# Patent

## Title
Engineered recombinant enzymes for methane oxidation

## Abstract

Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided.

## Background
This invention was made with government support under grant number GM070473 AND awarded by the National Institutes of Health and 1346523 (STTR Grant) awarded by the National Science Foundation. The government has certain rights in the invention.


FIELD
Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided.
BACKGROUND
Methane, the primary component of natural gas, is a cheap and abundant feedstock that is costly to transport and requires significant capital expenditures to convert to higher value products (refs. 1, 2; incorporated by reference in their entireties). As a result, natural gas produced in remote locations such as the Bakken Shale is flared, leading to over $18 billion worth of methane being wasted per year (ref 3; incorporated by reference in its entirety). One potential solution is to use biological systems for methane conversion. These systems are predicted to require lower capital expenditures per barrel than traditional gas-to-liquid Technology (refs. 1, 2; incorporated by reference in their entireties).
In nature, methane is aerobically oxidized by bacteria known as methanotrophs, which utilize it for energy production and carbon fixation (ref. 4; incorporated by reference in its entirety). Methane enters the methanotroph metabolic pathway by the action of methane monooxygenases (MMOs), which oxidize methane to methanol (ref. 4; incorporated by reference in its entirety).

R—H+O2+2H++2e−→R—OH+H2O

Nature employs two types of MMOs: soluble MMO (sMMO), which utilizes a diiron cofactor; and particulate MMO (pMMO), which utilizes a dicopper cofactor (FIG. 1) (refs. 5, 6; incorporated by reference in their entireties). A major issue limiting the development of biological gas-to-liquid technology is the inability to express sMMO or pMMO in an industrially relevant host organism (refs. 1, 2; incorporated by reference in their entireties).

SUMMARY
Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided.
In some embodiments, provided herein are compositions comprising a polypeptide capable of converting an alkane into an alkanol and/or an alkene to an epoxide. In some embodiments, the polypeptide is capable of converting methane into methanol. In some embodiments, the polypeptide is capable of converting propylene into propylene oxide. In some embodiments, the polypeptide carries out the hydroxylation of the alkane. In some embodiments, the polypeptide carries out the hydroxylation of the methane. In some embodiments, the polypeptide comprises a metal dependent oxygenase with an engineered active site. In some embodiments, the polypeptide is expressed in an industrially-relevant host organism and at a commercially-relevant scale. In some embodiments, the polypeptide comprises two domains, each with at least 60% sequence identity (e.g., >60%, >70%, >75%, >80%, >90%, >95%, >98%, >99%) with a domain of pmoB. In some embodiments, the polypeptide comprises S1-D1-linker-D2-S2, S1-D1-linker-D2, D1-linker-D2-S1, wherein S1 is a first soluble peptide, D1 is a polypeptide segment with at least 60% sequence identity (e.g., >60%, >70%, >75%, >80%, >90%, >95%, >98%, >99%) with a first soluble domain of pmoB, linker is a soluble peptide linker, D2 is a polypeptide segment with at least 60% sequence identity (e.g., >60%, >70%, >75%, >80%, >90%, >95%, >98%, >99%) with a second soluble domain of pmoB, and S2 is a second soluble peptide. In some embodiments, the polypeptide comprises at least 60% sequence identity (e.g., >60%, >70%, >75%, >80%, >90%, >95%, >98%, >99%) with spmoB7, sumo-spmoB7, and/or sumo-spmoB7 8pt3.
In some embodiments, provided herein are methods of converting an alkane (e.g., methane) into an alkanol (e.g., methanol) comprising exposing the alkane to a polypeptide described herein.
In some embodiments, provided herein are cells expressing a polypeptide described herein.
In some embodiments, provided herein are methods of biofuel production comprising exposing an alkane (e.g., methane) to a polypeptide described herein to produce an alkanol (e.g., methanol).
In some embodiments, provided herein are soluble polypeptides capable of converting an alkane into an alkanol comprising a core sequence comprising at least 60%, but less than 100% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, ranges there between) with SEQ ID NO: 23, the core sequence being C-terminally- and/or N-terminally-flanked by one or more soluble peptide segments. In some embodiments, the polypeptide is capable of converting methane into methanol. In some embodiments, the polypeptide carries out the hydroxylation of the alkane. In some embodiments, the polypeptide comprises a copper oxidase with an engineered active site. In some embodiments, the polypeptide is expressed in an industrially-relevant host organism and at a commercially-relevant scale. In some embodiments, the one or more soluble peptide segments are selected from peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ranges therein) sequence identity with one or SEQ ID NOS: 16, 17, 18, 21, and 22. In some embodiments, the soluble polypeptide comprises at least 60% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ranges therein) with spmoB7 (SEQ ID NO: 13), sumo-spmoB7 (SEQ ID NO: 14), and/or sumo-spmoB7 8pt3 (SEQ ID NO: 13), but less than 100% sequence identify from a naturally-occurring sequence.
In some embodiments, provided herein are system comprising the soluble polypeptides capable of converting an alkane into an alkanol described herein. In some embodiments, systems comprise a fixed support selected from the list consisting of: a yeast cell, a phage, and a functionalized bead.
In some embodiments, provided herein are bioreactors comprising the soluble polypeptides capable of converting an alkane into an alkanol described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. (A) Crystal structure of sMMO (PDB code 4GAM). (B) Crystal structure of pMMO (PDB code 3RGB). Iron (A) and copper (B) ions are shown as spheres.
FIG. 2. SpmoB7 activity assays and controls.
FIG. 3. Sequence alignments of variants.
FIG. 4. Improvements in soluble expression of N- and C-terminal fusions of spmoB. (A) N-terminal fusions. (B) C-terminal fusions.
FIG. 5. Improvement in soluble expression of multi-domain fusions of spmoB.
FIG. 6. Hydrophobic patches identified on spmoB by TRIAD using the SAP algorithm. Patch 1: V293, P294, F413, M414; Patch 2: I380, Y381, P383; Patch 3: P408, P411, I410; Patch 4: P280, L277, A279, P278; Patch 5: L109, P111; Patch 6: P169, V170; Patch 7: L175; Patch 8: P94, P96, G95.
FIG. 7. Improvement in soluble expression of combinatorial mutants of spmoB. All of the combinatorial mutants shown here were built using D1-D2-set12, as in the library screening experiments.
FIG. 8. Ancestry of variants by round of engineering. The starting template is spmoB. Combination mutants FC8pt1-4 are descended from a variant of spmoB with the Mc-6 linker, set12 C-terminal domain fusion, and M298F/M300C base double mutation.
FIG. 9. 13C methanol production by selected variants



