Patent Application
20230399666
The invention described and claimed herein was made utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-ACO2-05CH11231. The government has certain rights in this invention.
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 21, 2023, is named “2019-167-02 Sequence Listing.xml” and is 28 kilobytes in size.
The present invention is in the field of production of rhamnolipid.
Production of surfactants and detergents is $41.3 billion dollar global industry1. Dominating this field are petroleum derived chemicals with surfactant properties. Biosurfactants are an attractive class of biomolecules that are sustainable replacements for petroleum derived surfactants2-4. In this group, rhamnolipids (RLs) have been classified as the next generation biosurfactants3 because they are sustainably produced from renewable resources, are biodegradable, exhibit low toxicity and are highly reactive as emulsifiers3,5. RLs find application in oil recovery and remediation, as anti-microbial and/or antifungal agent, in detergent, cleaners, agriculture and cosmetics industry2,6. RLs belong to the class of microbial glycolipids and are predominantly produced at high titer by the opportunistic pathogen Pseudomonas aeruginosa7,8 therefore, rhamnolipid biosynthesis, regulation and bioprocess development has been extensively studied in P. aeruginosa2,6,7,9,10.
RLs are synthesized by diverting intermediates of bacterial fatty acid synthesis or β-oxidation to lipids and subsequently attaching L-rhamnose (sugar) moieties to the lipid chain, synthesizing the glycolipid11. The trans-2-alkanoyl-CoA intermediate of β-oxidation/fatty acid synthesis is first hydrated and isomerized to R-3-hydroxyalkanoyl-CoA by R-specific rhlY, rhlZ encoding enoyl-CoA hydratase/isomerase (
Methane is an abundantly available and low-cost feedstock. It is produced from a fossil source, natural gas, as well as from renewable source, biogas. Considering that methane is a highly potent greenhouse gas (GHG)25,26 and one of the main target for climate-change mitigation, novel technologies for methane utilization are becoming the must element for all industries that produce methane as a by-product. Biogas, a mixture of CH4 and CO2, is the product of anaerobic digestion, whereas natural gas is found in abundance in the subsurface, and is comprised of >90% methane with impurities of volatile higher alkanes26,27. Since the U.S. has substantial reservoirs of natural gas (EIA 2018)28 and an increasing capability to produce biogas29,30, there is recent interest in methane as a feedstock for microbial conversion27. Methanotrophs are bacteria that are able to use methane as a sole carbon source for growth31,32. Recently, some methanotrophs, in particular, Methylococcus capsulatus, and Methylotuvimicrobium buryatense have emerged as microbial platforms for methane conversion to bio-based chemicals33-35. Methylotuvimicrobium alcahphilum (syn. Methylomicrobium alcahphilum) is an attractive methanotrophic host. M. alcaliphilum is a Gram-negative, haloalkaliphilic, obligate methanotroph that has been sequenced and for which basic genetic tools for engineering and gene expression have been developed36-38.
Rhamnolipids, produced by the human opportunistic pathogen Pseudomonas aeruginosa, are glycolipid biomolecules that have been shown to be particularly effective bio-surfactants in applications from petroleum recovery to crop protection, soil treatment, pharmaceutical and food processing. Rhamnolipids have been discussed as a replacement for currently produced synthetic surfactant. However, high purity rhamnolipids are produced from organic substrates and are too costly to be employed on large scale. Recent studies of P. aeruginosa have established that medium chain (C8-C14) β-hydroxyacyl-CoAs, products of free fatty acid β-oxidation are precursors for rhamnolipids. rhlY, rhlZ, rhlA and rhlB encoding enoyl Co-A hydratase, isomerase and rhamnosyl transferase, respectively, are involved in the biosynthesis of rhamnolipids in P. aeruginosa.
Since pathogenic trait of P. aeruginosa possesses biosafety hazard for industrial processes and limits its commercialization in food, pharma and cosmetics, some attempts have been made previously in testing this pathway (rhlYZAB) in GRAS bacterial hosts. Heterologous production of rhamnolipids by expression of rhlYZAB gene cassette of P. aeruginosa in other host platforms like Escherichia colt and Pseudomonas putida has been plausible but with titer limitations and costly substrate supplementation. In recent years, methane is gaining popularity as a lucrative carbohydrate for microbial catalyzed bio-based chemical production due to its abundance in availability (renewable-waste land; non-renewable-natural gas) and lower cost compared to sugar based industrial processes and derived products.
