30 ml culture volume in 160 ml sealed serum vials supplemented with 20% CH4 and 20% CO2 in the headspace. Continuous reactor cultures were cultured as 100 ml culture volume in 160 ml borosilicate glass test tubes purchased from Kimble Kimax (Vineland, N.J.) continuously bubbled with 20% CH4 and 2% CO2 as described earlier. Escherichia coli Stellar (Takara Bio USA, Mountain View, Calif.) was used for cloning and plasmid propagation, and E. coli S17-1λ pir was used as the conjugation donor strain. E. coli strains were grown at 37° C. in Luria-Bertani (LB) broth supplemented with 50 ug/mL of kanamycin. Growth of E. coli or M. trichosporium OB3b was monitored by measuring the OD600 using a spectrophotometer in a 1 ml cuvette.
Plasmid Construction and Strain Generation.
Heterologous genes reference or codon optimized sequences are depicted in Table 2.
Methylosinus trichosporium OB3b phaA
Methylosinus trichosporium OB3b phaB
Methylosinus trichosporium OB3b phaC
Methylosinus trichosporium OB3b phaZ1
Methylosinus trichosporium OB3b phaZ2
Methylosinus trichosporium OB3b aacS
Methylosinus trichosporium OB3b bdh
Clostridium butyricum ptb
Clostridium butyricum buk
C.
butyricum
Strains and plasmids used in this study are presented in Table 4.
Methylosinus trichosporium
Methylosinus trichosporium
Methylosinus trichosporium
E. coli Stellar (an E. coli
E. coli S17-1 λpir
M. trichosporium OB3b
M. trichosporium OB3b
M. trichosporium OB3b
Clostridium butyricum
M. trichosporium OB3b
Plasmids for heterologous gene expression were constructed using 5× InFusion HD Enzyme Mix from Takara Bio USA, Inc. (Mountain View, Calif.) following the manufacturers protocol. Polymerase chain reactions were performed using Q5 High-Fidelity Polymerase from New England Biolabs and primers (Table 5) purchased from Integrated DNA Technologies (Coralville, Iowa). Heterologous genes under the control of the M. trichosporium OB3b mxaF (locus tag CQW49_RS14460 in NCBI Reference Sequence: NZ_CP023737.1) upstream region (PmxaF) were inserted into a modified IncP-containing pAWP78 backbone. Final constructs were confirmed by sequence analysis (Genewiz, South Plainfield, N.J.).
tataaaaaATGGCATGGGGCTTGC
gttataaaaATGTCTCTCGCCAAACGC
aggttttttATGACCGCAGGACGCCGC
C. butyricum ptb
C. butyricum
Plasmid constructs were transformed into M. trichosporium OB3b via conjugation as previously described with the following modifications: an overflowing 10 ul inoculation loop of M. trichosporium OB3b was spread on NMS mating plates and grown overnight. An equal volume of Escherichia coli S17-1 λpir donor biomass containing the vector of interest was then added to the plate, and the resulting mixture was incubated at 30° C. for 2 days. The resulting biomass was collected, resuspended in NMS liquid medium and a fraction was plated on selective NMS plates supplemented with 10 μg/ml nalidixic acid to remove S17 donor cells. Stock solutions of nalidixic acid were prepared at 10 mg/ml in H2O. After adjusting the pH to 11, the solution was sterile filtered and aliquots were stored at −20° C. Genomic knock-outs of M. trichosporium OB3b cells were generated by removing the coding sequence via homologous recombination induced by introduction of a linear DNA fragment via electroporation as described previously. The linear DNA fragments, including 1 kb DNA regions upstream and downstream of the gene of interest and a gentamicin resistance cassette, were constructed via fusion PCR as described previously, whereas overlapping ends were only added to the homology region to ease reusability of the selection marker. If fusion PCR was not successful the respective linear DNA fragment was ordered as a clonal gene from Twist Biosciences, San Francisco, Calif., and amplified from the vector via high fidelity PCR. KO background strains were confirmed via high fidelity PCR for specific target regions (see
Relative PHB Quantification Via Staining Cells with Nile Red.
