Patent Grant
12012434
This application contains a Sequence Listing submitted as an electronic text file entitled “NREL 20-53_ST25_V2.txt” having a size in bytes of 82 kb and created on Dec. 19, 2023. Pursuant to 37 CFR § 1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.
During the pendency of this application, access to the invention will be afforded to the Commission upon request. Upon granting of the patent the strain will be available to the public under the conditions specified in 37 CFR 1.808. The deposit will be maintained in a public repository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer. The deposit will be replaced if it should ever become unavailable.
As a dicarboxylic acid, terephthalic acid (TPA) cannot freely diffuse through the cell membrane. Therefore, among other things, efficient transport of TPA into the cell is important in order to engineer bacteria for the degradation of this product, and its potential biological conversion into value-added products. Furthermore, while there are enzymes known to be capable of breaking down the recalcitrant PET polymer into its constituents, TPA and ethylene glycol, these perform poorly. Thus, there remains a need for developing engineered microorganisms and/or enzymes having, among other things, improved TPA-transport capabilities.
An aspect of the present disclosure is a non-naturally occurring microorganism that includes a gene encoding a MucK transporter protein, where the microorganism is capable of catabolizing terephthalic acid (TPA). In some embodiments of the present disclosure, the gene encoding the MucK transporter protein may contain at least one mutation, relative to a reference gene encoding a reference MucK transporter protein.
In some embodiments of the present disclosure, the reference MucK transporter protein may be at least 90% identical to SEQ ID NO: 2. In some embodiments of the present disclosure, the gene encoding the reference MucK transporter protein may be at least 90% identical to SEQ ID NO: 1. In some embodiments of the present disclosure, the mutation to the MucK transporter protein may include at least one point mutation. In some embodiments of the present disclosure, the point mutation may be present at an amino acid located at at least one of positions 34, 53, 133, 341, or 342 on SEQ ID NO: 2. In some embodiments of the present disclosure, the point mutation may include at least one of M34L, M34I, Y133C, T342I, or E53G on SEQ ID NO: 2.
In some embodiments of the present disclosure, the non-naturally occurring microorganism may further include a deletion of an endogenous gene encoding a MucK transporter protein. In some embodiments of the present disclosure, the non-naturally occurring microorganism may be capable of growing on TPA. In some embodiments of the present disclosure, the non-naturally occurring microorganism may be characterized by a TPA consumption rate between greater than zero g TPA/L/hr and about 0.2 g/L/hr. In some embodiments of the present disclosure, the non-naturally occurring microorganism the microorganism may be grown in a liquid media at a temperature between about 25° C. and about 35° C. In some embodiments of the present disclosure, the liquid media may be maintained at pH between about 6 and about 7.
In some embodiments of the present disclosure, the non-naturally occurring microorganism comprises at least one of a bacterium, a yeast, or a fungus. In some embodiments of the present disclosure, the non-naturally occurring microorganism may be a bacterium. In some embodiments of the present disclosure, the bacterium may include a strain from at least one of A. baylyi, P. putida, P. fluorescens, and/or P. stutzeri. In some embodiments of the present disclosure, the bacterium is A. baylyi. In some embodiments of the present disclosure, the bacterium is A. baylyi ADPI. In some embodiments of the present disclosure, the non-naturally occurring microorganism may further include the deletion of an endogenous gene encoding a transcriptional regulator. In some embodiments of the present disclosure, the transcriptional regulator may be a DcaS transcriptional regulator.
Some embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.
The present disclosure may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that some embodiments as disclosed herein may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the embodiments described herein should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.
A “vector” or “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A vector may be suitable for use in cloning, sequencing, or otherwise manipulating one or more nucleic acid sequences of choice, such as by expressing or delivering the nucleic acid sequence(s) of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules.
A vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant host cell. The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of choice. An integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. A recombinant vector can contain at least one selectable marker.
The term “expression vector” refers to a recombinant vector that is capable of directing the expression of a nucleic acid sequence that has been cloned into it after insertion into a host cell or other (e.g., cell-free) expression system. A nucleic acid sequence is “expressed” when it is transcribed to yield an mRNA sequence. In most cases, this transcript will be translated to yield an amino acid sequence. The cloned gene is usually placed under the control of (i.e., operably linked to) an expression control sequence. The phrase “operatively linked” refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule can be expressed when introduced (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell.
Vectors and expression vectors may contain one or more regulatory sequences or expression control sequences. Regulatory sequences broadly encompass expression control sequences (e.g., transcription control sequences or translation control sequences), as well as sequences that allow for vector replication in a host cell. Transcription control sequences are sequences that control the initiation, elongation, or termination of transcription. Suitable regulatory sequences include any sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced, including those that control transcription initiation, such as promoter, enhancer, terminator, operator and repressor sequences. Additional regulatory sequences include translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. The expression vectors may contain elements that allow for constitutive expression or inducible expression of the protein or proteins of interest. Numerous inducible and constitutive expression systems are known in the art.
Typically, an expression vector includes at least one nucleic acid molecule of interest operatively linked to one or more expression control sequences (e.g., transcription control sequences or translation control sequences). In one aspect, an expression vector may comprise a nucleic acid encoding a recombinant polypeptide, as described herein, operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of polypeptide to be expressed.
Expression and recombinant vectors may contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene allows growth of only those host cells that express the vector when grown in the appropriate selective media. Typical selection genes encode proteins that confer resistance to antibiotics or other toxic substances, complement auxotrophic deficiencies, or supply critical nutrients not available from a particular media. Markers may be an inducible or non-inducible gene and will generally allow for positive selection. Non-limiting examples of selectable markers include the ampicillin resistance marker (i.e., beta-lactamase), tetracycline resistance marker, neomycin/kanamycin resistance marker (i.e., neomycin phosphotransferase), dihydrofolate reductase, glutamine synthetase, and the like. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts as understood by those of skill in the art.
Suitable expression vectors may include (or may be derived from) plasmid vectors that are well known in the art, such as those commonly available from commercial sources. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, and one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements or to other amino acid encoding sequences can be carried out using established methods. A large number of vectors, including bacterial, yeast, and mammalian vectors, have been described for replication and/or expression in various host cells or cell-free systems, and may be used with the sequences described herein for simple cloning or protein expression.
“Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides. This includes single- and double-stranded molecules (i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library) and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated.
Nucleic acids referred to herein as “recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures that rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning or assembling into a vector using restriction enzymes.