DEFINITIONS
As used herein, the term “peptide” refers a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 50 amino acids or less in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids.
As used herein, the term “polypeptide” refers a polymer of amino acids, linked together by peptide bonds, that is over about 50 amino acids or less in length. A polypeptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids.
As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another:
1) Alanine (A) and Glycine (G);
2) Aspartic acid (D) and Glutamic acid (E);
3) Asparagine (N) and Glutamine (Q);
4) Arginine (R) and Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V);
6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W);
7) Serine (S) and Threonine (T); and
8) Cysteine (C) and Methionine (M).
Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.
In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and/or amino acid analogs.
Non-conservative substitutions (e.g., not conservative or semi-conservative) involve the exchange of an amino acid of one class or group for an amino acid from another class or group.
As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) differ only by conservative (e.g., “conservative sequence similarity”) and/or semi-conservative (e.g., “semi-conservative sequence similarity”) amino acid substitutions. The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.
As used herein, the term “physiological conditions” encompasses any conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, chemical makeup, etc. that are compatible with living cells.
The term “soluble”, particularly when used in reference to peptide, polypeptide or protein, as used herein refers to the characteristic of being substantially, completely dissolvable in aqueous solution, under, for example physiological conditions. For example, soluble polypeptide typically lacks any transmembrane segments.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Sample may also refer to cell lysates or purified forms of the peptides and/or polypeptides described herein. Cell lysates may include cells that have been lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Sample may also include cell-free expression systems. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
As used herein, the term “bioreactor” refers to any vessel in which a chemical process or reaction (e.g., conversion of an alkane (e.g., methane) into an alkanol (e.g., methanol), etc.) is carried out which involves organisms or biochemically active substances (e.g., enzymes or polypeptides capable or advancing a chemical reaction without turnover). Process within a bioreactor may be aerobic or anaerobic. A bioreactor can be of any size so long as it is useful for the culturing of cells and/or the performance of the desired chemical reaction. Typically, the bioreactor will be at least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. In some embodiments, the internal conditions of the bioreactor, including, but not limited to pressure, pH and temperature, are optionally controlled during the culture and/or reaction period. A bioreactor is composed of any material that is suitable for holding the bioreactive components (e.g., media, reactants, products, etc.) under appropriate conditions, such as glass, plastic or metal. A bioreactor may also comprise one or more ports, vents, valves, etc. for the additional and/or removal (e.g., selective additional and/or removal) or products, reactants, etc.
DETAILED DESCRIPTION
Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided.
pMMO is a membrane bound α3β3γ3 homotrimer with protomers of three polypeptides: pmoA, pmoB, and pmoC (ref. 7; incorporated by reference in its entirety). The active site of pMMO is a dicopper site that resides in the N-terminal soluble domain of pmoB (ref. 5; incorporated by reference in its entirety) pmoB contains two soluble domains, an N-terminal cupredoxin domain and a C-terminal cupredoxin domain, connected in the middle by two transmembrane helices (ref 7; incorporated by reference in its entirety). It has been demonstrated that the two-transmembrane helices of pmoB can be replaced with an artificial linker and expressed in E. coli (ref. 5; incorporated by reference in its entirety). This protein, commonly referred to as spmoB, expresses as insoluble protein that can be refolded in the presence of copper to obtain active protein. The refolded protein is relatively unstable and obtained in extraordinarily low yields (ref 5; incorporated by reference in its entirety). In some embodiments, a protein within the scope herein does not require in vitro refolding and/or in vitro metal loading. In some embodiments, protein within the scope herein is active inside of an industrially relevant host organism (e.g., E. coli).
Using spmoB as a template, several computational protein design strategies were used to design in silico libraries to improve protein solubility. These strategies included: (1) designing the composition, length, and cutpoints for the linker between the two fragments of spmoB for improved stability and solubility, (2) stabilizing the hydrophobic core by identifying buried cavities and reducing their number and size, (3) redesigning the surface to reduce the number and size of hydrophobic patches, and (4) designing soluble fusion partners for N-terminus and C-terminus. Libraries encoding 2000 variants were screened for solubility and 5 variants with estimated expression yields of more than 10 mg/L in plasmid-based E. coli-based expression systems were obtained. Variants with expression levels as high as 30 mg/L were obtained. Purification of a variant referred to as spmoB7 was carried out and activity assays indicate this variant oxidizes methane to methanol. Additional variants, sumo-spmoB7 and sumo-spmoB7 8pt3, were also shown to be active using isotopically-labeled methane. Reconstitution of the copper cofactor is carried out using an in vivo copper loading method (see Examples). spmoB7 is isolated from the soluble fraction of an E. coli lysate (no refolding necessary) and no additional modifications of the protein are needed to obtain activity. In some embodiments, the protein is in an active, functional, and/or folded state in vivo. In some embodiments, this variant is routinely isolated at expression levels above 5 mg/L (e.g., >10 mg/L>20 mg/L, >30 mg/L, >50 mg/L, or more).
Experiments conducted during development of embodiments of the present invention demonstrate that computational and screening methods used herein produced protein sequences resulting in active soluble fragments of pMMO, and indicate that other variants with sequences described below also oxidize methane (See Examples).
Embodiments described herein find use in a variety of applications, not limited to the following:

    
    
        Methanol production from methane—proteins described herein are used, for example, in conjunction with other biochemical pathways to enable utilization of methane via a methanol-dependent pathway for biofuel production in either cell-free systems or in vivo systems.
        General oxidation chemistries—Since the chemistry performed by the protein described herein has not been previously demonstrated using recombinant proteins without in vitro refolding or metal loading, there are many alternative applications that stem from the construction of these polypeptides.
    
    


