Production of isoprene by methane-producing archaea

Abstract

Plasmid vectors and use of plasmid vectors in methods for producing methane and isoprene using Archaea are disclosed. Particularly, plasmid vectors that express isoprene synthase (ispS) are prepared and inserted into methanogens, such as Methanosarcina acetivorans, to allow for co-production of methane and isoprene. In one embodiment, the methods of the present disclosure can be used for wastewater management.

Claims

1. A transformed host cell comprising a vector comprising a nucleic acid encoding isoprene synthase having one of SEQ ID NO:1 and SEQ ID NO:2.

2. The transformed host cell of claim 1 wherein the cell is a microorganism selected from the group consisting of methanogen, methanotroph, and acetogen.

3. The transformed host cell of claim 2 wherein the cell is a Methanosarcinales strain.

4. The transformed host cell of claim 3 wherein the cell is selected from the group consisting of Methanosarcinales acetivorans and Methanosarcinales barkeri.

5. A vector comprising one of a nucleic acid of SEQ ID NO:1 and SEQ ID NO:2 encoding isoprene synthase.

6. A method of preparing a transformed microbial strain capable of producing isoprene, the method comprising: preparing a vector comprising a nucleic acid encoding a Populus alba isoprene synthase, wherein the nucleic acid is one of SEQ ID NO:1 and SEQ ID NO:2 by preparing a primer comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; amplifying a template Populus alba isoprene synthase cDNA with the primer to obtain an isoprene synthase cDNA; annealing the isoprene synthase cDNA with a plasmid to obtain the vector; introducing the vector into a host cell from a microorganism selected from the group consisting of methanogen, methanotroph and acetogen; and anaerobically culturing the transformed host cell including the vector in a culture medium comprising a carbon source.

7. The method of claim 6 wherein the host cell is a cell selected from the group consisting of Methanosarcinales acetivorans and Methanosarcinales barkeri.

8. The method of claim 6 wherein culturing the transformed host cell including the vector comprises culturing the transformed host cell anaerobically in high salt medium at 35 C. until OD.sub.600 reaches between about 0.5 and about 0.7.

9. The method of claim 6 wherein the carbon source is selected from the group consisting of carbon dioxide, methanol, methylamines, methylsulfides, methylated metal/metalloids, formate, carbonate, graphite, carbon monoxide, acetate, methane, and combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

(3) FIG. 1A depicts a plasmid vector map of one exemplary plasmid vector for use in the methods of the present disclosure.

(4) FIG. 1B depicts a plasmid vector map of another exemplary plasmid vector for use in the methods of the present disclosure.

(5) FIG. 2 depicts increased enzymatic activity in M. acetivorans cells, which have been transformed with the exemplary plasmid when grown on TMA, MeOH, or acetate as the carbon source.

(6) FIG. 3A depicts M. acetivorans growth on TMA as analyzed in Example 1.

(7) FIG. 3B depicts M. acetivorans growth on MeOH as analyzed in Example 1.

(8) FIG. 4 depicts biomass produced by M. acetivorans growth on MeOH as analyzed in Example 1.

(9) FIG. 5 depicts methane production by M. acetivorans grown on MeOH or TMA as analyzed in Example 1.

(10) FIG. 6 depicts M. acetivorans methane production on TMA with tetracycline as analyzed in Example 1.

(11) FIG. 7 depicts isoprene production by M. acetivorans grown on MeOH, TMA, or acetate as analyzed in Example 1.

(12) FIG. 8 depicts methane production by M. barkeri grown on MeOH as analyzed in Example 1.

(13) FIG. 9 depicts double-blind confirmation of isoprene production by M. acetivorans grown on MeOH as analyzed in Example 1.

(14) FIG. 10 depicts growth of M. acetivorans in synthetic wastewater as analyzed in Example 1.

(15) FIG. 11A depicts M. acetivorans growth on methane supplemented with sodium sulfide as analyzed in Example 1.

(16) FIG. 11B depicts microscopy confirming growth of M. acetivorans on methane.

(17) FIGS. 12A-12D depict the technoeconomic analysis of using methanogens to produce bioisoprene and other renewable products.

DETAILED DESCRIPTION

(18) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.

(19) The present disclosure is generally directed to methods for producing methane and isoprene using Archaea. More particularly, the present disclosure is directed to the use of methanogens, and in particularly suitable embodiments, Methanosarcina acetivorans or Methanosarcina barkeri, transfected with engineered plasmid vectors that allow for the methanogens to co-produce isoprene and methane. The plasmid vectors can be stably integrated into the methanogen chromosome to reduce the probability that the isoprene-production trait could be lost.