Recombinant nucleic acids also include those that result from recombination events that occur through the natural mechanisms of cells but are selected for after the introduction to the cells of nucleic acids designed to allow or make probable a desired recombination event. Portions of isolated nucleic acids that code for polypeptides having a certain function can be identified and isolated by, for example, the method disclosed in U.S. Pat. No. 4,952,501.
A nucleic acid molecule or polynucleotide can include a naturally occurring nucleic acid molecule that has been isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a polypeptide or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies.
Unless so specified, a nucleic acid molecule is not required to encode a protein having enzyme activity. A nucleic acid molecule can encode a truncated, mutated or inactive protein, for example. In addition, nucleic acid molecules may also be useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules, independent of a protein-encoding function.
Suitable nucleic acids include fragments or variants that encode a functional enzyme. For example, a fragment can comprise the minimum nucleotides required to encode a functional enzyme. Nucleic acid variants include nucleic acids with one or more nucleotide additions, deletions, substitutions, including transitions and transversions, insertion, or modifications (e.g., via RNA or DNA analogs). Alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
In certain embodiments, a nucleic acid may be identical to a sequence represented herein. In other embodiments, the nucleic acids may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence represented herein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequences represented herein. Sequence identity calculations can be performed using computer programs, hybridization methods, or calculations. Exemplary computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLAST programs are publicly available from NCBI and other sources. For example, nucleotide sequence identity can be determined by comparing query sequences to sequences in publicly available sequence databases (NCBI) using the BLASTN2 algorithm.
Nucleic acids may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
Also disclosed herein are recombinant vectors, including expression vectors, containing nucleic acids encoding enzymes. A “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A recombinant vector may be suitable for use in cloning, assembling, sequencing, or otherwise manipulating the nucleic acid sequence of choice, such as by expressing or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules.
The nucleic acids described herein may be used in methods for production of enzymes and enzyme cocktails through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell but can be used interchangeably with the term “transfection”.
Non-limiting examples of suitable host cells include cells from microorganisms such as bacteria, yeast, fungi, and filamentous fungi. Exemplary microorganisms include, but are not limited to, bacteria such as E. coli; bacteria from the genera Pseudomonas (e.g., P. putida or P. fluorescens), Bacillus (e.g., B. subtilis, B. megaterium or B. brevis), Caulobacter (e.g., C. crescentus), Lactoccocus (e.g., L. lactis), Streptomyces (e.g., S. coelicolor), Streptococcus (e.g., S. lividans), and Corynybacterium (e.g., C. glutamicum); fungi from the genera Trichoderma (e.g., T. reesei, T. viride, T. koningii, or T. harzianum), Penicillium (e.g., P. funiculosum), Humicola (e.g., H. insolens), Chrysosporium (e.g., C. lucknowense), Gliocladium, Aspergillus (e.g., A. niger, A. nidulans, A. awamori, or A. aculeatus), Fusarium, Neurospora, Hypocrea (e.g., H. jecorina), and Emericella; yeasts from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (e.g., P. pastoris), or Kluyveromyces (e.g., K. lactis). Cells from plants such as Arabidopsis, barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switch grass, alfalfa, miscanthus, and trees such as hardwoods and softwoods are also contemplated herein as host cells.
Host cells can be transformed, transfected, or infected as appropriate by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells.
Vectors may be introduced into host cells such as those from bacteria or fungi by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by bacteria or fungi or other host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by recombination.
Host cells carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing or expressing the polypeptides described herein. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing bacteria or fungi, for example, are available from ATCC. Media may be supplemented with aromatic substrates like guaiacol, guaethol or anisole for dealkylation reactions.
The nucleic acid molecules described herein encode the enzymes with amino acid sequences such as those represented by the SEQ ID NOs presented herein. As used herein, the terms “protein” and “polypeptide” are synonymous. “Peptides” are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity as the complete polypeptide sequence. “Isolated” proteins or polypeptides are proteins or polypeptides purified to a state beyond that in which they exist in cells. In certain embodiments, they may be at least 10% pure; in others, they may be substantially purified to 80% or 90% purity or greater. Isolated proteins or polypeptides include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides that are isolated. Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
Proteins or polypeptides encoded by nucleic acids as well as functional portions or variants thereof are also described herein. Polypeptide sequences may be identical to the amino acid sequences presented herein or may include up to a certain integer number of amino acid alterations. Such protein or polypeptide variants retain functionality as enzymes, and include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides and mutants comprising one or more modified residues. The variant may have one or more conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). Alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
In certain embodiments, the polypeptides may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences presented herein and possess enzymatic function. Percent sequence identity can be calculated using computer programs (such as the BLASTP and TBLASTN programs publicly available from NCBI and other sources) or direct sequence comparison. Polypeptide variants can be produced using techniques known in the art including direct modifications to isolated polypeptides, direct synthesis, or modifications to the nucleic acid sequence encoding the polypeptide using, for example, recombinant DNA techniques.
Polypeptides may be retrieved, obtained, or used in “substantially pure” form, a purity that allows for the effective use of the protein in any method described herein or known in the art. For a protein to be most useful in any of the methods described herein or in any method utilizing enzymes of the types described herein, it is most often substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in the method (e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein.
Among other things, the present disclosure relates to non-naturally occurring microorganisms that include a gene encoding a variant of a MucK transporter protein, where the microorganism is capable of at least one of growing on terephthalic acid (TPA), catabolizing TPA, and/or transporting TPA. In some embodiments of the present disclosure, a microorganism may further include the deletion of an endogenous gene encoding a DcaS transcriptional regulator. In some embodiments of the present disclosure, the microorganism may include at least one of a bacterium, a yeast, and/or a fungus. In some embodiments of the present disclosure, the microorganism may include a bacterium. In some embodiments of the present disclosure, the bacterium may include a strain from at least one of Acinetobacter baylyi (A. baylyi), Pseudomonas Putida (P. putida), Pseudomonas fluorescens (P. fluorescens), and/or Pseudomonas stutzeri (P. stutzeri). As described herein, in some embodiments of the present disclosure, a biosensor was incorporated into non-naturally occurring micro-organisms to identify strains capable of at least one of growing on TPA, catabolizing TPA, and/or transporting TPA.