Provided herein are soluble engineered polypeptides for oxidizing hydrocarbons, and methods of use, manufacture, and design thereof. In particular, soluble, polypeptides capable of oxidizing methane to methanol (e.g., hydroxylation) are provided.
In some embodiments, soluble engineered polypeptides comprise a first polypeptide having at least 60% sequence identity with a first soluble domain of pmoB linked (via a linker peptide) to a second polypeptide having at least 60% sequence identity with a first soluble domain of pmoB. In some embodiments, the first and second polypeptides are artificial sequences (e.g., not naturally-occurring) having less than 100% sequence identity with a naturally occurring pmoB sequence.
In some embodiments, soluble engineered polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the pmoB-like polypeptide domains. In some embodiments, a soluble peptide is attached to the C-terminus and/or N-terminus of the soluble engineered polypeptides.
In some embodiments, soluble engineered polypeptides comprise, a first soluble peptide portion (S1) (SEQ ID NOs: 16, 17, 18, 21, and/or 22; or variants thereof), two linked pmoB-like polypeptide domains (D1-linker-D2) (SEQ ID NO: 23; or variants thereof), and a second soluble peptide portion (S2) (SEQ ID NOs: 16, 17, 18, 21, and/or 22; or variants thereof). Polypeptides may comprise additional soluble peptides and/or linkers. In some embodiments, a soluble engineered polypeptide comprises S1-D1-linker-D2-52.
In some embodiments, soluble engineered polypeptides comprise a portion with at least 60% but less than 100% sequence identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) with SEQ ID NO: 23. In some embodiments, the polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the C-terminus and/or N-terminus of SEQ ID NO: 23. In some embodiments, the soluble peptide sequences comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) sequence identity with one of SEQ ID NOs: 16, 17, 18, 21, or 22.
In some embodiments, soluble engineered polypeptides comprise a portion with at least 60% but less than 100% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence similarity (e.g., semi-conservative or conservative) with SEQ ID NO: 23. In some embodiments, the polypeptides further comprise one or more soluble peptide sequences (e.g., sumo, set12, mbp, etc.) linked (directly or via a linker peptide) to the C-terminus and/or N-terminus of SEQ ID NO: 23. In some embodiments, the soluble peptide sequences comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence similarity (e.g., semi-conservative or conservative) with one of SEQ ID NOs: 16, 17, 18, 21, or 22.
In some embodiments, all or a portion of the polypeptides and peptide segments within the scope herein comprise at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence identity and/or similarity with one or more of SEQ ID NOS:10-23. In some embodiments, a soluble engineered polypeptide or a portion thereof comprises at least one substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or ranges there between) relative to one of SEQ ID NOS:10-23.
EXAMPLES
Methane/Propylene Oxidation Assay Results
Experiments were conducted during development of embodiments of the present invention to demonstrate monooxygenase activity by detecting the oxidation of propylene to propylene oxide (PO). In MMO activity assays, PO is frequently detected instead of methanol because of PO's low background in typical biological solutions and its relative ease of detection (ref 5; incorporated by reference in its entirety). Table 1 shows that several variants were capable of oxidizing propylene. SpmoB7 was subsequently assayed for methane oxidation and was found to be capable of producing methanol as well (FIGS. 2 and 9, Table 2, and SEQUENCES). The methane oxidation measurements have considerable measurement error due to the difficulty of measuring methanol production at low levels. To overcome this obstacle and to conclusively show methanol is being produced from methane, activity of an spmoB7 variant with an N-terminally encoded sumo tag and a similar construct with additional point mutations were measured using isotopically labeled methane (FIG. 9). The spmoB7, sumo-pmoB7, and sumo-spmoB7 8pt3 variants are the first examples of engineered fragments of pmoB that express solubly in E. coli and are active without refolding.







TABLE 1







Summary of purified spmoB data collected during the granting period.













Soluble
Propylene 
Methane



Cu ions 
expression 
oxidation 
oxidation 



per
yield
activity
activity


Variant
protein
(mg/L)
(min−1)
(min−1)





parent spmoB
5.4
Negligible
0.086 ± 0.037
 0.20 ± 0.020*



(n = 2)





spmoB2
—
11.4
0.035 ± 0.0035
—


spmoB5
2.5 ± 0.3 
32.1
0.022 ± 0.0074
—



(n = 2)





spmoB7
0.7 ± 0.04
17.3
0.028 ± 0.014
0.057 ± 0.051



(n = 3)





spmoB8
—
Negligible
—
—


spmoB10
—
6.0
—
—


spmoB12
—
15.1
0.031 ± 0.029
—


sumo spmoB7
—
6.5
—
See FIG. 9


sumo spmoB7
—
5.4
—
See FIG. 9


8pt3





*Literature values reported under different conditions.













TABLE 2







Glossary of spmoB variant labels. The second column (“Nomenclature”)


defines the variant architecture using a common nomenclature 


that is read from N-terminus to C-terminus, “D|1” and 


“D2” correspond to domains 1 and 2, respectively, “Mc-6” and 


“pmoA-gb1” are linker constructs that are defined in Table 3. “mbp,”


“set12,” and “sumo” are domain fusions; if a domain fusion label 


appears on the left of “D1,” then it is an N-terminal fusion, and if on the 


right of “D2,” a C-terminal fusion.









Label
Nomenclature
Description/Notes





spmoB
D1-D2
pmoB subunit of M. capsulatus




(Bath) pMMO


Mc-6
D1-Mo-6-D2
spmoB with GEPSGEPS 




linker (SEQ ID NO: 1)


pmoA-gb1
D1-pmoA-gb1-D2
spmoB with GEPS-gb1-




GE-pmoA-GS 




linker (SEQ ID NO: 2)


set12
D1-Mc-6-D2-set12
Mc-6 with C- terminal fusion


spmoB2
D1-pmoA-gb1-D2-set12
pmoA-gb1- with 




C-terminal fusion; measured 




PO and MMO activity


spmoB5
mbp-D1-pmoA-
pmoA-gb1 with double fusion; 



gb1-D2-set12
measured PO and MMO activity


spmoB7
set12-D1-pmoA-
pmoA-gb1 with double fusion; 



gb1-D2-set12
mearured PO and MMO activity


spmoB8
set12-D1-pmoA-
pmoA-gb1 with double fusion; 



gb1-D2-sumo
measured PO and MMO activity


spmoB10
mbp-D1-Mo-6-D2-sumo
Mc-6 with double fusion; 




measured PO and MMO activity


spmoB12
set12-D1-Mc-6-D2-sumo
Mc-6 with double fusion; 




measured PO and MMO activity


FC
D1-Mc6-D2-set12 + 
set12 with M298F/M300C



M298F/M300C



sumo 
set12-sumo-D1-pmoA-
spmoB7 with additional 


spmoB7
gb1-D2-set12
N-terminal fusion









Generation of Models of Parent spmoB

The original parent spmoB construct (ref. 5; incorporated by reference in its entirety), is derived from the pmoB subunit of Methylococcus capsulatus Bath (Mc) pMMO. The spmoB construct contains residues 22-172 and 256-414 of pmoB connected by a flexible Gly-Lys-Leu-Gly-Gly-Gly (GKLGGG) linker (SEQ ID NO: 3). Because no crystal structure of spmoB exists, a structural model for spmoB was generated using the loop modeling feature of TRIAD, Protabit's proprietary computational protein design software suite. The model was constructed by removing the two transmembrane helices (residues 172-265) from the coordinate file of pmoB derived from the crystal structure of holo-pMMO (PDB ID: 3RGB) (ref. 8; incorporated by reference in its entirety) and modeling the flexible linker into the resulting gap; TRIAD was used to find the lowest energy conformation and placement of the linker relative to the rest of spmoB.
Linker Designs for Improved Stability/Solubility
The initial linker inserted between the two domains of the parent spmoB is a six residue GKLGGG linker (SEQ ID NO: 3), which was designed by visual inspection of the holo-pMMO structure (ref. 5; incorporated by reference in its entirety). Detailed modeling of the spmoB linker region identified several opportunities for improvement. First, molecular dynamics (MD) analysis of the parent spmoB predicts that the linker is the most flexible part of the construct; a highly flexible linker may contribute to the low soluble expression of spmoB. To reduce flexibility, linkers with more complex sequences are used. Several higher-complexity sequences including Gly, Ser, Glu, and Pro amino acid types and also small proteins or protein fragments were tested (Table 3). Second, if the regions on either side of the linker are considered in addition to the linker itself, a flexible 14-residue tether connects the two structured regions of spmoB. In some embodiments, the flexibility of this region is reduced overall by decreasing the size of the tether. Thus, in addition to the cutpoints for the original linker at residues 172 and 265, a second set of cutpoints was selected at residues 169 and 267, reducing the length of the tether by 5 residues.