(20) Generally, the present disclosure provides plasmid vectors including a nucleic acid encoding an isoprene synthase (ispS). Isoprene synthase (ispS) is an enzyme that converts dimethylallyl diphosphate into isoprene. In one aspect, the codons of the ispS protein sequence can be semi-optimized such that most codons are adjusted to match the Methanosarcina tRNA abundances. In several positions low-frequency tRNA can be selected to decrease gene expression so that isoprene production is not lethal. Suitable primers for use in providing the plasmid vectors include the primers with nucleotide sequences represented by SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO:5.

(21) Further, in one embodiment, the plasmid vectors of the present disclosure include the nucleic acid having (i) the nucleotide sequence represented by SEQ ID NO:1, or (ii) the nucleotide sequence represented by SEQ ID NO:2, which is the nucleotide sequence consisting of the nucleotide residues at positions 1-3701 and 3813-6382 in the nucleotide sequence represented by SEQ ID NO:1. The nucleotide sequence consisting of the nucleotide residues at positions 3702-3812 can encode a putative chloroplast localization signal (CLS).

(22) In another embodiment, the nucleic acid includes a nucleotide sequence having 90% or more identity to the nucleotide sequence of (i) or (ii) above, and encodes a protein having an isoprene synthase activity. The percent identity to the nucleotide sequence may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. The isoprene synthase activity, as used herein, refers to an activity to form isoprene from dimethylallyl diphosphate (DMAPP) or isopentenyl pyrophosphate.

(23) The percent identity of the nucleotide sequences can be determined using algorithm BLAST (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) by Karlin and Altschul, and FASTA (Methods Enzymol., 183, 63 (1990)) by Pearson. The programs referred to as BLASTP and BLASTN were developed based on this algorithm BLAST (see, www.ncbi.nlm nih.gov). Thus, the percent identity of the nucleotide sequences may be calculated using these programs with default setting. The lowest value among the values derived from these calculations may be employed as the percent identity of the nucleotide sequences.

(24) Exemplary plasmid vectors prepared using the methods of the present disclosure are shown in FIGS. 1A & 1B. As shown, the plasmid vectors of FIGS. 1A & 1B include the strong constitutive promoter PmcrB. The difference in the two plasmid vectors is that the plasmid vector shown in FIG. 1A includes CLS, while the plasmid vector shown in FIG. 1B does not include CLS. Additionally suitable plasmid vectors include vectors similar to those shown in FIGS. 1A & 1B with the exception of using a tetracycline-inducible promoter, Ptet.

(25) The present disclosure further provides a transformant comprising the plasmid vector. The transformant of the present disclosure is one obtained by introducing the plasmid vector of the present disclosure into a host chromosome. The host used for the present invention is preferably a microorganism such as an Archaea, a bacterium (both gram-positive or gram-negative) or a fungus. In particularly suitable embodiments, the host includes methanogens. Methanogens are grown in HS+MeOH media until OD.sub.600 reaches about 0.5 to 0.7 and then transfected with the plasmids using conventional methods. While described herein as using M. acetivorans and M. barkeri, it should be understood that any Methanosarcina strains can be used as the host cells without departing from the scope of the present disclosure. Further, it is believed that the plasmids and vector system should be capable of ispS functionality in any methanogen, methanotroph, or acetogen.

(26) Advantageously, the methanogens can use any carbon source known in the art for making isoprene. In some embodiments, methanogens (e.g., M. acetivorans and M. barkeri) can use syngas (i.e., gaseous mixtures of any one or several of: CO, CH.sub.4, H.sub.2+CO.sub.2), such as derived from cellulosic biomass by thermal cracking or coal production, as a growth substrate. Other suitable growth substrates include for example, methanol (and methoxy compounds), methylamines (monomethylamine, dimethylamine, trimethylamine, and derivatives), methylsulfides (methanethiol, dimethylsulfide and derivatives), methylated metal/metalloids, formate, carbonate, graphite, carbon monoxide, acetate, and the like, and combinations thereof.

(27) It was found that once transfected, the methanogen host cells co-produce methane and isoprene. It was expected that isoprene production would result in a decrease in methane as carbon should be directed towards isoprene. As shown in the Examples below, expression of ispS had a 30% decrease in methane production in M. barkeri, which indicates these adapted cells may be producing as much as 6% isoprene per mole basis. Further, a surprisingly large effect on methane production was seen in the ispS-producing M. acetivorans strains, which were missing 87% of the carbon, which would translate to a 17.6% isoprene production efficiency per mol basis (FIGS. 3A & 3B).