Among other things, adaptive laboratory evolution was performed on an Acinetobacter baylyi ADP1 engineered strain for growth on the xenobiotic compound terephthalic acid, a component of the plastic polyethylene terephthalate (PET). Sequencing revealed that the native muconate transporter MucK had acquired mutations in several of the evolved clones, and that mutations that could inactivate a putative repressor of expression of MucK (i.e. DcaS) had also been selected. Using a transcription-factor based TPA fluorescent biosensor, it was demonstrated that TPA uptake in ADP1 strains expressing mutated versions of MucK and/or with dcaS gene deleted was more efficient than in wild-type ADP1. In particular, the MucK variants with improved performance contained the following amino acid mutations: i) M34I and E53G, ii) M34L and T342L, and iii) Y133C (see
As described herein, TPA conversion was engineered in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, ADP1-derived strains were derived capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ˜0.2 g/L/h (or ˜1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, a TPA biosensor was used that included the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression.
Results:
Heterologous Expression of Genes Encoding TPA Transport and Catabolism in ADPI:
To confer growth on TPA, genes needed to convert this substrate to PCA were introduced, a metabolite that is consumed via the native P-ketoadipate pathway, into ADP1 (see
Initially, high expression of all genes was targeted by replacing the native promoter with a constitutive tac promoter (Ptac) and by inserting synthetic ribosome binding site (RBS) sequences with high predicted translation initiation rates (TIR, ˜10,000 arbitrary units, a.u.). However, attempts to integrate this large cassette into the ADP1 chromosome were unsuccessful. This fragment had 9 kbp of synthetic sequence, flanked on either side by 2 kbp of DNA identical to the chromosomal target for integration downstream of pobA. To reduce the size of the transforming DNA, the synthetic cassette was split into three fragments for use in a stepwise integration plan (see
Next, we evaluated the effect of pH on TPA consumption by wild-type ADP1, IP130, and a strain lacking tpiBA (IP115), (see
Gene Amplification and Adaptive Laboratory Evolution by EASy:
With the aim of developing Tpa+ strains (i.e., capable of growing on TPA as sole carbon and energy source), we next amplified the chromosomal gene dosage of the tph genes to initiate adaptive laboratory evolution (ALE). Given the potential toxicity to the cell of synthesizing the transmembrane proteins at a high level, we first reorganized the synthetic operon so that the tpiBA genes would not be part of the amplicon (boundaries shown for IP148 in
IP148 was transformed with the SBF, which serves as a platform for homologous recombination and precise duplication of chromosomal segments. This duplication enables changes in the number of tandemly arrayed amplicon copies under selective pressure. In this way, transformants with increased gene dosage were first selected on MMP plates with high Km. Selective pressure was then changed to growth on TPA as the sole carbon source, and Tpa+ colonies arose after ˜10 days (in the absence of antibiotics). When these colonies were re-streaked, Tpa+ colonies appeared more rapidly, after only 2-3 days. Although individual colonies each represent a clonal population, the proclivity of the tandem copies to increase or decrease via recombination suggests that different cells within the colony may differ in amplicon copy number. Frequent recombination also promotes additional genetic change, so that all cells in any Tpa+ colony may not be genetically identical. Therefore, we hereafter refer to these Tpa+ mutants as isolates.
Four isolates, designated TPA_1 to TPA_4, were selected to initiate ALE. These were grown in liquid MM with 5 mM TPA, and cultures were serially transferred to enrich for mutations enabling faster growth. Given that lower pH values could advantageously favor diffusion of TPA into the cell, serial transfers for ALE with these isolates were conducted at pH 6 and pH 7 in parallel (8 lineages, designated by 0.6 or 0.7 after the isolate name to indicate the pH of the medium used for serial transfer (see
In parallel with ALE, we also sequenced PCR products amplified from the tpiBA genes from IP148 and isolates TPA_1 to TPA_4, to test whether early acquisition of beneficial mutations in the transporter genes could have enabled growth on TPA. Sequencing revealed that all of them, including parent-strain IP148, carried an unexpected mutation in tpiA that would result in a premature stop codon (GAG→UAG (see
Use of a Fluorescent Biosensor to Evaluate TPA Transport:
The results from sequencing raised the question of whether the TPA-TTT was still functional in IP148 and the four Tpa+ isolates, despite the mutation in tpiA. Two possibilities were contemplated. The first was that, due to the internal homology in TpiA, two TpiA(W366*) peptides, each encompassing TMHs 1-6, could form a homo-dimer that enabled TPA transport. Evolutionary studies of TTTs have shown that in homologs of TpiA, the transmembrane helices 1-6 are homologous to TMHs 7-12, suggesting that these proteins originated as a result of gene duplication and fusion. The second possibility was that, if re-initiation of translation were to occur downstream of the premature stop codon, the two individually translated peptides, TpiA(W366*) and TpiA(Δ1-370), could associate to restore function. We sought to evaluate TPA uptake in strains expressing different alleles of tpiA. To evaluate TPA transport, we required a rapid assay that would allow us to screen multiple mutants at the same time. Hence, we employed a biosensor for intracellular TPA based on the transcription factor TphR, an IclR-family member which regulates expression of the tph operon in Comamonas sp.
A total of three biosensors were tested, referred to herein as pTPA1, pTPA2, and pTPA3. Testing of all three sensors, not discussed herein, and out of the scope of the present disclosure, showed a negligible response towards aromatic compounds similar to TPA, such as benzoate, 4-hydroxybenzoate, PCA, and catechol, confirming the high specificity of the biosensor for TPA. Ultimately a single biosensor, pTPA3 was chosen for all subsequent work. We then re-evaluated A. baylyi strains encoding the different variants of the TPA-TTT using the pTPA3 biosensor (see
In all, no significant differences were found between ADP1-derived strains expressing different alleles of tpiA (i.e. IP297, IP313, and IP348). Furthermore, their fluorescent response was barely above that of wild type. We also note that no differences in growth rates were observed for any of the strains at the TPA concentrations tested. These results suggest that the heterologous TPA-TTT would have a minimal role in TPA uptake in A. baylyi, and that the evolved TPA+ isolates could instead be importing this substrate through an unidentified, native transporter.
