WO2016019249A1 - Formaldehyde sensing protein - Google Patents

Abstract

The present invention relates to genetically modified methylotrophic bacteria expressing exogenous efgA and use of the genetically modified bacteria to produce a target compound. Embodiments of the present disclosure are directed to the identification of proteins and pathways involved in HCHO sensing and/or detoxification. Embodiments of the present disclosure are directed to transgenic microorganisms (e.g., bacteria) modified to express exogenous EfgA (e.g., as a formaldehyde sensor) such that they are sensitive and responsive to HCHO levels.

Description

FORMALDEHYDE SENSING PROTEIN

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Patent Application No. 62/031,287, filed on July 31, 2014 and is hereby incorporated herein by reference in its entirety for all purposes.

FIELD

The present invention relates in general to genetically modified bacteria, such as Methylobacterium, having an increased biomass producing capability. Such genetically modified bacteria can be used to produce useful chemical compounds from single-carbon (Ci) compounds such as methanol (CH₃OH) and methane (CH₄).

BACKGROUND

Methylotrophy is the ability of an microorganisms, such as bacteria and certain fungi, to grow at the expense of reduced carbon compounds containing one or more carbon atoms but containing no carbon-carbon bonds, using Ci compounds like CH₄ and CH₃OH as the sole carbon and energy source. As some Ci assimilation pathways are highly inefficient, recombinant microorganisms adapted to be robust in the presence of HCHO waste production (a by-product of using methanol as a carbon source) would be useful for the production of beneficial carbon-based compounds. Therefore, genetically modified microorganisms optimized to respond to HCHO waste are desirable. SUMMARY

Embodiments of the present disclosure are directed to the identification of proteins and pathways involved in HCHO sensing and/or detoxification. Embodiments of the present disclosure are directed to transgenic microorganisms (e.g., bacteria) modified to express exogenous EfgA (e.g., as a formaldehyde sensor) such that they are sensitive and responsive to HCHO levels. Further embodiments of the present disclosure are also directed to modification of pathways within organisms (e.g., bacteria) that cannot grow in the presence of HCHO to enable their growth using HCHO as a carbon source.

The present invention is based in part on the discovery that EfgA acts as a watchdog for HCHO buildup. By expressing EfgA in recombinant microorganisms that use methanol or methane as a feed stock (but lack EfgA), these microorganisms will be live, grow and/or divide in the presence of formaldehyde that is produced as a by-product of this process. Accordingly, the presence of EfgA will increase the efficiency of conversion of single-carbon (Q) compounds, e.g., CH₃OH, CH₄ and the like, to a desired commodity, e.g., butanol, carotenoids and the like. In certain exemplary embodiments, genetically modified microorganisms described herein have an increased substrate yield (grams carbon into biomass vs. grams carbon to CO₂) or growth rate (grans biomass per time) compared to microorganisms without the genetic modifications, including wild-type microorganisms. In this aspect, biomass production is increased per unit substrate due to the increased efficiency of the genetically modified microorganisms, or increased per unit time due to the increased growth rate of the genetically modified microrganisms. According to an alternate aspect, genetically modified microorganisms described herein produce less carbon dioxide and more carbon-based biomass compared to microorganisms without the genetic modification, including wild-type microorganisms. In this aspect, biomass production is increased per substrate.

In certain exemplary embodiments, a recombinant microorganism expressing an exogenous efgA gene is provided. In certain aspects, the recombinant microorganism is a bacterium (e.g., a methylotropic bacterium). In certain aspects, the exogenous efga gene is exemplified by the many examples associated with the clade of efga genes indicated in Fig. 7. In certain aspects, the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO:l across the length of SEQ ID NO:l. In other aspects, the exogenous efgA gene has at least 90%o sequence identity to the nucleic acid of SEQ ID NO:l, that is at least 391 consecutive nucleic acids in length. In certain aspects, the exogenous efgA gene provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock. In yet other aspects, wherein the recombinant microorganism has an increased ability to produce a desired commodity product (e.g., butanol, one or more carotenoids or the like) as compared to a wild-type microorganism not expressing the exogenous efgA gene. In still other aspects, the recombinant microorganism can use formaldehyde as a carbon source for growth.

In certain exemplary embodiments, a recombinant microorganism expressing an exogenous EfgA polypeptide is provided. In certain aspects, the recombinant microorganism is a bacterium (e.g., a methylotropic bacterium). In certain aspects, the exogenous EfgA polypeptide has at least 90%o sequence identity to the amino acid of SEQ ID NO:2 across the length of SEQ ID NO:2. In other aspects, the exogenous EfgA polypeptide has at least 90%o sequence identity to the amino acid of SEQ ID NO:2, an example of which is at least 130 consecutive amino acids in length. In certain aspects, the exogenous EfgA polypeptide provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock. In yet other aspects, wherein the recombinant microorganism has an increased ability to produce a desired commodity product (e.g., butanol, one or more carotenoids or the like) as compared to a wild-type microorganism not expressing the exogenous EfgA polypeptide. In still other aspects, the recombinant microorganism can use formaldehyde as a carbon source for growth.

In certain exemplary embodiments, a recombinant methylotrophic bacterium expressing an exogenous efgA gene, wherein the efgA gene encodes a polypeptide that acts as a formaldehyde sensor is provided.