The present invention provides for a method for producing a rhamnolipid, the method comprising: (a) providing a genetically modified host cell comprising one or more of RhlYZAB, or homologous enzyme(s) thereof, capable of expression in the host cell in a growth or culture medium; (b) growing or culturing the host cell such that the one or more of RhlYZAB, or homologous enzyme(s) thereof, are expressed and a rhamnolipid is produced; and (c) optionally recovering the rhamnolipid from the host cell or from the growth or culture medium. In some embodiments, the host cell is a methanotroph. In some embodiments, the growing or culturing step results in the host cell or methanotroph producing more rhamnolipid and/or fatty acids than the host cell or methanotroph in its unmodified form. In some embodiments, the growing or culturing step results in the host cell or methanotroph producing rhamnolipid at a concentration equal to or less than about 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, 4.0 g/L, 4.5 g/L, or 5.0 g/L, or within a range of any two preceding values.
The present invention provides for a genetically modified host cell comprising one or more of RhlYZAB, or homologous enzyme(s) thereof, and one or more mutations in one or more endogenous genes described herein.
The present invention provides for a growth or culture medium comprising a genetically modified host cell of the present invention. In some embodiments, the growth or culture medium comprises methane. In some embodiments, methane is the sole carbon source in the growth or culture medium.
In some embodiments, the host cell comprises a nucleic acid encoding one or more of rhlYZAB, or homologous enzyme(s) thereof, operatively linked to one or more promoters capable of expressing rhlYZAB, or homologous enzyme(s) thereof, in the host cell. In some embodiments, the rhlYZAB is Pseudomonas aeruginosa rhlYZAB, or homologous enzymes thereof. In some embodiments, the rhlYZAB is in a rhlYZAB rhamnolipid cassette. In some embodiments, the rhlYZAB or rhlYZAB rhamnolipid cassette is on a vector, plasmid, or plasmid-based system. In some embodiments, the rhlYZAB gene(s), or homologous enzyme(s) thereof, are codon optimized for the host cell. In some embodiments, the host cell in the unmodified form does not produce rhamnolipid. In some embodiments, the host cell does not have any native rhlYZAB.
In some embodiments, the host cell is a methanotroph. A methanotroph is a bacterium that is able to use methane as a sole carbon source for growth. In some embodiments, the methanotroph is a Gram-negative, haloalkaliphilic, and/or obligate methanotroph. In some embodiments, the host cell is a methanotroph is a Methylococcus or Methylotuvimicrobium cell. In some embodiments, the Methylotuvimicrobium cell is a Methylotuvimicrobium buryatense or Methylotuvimicrobium alcahphilum (syn. Methylomicrobium alcahphilum). M. alcaliphilum is a Gram-negative, haloalkaliphilic, obligate methanotroph that has been sequenced and for which basic genetic tools for engineering and gene expression have been developed.
The present invention provides for a methanotroph comprising one or more, or all, of the mutations indicated in the Appendix of U.S. Provisional Patent Application Ser. No. 63/346,788, filed May 27, 2022, which is incorporated by reference in its entirety (hereafter “the Appendix”). In some embodiments, the host cell or methanotroph comprises one or more, or all, of the mutations indicated in the Appendix. In some embodiments, the host cell or methanotroph comprises mutations in the genes of one or more, or all, of the following: IS3 family transposase (gene-MEAL Z_RS03460, gene-MEALZ_RS08615, gene-MEALZ_RS11580); autotransporter outer membrane beta-barrel domain-containing protein (gene-MEALZ_RS22650); Crp/Fnr family transcriptional regulator (gene-MEALZ_RS12360); multicopper oxidase domain-containing protein (gene-MEALZ_RS14455); type IV pilus secretin PilQ (pilQ; gene-MEALZ_RS15795); and, FAD-dependent oxidoreductase/hypothetical protein/NAD(P)/FAD-dependent oxidoreductase (gene-MEALZ_RS18045). In some embodiments, the host cell or methanotroph comprises endogenous genes of one or more, or all, of the genes/proteins indicated in the Appendix, or for IS3 family transposase; autotransporter outer membrane beta-barrel domain-containing protein; Crp/Fnr family transcriptional regulator; multicopper oxidase domain-containing protein; type IV pilus secretin PilQ; and, FAD-dependent oxidoreductase/hypothetical protein/NAD(P)/FAD-dependent oxidoreductase.
In some embodiments, the host cell or methanotroph comprises one or more, or all, of the mutations indicated in Table 4. In some embodiments, the host cell or methanotroph comprises mutations in one or more of the following genes: MEALZ_RS01195, MEALZ_RS01280,MEALZ_RS01285, MEALZ_RS01290,fabG, MEALZ_RS01300, MEALZ_ RS01305, MEALZ_RS02270, MEALZ_RS02405, murA, MEALZ_RS04765, dxs, tssJ, MEALZ_RS06900,MEALZ_RS06905,folD, MEALZ_RS08615, MEALZ_RS11580, MEA LZ_RS16020, MEALZ_RS17985, MEALZ_RS18045, MEALZ_RS18060, MEALZ_RS18075, MEALZ_RS18090, MEALZ_RS18120, MEALZ_RS18840, tkt, and MEALZ_RS21105. In some embodiments, the host cell or methanotroph comprises mutations in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, or more, of the preceding listed genes.