Cellular PHB concentration was quantified relative to a WT sample by staining WT and mutant strain with nile red. Cells equivalent to an OD600 of 5 were harvested and the resulting pellet was resuspended in 900 μl NMS+100μ nile red solution (250 μg/ml nile red in dimethyl sulfoxide). After 15 min incubation, 200 μl aliquots were assayed for fluorescence at excitation (535 nm) and emission (605 nm) wavelength at a plate reader (Infinite M PLEX by Tecan Trading AG, Switzerland).
Acid Quantification in the Supernatant
To quantify acid levels in culture supernatant, an aliquot of culture broth was taken, filtered through a 0.2 μm syringe filter (SY25GN by mdi Membrane Technologies INC., Harrisburg, Pa.). The resulting clear supernatant was either analyzed by HPLC or 3-HB levels were determined using the β-Hydroxybutyrate (Ketone Body) Colorimetric Assay Kit by Cayman Chemical, Ann Arbor, Mich. following the manufacturers protocol. According to the manufacturer, the kit only detects the 3-HB D-isoform—which appears physiologically in humans and other animals—due to enzyme stereo-specificity. Succinic, lactic, formic and acetic acid levels were quantified via HPLC as described earlier.
Dry Cell Weight Measurement
Microbial cultures (7-90 ml) were centrifuged to collect biomass in tubes of known weight. Cell pellets were dried in those tubes in a 45° C. vacuum oven for at least 48 h prior to weighing them again. DCW was determined by subtracting the empty tube weight from the tube weight plus dried biomass and dividing by the volume of biomass harvested. The calculated DCW (mg/L) was plotted over OD600 and a linear model using the lm( ) function in R yielded a linear regression curve with y-intercept=−8.058, slope=228.629, p-value: 2.2×10−16.
NAD+/NADH Quantification.
To determine NAD+/NADH or NADP+/NADPH levels in M. trichosporium cells, mid-log culture was harvested to yield 3.33×107 cells/well of a 96 well plate and analyzed with the NAD/NADH-Glo™ or NADP/NADPH-Go™ kit by Promega Corporation (Madison, Wis.). The cell pellet was resuspended in 300 ul high pH Bicarbonate Buffer+1% DTAB and either processed immediately or, if necessary, stored at −80° C. 100 μl samples were acid/base treated, neutralized and 50 μl of the final reagent mixture was analyzed with the detection reagent following the manufacturers protocol. Relative luminescence units were determined after 60 min using white, flat bottom 96-well microplates procured from Tecan Trading AG, Switzerland, in an Infinite M PLEX plate reader (Tecan Trading AG, Switzerland).
RNA Isolation and RT-qPCR
To compare transcript levels to the wild-type levels, 1 ml of mid-log culture (corresponding to an OD600 between 3.5 and 10) from the continuous bioreactor was harvested. RNA levels were stabilized by resuspending the culture in 1 ml RNAlater (Thermo Fisher Scientific Inc., Waltham, Mass.) and samples were stored at −80° C. till further processing. RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) along with on-column DNase Digestion using the RNAse-Free DNase Set (Qiagen, Hilden, Germany) following the manufacturer's procedures. RNA levels were determined using the iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad, Hercules, Calif.) in a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Waltham, Mass.). Relative expression levels of target genes in a sample relative to a wild-type control were calculated using the ΔΔCt method with the M. trichosporium OB3b RNA polymerase sigma factor rpoD housekeeping gene (locus tag CQW49_RS04780 in CP023737.1) as reference gene.
The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting.
This application claims priority under 35 U.S.C. § 119 to U.S. provisional patent application No. 63/170,257 filed on 2 Apr. 2021, the contents of which are hereby incorporated in their entirety.
The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.
| Number | Date | Country | |
|---|---|---|---|
| 63170257 | Apr 2021 | US |