TABLE3







Linkers designed and tested.











Name
Cutpoints
Linker
Length
Type














Parent spmoB
173-265
GKLGGG (SEQ ID NO: 3)
6
Original linker


 


Mc-1
172-263
GSGSG (SEQ ID NO: 4)
5
Gly/Ser


 


Mc-2
172-263
GSGSGS (SEQ ID NO: 5)
6
Gly/Ser


 


Mc-3
172-263
GSGSGSG (SEQ ID NO: 6)
7
Gly/Ser


 


Mc-4
169-267
GSGSGSG (SEQ ID NO: 6)
7
Gly/Ser


 


Mc-5
169-267
GSGSGSGS (SEQ ID NO: 7)
8
Gly/Ser


 


Mc-6
169-267
GEPSGEPS (SEQ ID NO: 1)
8
Gly/Ser/Glu/Pro


 


Mc-7
169-267
GSGEPSGS (SEQ ID NO: 8)
8
Gly/Ser/Glu/Pro


 


pmoA-gb1
182-267
GEPS-gb1-GE-pmoA-GS (SEQ
96
pmoA + domain




ID NO: 2)




 


pmoA-b2m
182-267
GEPS-b2m-GE-pmoA-GS (SEQ
139
pmoA + domain




ID NO: 2)




 


pmoA-b2mL
182-267
GEPS-b2mL-GE-pmoA-GS (SEQ
189
pmoA + domain




ID NO: 2)





* the sequence of the pmoAlinker is PVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEK(SEQ ID NO: 9)






Several Gly/Ser and Gly/Ser/Glu/Pro linkers ranging in length from 4 to 11 residues were evaluated with TRIAD's loop modeling feature, which uses inverse kinematics (ref. 9; incorporated by reference in its entirety) and multiple rounds of relaxation to predict the most likely conformation and position of each linker relative to the rest of spmoB. Linkers containing less than 5 residues were found to be strained or not able to span the distance between the fixed cutpoints. Linkers of length 6 and higher did not show signs of strain and some were even predicted to form short secondary structure elements.
Seven short Gly/Ser and Gly/Ser/Glu/Pro linkers were constructed and evaluated for their effect on the soluble expression of spmoB (Table 2, Mc-1 to Mc-7). Soluble expression screening with the split-GFP assay (described below (ref 10; incorporated by reference in its entirety)) indicated that construct Mc-6 had the highest soluble expression of the group, with a marginal improvement in soluble expression over the parent. Further rounds of engineering were executed using Mc-6 as the background template.
A simple structure-based approach was used to recapitulate a more native-like linker between the two domains of spmoB. Using the crystallographic information from 3RGB and 3RFR (pMMO from Methylocystis sp. strain M) as a guide, a sequence from a soluble region of pmoA that interacts with pmoB was selected and incorporated as a natural linker between the two domains in the pmoB subunit. To achieve this, the cutpoint of pmoB was moved from residue 169 to 182, the construct was appended with a Gly-Glu-Pro-Ser linker, and an additional Gly-Ser linker was attached N-terminally to the cutpoint at residue 267.
The new linker and cutpoints create an empty 15 Å space from residue 182 of pmoB to where the pmoA subunit interacts with pmoB for 33 residues. Three different molecular entities were sampled at the 15 Å space: the soluble GB1 domain (ref 10; incorporated by reference in its entirety), beta-2-microglobulin (b2m) (ref 11; incorporated by reference in its entirety), and an extended b2m linker (ref 11; incorporated by reference in its entirety). The GB1 domain proved to be the most soluble of the three by the split-GFP assay on the spmoB Mc-6 template. This construct was named spmoB pmoA-gb1, and was later incorporated into downstream versions of the protein.
N- and C-Terminal Fusion Designs
A wide range of soluble protein domains were investigated for their effects on spmoB expression at both the N- and the C-termini. These domains included GB1 (ref 10; incorporated by reference in its entirety), b2m (ref. 11; incorporated by reference in its entirety), maltose binding protein (mbp), N-utilising substance A (NusA) (ref. 12; incorporated by reference in its entirety), small ubiquitin-like modifier (sumo) protein (ref. 12; incorporated by reference in its entirety), and two versions of highly charged peptides originating from the bacteriophage T7 minor capsid protein 10B (set6 and set12) (ref 12; incorporated by reference in its entirety). These domains were selected for their known performance as solubility-enhancing factors either from the literature (refs. 10-12; incorporated by reference in their entireties) or from in-house experiments. A split-GFP soluble expression assay showed that fusions containing GB1, sumo, mbp, and set12 yielded the best results overall (FIG. 5), and fusions to the C-terminus were, in general, more effective than fusions to the N-terminus.
This first round of data informed a second round of domain fusion experiments, in which two fusion protein domains were attached to the parent spmoB protein, one at each terminus (FIG. 6). Four double-fusion constructs were made, featuring either mbp or set12 at the N-terminus and set12 or sumo at the C-terminus. An additional four constructs were made with identical N- and C-terminal fusions, but with the Mc-6 linker replaced with the pmoA-gb1 linker described above. These combination constructs showed the further improved soluble expression of the spmoB variants (FIG. 6), and six of these variants were selected for large-scale expression and further characterization.
Designs to Improve the Stability of spmoB
A strategy for improving the stability of spmoB was to optimize packing within the protein's core. Poorly packed residues were identified using three analysis methods in TRIAD. First, statistical residue energy (SRE) analysis, a proprietary multicomponent scoring function, was used to identify residues that are not compatible with their environment in the protein. This method also identifies sub-optimal residues on the protein's surface. Second, the interstitial space between the atoms in the solvent-excluded interior of the protein (solvent excluded volume) for each residue was compared to a statistical distribution of volumes from a database of thermophilic and mesophilic protein structures (ref. 13; incorporated by reference in its entirety). Third, positions where point mutations are predicted by molecular mechanics force fields to improve the stability of the protein were identified with a ΔΔG scan using FoldX (ref. 14; incorporated by reference in its entirety) and TRIAD ΔΔG with the Rosetta (ref. 16; incorporated by reference in its entirety) and Phoenix (ref. 17; incorporated by reference in its entirety) forcefields.
The residues identified by the three methods were probed by single or double site-saturation mutagenesis as shown in Table 4. Subsequently, combinatorial sequence designs were carried out to optimize regions surrounding poorly packed “seed” residues within the core of spmoB, which were identified based on a consensus of packing quality based on SRE and solvent excluded volume (Table 5).







TABLE 4







Summary of spmoB single and double site stability libraries tested. 


Positions were identified by core packing, SRE, or ΔΔG analysis.