In some embodiments, the host cell or methanotroph is not a natural rhamnolipid producer. In some embodiments, the host cell or methanotroph in its unmodified form is sensitive, or unable to grow, in a medium having rhamnolipid at a concentration equal to or more than about 0.05 g/L, 0.06 g/L, 0.07 g/L, 0.08 g/L, 0.09 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1.0 g/L, or 1.5 g/L, or within a range of any two preceding values. In some embodiments, the host cell or methanotroph when modified is resistant, or able to grow, in a medium having rhamnolipid at a concentration equal to or less than about 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, 4.0 g/L, 4.5 g/L, or 5.0 g/L, or within a range of any two preceding values. In some embodiments, the host cell or methanotroph when modified is capable of producing more rhamnolipid and/or fatty acids than the host cell or methanotroph in its unmodified form. In some embodiments, the methanotroph can produce more rhamnolipid and/or fatty acids using methane as the sole carbon source.
Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.
The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host cells, microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “expression vector” includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to “cell” includes a single cell as well as a plurality of cells; and the like.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
The terms “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.
The term “about” as used herein means a value that includes 10% less and 10% more than the value referred to.
The terms “host cell” and “host microorganism” are used interchangeably herein to refer to a living biological cell, such as a microorganism, that can be transformed via insertion of an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.
The term “heterologous DNA” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. The term “heterologous” as used herein refers to a structure or molecule wherein at least one of the following is true: (a) the structure or molecule is foreign to (i.e., not naturally found in) a given host cell; or (b) the structure or molecule may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present invention describes the introduction of an expression vector into a host cell, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is not normally found in a host cell. With reference to the host cell's genome, then, the nucleic acid sequence that codes for the enzyme is heterologous.
The terms “expression vector” or “vector” refer to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An “expression vector” contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.
The term “transduce” as used herein refers to the transfer of a sequence of nucleic acids into a host cell or cell. Only when the sequence of nucleic acids becomes stably replicated by the cell does the host cell or cell become “transformed.” As will be appreciated by those of ordinary skill in the art, “transformation” may take place either by incorporation of the sequence of nucleic acids into the cellular genome, i.e., chromosomal integration, or by extrachromosomal integration. In contrast, an expression vector, e.g., a virus, is “infective” when it transduces a host cell, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.
As used herein, the terms “nucleic acid sequence,” “sequence of nucleic acids,” and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; intemucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., arninoalklyphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).
The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
In some embodiments, the methanotroph is an obligate methane consumer, Methylomicrobium alcaliphilum strain DSM19304 into a recombinant rhamnolipid producer. The parent M. alcaliphilum DSM19304 cannot withstand higher levels of rhamnolipids (MIC 0.5 g/L). So, to alleviate product toxicity, M. alcaliphilum DSM19304 is evolved over a period of about 4 months. The evolved strain M. alcaliphilum DASS can tolerate up to about 4 g/L rhamnolipids. In some embodiments, the methanotroph can tolerate up to about 1, 2, 3, or 4 g/L rhamnolipids. In some embodiments, the methanotroph is the strain DASS described herein. In some embodiments, the methanotroph is engineered by introducing a rhlYZAB rhamnolipid cassette of P. aeruginosa on a plasmid based system.
In some embodiments, the host cell engineered by introducing a rhlYZAB produces a higher titer of fatty acids than a modified host cell, and/or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/L of mono-rhamnolipids. In some embodiments, the host cell is an engineered M. alcaliphilum strain that produces a higher titer of fatty acids than a parent M. alcaliphilum strain DSM19304, which is a GRAS characterized strain for industrial purposes as compared to P. aeruginosa (native producer). In some embodiments, the host cell is capable of growing at a pH equal to or more than about 9.0, and/or in a medium with equal to or more than about 3M NaCl. M. alcaliphilum strains are known for their halo-alkaline nature, wherein they can grow at a pH >9.0 and 3M NaCl in a chemically defined mineral salts media. Rhamnolipid production from low cost methane, low cost media and no added carbon or nitrogen supplementation makes the overall process cost-effective over existing industrial bioprocesses.