In certain exemplary embodiments, a methylotrophic bacterium expressing an exogenous EfgA polypeptide, wherein the EfgA polypeptide acts as a formaldehyde sensor is provided.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawing in which:

Fig. 1 schematically depicts common toxic metabolites in bacteria.

Fig. 2 schematically depicts pathways involved in methylotrophy. Fig. 3 schematically depicts a metabolic paradox regarding methylotrophy. Although it is essential for certain bacterial cells to produce intracellular formaldehyde, they cannot grow on it. Methylobacterium extorquens PAl cannot survive in the presence of 10 mM HCHO for greater than 60 minutes.

Fig. 4 graphically depicts data from the weaning experiments used to identify mutant bacteria that could grow in the presence of HCHO.

Fig. 5 graphically depicts the genetic basis of adaptation to grow in the presence of HCHO. Two uncharacterized genes were identified that incurred mutations across replicate populations: efgA and efgB. efgB is involved in a novel stress response specific to aldehydes. Fig. 6 graphically depicts the efgA (evolved formaldehyde growth A) gene, which encodes the conserved protein of unknown function, DUF336. The graph depicts three point mutations identified that allowed growth in the presence of HCHO.

Fig. 7 depicts an efgA phylogeny tree using amino acid alignment. The tree reveals a role for efgA in methylotrophy.

Figs. 8A-8B show that efgA prevents formaldehyde growth. Fig. 8A shows that efgA^Ev° - expressing strains could grow on 5 mM formaldehyde. These strains demonstrated loss of function. Fig. 8B shows that the growth rate of evolved (e/g^^Ev0'-expressing) strains had a greater growth rate of HCHO than on C¾OH. Figs. 9A-9B graphically depicts phenotypic differences between efgA^¹ and AefgA. Fig. 9A shows that efgA^wl leads to an 8% increase in growth rate using methanol as a substrate. Fig. 9B shows that efgA^WT allows cells to better survive HCHO shock (50 mM HCHO).

Fig. 10 depicts a model of EfgA structure, which reveals a regulatory role. EfgA is structurally similar to Streptomyces reticuli HbpS, even though the two proteins have very little amino acid similarity. HbpS undergoes a structural change during oxidative stress. It is involved with regulation of the oxidative stress response. EfgA is involved in sensing HCHO and regulating a response by the cell upon exposure to HCHO.

Figs. 11A-11B depict the relevance and mechanism of efgA. Fig. 11A depicts Methylobacterium extorquens PA1 growth. It metabolizes 1.5 - 45.7 μg of methanol per g^"1 h^"1. Fig. 1 IB depicts that during steady flux, intermediate concentrations of HCHO in an artificial environment makes efgA redundant.

Fig. 12 depicts particular portions of the pathways involved in methylotrophy that will be investigated to learn more about the regulatory framework, protein partners, substrate binding and key residues involved in HCHO detoxification. Fig. 13A depicts the efgA gene sequence. Fig. 13B depicts the EfgA protein sequence.

Fig. 14A depicts the def gene sequence. Fig. 14B depicts the Def protein sequence.

Figs. 15A-15B depict efgA mutant data. Fig. 15A shows a list of mutations with the mutations in Mext_4158 notated. Fig. 15B shows that EfgA functions as a formaldehyde sensor that responds to formaldehyde shock across distantly related organisms. Survival ratio equals (#Cells after formaldehyde shock/ # cells before formaldehyde shock) for 20 replicates per strain.

Fig. 16 is a schematic of an experimental protocol designed to detect binding of EfgA to PDF under certain conditions of formaldehyde administration. Notably, formaldehyde can be added at either in vivo step, or in vitro, and either the wild-type proteins or mutant versions can be tested. By using anti-FLAG antibody for detection on the Western blot, this requires PDF-FLAG to have co- purified with EfgA on the nickel column.

Fig. 17 depicts certain experiments using wild type EfgA and PDF under certain conditions of formaldehyde administration and resulting binding of EfgA to PDF or lack thereof. Formaldehyde additions refer to the experimental steps outlined in Figure 16.

Fig. 18A depicts certain experiments using wild type and H2A and SI 14N mutant EfgA and wild type PDF under certain conditions of formaldehyde administration and resulting binding of EfgA to PDF or lack thereof.

Fig. 18B depicts certain experiments using wild type EfgA and wild type and V54G mutant PDF under certain conditions of formaldehyde administration and resulting binding of EfgA to PDF or lack thereof.

DETAILED DESCRIPTION

Embodiments of the present disclosure include a recombinant host microorganism that includes one or more genetic modifications which result in an increased ability to survive and/or grow in the presence of formaldehyde (HCHO), optionally having a concomitant increase in flow to biomass or by-product production. Embodiments of the present disclosure include a recombinant host microorganism that includes one or more genetic modifications that result in increased rate of cell proliferation. Embodiments of the present disclosure include a recombinant host microorganism that includes one or more genetic modifications that result in increased biomass production per substrate. Embodiments of the present disclosure include a recombinant host microorganism that includes one or more genetic modifications that result in increased fitness and/or survival of the microorganism in the presence of HCHO.

Embodiments of the present disclosure include recombinant host microorganisms described herein that have been further genetically modified to include a biosynthetic pathway for a target carbon- containing compound. According to one aspect, the recombinant host microorganisms have been genetically modified to reduce use of a carbon feedstock to produce carbon dioxide resulting in an increased production of carbon biomass by the recombinant host microorganisms. When a recombinant host microorganism has been further genetically modified to include a biosynthetic pathway for a target carbon- containing compound, the recombinant host microorganism produces the target compound in a greater amount compared to a host organism that has not been recombinantly modified as described herein.