Degenerate
Amino acid types
Design 


Positions
codon
represented
strategy





K36
NNS
all
SRE


E66
NNS
all
ΔΔG


G76
NNS
all
SRE


T80
NNS
all
SRE/ΔΔG


L89
DKS
R, C, G, I, L, M, F, S, W, V
Core


P96
NNS
all
SRE


G107
NNS
all
SRE


L109
NNS
all
SRE


D123
NNS
all
ΔΔG


D135
NNS
all
SRE


H137
NNS
all
ΔΔG


T140
NNS
all
Core/ΔΔG


N143
NNS
all
ΔΔG


Q145
NNS
all
SRE


G146
NNS
all
SRE/ΔΔG


G148
NNS
all
SRE


M163
NNS
all
SRE


T281
NNS
all
ΔΔG


D288
NNS
All
SRE/ΔΔG


M298
DKS
R, C, G, I, L, M, F, S, W, V
Core


M300
DKS
R, C, G, I, L, M, F, S, W, V
Core


N306
NNS
all
ΔΔG


S321
NNS
all
ΔΔG


I380
NNS
all
SRE


S385
NNS
all
ΔΔG


R400
NNS
All
SRE


D406
NNS
all
SRE/ΔΔG


L89/T140
DKS/DBS
R, C, G, I, L, M, F, S, W, V/A, 
Core




R, C, G, I, L, M, F, S, T, W, V



M298/M300
DKS/DKS
R, C, G, I, L, M, F, S, W, V/R, 
Core




C, G, I, L, M, F, S, W, V
















TABLE 5







Seed positions and nearby residues


included in combinatorial sequence design


calculations.










Seed
Nearby residues included in



residues
design calculations|







L52
W49, W54, F124, Y336



W54
L52, I67, Y336



I67
W54, V65, V126, L128, T140



L89
W49, F124, V126, T140, M142



T140
I67, L89, F124



M298
A289, M300, A366, W371, L409



W371
M298, F324, L376










For each seed position, the surrounding positions within 3 Å that interact with the sidechain of the seed position were chosen for design (Table 4). The design positions were allowed to retain their wild-type identities or mutate to any nonpolar amino acid identity. For all sequence design calculations, special care was taken to avoid designing positions that are known to coordinate the monocopper site (H48, H72) or the dicopper active site (H33, H137, and H139).
Using TRIAD, combinatorial sequence designs were carried out with variable design parameters, and the resulting sequences were converted into degenerate codon libraries with a target size of 250 variants. These variant sequences were also evaluated and ranked by solvent excluded volume to identify variants with improved packing quality. Sequence designs with seeds L89, M298, and W371 were chosen for further analysis because these designs yielded variants with the best solvent excluded volume. The resulting degenerate codon libraries are shown in Tables 6, 7, and 8. In the first round of engineering, testing focused on the single and double mutants in Table 4, which yielded several hits (see Table 9). The combinatorial sequence design libraries in Tables 6, 7, and 8 are evaluated in subsequent rounds of laboratory screening.







TABLE 6







Degenerate codon library 1.











Degenerate


Position
Library AAs
codon





M298
A, I, L, M, P, T, V
VYS


F324
R, Y
TWC


L376
A, C, D, E, F, G, H, I, K, L, M, 
NBS + VNS



N, P, Q, R, S, T, V, W
















TABLE 7







Degenerate codon library 2.











Degenerate


Position 
Library AAs
codon





W49
F, W
TTC + TGG


L89
I, M
ATS


V126
F, I, L, M, V
NTS


T140
I, M, V|
RTS


M142
I, L, M, V
VTS
















TABLE 8







Degenerate codon library 3.











Degenerate


Position 
Library AAs
codon





M298
F, I, M, V
NTS


M300
F, I, L, M, V
NTS


W371
C, F, L, R, W
YKS


L409
L, M
MTG

























Library
#
#
# 
Hit amino 


Library
size
screened
sequenced
hits
acid identity




















K36
20
96
12
5
Q, V, P, E, M


E66
20
96
18
0
—


G76
20
96
34
2
R, E


T80
20
96
9
1
E


P96
20
96
8
2
G, A


G107
20
96
5
1
W


L109
20
96
4
1
D


D123
20
96
6
0
—


H137
20
96
5
2
E, D


T140
20
96
5
0
—


N143
20
96
23
2
G, S


G146
20
96
7
1
D


G148
20
96
13
1
L


M163
20
96
12
4
G, C, R, L


T281
20
96
4
0
—


D288
20
96
16
3
S, E, N


V293
18
96
21
1
A


S321
20
96
8
2
G, H


I380
20
96
4
0
—


S385
20
96
17
1
G


R400
20
96
6
0
—


D406
20
96
9
0
—


I410
18
96
8
0
—


L277/A279 
324
768
28
1
L/P


M298/ M300
144
384
5
3
F/C, F/L, F/V


I380/Y381
324
768
38
6
A/E, E/S, G/S, 







T/D, T/G, G/D


F413/ M414
324
768
41
7
P/S, A/M, R/A, H/H,  







R/N, P/P, A/D









Designs to Remove Hydrophobic Patches on the Surface of spmoB

To improve the solubility of spmoB, hydrophobic patches on the surface of spmoB were identified using the structure-based spatial aggregation propensity (SAP) algorithm (ref 17; incorporated by reference in its entirety). SAP calculations yielded eight hydrophobic patches as shown in FIG. 6. Many of the patches included prolines, which often serve important structural roles in proteins. Thus, proline positions were not mutated in any of the libraries. Three of the patches were excluded from consideration because of their location near or on the flexible linker that is being replaced in other variants (Patches 6 and 7), or because the patch is made up only of prolines and glycines, which often serve important structural roles and may be poor choices for design (Patch 8).
For each of the remaining patches, the degenerate codon VVW was chosen to replace the wild type residue at each position in the patch as shown in Table 10. This degenerate codon encodes 12 amino acids, which include all of the polar or charged amino acid types and was expected to improve the hydrophilicity at these positions on the surface of spmoB. Each position was mutated individually, resulting in 9 single site libraries; three combinations were also constructed for patches that have residues close in primary sequence (Table 10). L109 and 1380 were replaced with NNS instead of VVW because they were also identified by SRE as described above. Finally, residues F413 and M414 are located at the C-terminus of spmoB, which is exposed to the solvent. In addition to mutating these residues with the degenerate codon VVW, spmoB was truncated to remove either M414 or F413/M414.







TABLE 10







Summary of spmoB single and double site solubility 


mutation libraries tested.