Rhamnolipids (RLs) are detergent/emulsifiers and can be toxic to other microbes. This glycolipid is natively produced by virulent strains of P. aeruginosa at high titer. Recombinant production of rhamnolipids by expression of rhlYZAB gene cassette of P. aeruginosa in other host platforms has not been commercially successful due to costly substrate supplementation, media composition and titer limitations making the process and targeted product costly. This gap requires us to think of an alternate and comparatively low-cost substrate and a platform host that can be engineered to produce this product, rhamnolipid. Methanotroph Methylomicrobium alcahphilum (obligate methane assimilating gram-negative bacteria) is an interesting microbial host to pursue recombinant RL synthesis. M. alcaliphilum strain DSM19304 is not a natural rhamnolipid producer and it cannot tolerate rhamnolipids surfactant effects at concentrations as low as 0.5 g/L. The present invention comprises or is one or more of the following: (a) a M. alcaliphilum strain that adapted to grow and tolerate 4 g/L rhamnolipids. (b) The strain (DASS) produces comparatively higher amount of free fatty acids than the native strain DSM19304. Secretion of free fatty acids has been of biotechnological relevance in many recent studies, since the free fatty acids can be chemically processed into many other products, for example, lubricants, surfactants, polymer additives, or the like. (c) Expression of codon optimized rhamnolipid pathway harboring plasmid (pDA21) in the wild type (WT) and adapted (DASS) strain had a very distinct characteristic. The M alcahphilum WT strain that harbored the rhamnolipid expression plasmid (pDA21) showed very poor and slow growth, as expected from the low MIC of rhamnolipid on WT strain. In contrast, the adapted strain produces about 10 mg/L rhamnolipids from methane as the sole carbon source.
Enzymes, and nucleic acids encoding thereof
A homologous enzyme is an enzyme that has a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any one of the enzymes described in this specification or in an incorporated reference. The homologous enzyme retains amino acids residues that are recognized as conserved for the enzyme. The homologous enzyme may have non-conserved amino acid residues replaced or found to be of a different amino acid, or amino acid(s) inserted or deleted, but which does not affect or has insignificant effect on the enzymatic activity of the homologous enzyme. The homologous enzyme has an enzymatic activity that is identical or essentially identical to the enzymatic activity any one of the enzymes described in this specification or in an incorporated reference. The homologous enzyme may be found in nature or be an engineered mutant thereof.
The nucleic acid constructs of the present invention comprise nucleic acid sequences encoding one or more of the subject enzymes. The nucleic acid of the subject enzymes are operably linked to promoters and optionally control sequences such that the subject enzymes are expressed in a host cell cultured under suitable conditions. The promoters and control sequences are specific for each host cell species. In some embodiments, expression vectors comprise the nucleic acid constructs. Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.
Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3′-blocked and 5′-blocked nucleotide monomers to the terminal 5′-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5′-hydroxyl group of the growing chain on the 3′-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature (e.g., in Matteuci et al. (1980) Tet. Lett. 521:719; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637). In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired nucleic acid sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No. 4,683,195).
Each nucleic acid sequence encoding the desired subject enzyme can be incorporated into an expression vector. Incorporation of the individual nucleic acid sequences may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, Hhal, Xhol, Xmal, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired nucleic acid sequence are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the nucleic acid sequence are complementary to each other. In addition, DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.
A series of individual nucleic acid sequences can also be combined by utilizing methods that are known to those having ordinary skill in the art (e.g., U.S. Pat. No. 4,683,195).
For example, each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3′ ends overlap and can act as primers for each other Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are “spliced” together. In this way, a series of individual nucleic acid sequences may be “spliced” together and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is effected.
Individual nucleic acid sequences, or “spliced” nucleic acid sequences, are then incorporated into an expression vector. The invention is not limited with respect to the process by which the nucleic acid sequence is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression vector. A typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine et al. (1975) Nature 254:34 and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, N.Y.
Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. An example includes lactose promoters (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operator). Another example is the tac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. Sci. USA, 80:21-25.) As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.
Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSC101, pBR322, pBBR1MCS-3, pUR, pEX, pMR100, pCR4, pBAD24, pUC19; bacteriophages, such as M13 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.
The expression vectors of the invention must be introduced or transferred into the host cell. Such methods for transferring the expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Also, microinjection of the nucleic acid sequencers) provides the ability to transfect host cell. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.
For identifying a transfected host cell, a variety of methods are available. For example, a culture of potentially transfected host cells may be separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of the desired nucleic acid sequence. In addition, when plasmids are used, an often-used practice involves the selection of cells based upon antimicrobial resistance that has been conferred by genes intentionally contained within the expression vector, such as the amp, gpt, neo, and hyg genes.
In some embodiments, the host cells are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene(s) encoding the enzyme(s) may be heterologous to the host cell or the gene may be native to the host cell but is operatively linked to a heterologous promoter and one or more control regions which result in a higher expression of the gene in the host cell.