Phenotypic Characterization of Evolved A. baylyi Isolates:
After ˜100 transfers of ALE (˜750 generations), the copy number of the amplicon stabilized at 15-25 copies without substantial changes in growth rates (see
We also compared the evolved A. baylyi isolates to the native TPA-utilizing bacteria Comamonas sp. E6 and R. jostii RHA1, both grown at the standard pH 7 (see
X1
X1
X2
X2
As observed in the phenotypic characterization of the evolved isolates, there were no clear trends in the mutations found for the lineages evolved at pH 6 or at pH 7, except for an in-phase tandem repeat in the gene coding for an EpsG family protein (ACIAD_RS00385) that was present in 3 out of the 4 isolates evolved at pH 6 and absent in all those evolved at pH 7 (see Table 2A). The mutation in tpiA, initially identified in the parent-strain IP148 and the amplified derivatives that were used to initiate ALE (TPA_1 to TPA_4), was maintained in all evolved isolates. This fact suggests that this mutation, potentially encoding a truncated or split TpiA, was not disadvantageous for growth on TPA. Unexpectedly, no mutations were found in the tph genes, except for a single nucleotide change in the synthetic RBS sequence preceding tphA3 in IP251 and IP254, both derived from TPA_3. Nevertheless, it should be noted that the short reads provided by the Illumina sequencing method do not allow the detection of mutations that might be present in only one of the multiple copies of the amplicon and that could be beneficial for growth on TPA.
Another mutation identified in the parent-strain IP148 encoded a variant of the RNA polymerase sigma-70 factor, RpoD(A87E). This mutation was maintained in all evolved isolates except IP246 (see Table 2A). A different mutation in rpoD results in a growth deficit in ADP1. However, a growth competition experiment showed that RpoD(A87E) was only minimally detrimental to growth. Residue A87 is located in region 1.1 of RpoD, which is involved in promoter binding. Therefore, this mutation could potentially affect transcription efficiency in the cell. Indeed, when the mutated rpoD was replaced by the wild-type allele in IP148 derivatives, the high baseline fluorescence observed when transformed with the biosensor plasmid pTPA3 was reduced (see
Further analysis of WGS data revealed that 4 out of the 8 isolates (derived from TPA_1 and TPA_4) had mutations in mucK (ACIAD_RS07750) in combination with mutations in dcaS (ACIAD_RS07760), which are two genes in close proximity in the chromosome (see
A DcaS homolog from Brucella abortus, also an IclR-type regulator, has been identified as a repressor of adipic acid metabolism therein, and its crystal structure has been solved. Using this structure as template (PDB 5WHM, 62% sequence identity), three-dimensional structure models of DcaS were built to examine the location of the amino acid replacements encoded by the different evolved isolates (see
While there were conserved dcaS mutations in evolved isolates derived from the same initial Tpa+ mutant, mucK mutations were different in all cases, suggesting that mutations in dcaS appeared before those in mucK (see
A similar combination of mutations in genes encoding a transcriptional regulator and a MFS transporter is found in evolved isolates IP247 and IP250 (both derived from TPA_2). These isolates share a ˜100 bp deletion in a gene encoding a FadR-family transcriptional regulator (ACIAD_RS00620 locus). In contrast, they have two different mutations in a neighboring gene predicted to encode a glucarate/galactarate MFS transporter (gudP, ACIAD_RS00595 locus). These mutations lead to amino acid replacements R289C in IP247 and R447L in IP250 (see Table 2A). However, we decided to focus on mucK and dcaS for further evaluation, as they had apparently co-evolved in separate lineages.
Evaluation of MucK as a TPA Transporter and its Regulation by DcaS:
To test our hypothesis that MucK was importing TPA, we first attempted to knock out its coding gene in the eight evolved isolates. However, due to a presumed loss of natural competency, we were only able to knock out mucK in IP243 and IP255, 2 out of the 4 isolates that had mutations in this gene. Consistent with our hypothesis, these mutants lost the ability to grow on either muconate (Muc− phenotype) or TPA (see
We then tested whether the deletion of dcaS and/or replacement of wild-type mucK with the four alleles selected during ALE enabled growth of the IP148 parent strain on TPA (see Table 3). The resulting IP148-derived mutants were inoculated in MM with 10 mM TPA as a sole carbon and energy source. After an incubation of 1-2 weeks, 8 out of 9 mutant cultures started growing and reached saturation in 24-48 h, indicating that each of these genetic modifications was sufficient to enable growth on TPA. The exception was strain IP402, carrying the allele encoding MucK(W150C I403T), which unexpectedly also presented a Muc− phenotype. However, when this allele was expressed in a ΔdcaS background (strain IP417), Muc+ and Tpa+ phenotypes were observed, indicating that MucK(W150C I403T) was still functional. As expected, control mutants IP367 and IP387 with mucK knocked out were Tpa and Muc−, even if dcaS was deleted in the latter. Consistent with previous observations, no growth on TPA was observed for parent-strain IP148 after 2 weeks of incubation.
Given the natural propensity of bacteria to undergo spontaneous gene duplication and amplification to increase gene expression under selective pressure, we tested whether these IP148-derived Tpa+ mutants still retained a single copy of the synthetic tph operon or if it had been duplicated. For this purpose, we collected the Tpa+ cells from the TPA cultures indicated in Table 4 to evaluate the copy number by qPCR. Unlike the case of the Tpa+ strains obtained by transformation with an SBF to delimit the amplicon, it was possible that in these new mutants an amplified chromosomal region may not include the KmR gene. Therefore, we used primers and a probe specific for tphA2. We found that, indeed, all Tpa+ mutants had 6-14 copies of tphA2 (See
The apparent sequential emergence of dcaS and mucK mutations in the EASy lineages derived from TPA_1 and TPA_4, and the observation that deletion of dcaS alone enabled a Tpa+ phenotype in IP148-derived mutants, strongly suggested that DcaS acts as a repressor of the transcription of mucK. To test this hypothesis, we used RT-qPCR in wild-type ADP1 and a Δ dcaS mutant (IP461) grown on 20 mM pyruvate, 10 mM muconate, or 10 mM PCA. In wild-type ADP1, transcription of mucK is induced in the presence of muconate when compared to an unrelated carbon source, i.e. pyruvate (see
Next, we evaluated how deletion of dcaS affected TPA uptake using our biosensor. For this assessment, we transformed the third-generation TPA biosensor pTPA3 into IP461 and compared the fluorescent response to increasing TPA concentrations to that of wild-type ADP1 (see
Finally, we sought to assess how mutations in mucK affected uptake of TPA in the presence or absence of DcaS. Hence, we replaced the native mucK gene in either wild-type ADP1 or IP461 backgrounds, transformed all strains with pTPA3, and evaluated fluorescence in the presence or absence of TPA. In order to detect differences in uptake efficiency between the different MucK variants, we induced expression of the sfGFP gene with 0.01 mM TPA, the lowest concentration tested at which wild-type ADP1 did not exhibit a fluorescent response but IP461 did (see
Discussion:
In this work, our goal was to engineer A. baylyi ADP1 to grow on TPA as a sole carbon and energy source using EASy with the tph catabolic genes from Comamonas sp. E6, a native TPA-utilizing bacterium. To that end, we successfully evolved A. baylyi strains for improved growth and consumption rates on this substrate. ALE through serial transfer did not lead to the isolation of Tpa+ strains with less than −15 copies of the tph catabolic operon. While WGS [whole genome sequencing] of evolved isolates identified beneficial mutations that enabled growth of ADP1 mutants on TPA (i.e. in dcaS and mucK), none of these were found to lie within the exogenous catabolic or transporter genes. Furthermore, the reintroduction of these beneficial mutations in the parent strain IP148 still led to the spontaneous amplification of the tph genes upon selection for growth on TPA (see Table 4 and
Interestingly, the TPA catabolic gene clusters in TPA-utilizing bacteria Comamonas sp. E6, R. jostii RHA1, and Rhodococcus sp. DK17 are duplicated in the genomes of these organisms. Additionally, the presence of transposable elements and DNA modifying genes (integrases, recombinases, and reverse transcriptases) in the immediate vicinity of the TPA operons in these three bacteria and in D. tsurhuratensis (de visu inspection of publicly available sequences) suggest that these may have been acquired through horizontal gene transfer. Considering the xenobiotic nature of TPA, it is likely that TPA catabolism has appeared recently, and that it has not yet fully evolved to be as efficient as the catabolism of other aromatic compounds that are ubiquitous in nature, such as those derived from lignin. In this sense EASy, by combining increased gene dosage and ALE, mimics how new catabolic pathways can evolve in nature: acquisition of exogenous genes through horizontal gene transfer, duplication of the acquired genes to overcome limitations in expression, and/or selection of mutations in the acquired or native genes that enable new functions.