(28) It is believed that the plasmid vectors engineered herein and used in the methods of the present disclosure would be advantageous in many industries, including municipal, agricultural, and industrial waste treatment, and in a particularly suitable aspect, the methods are used in wastewater treatment plants. Conventionally, isoprene can be produced by engineered E. coli, yeast or algae, but because they are different organisms, each has different requirements for feedstocks. Depending on the organism, each has different requirements for a production facility: algae must be aerated, while E. coli and yeast can be grown under aerated or anaerobic conditions using glucose or protein feedstocks. As such, methanogen technologies, such as the methods of the present disclosure, can be used in anaerobic digesters and anaerobic fermentation technologies using E. coli or yeasts to improve yield of upstream value-added bioproducts. The methanogens will increase efficiency and overall yield of microbial conversion technologies and improve the overall economic profit margins for those technologies (see FIGS. 9A-9D). The lack of specialized facilities to grow methanogens and collect bioproducts is a significant advantage versus algae technologies or aerobic microbial technologies, which require effort, energy, and cost to add oxygen.

(29) Other industries that are drop-in compatible with the methods of the present disclosure include, by way of example only, ethanol, butanol and other transportation or renewable solvent or biodiesel production facilities, syngas bioproduct facilities, renewable bioplastics, vitamins and amino acids and the like produced by microbial fermentation (e.g., animal feed additives, human food additives, plastics, cosmeceuticals), and the like. One further industry includes the coal industry.

EXAMPLES

Example 1

(30) In this Example, plasmid vectors were engineered and cells were transfected with the plasmid vectors. The ability of the cells to produce isoprene and methane was then analyzed.

(31) Initially, cDNA of the isoprene synthase from Populus alba was obtained by RT-PCR with the total RNA obtained as a template using primers designed based on the analyzed nucleotide sequence information of the isoprene synthase gene from Populus alba. Particularly, plasmids were engineered using the protocol adapted from Li et al., Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC, Nat Meth 4, 251-256 (2007), in which the primers are designed with a 25-basepair homology with the parental plasmid (i.e., pNB730) following digestion (insertion into multiple cloning site (MCS)) as well as a 15-20 basepair homology to isoprene synthase (ispS) from P. alba. Particularly, 2 g of parental plasmid was digested with restriction enzymes. The primers have the nucleotide sequences of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

(32) After digestion, linearized parental plasmid was purified following PCR Clean up protocol (commercially available as WIZARD SV Gel and PCR Clean-Up System (Promega, Madison, Wis.) Ample amount of restriction enzyme as well as enough time for complete digestion (e.g., at least 3 hours) was provided. DNA from the Clean up column was eluted with ddH.sub.2O. ispS was PCR-amplified following the Phusion Flash protocol (commercially available as Phusion High-Fidelity DNA Polymerase, Thermo Scientific, Lenexa, Kans.) using the primers as designed above. The amplified gene of interest was purified using PCR Clean up protocol, and DNA was eluted from the Clean up column using ddH.sub.2O.

(33) T4 Treatment

(34) Next, 1 g of digested parental plasmid and 1 g of the amplified ispS were separately treated with 0.5 U of T4 DNA polymerase in 1NEBuffer 2 plus BSA in a 20 l reaction at room temperature for 30 minutes. The reaction was stopped by adding 1/10 total volume of 10 mM dCTP and left on ice.

(35) Annealing Mixture

(36) After the T4 treatment was completed, a 10 L annealing reaction was conducted using a 1:1 ispS to parental plasmid molar ratio. The amount of parental plasmid was 150 ng. 1 ligation buffer (NEB), appropriate amount of T4 treated ispS (1:1 molar ratio between parental T4 treated plasmid and T4 treated ispS), and water were then added to the parental plasmid. The mixture was incubated in a 37 C. water bath for 30 minutes. After the reaction, the mixture was returned to ice until further use.

(37) Transformation

(38) 5 L of the annealed mixture was added to 150 L of chemically competent cells (E. coli), the mixture was incubated on ice for 30 minutes, heat shocked at 42 C. for 45 seconds, and then returned to ice for 2 minutes. 0.9 mL of super optimal broth with catabolite repression (SOC) was added to the mixture, the cells were allowed to recover at 37 C. for 1 hour. After recovery, cells were plated on LB agar plates supplemented with 100 mg/L ampicillin and incubated overnight at 37 C.