The enzyme can be native or heterologous to the host cell. Where the enzyme is native to the host cell, the host cell is genetically modified to modulate expression of the enzyme. This modification can involve the modification of the chromosomal gene encoding the enzyme in the host cell or a nucleic acid construct encoding the gene of the enzyme is introduced into the host cell. One of the effects of the modification is the expression of the enzyme is modulated in the host cell, such as the increased expression of the enzyme in the host cell as compared to the expression of the enzyme in an unmodified host cell.
References cited herein:
It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4
Rhamnolipids (RL) are well-studied biosurfactants naturally produced by pathogenic strains of P. aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low-cost and may mitigate greenhouse gas emissions. Herein is demonstrated RL production from CH4 by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth at low concentrations (<0.05 g/L). Adaptive laboratory evolution is performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of g/L of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared to the parent strain. Expression of plasmid borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes show no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared to the parent strain, solely from CH4 with no added supplementations. This work has promise for developing technologies to produce fatty acid- derived bioproducts, including biosurfactants, from CH4.
M. alcaliphilum is engineered to produce rhamnolipids from CH4 without additional mixed or expensive substrate supplementation. The wild type M. alcaliphilum strain exhibited inhibited growth when the P. aeruginosa rhl genes are expressed; however, adaptation of M. alcaliphilum to grow in the presence of RLs produced an evolved strain tolerant to RLs and is able to produce up to 1 μM mono-rhamnolipid from methane.
RESULTS AND DISCUSSION 2.1 Impact of rhamnolipids on growth of M. alcaliphilum
M. alcaliphilum converts methane by sequential oxidation to formaldehyde, which enters the central carbon metabolism through the RUMP pathway39. M. alcaliphilum produces high amounts of glycogen, sucrose and ectoine with smaller amounts of lactate, formate, succinate and no known reports of RLs38,40,41. Rhamnolipids are used as biocontrol/anti-microbial agents, and increasing rhamnolipid concentrations are found to negatively impact growth of Gram-negative and Gram-positive heterologous hosts, E. coli, Bacillus subtilis and Corynebacterium glutamicum23. Therefore, M. alcaliphilum growth is tested in the presence of RLs. Compared to the maximum optical density of M. alcaliphilum after 36 hours of culture, a 50% reduction in final optical density is observed when the medium is amended with 0.1 g/L RL and almost complete inhibition is observed with 1 g/L RL amendment (
2.2 Adaptive laboratory evolution of M. alcaliphilum
A course of adaptive laboratory evolution to allow M. alcaliphilum to grow on CH4 in the RLs is followed for four months. During this adaptation, M. alcaliphilum strain DSM19304 (hereafter referred as WT, wild type) is subjected to gradually increasing RL concentration starting from 0.5 g/L to 5 g/L (
2.3 Strain characterization
To discern the phenotypic difference between the WT and DASS strains, gas chromatography/mass spectrometry (GC/MS) analysis of fatty acids, proteomics and targeted metabolomics are performed on both strains grown on CH4 in the absence of rhamnolipids. The results of these experiments are discussed here.
2.3.1 Fatty acid assessment
Fatty acids are a vital component of microbial cells, which are used as building blocks to construct cell membranes, as well as to provide precursors for synthesis of storage, energy and signaling molecules42. Surfactants and detergents solubilize the lipids of the membrane and disrupt cell structure43. Therefore, M. alcahphilum DASS may have alterations in its fatty acid and/or lipid biosynthesis that enabled the strain to tolerate higher RL concentrations relative to the WT strain. The approach is to establish preliminary evidence for this hypothesis by quantifying long chain fatty acids produced by the strains grown on CH4. Long chain (LC) fatty acids (>C12) are known precursors to phospholipids (PL) and lipopolysaccharides (LPS) that constitute the cell membrane42. Moreover, type-I methanotrophs, including M. alcahphilum are known to contain mainly 16:0 and 16:1 fatty acids44,45. GC/MS analysis is performed at 24 and 48 hours for the cell pellets and supernatants and focused on C16 and C18 fatty acids that are involved in PL and LPS synthesis (Table 1).