In particular, here we have identified variants of the ADP1 muconate MFS transporter MucK that are more efficient at importing TPA. It should be stressed that this discovery would not have been possible without amplification of the tph catabolic genes by EASy, which enabled the growth on TPA that was needed to initiate ALE. Of note, WGS data hint that GudP, another MFS [major facilitator superfamily] transporter predicted to transport glutarate/galactarate, could have also evolved to transport TPA in the engineered strains. The fact that both transporters are involved in the uptake of dicarboxylic acid suggests that they have a latent activity towards TPA that was improved through ALE. As there are no predicted TTTs in ADP1, it is possible that the transmembrane proteins TpiBA from Comamonas sp. E6 are not properly folded or sufficiently active in the heterologous host, and that ADP1 would instead favor MFS transporters to import TPA. However, alignments for MucK and GudP against known TPA MFS transporters (i.e. TpaK) from Rhodococcus sp. or P. xenovorans show sequence identities below 26% (see
In this work, we have also discovered that the transcription factor DcaS represses expression of MucK and that PCA, a metabolite generated during TPA catabolism, could also inhibit the expression of this transporter. Cross-regulation between the catechol (cat gene cluster) and PCA (pca gene cluster) branches of the P-ketoadipate pathway in ADP1, which will favor the former, are well documented in the literature (Bleichrodt et al., 2010; Brzostowicz et al., 2003; Siehler et al., 2007). However, their interaction with the dca gene cluster, involved in the catabolism of saturated C6-C10 dicarboxylic acids (Parke et al., 2001), has not been extensively studied. It should be noted that the dca gene cluster encodes another IclR-type regulator, DcaR (Fischer et al., 2008), that could add another layer of regulation that has not been elucidated in the present study (see
Materials and Methods:
Strains and Culture Media:
A. baylyi ADP1 (American Type Culture Collection ATCC 33305) and derived strains were routinely grown aerobically at 30° C. in minimal medium (MM) (Shanley et al., 1986), consisting of 0.5 M KH2PO4, 0.5 M Na2HPO4, 10% (NH4)2SO4, and 1 mL concentrated base solution. Concentrated base solution contained (per liter): 20 g nitriloacetic acid (dissolved in 600 mL H2O with 14.6 g KOH); 28.9 g MgSO4; 6.67 g CaCl2·2H2O; 18.5 mg Mo7O24·4H2O; 198 mg FeSO4·7H2O; and 100 mL of Metals 44 solution. The Metals 44 solution contained (per liter): 2.5 g EDTA; 10.95 g ZnSO4·7H2O; 5 g FeSO4·7H2O; 1.54 g MnSO4∜7H2O; 392 mg CuSO4·5H2O; 250 mg Co(NO3)2·6H2O; and 177 mg Na2B4O7·10H2O. Minimal medium was supplemented with 20 mM pyruvate (hereafter MMP) or 5 or 10 mM TPA as carbon sources, unless otherwise indicated. For growth on plates, 1.5% w/v agar was added. When noted, the media was adjusted to pH 6 by changing the ratio of phosphate salts. Comamonas sp. E6 (Biological Resource Center, NITE, strain number 107749) and Rhodococcus jostii RHA1 (kindly provided by Dr. Lindsay Eltis from the University of British Columbia) were grown in lysogeny broth (LB) or MM supplemented with 10 mM TPA. Escherichia coli NEB 5-alpha F′Iq and NEB 5-alpha cells (New England Biolabs) were grown in LB broth. Antibiotic concentrations used for ADP1 were 25 μg/mL (standard) or 1 mg/mL (high) kanamycin (Km), and 25 μg/mL streptomycin (Sm). For E. coli, 100 μg/mL ampicillin (Ap), and 50 μg/mL Km or Sm were used.
Plasmid Construction:
Routine PCR amplifications were carried out using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers synthesized by Integrated DNA Technologies (IDT) or Eurofins. Synthetic, double-stranded DNA fragments (gBlocks) were also synthesized by IDT. Plasmids were constructed either by NEBuilder HiFi DNA assembly or by ligation with T4 DNA ligase (both from New England Biolabs). Plasmids containing Ptac were transformed and maintained in NEB 5-alpha F′Iq cells. All plasmid inserts were verified by Sanger sequencing, performed by GENEWIZ. Details on plasmid construction and primer and gBlock sequences can be found in Tables 5-7.