Aspects of the present disclosure relate to recombinant methylotrophic microorganisms being genetically modified to increase overall biomass production from lower carbon-containing compounds, such as Ci compounds. Methylotrophic microorganisms are those, such as bacteria and certain fungi, which grow on Ci compounds like CH₄ and CH₃OH as the carbon and energy source. Methylotrophic microorganisms may also include those that, in addition to Ci compounds, grow on lower carbon compounds such as C₂ to C₅ carbon compounds.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., (1989) and by Silhavy, T.J., Bennan, M.L. and Enquist, L.W., Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., (1984); and by Ausubel, F.M. et. al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience (1987) each of which are hereby incorporated by reference in their entireties.

Additional useful methods are described in manuals including Advanced Bacterial Genetics (Davis, Roth and Botstein, Cold Spring Harbor Laboratory, 1980), Experiments with Gene Fusions (Silhavy, Berman and Enquist, Cold Spring Harbor Laboratory, 1984), Experiments in Molecular Genetics (Miller, Cold Spring Harbor Laboratory, 1972) Experimental Techniques in Bacterial Genetics (Maloy, in Jones and Bartlett, 1990), and A Short Course in Bacterial Genetics (Miller, Cold Spring Harbor Laboratory 1992) each of which are hereby incorporated by reference in their entireties.

Methylotropic microorganisms may be genetically modified to incorporate genes or delete genes by methods known to those of skill in the art. Genes, or homologs thereof, to be added to the genome of the methylotropic microorganisms are known to those of skill in the art or may be identified and obtained using methods known to those of skill in the art. Genes expressed by the methylotropic microorganisms to be inhibited are known to those of skill in the art or may be determined using methods known to those of skill in the art.

Certain aspects of the invention pertain to vectors, such as, for example, expression vectors. As used herein, the term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. By way of example, but not of limitation, a vector of the invention can be a single-copy or multi-copy vector, including, but not limited to, a BAC (bacterial artificial chromosome), a fosmid, a cosmid, a plasmid, a suicide plasmid, a shuttle vector, a PI vector, an episome, YAC (yeast artificial chromosome), a bacteriophage or viral genome, or any other suitable vector. The host cells can be any cells, including prokaryotic or eukaryotic cells, in which the vector is able to replicate.

Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Vectors and plasmids useful for transformation of some host cells may be common and commercially available from companies such as Invitrogen Corp. (Carlsbad, CA), Stratagene (La Jolla, CA) and New England Biolabs, Inc. (Beverly, MA). Other organisms will require broad- host-range vectors such as the pCM series generated from a minimal IncP replicon (Marx and Lidstrom, Microbiology 147-2065-2075 (2001)), or that take advantage of conjugal transfer to introduce suicide vectors (Marx and Lidstrom, BioTechniques 33:1062-1067 (2002); Marx, BMC Research Notes 1 :1 (2008)). In certain exemplary embodiments, an exogenous nucleic acid described herein (e.g., a nucleic acid sequence encoding efgA) is expressed in bacterial cells using a bacterial expression vector such as, e.g., a fosmid. A fosmid is a cloning vector that is based on the bacterial F-plasmid. The host bacteria will typically only contain one fosmid molecule, although an inducible high-copy ori can be included such that a higher copy number can be obtained (e.g., pCClFOS™, pCC2FOS™). Fosmid libraries are particularly useful for constructing stable libraries from complex genomes. Fosmids and fosmid library production kits are commercially available (EPICENTRE^® Biotechnologies, Madison, WI). For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence in a form suitable for expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the foreign nucleic acid sequence encoding a plurality of ribonucleic acid sequences described herein is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid sequence. In certain aspects, operably linked nucleic acid sequences are physically linked, using e.g., fusion RNAs and/or fusion proteins without splicing and/or cleavage of the endogenous product and recombinant nucleic acid sequences. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Foreign nucleic acids (i.e., those which are not part of a cell's natural nucleic acid composition) (e.g., expressing one or any combination of efgA, EfgA, def and Def) may be introduced into a cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transduction, infection (e.g., viral transduction), injection, microinjection, gene gun, nucleofection, nanoparticle bombardment, transformation, conjugation, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.

As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTESf® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

Typically, the vector or plasmid contains sequences directing transcription and translation of a relevant gene or genes, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the species chosen as a production host.

Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, lac, ara, tet, trp, IP_L, IP_R, T7, tac, and trc (useful for expression in Escherichia coli and Pseudomonas); the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, and Bacillus licheniformis; nisA (useful for expression in gram positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic PI 1 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152: 101 1 -1019 (2006)). Termination control regions may also be derived from various genes native to the preferred hosts.

The various genes for a desired biosynthetic or other desired pathway may be assembled into any suitable vector, such as those described above. The codons can be optimized for expression based on the codon index deduced from the genome sequences of the host strain, such as for a methylotrophic microorganism (Agashe et al., Mol. Biol. Evol. 30:549-560 (2013)).