Relatively high abundance of C16:0 fatty acid is observed in the cell pellets of both strains, WT and DASS (Table 1), which are consistent with previous findings of other type-I methanotrophs44. However, when strains WT and DASS are compared to each other, C16:0 concentrations are ˜2x higher in strain DASS in the cell pellet at 48 h. The C16:1 fatty acid concentration is found 1.5x- higher in cell pellets and 5-6x higher in supernatant of strain DASS compared to WT (Table 1). Also, C18:1 is undetected in the supernatant of the WT strain but found at similar abundance to the C16:1 fatty acid in the DASS strain. Therefore, the DASS strain produces higher amounts of fatty acids than the WT strain and secretes them at higher levels into the medium. Excretion of free fatty acids is not a regular occurrence in methanotrophic bacteria32 It is proposed, in strain DASS, to maintain cell membrane integrity from solubilizing in surfactant, a high rate of fatty acid synthesis must be maintained to continually replenish phospholipids and lipopolysaccharide layers of cell membrane, as suggested by the observed high C16:0, C16:1, C18:1 fatty acid cell pellet level (Table 1). At the same time, to maintain normal lipid to protein ratio for cell homeostasis, excess fatty acids must be secreted out or stored as intracellular granules (like, PHAs)46. Since, type-1 methanotrophs are known to accumulate glycogen and not PHAs, the outlet of fatty acids in this host perhaps becomes excretion. The possibility of enhancing the secretion of free fatty acids has been explored by engineering many microbial platforms47. M. alcahphilum DASS is innately capable of improved fatty acid production and could serve as a foundational strain for further development of fatty acid-based biofuel/chemical production platform from CH4.
2.3.2 Metabolite and proteome analysis of M. alcahphilum strains DASS and WT
To study the physiological variations that have occurred due to the surfactant-tolerance in the newly adapted strain DASS with respect to its parent, the metabolome and proteome of strain DASS is analyzed and compared with strain WT. Quantification of select metabolites is performed by LC/MS for both intracellular and extracellular fractions, at 24 hours of growth. Presented in
Other secreted products included lactate as well as sucrose and ectoine. Another key metabolite, rhamnose is also evaluated since it is a native precursor of interest for heterologous rhamnolipid synthesis as well as being involved in LPS biosynthesis. Lactate is undetected in the WT strain but present at ˜30 μM in the extracellular fraction from the strain DASS (
Whole cellular proteome of the strains is evaluated and a total of 725 expressed proteins are detected at the two experimental time points. Out of the total, 118 proteins are observed to be downregulated and 102 are found upregulated, however, after qualifying p≤0.05 and log2FC≥0.32 value significance test, only 30 proteins are characterized as significantly down and up regulated, respectively at 24 h. The fold change in NSAF of proteins in strains DASS to WT at 24 h, is listed and represented as heat map in
Based on the metabolomic and proteomics data, at 24 h of cultivation, the DASS strain shifted central carbon processing from EMP to ED, simultaneous activity of both pathways contributed to higher pyruvate pools. The observation of lactic acid secretion by strain DASS is likely results from the increased internal pyruvate pool. This work on strain DASS characterization identifies the unique metabolic changes due to surfactant acclimatization, reinforced evidence of the increased pool of fatty acids and rhamnose, which is a positive outcome for engineering this strain for rhamnolipid biosynthesis.
2.4 Rhamnolipid biosynthesis
M. alcahphilum is not known to produce RLs, so it is essential to identify the availability of precursors for heterologous RL synthesis in this host. Including the four gene (rhlYZAB) enzyme cassette from P. aeruginosa, the pre-requisites for RL production are fatty acid biosynthesis/(3-oxidation and an available pool of rhamnose. Fatty acid biosynthesis is well characterized for ts. buryatense 5 GB(1), a methanotroph closely related to M. alcahphilum52; however, reports of R-3-hydroxydecanoyl-CoA (direct precursor to RL) and enzymes for RL synthesis are not known. Internal rhamnose pools have been reported earlier in M. alcahphilum and rhamnose pools in the strains are also observed during strain characterization (
2.4.1 Heterologous rhamnolipid production in M alcahphilum strains WT (parent)
Codon optimized rhlYZAB are cloned in shuttle vector, pCAH01 under inducible (Ptet: tetracycline; pDA17) and constitutive (Psps: sucrose phosphate synthase; pDA21) promoters (Table 2). The inducible Ptet promoter has been shown to express heterologous ldh (lactate dehydrogenase) in Type-1 methanotrophs for lactic acid production33, and the constitutive mxaF (methane monooxygenase, MMO) promoter has been used for heterologous production of 2,3- butanediol53. In this work, for pDA21, P. aeruginosa rhlYZAB expression is controlled by the constitutive M. alcahphilum sucrose phosphate synthase promoter (Psps), since M. alcahphilum accumulates high amounts of sucrose in their environment in response to maintaining osmotic balance (
E. coli TOP10
E. coli S17-1
Methylotuvimicrobium
alcaliphilum 20Z
Methylotuvimicrobium
alcaliphilum DASS
aPtet promoter;
bPsps promoter driving rhlABYZ expression.