GAATTC
CTGCAG
TCTAGA
GGATCC
ctagcttcacgctgccgcaag
EcoRI
, PstI, XbaI, and
BamHI
sites (F)
acagctatgaccatgattacgccAAGCTTGagtgcttggattctcaccaa
pUC19 overlap and
HindIII site
cgttttatttgatgtctgqttagctggcatgttttaaatagtcaag
overlap
gactatttaaaacatgccagctaaccagacatcaaataaaacg
GTGAATTCGAGCTCGGTACCCGGGGATCC
GTTTAAACCAAATTACGCAGCTCA
overlap and PmeI site
gctctctttttgtttta
ACTAGTtcaatcttctacaaaggcc
overlap with downstream
pobA sequence
ggcctttgtagaagattga
ACTAGTtaaAACAAAAAGAGAGCGATTAG
overlap and SpeI site
aacagctatgaccatgattacgccaagctt
GTTTAAACAGGCATAAGGATATT
PmeI site
corrects c590 deletion in
tphA2
corrects c590 deletion in
tphA2
corrects t1013 deletion in
tphA2
corrects t1013 deletion in
tphA2
ggttcgcttgctgtccattcatgcctgcatttcttgtc
cgcaaaagaaaatgccgatgttaaaacaaaaagagagcgattag
overlap (F), for stepwise
cctacaggtcaccactag
CGGAACGGCGAT
g
tgaaaattaaaagtcaaaaag
RBS with 1952.24 TIR
overlap with tphA2
ctttttgacttttaattttcac
ATCGCCGTTCCGctagtggtgacctgtagg
synthetic RBS with
1952.24 TIR upstream
overlap with tpiB
catttatcgcgggttaa
CCGGTAAGCGGCatggatcttattcaaaac
synthetic RBS with
496.23 TIR upstream
with tpiB
gttttgaataagatccat
GCCGCTTACCGGttaacccgcgataaatg
RBS with 496.23 TIR
overlap with tpiA
cgttttatttgatgtctggcgataccgtcgacctc
contains overlap with 5′-
end of Ptac:tph cassette
gaggtcgacggtatcgccagAcatcaaataaaacg
contains overlap with 3′-
end of ΩKmR cassette
gagcggataacaatttcacac
TGGAGCGCACACgtgaaaattaaaagtcaaaa
synthetic RBS with
1705.68 TIR
gctactagtaagcttctccttttcaatcttctacaaaggcctc
GGGCCCctagtggtgacctgtagg
tcctacaggtcaccactag
GGGCCCgagctgttgacaattaatcatC
pIP031 overlap
tcgctctctttttgttttaactagttcaatcttctacaaaggcctc
gccctgaggcctgcagcggccgc
TACTAGTttacctaggtgtgaattcagaac
site and pBAV overlap
gatgattaattgtcaacagctc
GGGCCCttaaagttttacgtttgctgc
agatctaagcttctgcaggtcgac
TCTAGAcggatccccctcaagtc
tcgctctctttttgttttaACTAGTTacatgcttgcaataagacc
overlap with downstream
pobA sequence
gatccctagcttcacgctgccctgttatccctactcgag
gaaccccagagtcccgctcatttgccgactaccttgg
taaaaacgcaaaagaaaatgccgatgtctagctatcgccatg
taacagggcagcgtgaagctagggattaaagttttacgtttgctgc
GTGAATTCGAGCTCGGTACCCGG
GGATCC
GTTTAAAC
cactgtcaaagctcaa
Pme
I
sites, and pUC19
overlap
AACAGCTATGACCATGATTACGCC
AAGCTT
GTTTAAAC
ttattggcatctttg
Pme
I
sites, and pUC19
overlap
gcagcgtgaagctagggacataggaaagagtatactcaactc
with SmR
cgcaaaagaaaatgccgattaaaaaatatcgcaaaatgcgtac
with sacB
attttgcgatattttttacataggaaagagtatactcaactc
with downstream
targeting region
gagtatactctttcctatgtaaaaaatatcgcaaaatgcgtac
with upstream targeting
region.
gtgaattcgagctcggtacccgg
GGATCC
GTTTAAAC
agatactgtttgatca
PmeI
sites, and pUC19
overlap
aacagctatgaccatgattacgcc
AAGCTT
GTTTAAAC
caggtactttacctg
overlap
gcagcgtgaagctagggataacttataaatgcttatacacttc
with SmR
cgcaaaagaaaatgccgatcatagctatattcctttagcaaag
assembly with sacB
ctcagctttaatactgtttaaactataagagagcaaTatcaggtctgctc
contains overlap with
oIP428.
ttaaacagtattaaagctgagtttaatttaagtacagttgGagctggaatg
contains overlap with
oIP427.
gaggctcgtcctgaatgatatcttacctaggtgtgaattcagaac
overlapping sequence
with pBTL-2 backbone
AAGGAGAtatacatatggctagcaaaggagaagaac
RBS site at the 5′ end
tttgctagccat
atgtataTCTCCTTcttgtgtggggaactgcag
with sfGFP gene and
canonical RBS (R)
tgctatggaggtcaggtatgattctacaacccctgcggat
overlapping sequence
with pBTL-2 backbone
tgtcgtactttg
with oRJ147
atcct
oRJ146
GTGAATTCGAGCTCGGTACCCGGGCCAGACATCAAATAAAACGAAAGGCTCAG
TGTGGAATTGTGAGCGGATAACAATTTCACAC
AGGAGAGTCTATAT
atgCGTA
CCTTGATGCAGTAATCGAAATGGGCGGTGATCATGAAGGCAGCTCTGAAGGTC
ATAAGTGG
CTTCCATCAGCTATCGATAAGTGGCGTAAGGTATGGCCACAATTCCGTTACAT
AATGAGAATATCACC
Strain Construction:
Chromosomal modifications were engineered in A. baylyi by natural transformation of recipient strains with linear DNA fragments. These DNA fragments were obtained by PCR or from restriction enzyme digestion of plasmids. To increase transformation efficiency, cells were grown in MMP instead of LB broth. To facilitate efficient homologous recombination, the transforming DNA carried 1-2 kbp of sequence that was identical to the chromosomal target on each side of the mutated region. A. baylyi mutants were selected on MMP plates with the appropriate antibiotic, or on YT+25% sucrose (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, and 18 g/L agar) in the case of sacB counterselection. Genotypes were confirmed by colony PCR with MyTaq HS Red Mix (Bioline) and, in some cases, Sanger sequencing of localized regions. A brief description of the strains used in this study is provided in Table 3 and
A. balylyi strains, isolates and lineages used in this study. ADP1-derived strains
Chromosomal Gene Amplification and Adaptive Laboratory Evolution by EASy:
Chromosomal amplification was achieved by natural transformation of IP148 with a synthetic bridging fragment (SBF) and selecting on high-Km. The SBF defines the chromosomal region to be amplified and promotes duplication and further amplification through homologous recombination. For the construction of a SBF, the first and last ˜1000 bp of the synthetic Ptac:tphCA2A3BA1:Δ KmR cassette were amplified by PCR and fused tail-to-head by overlap extension PCR. The resulting ˜2 kbp SBF was transformed into strain IP148 and mutants with increased gene copy number were selected on MMP plates supplemented with 1 mg/mL Km. Growth on high-Km is presumably due to multiple copies of the KmR gene, resulting from the chromosomal amplification of a region encompassing it. This region (the amplicon) also encompasses the genes needed for TPA consumption. Individual colonies confirmed by colony PCR to have integrated the SBF were re-streaked on a new high-Km plate and grown at 30° C. By scraping cells from the high-Km plate and streaking on a MM plate with 5 mM TPA as the carbon source (and no antibiotic pressure), selection for optimal copy number of the tandemly arrayed amplicon is altered, and changes in gene dosage can thereby enable the new phenotype, i.e. growth on TPA (Tpa+). In this fashion, four Tpa+ colony isolates, designated TPA_1 to TPA_4, were selected and confirmed to contain the SBF by colony PCR.