Methylotrophic microorganisms which may serve as host cells and which may be genetically modified to produce recombinant methylotrophic microorganisms include methylotrophic bacteria and methylotrophic fungi. Methylotrophic microorganisms which may serve as host cells and which may be genetically modified to produce recombinant methylotrophic microorganisms as described herein may include one or more members of the genera Clostridium, Rhodobacter, Xanthobacter, Brevibacterium, Mycobacterium, Amycolaptosis, Bacillus, Methylobacillus, Methylomicrobium, Methylotenera, Paracoccus, Methylocella, Methylacidiphium, Methylobacterium, Methylococcus, Methylobacter, Methylibium, Leisingera, Methylophilus, Methylosulfonomonas, Hyphomicrobium, Methylocystis, Methylosinus, Methylomirabilis, Methylophaga and Methyloversatilis.

Methylotrophic microorganisms which may serve as host cells and which may be genetically modified to produce recombinant methylotrophic microorganisms as described herein may include Methylobacterium spp. such as M. extorquens AMI, M. extorquens PA1 , M. extorquens DM4, M. extorquens CM4, M. populi BJ001 , M. nodulans ORS 2060, Methylobacterium spp. 4-46, M. radiotolerans JCM 2831, Paracoccus denitrificans PD122, Xanthobacter autotrophicus Py2, and Hyphomicrobium denitrificans.

Methylobacterium extorquens AMI is a well-characterized bacterium and is described in (Peel and Quayle, Biochem. Journal 81 (3): 465-469 (1961)). The genes encoding enzymes involved in Ci growth of Methylobacterium extorquens AMI are described in (Chistoserdova et al, J. Bacteriol. 185(10): 2980-2987 (2003)). Methylobacterium extorquens PA1 is a well-characterized bacterium and is described in (Knief et al., Microb. Ecol. 60: 440-452 (2010)). The genes encoding enzymes involved in Ci growth of Methylobacterium extorquens PA1 are described in (Marx et al., J. Bacteriol. 194: 4746-4748 (2012)). Certain embodiments of the subject invention are directed to one or more polypeptide sequences having one or more EfgA activities. As used herein, the term "EfgA activity" refers to the ability of a polypeptide sequence to confer to a cell an increased ability to survive, grow and/or divide in the presence of HCHO compared to a cell not expressing a polypeptide sequence having one or more EfgA activities. An EfgA activity further includes the ability of a cell expressing EfgA to better survive HCHO shock than a cell that doesn't express EfgA. A polypeptide having one or more EfgA activities thus act as a formaldehyde sensor.

Certain embodiments of the subject invention are directed to a first nucleic acid (e.g., a nucleic acid sequence encoding an efgA (e.g., SEQ ID NO: l)) or polypeptide sequence (e.g., an EfgA (e.g., SEQ ID NO:2)) having a certain sequence identity or percent homology to a second nucleic acid or polypeptide sequence, respectively. Certain embodiments of the subject invention are directed to a first nucleic acid (e.g., a nucleic acid sequence encoding a def (e.g., SEQ ID NO:3)) or polypeptide sequence (e.g., a Def (e.g., SEQ ID NO:4)) having a certain sequence identity or percent homology to a second nucleic acid or polypeptide sequence, respectively.

Techniques for determining nucleic acid and amino acid "sequence identity" are known in the art. Typically, such techniques include determining the nucleotide sequence of genomic DNA, mRNA or cDNA made from an mRNA for a gene and/or determining the amino acid sequence that it encodes, and comparing one or both of these sequences to a second nucleotide or amino acid sequence, as appropriate. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI).

One method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found at the NCBI/NLM web site.

Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA sequences, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, at least about 85%-90%, at least about 90%-95%, or at least about 95%-98%, or about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity over a defined length of the molecules, as determined using the methods above.

In certain aspects, the sequence identity is determined across the length of a particular SEQ ID. In other aspects, the sequence identity is determined across a particular number of consecutive nucleic acids in length. In other aspects, the sequence identity is determined across a particular number of consecutive amino acids in length. For efgA, a nucleic acid sequence having a particular sequence identity can be at least about 348, 369, 391, 413, 417, 422, 426 or about 430 consecutive nucleic acids in length. For EfgA, an amino acid sequence having a particular sequence identity can be at least about 116, 123, 130, 138, 139, 140, 142 or about 143 consecutive amino acids in length. For def, a nucleic acid sequence having a particular sequence identity can be at least about 413, 438, 464, 490, 495, 500, 505 or about 511 consecutive nucleic acids in length. For Def, an amino acid sequence having a particular sequence identity can be at least about 137, 146, 155, 163, 165, 167, 168 or about 170 consecutive amino acids in length.

As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, NY; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press.

Two nucleic acid fragments are considered to "selectively hybridize" as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., supra). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent" conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization, Supra).

With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook et al., Supra).

As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% identical to each other typically remain hybridized to each other. In one aspect, the conditions are such that sequences at least about 70%, at least about 80%, at least about 85% or 90% or more identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50 °C, at 55 °C, or at 60 °C or 65 °C.

According to certain aspects, a carbon-containing compound is used as a feedstock or growth substrate for the recombinant microorganisms described herein. Suitable carbon containing compounds useful as a feedstock include any Ci compounds, or multi-C compounds that contain Ci units such as methylated amines, methylated sulfhydryls, methoxy groups, etc. Such carbon- containing compounds include methane, methanol, methylamine hydrochloride, sodium formate, betaine, sarcosine, methanethiol, and formaldehyde. Exemplary carbon- containing compounds useful as a feedstock include methane and methanol. Exemplary products produced by the recombinant microorganisms are butanol and carotenoids.