(39) Transfecting cells with the plasmids.

(40) All of the following methods were performed anaerobically in a 20% CO.sub.2/5% H.sub.2/75% N.sub.2 environment unless otherwise stated. Methanogens were grown in HS+MeOH media, 10 ml culture per transformation for M. acetivorans and 40 ml culture per transformation for M. barkeri. Cells were grown at 35 C. for two days or until OD.sub.600 reached between 0.5-0.7. Cells were concentrated via clinical centrifugation (15 minutes for M. acetivorans, 2 minutes for M. barkeri) and media removed. 2 g of Vector DNA was diluted to 50 l with sucrose and prepared with DOTAP reagent in sterile borosilicate glass test tubes according to the manufacturer's protocol. DNA and DOTAP was gently mixed and incubated at room temperature for 15 minutes. Cell pellets were resuspended in 1 mL of sucrose and added directly into the DNA/DOTAP solution. The solution was allowed to incubate for 4 hours at room temperature. The entire volume of the solution was then added to 25 mL of fresh high salt medium (HS)+MeOH (40 mM) media and incubated overnight (16 hours) at 35 C. (Sowers et al., 1993 Appl Env Microbiol. P3832-3839).

(41) M. acetivorans was plated on agar plates by applying the cells directly onto agar, whereas M. barkeri was first mixed with a 0.5% top agar then applied gently to the agar plate. Plates were incubated under H.sub.2S for 14 days and checked for colony formation. Isolated colonies were streaked for selection on agar plates containing puromycin.

(42) A M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans were grown anaerobically in 10 mL HS media containing either 125 mM methanol (MeOH), 40 mM trimethylamine (TMA), or 120 mM acetate. Cells were collected and the EnzCheck pyrophosphatase biochemical assay kit (ThermoFisher Scientific) was used to confirm the introduced ispS gene resulted in increased pyrophosphate production from isopentenyl-pyrophosphate in whole cell lysates. It should be understood that normal cells will produce pyrophosphate from isopentenyl-pyrophosphate as a part of normal cellular lipid synthesis. In this assay, higher pyrophosphate release from isopentenyl-pyrophosphate indicates either higher lipid synthesis or isoprene production from the isopentenyl-pyrophosphate substrate. As shown in FIG. 2, strains expressing ispS had higher enzymactic activity as compared to the vector only control (VOC) strains.

(43) A M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans were grown anaerobically in 10 mL HS media containing 40 mM trimethylamine (TMA). Optical density at 600.sub.nm was recorded using a Spectronic 20D+ (Thermo Scientific, Waltham, Mass.) at 4 hour increments. As shown in FIG. 3A, transfecting M. acetivorans cells with an ispS plasmid vector did not negatively affect the growth of M. acetivorans in TMA. Additionally, a M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans were grown anaerobically in 10 mL HS media containing 125 mM methanol (MeOH). Optical density at 600.sub.nm recorded using the Spectronic 20D+ at 4 hour increments. As shown in FIG. 3B, transfecting M. acetivorans cells with an ispS plasmid vector did not negatively affect the growth of M. acetivoran in MeOH.

(44) Endpoint methane production of the M. acetivorans vector only control (VOC), the parental plasmid, and ispS expressing strains of M. acetivorans, with and without CLS, grown anaerobically in 10 mL HS media containing 40 mM TMA was also analyzed. Methane production was measured by sampling headspace gasses using an Agilent Technologies 7890A Gas Chromatography system equipped with a flame ionization detector (FID). As shown in FIG. 5, methane production was decreased in both the ispS expressing strains as carbon should have been directed towards isoprene.

(45) Experimental data shows ispS expressing cells grew better than vector only control (VOC) strains. As shown in FIG. 4, dry weight biomass measurements of cultures show ispS expressing cells accumulated more biomass than VOC cultures.

(46) Endpoint methane production of the M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans without CLS grown anaerobically in 10 mL HS media containing 125 mM methanol was also analyzed. Methane production was measured by sampling headspace gasses using an Agilent Technologies 7890A Gas Chromatography system equipped with a flame ionization detector (FID). As shown in FIG. 5, methane production was decreased in the ispS expressing strain as, again, carbon should have been directed towards isoprene.

(47) A summary of the data shown in FIGS. 2-5 are shown in Table 1 below.