After growth on a second TPA plate, each isolate was used in adaptive laboratory evolution (ALE) conducted by serial transfer in MM with 5 or 10 mM TPA as the carbon source. Each isolate was evolved at pH 6 and pH 7 in parallel (8 lineages in total). Two-mL cultures (in 13 mm test tubes) were grown at 30° C. with shaking (225 rpm). When cultures reached stationary phase, cells were diluted 100 or 200-fold in 2 mL of fresh medium. Weekly, glycerol stocks were prepared, and genomic DNA from each culture was extracted with the Quick-DNA Miniprep Plus kit (Zymo Research) for quantitation of the average gene copy number.
Gene Copy Number Analysis by Quantitative PCR:
Quantitative PCR (qPCR) was carried out using 6FAM-MGBNFQ labelled TaqMan probes and TaqMan Gene Expression Master Mix (Thermo Scientific) in a Bio-Rad CFX96 thermocycler. Primer and probe sequences are provided in Table 9. To evaluate changes in amplicon copy number during ALE, relative amounts of the KmR gene were calculated with respect to rpoA, as previously described. For spontaneous amplification mutants not obtained by transformation with the SBF, primers and a probe specific for the synthetic tphA2 gene were used. All reactions were carried out with four technical replicates per genomic DNA sample, with single-copy parent strain IP148 included as control.
Whole-Genome Sequencing and Variant Analysis:
After ˜750 generations of ALE, cells from each individual lineage were diluted 107-fold and 100 μL plated on MM agar with 5 mM TPA. Two individual colonies from each lineage were selected and the amplicon copy number verified by qPCR. The clone with the lowest copy number from each linage was selected for whole-genome sequencing and phenotypic characterization. Approximately 1 μg of genomic DNA was fragmented by sonication to an average size of 300-500 bp. End repair, A-tailing, and adapter ligation reactions were performed on the fragmented DNA using the NEBNext Ultra II kit (New England Biolabs). Illumina paired-end sequencing was performed on a NextSeq500 device at the Georgia Genomics Facility (University of Georgia). Sequence analysis and variant calling (minimal frequency set at 0.25) was performed with Geneious Prime software against the theoretical genome sequence of IP148 parent strain and a modified sequence presenting two copies of the amplicon. The wild-type ADP1 genome sequence (National Center for Biotechnology Information accession number NC_005966) was used as reference.
Evaluation of Growth and TPA Consumption by Cultures Grown in Microtiter Plates and Shake-Flasks:
To evaluate growth of A. baylyi mutants, cultures in microtiter plates were analyzed with a Bioscreen C MBR plate-reader (Growth Curves USA). Cells for inoculation were grown overnight in MMP at 30° C. and 225 rpm. After collection by centrifugation, cells were washed with MM (no carbon source) and added to 300 μL media to an OD600 of 0.05 (per well). Cells were incubated at 30° C. with shaking and OD420-580 measured at 15-minute intervals.
For shake-flask cultures, cells for inoculation were grown overnight in MMP (A. baylyi) or LB broth (Comamonas sp. E6 and R. jostii RHA1) at 30° C. and 225 rpm. After collection by centrifugation, cells were washed with MM (no carbon source). For wild-type ADP1 and single-copy mutants, cells were inoculated to an OD600 of 0.02 in 25 mL MMP supplemented with 5 mM TPA (pH 6 and 7) in 125-mL flasks. For Tpa+ A. baylyi mutants, Comamonas sp.
E6, and R. jostii RHA1, cells were inoculated to an OD600 of 0.02 in 50 mL MM with 10 mM TPA (pH 6 or 7) in 250-mL flasks. In all cases, cells were grown at 30° C., 225 rpm, and sampled regularly by removing 1 mL aliquots for OD600 measurements and HPLC analysis. Standard curves were made to correlate cell dry weight to OD600 for strains A. baylyi IP148, Comamonas sp. E6, and R. jostii RHA1.
HPLC analysis: HPLC analysis of samples was performed on an Agilent 1260 LC system (Agilent Technologies) equipped with a G7117C diode array detector (DAD). All samples and standards were injected at a volume of 10 μL onto a Phenomenex Luna C18(2), 5 μm, 4.6×150 mm column. The column temperature was maintained at 30° C. and the buffers used to separate the analytes of interest were (A) 20 mM phosphate buffer in water and (B) methanol. The separation was carried out using a gradient program of: (A)=80% and (B)=20% at time t=0; (A)=35% and (B)=65% at time t=15 min; and (A)=80% and (B)=20% at t=15.01 min through 20 min. The flow rate was held constant at 0.6 mL/min for a total run time of 20 min. DAD wavelength of 240 nm was used for analysis of TPA while pyruvic acid signal was collected at 210 nm. Calibration curve concentration for each analyte varied between the ranges of 0.1-2500 μg/L. A minimum of 5-6 calibration levels was used with an R2 coefficient of 0.995 or better for each analyte. A check calibration standard was analyzed every 10-20 samples to ensure the integrity of the initial calibration.