According to certain aspects of the present disclosure a Methylobacterium is genetically modified to produce a recombinant Methylobacterium. According to certain aspects, a Methylobacterium is genetically modified to express exogenous EfgA as a sensor that better enables the genetically modified Methylobacterium to survive, grow and/or divide in the presence of HCHO.

According to certain aspects, recombinant host microorganisms described herein have been further genetically modified to include a biosynthetic pathway for a target carbon-containing compound. Certain useful target carbon-containing compounds include but are not limited to polyhydroxyalkanoates and related storage polymers (Borque et al., Appl. Microbiol. Biotechnol. 44:367-376 (1995)), polysaccharides (Oh et al., Biotechnol. Bioeng. 54:115-121 (1997)), amino acids such as serine (Sirirote et al., J. Fermen. Technol. 66:291-297 (1988)), liquid biofuels such as ethanol and butanol, 1,4-butanediol, isoprenoids (Van Dien et al., Appl. Environ. Microbiol. 69:7563-7566 (2003)), and plant growth hormones such as indole-3-acetic acid (Omer et al., Plant Growth Regul. 43:93-96 (2004)) trans-ze&tin (Koenig et al., J. Bacteriol. 184:1832-1842 (2002)). Furthermore, d -based protein production could be enhanced due to higher biomass efficiencies for active polypeptides such as enterocin P (Gutierrez et al., FEMS Microbiol. Lett. 248:125-131 (2005)), haloalkane dehalogenase (FitzGerald and Lidstrom, Biotechnol. Bioeng. 81 :263-268 (2003)), and esterase (Choi et al., Appl. Environ. Microbiol. 72:753-759 (2006)). Biosynthetic pathways for useful target carbon-containing compounds are known to those of skill in the art and include the pathway for the generation and incorporation of β-hydroxybutyrl-CoA into polyhydroxybutyrate or related polymers (Korotkova and Lidstrom, J. Bacteriol. 184:1750- 1758 (2001)), the biosynthetic pathway for conversion of isopentyl pyrophosphate into carotenoids (Van Dien et al., Appl. Environ. Microbiol. 69:7563-7566 (2003)), the isopentylation of adenines of some tRNAs by the miaA gene product to produce trans-zeatin (Koenig et al., J. Bacteriol. 184:1832-1842 (2002)), the pathway for indole-3 -acetic acid from tryptophan (Omer et al., Plant Growth Regul. 43:93-96 (2004)), enzymes such as pyruvate decarboxylase and alcohol dehydrogenase to generate ethanol (Deng and Coleman, Appl. Environ. Microbiol. 65:523-528 (1999)), butanol production from crotonyl-CoA via expression of enzymes such as Bed, EtfAB, and AdhE2 from Clostridium acetobutylicum (Shen et al., Appl. Environ. Microbiol. 77:2905-2915 (2011)), or 1,4-butanediol from succinyl-CoA or a-ketoglutarate via CoA-dependent succinate semialdehyde dehydrogenase or 2-oxoglutarate decarboxylase, then 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrl-CoA transferase, 4-hydroxybutyryl-CoA reductase, and alcohol dehydrogenase (Yim et al., Nat. Chem. Biol. 7:445-452 (2011)). Protein production can be accomplished via strong expression systems for methylotrophic bacteria, such as those dependent upon the methanol dehydrogenase promoter (Ρ_{η α}ρ) of M. extorquens AMI such as pCM80, pCMl lO, pCM160 (Marx and Lidstrom, Microbiology 147-2065-2075 (2001)), or inducer- regulated versions such as pHC 112 (Chou and Marx, Cell Reports 1 :1-8 (2012)).

It is to be understood that the embodiments of the present invention that have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures, tables and accompanying claims.

EXAMPLE I Materials and Methods

Experimental Evolution Methylobacterium extorquens PA1 is an alphaproteobacteria that can grow on reduced single carbon compounds like methanol as the sole source of carbon and energy. During growth on single carbon compounds, formaldehyde is produced as an essential metabolic intermediate. WT does not grow on formaldehyde concentrations > 1 mM. Mutant strains of M. extorquens PA1 that could grow on elevated concentrations of formaldehyde (20 mM) were screened by serially transferring three replicate populations of WT in minimal media with increasing concentrations of formaldehyde and decreasing concentrations of methanol (a "weaning technique"). After 30 transfers mutant strains were obtained that could grow on 20 mM formaldehyde as the sole carbon source. Genome Sequencing

The genome of three mutant strains were sequenced to elucidate the mutations that enabled growth on elevated concentrations of formaldehyde. Each of these three mutant strains had a different, non-synonymous mutation in the coding sequence of Mext_4158, a gene encoding a hypothetical conserved protein with a domain of unknown function of type 336 among other mutations (Fig. 15A).

The function of Mext_4158 (renamed efgA)

By using an allele exchange method, the mutant alleles of efgA were introduced into the WT strain. These strains were then able to grow on 5 mM formaldehyde as the sole carbon source. A clean knockout or null mutation in efgA in the WT genomic background was also observed to enable growth on 5 mM formaldehyde. (See Fig. 8.) Despite being able to grow on 5mM formaldehyde, the efgA knockout mutants were more susceptible to formaldehyde shock. (See Fig. 9.) Without intending to be bound by scientific theory, it was then postulated that EfgA works as a formaldehyde sensor and temporarily stalls core cellular functions during formaldehyde buildup.