Degenerate
Amino acid types


Position
Patch #
codon
represented





L109*
5
NNS
all


L277
4
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


A279
4
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


V293
1
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


I380*
2
NNS
all


Y381
2
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


I410
3
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


F413
1
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


M414
1
VVW
A, R, N, D, Q, E, G, H, K, P, S, T


L277/A279
4
VVW/VVW
A, R, N, D, Q, E, G, H, K, P, S, T/





A, R, N, D, Q, E, G, H, K, P, S, T


I380/Y381
2
VVW/VVW
A, R, N, D, Q, E, G, H, K, P, S, T


F413/M414
1
VVW/VVW
A, R, N, D, Q, E, G, H, K, P, S, T/





A, R, N, D, Q, E, G, H, K, P, S, T





*indicates that position was also identified by SRE (Table 3)






Construction and Cloning of Initial spmoB Gene

The parent spmoB gene was cloned into pY71A(lc) using Gibson assembly (ref. 18; incorporated by reference in its entirety). The spmoB gene was cloned in frame with a C-terminal tag composed of a TEV cleavage site, (Gly3 Ser)2 linker, β-strand 11 of GFP (ref 19; incorporated by reference in its entirety), a Gly3 Ser linker and a Strep-tag (ref 20; incorporated by reference in its entirety). This arrangement of purification and split-GFP tags on the C-terminus was selected from a collection of tag arrangements that still exhibited the desired functionality of each tag. The combined tag was abbreviated to “TGS” and referenced as such within this document.
Construction of spmoB Variant Libraries
Variant libraries of spmoB were constructed using either the Q5® SDM Kit (NEB Inc., Ipswich, Mass., USA) or through megaprimer mutagenesis (ref. 21; incorporated by reference in its entirety). After transformation into BL21 Gold DE3 cells, colonies were picked on a Genetix Qbot into 384-well glycerol stock plates. Libraries were oversampled by at least 3-fold, and in most cases almost 5-fold.
Soluble Expression Screen Development
To identify spmoB variants with improved solubility, an in vitro split-GFP system was employed (ref. 19; incorporated by reference in its entirety). 96-well 2 mL deep well plates containing 1 mL of autoinduction media were inoculated with individual library colonies. Cultures were grown for 16 hours at 28° C. with shaking at 350 RPM. Plates were then centrifuged and the pellets were stored at −20° C. Cells were lysed using a detergent-based lysis buffer. Clarified lysate containing the expressed spmoB variants (with a C-terminal β-strand 11 from GFP) was mixed with the GFP1-10 reagent, allowing the full-length GFP protein to become reconstituted and fluoresce. The detected GFP fluorescence is proportional to the amount of soluble protein; therefore, a brighter signal indicates more soluble spmoB is present in the lysate. The advantages of this assay include: (1) the minimal effect of the C-terminal GFP11 tag on solubility of the protein of interest, ensuring the response is due only to the protein of interest, and (2) the selectivity of the split-GFP interaction, allowing the assay to be performed in clarified cell lysate.
The split-GFP complementation assay was automated with a Tecan Evo liquid-handling robot, allowing for 768 unique clones to be examined in triplicate each day. Assay plates had a set of control wells containing clones with the empty pY71A(lc) plasmid, the parent spmoB-C-set12 gene, or an unrelated solubly-expressed protein as the negative, baseline, and positive controls, respectively. Over the course of the project, the GFP1-10 reagent requirement was reduced 4-fold through the use of high density microplates (384-well vs. 96-well). The cost of the lysis buffer was also reduced by 66% while maintaining effectiveness by decreasing the amount of a commercial lytic additive and supplementing it with an off-the-shelf detergent.
Soluble Expression Screen Results
After split-GFP analysis, potential hits were re-arrayed, re-cultured, and sent to Beckman Genomics for single pass sequencing. True hits were identified by matching results from the sequencing analysis and data from a secondary split-GFP assay. Results are shown in Table 9. Library construction and solubility screening
Large Scale Growth, Purification, and Activity Assay of spmoB7
Eight constructs identified from domain fusion studies were selected for growth scale-up and purification (Table 1). Plasmids were transformed into E. coli strain C41(DE3) or BL21(DE3) and grown overnight on LB-agar plates containing ampicillin. Individual colonies were used to inoculate 1 L cultures of autoinduction media, then grown at 37° C. with shaking at 225 RPM. The growth temperature was changed to 20° C. at an OD600 of approximately 0.5. After 10 hours of growth, cells were transferred to 1 L centrifuge bottles along with 5 mL of sterile sugar solution and 5 mL of 1 M CuSO4. The sugar solution contains 125 g glycerol, 12.5 g glucose, and 50 g α-lactose monohydrate brought to a final volume of 500 mL with water. Resuspended cells were incubated for two hours at room temperature in sealed bottles. Cells were harvested by centrifugation and the cell pellet was flash frozen and stored at −80° C.
Frozen cell pellets were re-suspended in wash buffer (150 mM NaCl and 50 mM Tris base, pH 8.0) and lysed by sonication or chemical additives. Cellular debris was removed by ultracentrifugation at 40,000 RPM for 30 minutes. The resulting supernatant was applied to a streptactin column equilibrated with wash buffer using an FPLC. After extensive washing, the purified protein was eluted from the column using buffer containing 150 mM NaCl, 50 mM Tris base, 2.5 mM desthiolbiotin, and 10 mM MgCl2 at pH 8.0. Eluted protein was then analyzed using SDS-PAGE. The extinction coefficient, calculated by the program Geneious (Biomatters, Auckland, New Zealand) at 280 nm was used for all protein quantitation. Results indicate that four of the six constructs express at levels of >10 mg/L, with one (spmoB5) expressing at >30 mg/L (Table 1).
Metal Content Analysis of Hits
The activity of spmoB is contemplated to be dependent on both the correct coordination of copper ions within the active sites of the protein and the concentration of copper in the in vitro assay during methane oxidation; although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention. The metal content of spmoB5 and spmoB7 were therefore determined using inductively coupled plasma-atomic emission spectroscopy (ICPAES). The average metal content of spmoB5 and spmoB7 shown in Table 1 is from 2 and 3 biological replicates (grown and purified independently), respectively. Subsequent activity assays indicate that there is not a direct correlation between metal content and activity.
Methane/Propylene Oxidation Assay Development
Duroquinol was prepared (22; incorporated by reference in its entirety) 20-100 μL of protein was placed in a gas chromatography (GC) vial with  ̃1-2 mg of solid duroquinol and sealed. Typical protein concentrations were between 5 and 100 μM. 1 mL of propylene, methane or 13C labeled methane was added using a syringe. Reactions were then incubated in a shaking water bath set at 20 to 45° C. After 1 to 24 hour(s), samples were moved to −20° C. for about 10 minutes. 500 μl of chloroform was added to samples containing propylene, shaken at 1800 RPM for 10 minutes, and centrifuged at 2,000×g for 2 minutes. The chloroform layer was then chromatographed with an Agilent 7890B gas chromatograph coupled to a 5977A MSD equipped with a 25 m×0.25 mm PorabondQ with particle traps. Single ion mode was used to quantitate the concentration of propylene oxide by monitoring the 58 m/z ion. Methylene chloride was used as an internal standard by monitoring the 49 m/z ion.
For samples containing methane, 500 μL of chloroform was added to each sample and shaken at 1800 RPM for 10 minutes. The samples were then centrifuged at 2,000×g for 2 minutes to clarify the emulsion. The chloroform layer was then transferred to a fresh vial and chromatographed as described above except that the 31 m/z ion and 33 m/z ion was used to quantitate the concentration of methanol and 13C labeled methane. The rate of methane oxidation is calculated after correcting for background methanol.
Combination of Mutations from Top Performing Hits
The best single and double mutations obtained from the first round of design (K36Q/V, T80E, P96G/A, G146D/G148L, V293A, M298F/M300C, I380A/Y381E) were combined into the backbone of the screening construct D1-D2-set12. As shown in FIG. 7, these mutations all increase the solubility of the D1-D2-set12 construct to varying degrees and show a similar fold improvement when compared to the multi-domain fusions in FIG. 5 and FIG. 6. The starting backbone was first mutated to include M298F/M300C, generating the FC construct, while the subsequent FC8pt mutations incorporated the following base mutations (T80E, G146D, G148L, V293A, I380A, Y381E), but differed in the combinations at K36Q/V and P96G/A (FC8pt1 K36Q/P96G, FC8pt2 K36Q/P96A, FC8pt3 K36V/P96G, FC8pt4 K36V/P96A). The best expressing variant based on the GFP assay was FC8pt3. Mutations in variant FC8pt3 were combined with sumo-SpmoB7 to generate sumo-SpmoB7 8pt3, which was capable of oxidizing 13C labeled methane to 13C methanol.
The number of positions considered presents numerous combinations for consideration when combining variants. Although all of the mutations in the FC8pt series are beneficial, we identified a smaller subset of mutations that provide similar levels of soluble expression. Additional variants include SS1 (K36V, P96G, G146D, M298F, M300C), SS2 (P96G, G146D, M298F, M300C) and the corresponding N-terminal sumo fusions.
Assessment of the Stability and Solubility of Best Variants
Variants of spmoB with increased solubility were expressed and purified to homogeneity using Streptactin resin followed by size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare Life Science, Piscataway, N.J.). The purified proteins showed similar secondary structure to refolded spmoB when examined with circular dichroism; however, no discernible melting transition was observed for any of the variants. Thermofluor assays (ref. 23; incorporated by reference in its entirety) were also performed; however none of the constructs exhibited a thermal transition. It is contemplated that the spmoB proteins exist as a soluble aggregate, which can still allow for significant activity despite having non-traditional tertiary structure (ref. 24; incorporated by reference in its entirety); although the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention.
Soluble spmoB Variants in Eukaryotic Expression Systems
To test the transferability of the soluble spmoB protein variants, a sampling of early hits from the fusion domain studies were cloned into a Pichia pastoris-compatible vector (ref. 25; incorporated by reference in its entirety) The variants were cloned in frame with the alpha mating factor secretion signal, ensuring secretion of the protein out of the cell and into the media. Despite potential difficulties not accounted for during solubility engineering, such as glycosylation, measurable amounts of protein was recovered for one of the variants tested. This shows for the first time the expression of the spmoB protein solubly by a eukaryotic microbe and secreted into the medium.
Similarly, soluble spmoB variants were cloned into the Saccharomyces cerevisiae plasmid pPNL6, in frame with the yeast surface protein aga2 to promote yeast display (ref. 26; incorporated by reference in its entirety). Under control of the galactose promoter, spmoB variants were successfully displayed on the surface of yeast as measured by flow cytometry. Roughly 40% of cells counted were fluorescent in response to labeling with an anti-cmyc primary antibody and an anti-IgG secondary antibody conjugated to phycoerythrin.