(48) TABLE-US-00001 TABLE 1 Summarized data from FIGS. 2-5 containing respective standard deviations and p-values Growth Growth Growth Methane % Rate Rate std Rate p vs production Methane Methane Strain (hr) dev VOC (nmol) std dev yield Methanosarcina acetivorans grown on 125 mM MeOH att::VOC 9.57 0.36 1 4948.06 146.01 100 att::ispS - CLS 9.01 0.42 0.77 4759.15 238.39 96.2 Methanosarcina acetivorans grown on 40 mM trimethylamine att::VOC 11.75 0.31 1 20512.20 654.69 100 att::ispS - CLS 12.94 1.92 0.472 2926.99 359.00 14.3

(49) Similarly, endpoint methane production of a M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans grown anaerobically in 10 mL HS media containing 100 mg/L tetracycline was analyzed. As shown in FIG. 6, methane production decreased in the ispS expressing strain.

(50) Further, endpoint isoprene production of a M. acetivorans vector only control (VOC) and an ispS expressing strain of M. acetivorans grown anaerobically in 10 mL HS media containing either 125 mM methanol (MeOH), 40 mM trimethylamine (TMA), or 120 mM acetate were analyzed. As shown in FIG. 7, isoprene production increased in the ispS expressing strain on all tested substrates.

(51) Endpoint methane production of the M. barkeri vector only control (VOC), the parental plasmid, and ispS expressing strains of M. barkeri, with and without CLS, grown anaerobically in 10 mL HS media containing 40 mM TMA was also analyzed. Methane production was measured by sampling headspace gasses using an Agilent Technologies 7890A Gas Chromatography system equipped with a flame ionization detector (FID). As shown in FIG. 8, methane production was decreased in both the ispS expressing strains as carbon should have been directed towards isoprene.

(52) FIG. 9 shows isoprene detected by gas chromatography and flame ionization detection using authentic isoprene standards (as in FIG. 7) is confirmed by gas chromatography mass spectrometry. Cells either expressing ispS or vector only control (VOC) were grown on 125 mM methanol. Samples from the cultures were collected and marked by number code. Data shown in FIG. 9 was collected by double-blinded sample submission to the University of Nebraska Biotechnology Core Facility. The mass to charge ratio of the dominant chemical species in the ispS expressing culture samples has a molecular mass of 67.1, which is consistent with a negative ion of isoprene (C.sub.5H.sub.7).

(53) It was determined that ispS expressing cells grow in synthetic wastewater, which has chemical and ionic composition very similar to authentic wastewater but with known components. The synthetic wastewater is composed of peptone (28 mg/L), meat extract (100 mg/L), urea (100 mg/L), K.sub.2HPO.sub.4 (28 mg), sodium chloride (7 mg/L), calcium chloride dehydrate (4 mg/L), magnesium sulfate septahydrate (2 mg/L), sodium bicarbonate (3.8 g/L), agarose (2.3 g/L), evaporated milk (30 mL), 125 mM methanol. As shown in FIG. 10, ispS expressing cells (blue) are capable of growing to high cell density in synthetic wastewater.

(54) Recent published literature suggests M. acetivorans must be engineered to express a methyl-coenzyme M reductase enzyme from anaerobic methanotrophs to grow on methane as a carbon source (Soo V. W. et al. 2016. PMID: 26767617). The ability for the parent strain to grow on methane was also determined to evaluate if ispS expressing strains might also be able to grow on methane. M. acetivorans was added to high-salt (HS) culture medium that did not contain a carbon source. Pure methane was added to the culture headspace at 15 psi and cultures were incubated at 35 C. FIG. 11A shows that growth was enhanced by adding sodium sulfide (Na.sub.2S). FIG. 11B shows that cultures contained viable growing cells (irregular spheres) at high cell density in addition to struvite crystals (rhombic shapes).

(55) Table 2 below summarizes the yields of isoprene produced by M. acetivorans and M. barkeri on various substrates in batch cultures.

(56) TABLE-US-00002 TABLE 2 Isoprene production of Methanosarcina barkeri and Methanosarcina acetivorans grown on various carbon sources Growth Substrate Isoprene per 1 L culture (mg) Methanosarcina barkeri H.sub.2:CO.sub.2.sup.a 23.64 125 mM Methanol.sup.a 2.45 Methanosarcina acetivorans Methane.sup.a 5.23 120 mM Acetate.sup.b 62.42 18.45 50 mM Trimethylamine.sup.b 69.48 16.89 125 mM Methanol.sup.b 65.01 16.08 120 mM Acetate:125 mM Methanol.sup.a 25.89 .sup.asingle culture measurement. .sup.baverage of three biological replicates