EfgA carries out the same function in E. coli as well

Next, whether EfgA would work as a formaldehyde sensor that responds to formaldehyde shock across distantly related organisms was tested. A vector that expressed efgA from M. extorquens PA1 on a constitutive promoter (Ptac) was introduced into E. coli K12. The resulting trans conjugant was significantly more resistant to formaldehyde shock (20 mM for 35 minutes) that a strain expressing the mutant allele of efgA^jm or a strain expressing an empty vector (Fig. 15B).

EXAMPLE II Data efgA (Gene id: Mext_4158 in Methylobacterium extorquens PA1) is a 435 bp long gene that is currently annotated as a "hypothetical protein of unknown function with a conserved domain of unknown function type 336." (See Fig. 13.) The nucleic acid sequence of the gene efgA is set forth as SEQ ID NO:l, and the amino acid sequence encoded by efgA is set forth as SEQ ID NO:2. A WT strain of M. extorquens PAl could not grow on >0.5 mM formaldehyde but strains with a null mutations in efgA (efgA^m]jmT) could grow on <5 mM formaldehyde as the sole carbon and energy source. Mutant strains of M. extorquens PAl lacking efgA were 15-fold more sensitive to formaldehyde shock (e.g., exposure to 50 mM formaldehyde for 35 minutes) than WT. def Gene id: Mext_1636 in Methylobacterium extorquens PAl) is a 516 bp long gene that encodes the peptide deformylase: a protein that is essential for translation in bacteria. (See Fig. 14.) The nucleic acid sequence of the def gene is set forth as SEQ ID NO:3, and the amino acid sequence encoded by def is set forth as SEQ ID NO:4.

A WT strain of M. extorquens PAl could not grow on >0.5 mM formaldehyde but strains with unique mutations (V45G and G143S) in def

could grow on <5 mM formaldehyde as the sole carbon and energy source. efgA^WJ and efgA^mTTANT were cloned from M. extorquens PAl and placed in front of a strong promoter (P_tac) on a plasmid encoding a kanamycin resistance cassette with a ColEl origin of replication (pDN145, pDN146 respectively). pDN145 was transformed into E. coli K12. WT E. coli K12 could grow in minimal media with 10 mM glucose and 1.5 mM formaldehyde (final OD₆oo -0.900). E. coli K12+ pDN146 (efgA "™*⁷) could grow in minimal media with 10 mM glucose, 1.5 mM formaldehyde and lO g/ml kanamycin (final OD₆oo -0.900). However, E. coli K12+ pDN145 (efgA^¹) grew very poorly in minimal media with 10 mM glucose and 1.5 mM formaldehyde and lO g/ml kanamycin (final OD₆oo -0.015). E. coli K12 + pDN145 could survive formaldehyde shock (e.g., exposure to 20 mM formaldehyde for 35 minutes) 73-fold greater than WT E. coli K12. E. coli K12 + pDN146 could survive formaldehyde shock (e.g., exposure to 20 mM formaldehyde for 35 minutes) 7-fold greater than WT E. coli K12.

EXAMPLE III Conclusions

These data indicate that EfgA, specifically in the presence of sub-inhibitory concentrations of formaldehyde, can inhibit bacterial growth. Strains of M. extorquens PA1 with a WT allele of efgA but with a mutant allele of def (encoding peptide deformylase) were able to grow on <5 mM formaldehyde as the sole carbon and energy source. Without intending to be bound by scientific theory, in the presence of formaldehyde, EfgA directly interacts with Def and inhibits protein translation. EXAMPLE IV

EfgA Binding to Formaldehyde and Peptide Deformylase (PDF of Def)

Experiments were carried out to confirm that EfgA expressed by a microorganism binds to formaldehyde and further binds to peptide deformylase. As shown in the general schematic of Fig. 16, a first colony of E. coli overexpressing EfgA tagged with 6X histidine was grown. A second separate colony of E. coli overexpressing PDF tagged with FLAG was grown. Each of the separate colonies were then separately lysed and the lysates were mixed and applied to a Ni⁺² column for binding to the 6X histidine tag. If EfgA is also bound to the FLAG tagged PDF, then the FLAG tagged PDF would also be bound to the Ni⁺² column. The bound EfgA is then removed from the column and analyzed for the presence of the FLAG indicating binding to PDF. As indicated in Fig. 16, in a first experiment, formaldehyde was added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1). Then the separate colonies were lysed and the lysates were mixed and analyzed for binding of EfgA to PDF. In a second experiment, formaldehyde was added in vivo to the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 2). Then the separate colonies were lysed and the lysates were mixed and analyzed for binding of EfgA to PDF. In a third experiment, formaldehyde was added in vitro to the mixture of lysates from the growing E. coli overexpressing EfgA tagged with 6X histidine and the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 3). Then the mixture was analyzed for binding of EfgA to PDF. In a fourth experiment, formaldehyde was added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1) and formaldehyde was added in vivo to the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 2). Then the separate colonies were lysed and the lysates were mixed and analyzed for binding of EfgA to PDF.

Fig. 17 depicts a table representing the experiments using the wild type gene for expressing EfgA and the wild type gene for expressing PDF and where formaldehyde was added as indicated by the numbers 1, 2 and 3. As shown in the upper blot, EfgA was present in each experiment where wild type EfgA was used. As shown in the lower blot, when 5 mM formaldehyde was added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1), EfgA bound to PDF. As shown in the lower blot, when 5 mM formaldehyde was added in vitro to the mixture of lysates from the growing E. coli overexpressing EfgA tagged with 6X histidine and the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 3), EfgA bound to PDF. Accordingly, EfgA directly binds to formaldehyde and also binds to PDF.