SEQUENCES















In some embodiments, sequences include a C-terminal TGS tag.


spmoB-C-set12 (SEQ ID NO: 10)


MHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFL


NVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQG


GGPIIGPGKWITVEGSMSEFRNGEPSGEPSGTMRGMKPLELPAPTVSVKVEDATYRVPG


RAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPL


APGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFM


ENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQGSGGGSTSRDH


MVLHEYVNAAGITGGGSAWSHPQFEK


 


spmoB2 (D1-pmoA-gb1-D2-set12)(SEQ ID NO: 11)


MHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFL


NVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQG


GGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAV


DAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYV


RTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGN


SPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDA


AWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGGEEASVTS


TEETLTPAQEAAETEAANKARKEAELEAETAEQGSGGGSTSRDHMVLHEYVNAAGITG


GGSAWSHPQFEK


 


spmoB5 (mbp-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 12)


MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP


DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY


NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGK


YDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAW


SNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA


VNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA


ASGRQTVDEALKDAQTNENLYFQGHGEKSQAAFMRMRTIHWYDLSWSKEKVKINETV


EIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFR


VVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENG


EPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE


GEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVE


DATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGL


SVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQID


APLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQGSGG


GSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK


 


spmoB7 (set12-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 13)


MEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGHGEKSQAAFM


RMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRK


ESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVE


GSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYA


NDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEK


GSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMIRMKLTITNHGNSPIRLGEFYTASV


RFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIY


DPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAA


ETEAANKARKEAELEAETAEQGSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK


 


sumo-spmoB7 (set12-sumo-D1-pmoA-gb1-D2-set12)(SEQ ID NO: 14)


MEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGLQDSEVNQEA


KPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGI


RIQADQAPEDLDMEDNDBEAHREQIGGHGEKSQAAFMRMRTIHWYDLSWSKEKVKIN


ETVEIKGKFHVFEGWPETVDEPDVAFLNVGMPGPVFIRKESYIGGQLVPRSVRLEIGKTY


DFRVVLKARRPGDWHVHTMMNVQGGGPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDL


ENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT


VTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSV


KVEDATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAE


DGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQV


VQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ


GSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK


 


sumo-spmoB7 FC8pt3 (SEQ ID NO: 15)


MEIASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQENLYFQGLQDSEVNQEA


KPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRKLMEAFAKRQGKEMDSLRFLYDGI


RIQADQAPEDLDMEDNDIIEAHREQIGGHGEVSQAAFMRMRTIHWYDLSWSKEKVKIN


ETVEIKGKFHVFEGWPEEVDEPDVAFLNVGMPGGVFIRKESYIGGQLVPRSVRLEIGKTY


DFRVVLKARRPGDWHVHTMMNVQDGLPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDL


ENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT


VTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRGMKPLELPAPTVSV


KVEDATYRAPGRAFRCKLTITNHGNSPIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAE


DGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIAEDPDSRFAGLLFFTDATGNRQV


VQIDAPLIPSFMENLYFQGGEEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ


GSGGGSTSRDHMVLHEYVNAAGITGGGSNWSHPFEK


 


set12 (SEQ ID NO: 16)


EEASVTSTEETLTPAQEAAETEAANKARKEAELEAETAEQ


 


ENLYFQG (SEQ ID NO: 17)


ENLYFQG


 


sumo (SEQ ID NO: 18)


LQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEM


DSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG


 


pmoA-gb1-D2 (SEQ ID NO: 19)


PVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDG


EWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVRTGTPEYIRMVEKGSGTMRG


MKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGNSPIRLGEFYTASVRFLDSDVY


KDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDAAWEVYRLSDIIYDPDSRFAG


LLFFFDATGNRQVVQIDAPLIPSFMENLYFQGG


 


D1 (SEQ ID NO: 20)


HGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLN


VGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGG


GPIIGPGKWITVEGSMSEFRN


 


TGS(SEQ ID NO: 21)


GSGGGSTSRDHMVLHEYVNAAGITGGGSAWSHPQFEK


 


Mbp (SEQ ID NO: 22)


MKIEEGKLVIWINGDKGYNGLQSGLLAEITPDKAKDTGIKVTVEHPDKLEEKFPQVAAT


GDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA


LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKY


ENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGP


WAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE


GLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA


VINAASGRQTVDEALKDAQTNENLYFQG


 


D1-pmoA-gb1-D2 (SEQ ID NO: 23)


HGEKSQAAFMRMRTIHWYDLSWSKEKVKINETVEIKGKFHVFEGWPETVDEPDVAFLN


VGMPGPVFIRKESYIGGQLVPRSVRLEIGKTYDFRVVLKARRPGDWHVHTMMNVQGG


GPIIGPGKWITVEGSMSEFRNPVTTLTGQTVDLENGEPSTYKLILNGKTLKGETTTEAVD


AATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGEPVEYNGMLMSIADIQGYNYVR


TGTPEYIRMVEKGSGTMRGMKPLELPAPTVSVKVEDATYRVPGRAMRMKLTITNHGNS


PIRLGEFYTASVRFLDSDVYKDTTGYPEDLLAEDGLSVSDNSPLAPGETRTVDVTASDA


AWEVYRLSDIIYDPDSRFAGLLFFFDATGNRQVVQIDAPLIPSFMENLYFQGG









REFERENCES
The following references, many of which are referenced above by number, are herein incorporated by reference in their entireties.

    1. Conrado, R. J. and R. Gonzalez, Chemistry. Envisioning the bioconversion of methane to liquid fuels. Science, 2014. 343(6171): p. 621-3.
    2. Haynes, C. A. and R. Gonzalez, Rethinking biological activation of methane and conversion to liquid fuels. Nat Chem Biol, 2014. 10(5): p. 331-9.
    3. Kjellstrom, T., et al., Public health impact of global heating due to climate change: potential effects on chronic non-communicable diseases. Int J Public Health, 2010. 55(2): p. 97-103.
    4. Trotsenko, Y. A. and J. C. Murrell, Metabolic aspects of aerobic obligate methanotrophy, in Adv Appl Microbiol, A. L. Laskin and S. Sariaslani, Editors. 2008, Elsevier Academic Press Inc: San Diego. p. 183-229.
    5. Balasubramanian, R., et al., Oxidation of methane by a biological dicopper centre. Nature, 2010. 465: p. 115-119.
    6. Tinberg, C. E. and S. J. Lippard, Dioxygen activation in soluble methane monooxygenase. Accounts Chem Res., 2011. 44: p. 280-288.
    7. Lieberman, R. L. and A. C. Rosenzweig, Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature, 2005. 434(7030): p. 177-82.
    8. Smith, S. M., et al., Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry, 2011. 50(47): p. 10231-40.
    9. Canutescu, A. A. and R. L. Dunbrack, Jr., Cyclic coordinate descent: A robotics algorithm for protein loop closure. Protein Sci, 2003. 12(5): p. 963-72.
    10. Bao, W. J., et al., Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization. Protein Expr Purif, 2006. 47(2): p. 599-606.
    11. Gastinel, L. N., N. E. Simister, and P. J. Bjorkman, Expression and crystallization of a soluble and functional form of an Fc receptor related to class I histocompatibility molecules. Proc Natl Acad Sci USA, 1992. 89(2): p. 638-42.
    12. Correa, A. and P. Oppezzo, Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J, 2011. 6(6): p. 715-30.
    13. Taylor, T. J. and Vaisman, I I, Discrimination of thermophilic and mesophilic proteins. BMC Struct Biol, 2010. 10 Suppl 1: p. S5.
    14. Guerois, R., J. E. Nielsen, and L. Serrano, Predicting changes in the stability of proteins and protein complexes: A study of more than 1000 mutations. Journal of Molecular Biology, 2002. 320(2): p. 369-387.
    15. Rohl, C. A., et al., Protein structure prediction using rosetta. Numerical Computer Methods, Pt D, 2004. 383: p. 66-+.
    16. Chica, R. A., et al., Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries. Proc Natl Acad Sci USA, 2010. 107(47): p. 20257-20262.
    17. Chennamsetty, N., et al., Prediction of Aggregation Prone Regions of Therapeutic Proteins. Journal of Physical Chemistry B, 2010. 114(19): p. 6614-6624.
    18. Gibson, D. G., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 2009. 6(5): p. 343-U41.
    19. Cabantous, S. and G. S. Waldo, In vivo and in vitro protein solubility assays using split GFP. Nature Methods, 2006. 3(10): p. 845-854.
    20. Skerra, A. and T. G. M. Schmidt, Use of the Strep-tag and streptavidin for detection and purification of recombinant proteins. Applications of Chimeric Genes and Hybrid Proteins, Pt A, 2000. 326: p. 271-304.
    21. Unger, T., et al., Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. Journal of Structural Biology, 2010. 172(1): p. 34-44.
    22. Zahn, J. A. and A. A. DiSpirito, Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath) (vol 178, pg 1018, 1996). Journal of Bacteriology, 1996. 178(9): p. 2726-2726.
    23. Phillips, K. and A. H. de la Pena, The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol, 2011. Chapter 10: p. Unit10 28.
    24. Garcia-Fruitos, E., et al., Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact, 2005. 4: p. 27.
    25. Lee, C. C., et al., An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid, 2005. 54(1): p. 80-85.
    26. Feldhaus, M. J. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol, 2003. 21, 163-170.

## Claims

1. A soluble polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 13 but less than 100% sequence identity from a naturally-occurring sequence, wherein the soluble polypeptide is capable of converting an alkane into an alkanol.

2. The soluble polypeptide of claim 1, wherein the polypeptide is capable of converting methane into methanol.

3. A method of converting an alkane into an alkanol comprising exposing the alkane to the soluble polypeptide of claim 1.

4. A cell expressing the soluble polypeptide of claim 1.

5. A method of producing a biofuel comprising exposing an alkane to a soluble polypeptide of claim 1.

6. A system comprising a soluble polypeptide of claim 1 attached to a fixed support.

7. The system of claim 6, wherein the fixed support is selected from the list consisting of: a yeast cell, a phage, and a functionalized bead.

8. A bioreactor comprising the soluble polypeptide of claim 1.

9. The bioreactor of claim 8, further comprising methane.

10. The bioreactor of claim 8, further comprising a cell expressing the soluble polypeptide.