2.4.2 Heterologous rhamnolipid production in M. alcahphilum strain DASS (tolerized)
The toxicity observed when the RL production genes are expressed in M. alcahphilum results suggested that the DASS strain might be more amenable to RL production. Expression of the rhlYZAB cassette in strain DASS containing pDA17 and pDA21 had negligible impact on cell growth (
The work presented here is a proof-of-concept study to produce RLs from CH4. This study demonstrated that rhamnolipids inhibit the growth of M. alcahphdum; however, after adaptive laboratory evolution of M. alcahphdum on gradually increasing RL concentrations, M. alcahphdum metabolism is able to grow in the presence of 10-fold higher concentrations of RLs compared to the parent strain. It is also established that the metabolic changes directly impacted fatty acid synthesis in the cells and strain DASS is found to have acquired natural ability to secrete ˜5-fold higher fatty acids in the medium than the parent strain. A strategy of adaptive laboratory evolution enables the newly generated strain DASS produce ˜600-fold high titer of RL compared to strain WT, where the latter failed to survive when expressing the recombinant RL biosynthetic pathway. The increased fatty acid biosynthesis and secretion by strain DASS suggests a route to develop methanotrophic strains with higher levels of fatty acid production from CH4. Genome sequencing will establish the causative mutations, which may be applied to developing strains that produce fatty-acid-derived fuels and bioproducts.
4.1 Bacterial strains, plasmids, and growth conditions
Escherichia coli and M. alcahphdum strains and plasmids used in this study are listed in Table 2. Luria-Bertani (LB) broth and agar plates are routinely used to culture E. coli cells at 37° C. For routine cultivation of M. alcahphilum strain WT (wild type) and its derivatives, Pi (π) media with 3% (w/v) NaCl is used as described in Collins and Kaluzhnaya.56 When needed, kanamycin (Kan) is added to the growth medium at 100 μg/mL for ts. alcahphilum and 50 μg/mL for E. coli cultures. Ampicillin is added to the growth medium at 100 μg/mL. M alcahphilum cell cultures are grown as batch cultures, either 4 mL culture in 20 mL anaerobic glass tubes or 10 mL culture in 50 mL serum vials, under a methane (99.9%; Airgas): air atmosphere (1:1). Cell cultures are incubated at 30° shaking at 220 rpm. Cell growth is measured as optical density (OD 600 nm) using Spectronic 200E spectroscope at time points mentioned in results and discussion. Single colony isolates and transformant selections are performed on Pi media agar plates incubated in anaerobic jars (Oxoid; Remel) under a methane-air atmosphere (1:1). For M. alcahphilum (pDA17) induction, antimicrobial activity of Ptet inducer, anhydrotetracycline (aTC) is first evaluated (
4.2 Plasmid construction and transformation
The rhamnolipid biosynthetic cassette from P. aeruginosa (GenBank RefSeq: NC_002516.2) containing the genes rhlY, Z, A and B encoding R-specific enoyl-CoA hydratase/isomerase, 3-hydroxyacyl-ACP-O-3 hydroxyacyltransferase and rhamnosyl transferase, respectively are codon optimized for optimal protein expression in M. alcahphilum and synthesized by Genscript (Table 5). The codon optimized rhl genes for M. alcahphilum are assembled in concatenation in a replicative expression plasmid, pET28b(+) with individual RBS upstream of each gene. Steps of assembly are illustrated in
All strains and plasmids developed in this work, along with their associated information have been deposited in the public instance of the JBEI Registry58 (webpage for: public-registry.jbei.org/folders/713).
4.3 Adaptive laboratory evolution and development of M alcahphilum strain DASS
M. alcahphilum strain DSM19304 is grown in batch cultures of 10 mL Pi media in 50 mL serum vials under a methane-air atmosphere at 30° C. with agitation at 200 rpm. To adapt the cells to grow in the presence of RLs, 0.5 g/L RL (90% mono-RL, Millipore Sigma) is supplemented to the starting cell culture. The concentration of RLs is increased gradually and stepwise (1, 1.5, 2, 3, 4 and 5 g/L) to achieve a final strain of M. alcahphilum tolerant to 5 g/L RL. A 0.1% inoculum is manually transferred from a growing batch culture to a fresh culture in 48-60h. The RL concentration in the media is increased to next higher concentration when the OD 600 nm at 48 h of the growing batch culture with RL reached similar OD600 to WT (>1.0) at the end of 48 h. After the adaptation, single colonies of M. alcahphilum strain DASS are isolated on Pi media agar plates. Multiple single colony isolates are confirmed to be M. alcahphilum via 16S rRNA sequencing to rule out possibility of co-contaminants. No differences are observed in growth of multiple single colonies that are tested, one clone is selected for further analysis and plasmid transformation.