Transformation of Biosensor Plasmid Libraries into A. baylyi:
For the transformation of biosensor plasmid libraries, the natural transformation protocol was used, with slight modifications. A. baylyi cultures were started from glycerol stocks in 1 mL MMP and grown overnight at 30° C. under constant shaking. A small volume of the overnight culture (70 μL) was then added to 1 mL fresh MMP and mixed with ˜100 ng of plasmid DNA. The cells were incubated at 30° C. under constant shaking for 2-6 h, spun down at 5000 rpm for 3 minutes in a tabletop centrifuge at ambient temperature, and the concentrated pellet plated on MMP+Sm. In order to cover a large library diversity, overnight cells were concentrated 10-fold. Then, 70 μL of the concentrated cells were mixed with up to 300 ng of plasmid DNA in 1 mL MMP and incubated as described above. Multiple parallel transformations were carried out for larger libraries. Plates were incubated overnight at 30° C. Colonies from plates were then scraped and resuspended in 2 mL of liquid media, rotated gently for 15 minutes for homogeneity, and subsequently saved as glycerol stocks.
Flow Cytometry and Cell Sorting:
A. baylyi mutants transformed with biosensor plasmid libraries were pooled by scraping from plates and diluted to an OD600 of ˜0.05. After growth for 2-4 hours, TPA was added at various concentrations (in the range of 0-3 mM) to induce expression of the sfGFP gene. The cultures were grown overnight at 30° C. and analyzed by fluorescence-activated cell sorting (FACS) on a FACSAria III flow cytometer (BD Biosciences), using the standard settings for GFP fluorescence (488 nm excitation laser and 530/30 nm bandpass emission filter). The cells were gated based on forward and side light scatter (FSC/SSC). Based on the theoretical diversity of the libraries, two to three rounds of sorting were performed. These rounds consisted of positive (top 1-3% fluorescent cells from an induced population) and negative (bottom 50-80% low fluorescent cells from an uninduced population) cell sorting. In any round of sorting, 25-50 thousand cells were collected. Finally, the sorted cells were grown on MMP+Sm plates and colonies picked for individual clone verification.
Individual colonies were inoculated into 600 μL of MMP in a 96 deep-well v-bottom plate (Agilent) and grown for 6 hours at 30° C. and 1000 rpm in a deep-well maximizer shaker (Taitec Bioshaker MBR-022UP). The cultures were then split into replicate wells, after which one of the two sets was provided with 0.3 mM TPA to induce expression of the sfGFP gene. After overnight growth, cells were diluted 20- to 50-fold in phosphate buffer saline (PBS) and analyzed on an Accuri C6 flow cytometer (BD Biosciences) under the standard settings for GFP measurements (excitation 488 nm and emission 533/30 nm). The cells were gated based on FSC/SSC, and the clones with the highest contrast ratios (induced/uninduced fluorescence response) were selected for further evaluation.
Dose Response and Specificity Testing of the TPA Sensor:
A. baylyi mutants were transformed with selected plasmids and grown on MMP+Sm plates. Three colonies were picked and grown overnight as seed cultures. These cultures were diluted 50-fold into 10 mL MMP+Sm and grown for 2-4 h in a 50-mL conical tube to an OD600 of ˜0.6. The culture was then split into triplicate wells in a 96 deep-well v-bottom plate. Into each well (containing 270 μL of culture), 30 μL of 10× stock solutions of possible inducers of sfGFP gene expression were added (in the range of 0-100 mM for each). Cultures were grown overnight and analyzed using an Accuri C6 flow cytometer (BD Biosciences), following the protocol described above.
Evaluation of TPA Transport in A. baylyi with the TPA Sensor:
A. baylyi mutants were naturally transformed with plasmids encoding the TPA biosensors. After selection on MMP+Sm plates, transformants were grown overnight in the same medium at 30° C. with shaking, collected by centrifugation, and washed with MM. Cells were then used to inoculate, to an OD600 of 0.05, 200 μL of medium per well of 96-well black, clear flat-bottom plates (Corning). Cultures contained MMP+Sm with varied concentrations of TPA. Plates were incubated at 30° C. with shaking in an Infinite® F500 Tecan plate reader for 24 hours. The OD600 and fluorescence at 520 nm (excitation at 488 nm) were measured at 15-minute intervals. The gain used for fluorescence reads was adjusted manually.
Gene Expression Analysis by Reverse Transcriptase-Quantitative PCR (RT-qPCR):
Wild-type ADP1 and Δ dcaS mutant IP461 were grown overnight in MMP at 30° C., 225 rpm. Cells were harvested by centrifugation, washed with MM, and inoculated in triplicate to an OD600 of 0.1 in 25 mL MM with either 20 mM pyruvate, 5 mM muconate, or 5 mM PCA as the carbon source, in 125-mL flasks. Cells were grown at 30° C., 225 rpm, to an OD600 of ˜0.6, after which cells were harvested by centrifugation, flash-frozen in liquid nitrogen, and stored at −80° C. For RNA extraction, cells were lysed by bead-beating and RNA purified with a QIAGEN RNeasy Mini kit. Genomic DNA was digested with a TURBO DNA-free kit (Thermo Scientific) and cDNA was synthesized with iScript Reverse Transcription Supermix using random primers (Bio-Rad). qPCR was performed in triplicate for each biological sample with 6FAM-MGBNFQ labelled TaqMan probes and TaqMan Gene Expression Master Mix (Thermo Scientific). Primer and probe sequences are provided in Table 9. Expression of mucK relative to rpoA was calculated using the 2−ΔΔCt method.
The foregoing discussion and examples have been presented for purposes of illustration and description. The foregoing is not intended to limit the aspects, embodiments, or configurations to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the aspects, embodiments, or configurations are grouped together in one or more embodiments, configurations, or aspects for the purpose of streamlining the disclosure. The features of the aspects, embodiments, or configurations, may be combined in alternate aspects, embodiments, or configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the aspects, embodiments, or configurations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment, configuration, or aspect. While certain aspects of conventional technology have been discussed to facilitate disclosure of some embodiments of the present invention, the Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect, embodiment, or configuration.
This application claims priority from U.S. Provisional Patent Application No. 63/077,173 filed on Sep. 11, 2020, the contents of which are incorporated herein by reference in their entirety.
This invention was made with government support under Contract No. DE-AC36-08GO28308 awarded by the Department of Energy. The government has certain rights in this invention.
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| 4952501 | Jasin et al. | Aug 1990 | A |
| 11091782 | Zhang et al. | Aug 2021 | B2 |
| 20170096689 | Burk | Apr 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2019222396 | Nov 2019 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20220089654 A1 | Mar 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 63077173 | Sep 2020 | US |