As shown in the lower blot, when 5 mM formaldehyde was added in vivo to the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 2), no binding between EfgA and PDF was detected. As shown in the lower blot, when 5 mM formaldehyde was added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1) and in vivo to the growing E. coli overexpressing PDF tagged with FLAG (indicated by the number 2), no binding between EfgA and PDF was detected. Without wishing to be bound by scientific theory, adding 5 mM formaldehyde in vivo to growing E. coli overexpressing PDF tagged with FLAG may alter the ability of the PDF to bind to EfgA.

As shown in Fig. 18A, similar experiments summarized in the left table were carried out using H2A and S114N mutants of efgA thereby producing altered forms of EfgA. Formaldehyde was either added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1) or added in vitro to the mixture of lysates from the growing E. coli overexpressing EfgA tagged with 6X histidine and the growing E. coli overexpressing PDF tagged with FLAG (indicated by number 3) as indicated. As shown in the lower gel, the experiments using the H2A and S114N mutants of efgA did not produce detectable binding of EfgA to PDF. Without wishing to be bound by scientific theory, the mutant form of the EfgA may prevent binding to PDF as the experiments using the WT forms of EfgA and PDF produced detectable binding of EfgA and PDF.

As shown in Fig. 18B, similar experiments summarized in the right table were carried out using a V54G mutant of def thereby producing altered forms of PDF. Formaldehyde was either added in vivo to the growing E. coli overexpressing EfgA tagged with 6X histidine (indicated by the number 1) or added in vitro to the mixture of lysates from the growing E. coli overexpressing EfgA tagged with 6X histidine and the growing E. coli overexpressing PDF tagged with FLAG (indicated by number 3) as indicated. As shown in the lower gel, the experiments using the V54G mutant def did not produce detectable binding of EfgA to PDF. Without wishing to be bound by scientific theory, the mutant form of the PDF may prevent binding to EfgA as the experiments using the WT forms of EfgA and PDF produced detectable binding of EfgA and PDF. EXAMPLE V

Experimental Protocols for Example IV

Overexpression of efgA (His-tagged EfgA) or def (FL AG-tagged PDF) The pET28a vector containing desired ORF in BL21DE3 (T7 overexpression strain ordered from NEB) was grown up in LB+Kan for about 12 hours. A 50-fold dilution (600 xL) of the saturated culture was added to 30 ml of LB+Kan. The culture was grown in a 30°C incubator shaking at 225 rpm. After about 2 hours, ImM IPTG was added to the culture. If required, 5mM formaldehyde was added 3 hours after induction. Cultures were harvested 4 hours after induction by spinning 50 mL falcon tubes at 3000 rpm for about 15 minutes at 4°C in a table-top centrifuge. The supernatant was decanted, the cell pellet was dried and frozen overnight at -80°C.

Cell Lysis (EfgA) The frozen cell pellets of strains overexpressing the His-tagged EfgA protein were thawed at room temperature for about 15 minutes. Cell pellets were resuspended in 1 mL of cold EfgA lysis buffer (50mM Tris-HCl at pH=7.5 and 250 mM NaCl). The cell suspension was transferred to Eppendorf tubes and centrifuged at 13000 rpm for 10 minutes. One protease cocktail tablet was added to 10 mL of the EfgA lysis buffer and the solution was vortexed vigorously. The supernatant of centrifuged cells was decanted and 1 mL of the protease cocktail solution was added to resuspend the cell pellets. The cell suspension was transferred to tubes with lysing matrix B. 10xL of a 0.1M stock of PMSF was added to each tube just before lysing. Cells were lysed by using the bead beater at 6.5 m/s for 1 minute. The lysate was centrifuged at 13000 rpm for 1 minute to collect beads at the bottom. The supernatant was transferred to Eppendorf tubes pre-chilled in a 4°C fridge (or on ice) and centrifuged at 13000 rpm for 20 minutes in a centrifuge at 4°C. The supernatant (or the soluble fraction) was transferred to Eppendorf tubes pre-chilled in a 4°C fridge (or on ice). The samples were saved at -80°C if not immediately proceeding to the next step.

Pulldowns

The nickel resin slurry was vortexed until the beads were completely in suspension. 35 xL of the slurry was added to a pre-chilled epi tube and kept on ice. 3.5 of a 1M imidazole stock solution was added (final concentration about 5 mM) to the soluble fraction of the cell lysates and transferred to the tubes with the nickel resin slurry. The tubes with the nickel resin slurry and the cell lysates were gently shaken on a rocker at 4°C for 1 hour. After shaking, the mixture was centrifuged at 13000 rpm for 1 minute. About 500 μL of the supernatant was decanted. 500 μL of EfgA-wash buffer (EfgA-lysis buffer + 35mM imidazole) was added to each tube and the solution was mixed gently. The tubes were centrifuged at 13000 rpm for 1 minute and about 500 μΣ, of the supernatant was decanted. The wash step was repeated once more (a total of 2 washes) if studying the interaction of EfgA and PDF. Two additional washes with the PDF-lysis buffer were performed if conducting interactions assays. The wash step was repeated three more times (a total of 4 washes) if stopping at pulldowns. After the last wash, all the supernatant (as possible without disturbing the nickel coated beads) was decanted.