4.4 Proteomic analysis
M. alcahphilum cell cultures are grown in batch in 10 mL Pi medium in 50 mL serum vials under a methane-air atmosphere. Cell cultures are incubated at 30° shaking at 220 rpm. Both strains are grown for proteomic analysis in triplicates. Cells are harvested at 24 h and 48 h and stored at −80° C. until use. Samples for proteomic analysis are processed and whole proteome is analyzed as described by Yan et.al. (website for: dx.doi.org/10.17504/protocols.io.b19xjr7n). Normalized Spectral Abundance Factor (NSAF) values obtained are processed to categorize upregulated and downregulated proteins of M alcahphilum strain DASS and WT. p value <0.05 for FC >0.32 are considered significant and are presented in heat map table or as specified.
4.5 Metabolite analysis
Growing M. alcahphilum cell cultures (4 mL in Pi medium) in 20 mL anaerobic glass tubes, are harvested at 24 h and 48 h of growth. M alcahphilum strains harboring plasmids are grown with antibiotic and inducer (anhydrotetracycline) as necessary, in the culture medium. 2 mL cell culture is centrifuged at 10,000 rpm for 1 min at room temperature (RT). Thereafter, 1 mL supernatant is stored in a separate tube and the rest is discarded. Cell pellets are immediately quenched with 4° C. cold 100% methanol. Both supernatant and pellets are stored at −20° C. until further processing. All strains, parents and harboring plasmids are grown in technical triplicates for analysis. To analyze central carbon metabolism and associated metabolites, the cells and supernatant are processed separately using aqueous methanol extraction method as described earlier by Baidoo et.al.59
Intracellular metabolites are analyzed via liquid chromatography-mass spectrometry (LC-MS; Agilent Technologies 1290 Infinity II UHPLC system and Agilent Technologies 6545 quadrupole time-of-flight mass spectrometer) on a ZIC-pHILIC column (150-mm length, 4.6-mm internal diameter, and 5 μm particle size). The UHPLC method used is as described by Baidoo et. al. and Kim et.al.59,60 For rhamnolipid analysis the cell pellets and supernatant is processed using acidic (HCl) methanol/chloroform precipitation method described previously by cakmak et.al.61 Total rhamnolipids are analyzed using the LC-MS method as described by Amer et.al.(webpage for: protocols.io/edit/lc-ms-analysis-of-rhamnolipid-bu5wny7e).
4.6 Analysis of fatty acids
Cell cultures are grown in 4 mL Pi media in 20 mL anaerobic glass tubes, under methane-oxygen at 30° C. and shaking at 220 rpm. 2 mL culture is aspirated at 24 and 48 hour and cell pellets are harvested by centrifugation at 8000 rpm for 10 mins at room temperature. Supernatant and pellets are stored in separate 2 mL Eppendorf tubes at −80° C. until further processing. Total cell fatty acids are analyzed as fatty acid methyl esters (FAMEs) using GC-MS. FAMEs are prepared by transesterification using 2% (v/v) sulfuric acid in methanol (90° C.; 2 h). FAMEs are subsequently extracted in 400 μL hexane, of which 1μL is analyzed on an Agilent 5973-HP6890 GC-MS using a 30 m DB-5 ms capillary column. Electron ionization (EI) GC/MS analyses are performed with a model 7890A GC quadrupole mass spectrometer (Agilent) with a DB-5 fused silica capillary column as described previously62.
4.7 Materials
All chemicals used in this study are analytical grade. Organic and inorganic chemicals are purchased from Fisher Scientific (Pittsburgh, Pa.). Biochemicals are from Sigma-Aldrich Co. (St. Louis, Mo.) and Millipore Sigma (Burlington, Mass.). Molecular biology reagents and supplies are from New England Biolabs (Ipswich, Mass.) and Thermo Fisher Scientific (Waltham, Mass.). Plasmid DNA extraction kit is from QIAGEN (Valencia, Calif.). DNA clean up kits are from QIAGEN (Valencia, Calif.). DNA oligonucleotides for PCR are from IDT (Coralville, Iowa).
Plasmid construction: Prior to assembling the rhlYZAB cassette under Ptet promoter, individual RBS (ribosome binding sequence) are added upstream to translation start sequence (ATG) of each enzyme. To add the RBS, individual genes rhlY , rhlZ, rhlA and rh/B are first amplified using primer with upstream Ndel attached to forward priming sequence (5′) and Nhel-Bamhl to reverse priming sequence (3′) with Phusion DNA polymerase (Thermo scientific)
alcaliphilum
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
This application claims priority to U.S. Provisional Patent Application Ser. No. 63/346,788, filed May 27, 2022, which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63346788 | May 2022 | US |