Cell Lysis (ΡΌΈ)

The frozen cell pellets of strains overexpressing the FLAG-tagged PDF protein was thawed at room temperature for about 15 minutes. 1 mL of cold PDF lysis buffer (50mM Tris-HCl at pH=7.5 and 40 mM NaCl) was added to each Falcon tube to resuspend the cell pellet and the cell suspension was transferred to Eppendorf tubes and centrifuged at 13000 rpm for 10 minutes. One protease cocktail tablet was added to 10 mL of the EfgA lysis buffer and the solution was vortexed vigorously. The supernatant of centrifuged cells was decanted and 1 mL of the protease cocktail solution was added to resuspend the cell pellets. The cell suspension was transferred to tubes with lysing matrix B. 10 of a 0.1M stock of PMSF was added to each tube just before lysing. Cells were lysed by using the bead beater at 6.5 m/s for 1 minute. The lysate was centrifuged at 13000 rpm for 1 minute to collect beads at the bottom. The supernatant was transferred to Eppendorf tubes pre-chilled in a 4°C fridge (or on ice) and centrifuged at 13000 rpm for 20 minutes in a centrifuge at 4°C. The supernatant (or the soluble fraction) was transferred to Eppendorf tubes pre- chilled in a 4°C fridge (or on ice). The samples were saved at -80°C if not immediately proceeding to the next step.

In vitro protein-protein interaction

The supernatant of the PDF cell lysates (or the soluble fraction) was added to the tubes with the nickel resin equilibrated with the PDF-lysis buffer. If formaldehyde was being added in vitro, then 4 of a 1M formaldehyde stock (about 5 mM) was added to the tube with the PDF cell lysates and the nickel resin. The tubes were incubated on a rocker in a 30°C incubator for 30 minutes. After 30 minutes, the tubes were centrifuged at 13000 rpm for 1 minute. About 500 μΐ, of the supernatant was decanted. 500 μΐ, of PDF-wash buffer (PDF-lysis buffer + 35mM imidazole) was added to each tube and the solution was mixed gently. The tubes were centrifuged at 13000 rpm for 1 minute and about 500 μΐ, of the supernatant was decanted. The wash step was repeated three more times (a total of 4 washes). After the last wash, all the supernatant (as possible without disturbing the nickel coated beads) was decanted.

SDS-PAGE Gels 12.5% SDS-PAGE gels were prepared. 25 xL of the 2X sample buffer (with beta- mercaptoethanol) were added to each sample. Samples were boiled 100°C for 3 minutes and centrifuged at 13000 rpm for 1 minute. 7.5 μΐ, of the sample was loaded in each well and 5 μΐ, of the prestained protein ladder was added in the right most corner well. The 12.5% gel was run at 200V for about 45 minutes or until the dye front reached the bottom of the gel. The glass case was removed gently and the stacking gel was discarded.

For Coomassie gels, the gel was transferred to a box with R-250 Coomassie stain solution and shaken on a rocker at room temperature for 20 minutes. The Coomassie stain was drained and the destain solution was added and the gel was shaken on a rocker at room temperature for 5 minutes. The destain solution was drained and fresh destain solution was added and the gel was shaken on a rocker at room temperature for 30-45 minutes until the background was almost clear. The destain solution was drained and deionized water was added and the gel was shaken on a rocker at room temperature for 5 minutes. The deionized water was drained. For Western blots, the gel was carefully transferred to a box with cold transfer buffer.

Transfer proteins from gel to a nitrocellulose membrane

A 5-10 inch tray was filled with cold transfer buffer. To the sandwich cassette, first two sponges were added and rolled with a test tube to squeeze out any air bubbles. Next, a layer of Whatman paper (cut to 3.5x4.0 inches) was added and rolled with a test tube to squeeze out any air bubbles. Next, the gel was arranged on the Whatman paper and then the nitrocellulose membrane was added to completely cover the gel. Another layer of Whatman paper was added and rolled with a test tube to squeeze out any air bubbles. A final layer of sponge was added and rolled with a test tube to squeeze out any air bubbles. The sandwich cassette was sealed tightly and then the transfer buffer was drained by shaking. The transfer sandwich was arranged in the right orientation in the gel box (black end goes against the black side of the transfer box), an ice block was added to the gel box and then ice cold transfer buffer was added until it completely filled the gel box. The gel was run at 100V for 1 hour.

Blocking and incubating with antibodies

Using tweezers, the membrane was carefully transferred in a box with 3% milk buffer (also known as blocking buffer; 1.5 gms of nonfat dry milk in 50 mL of Western buffer) and shaken on a rocker at room temperature for 20 minutes. The blocking buffer was drained and the membrane was incubated on a rocker at room temperature with 8-10 mL of a 1 :500 dilution (in Western buffer) of lmg/mL M2 anti-FLAG antibody sold by Sigma. The membrane was washed with Western buffer for 5 minutes. The wash step was repeated two more times for a total of three washes. To 20 mL of the Western buffer, \ xL of the mouse secondary antibody was added and mixed. This solution was added to the tray containing the membrane and incubated on a rocker at room temperature for 20 minutes. The membrane was washed with Western buffer for 5 minutes. The wash step was repeated three more times for a total of four washes. The Western buffer was drained and lmL of the chemiluminescent substrate was added and the membrane was gently agitated for 2 minutes. The membrane was dried completely and wrapped in polyvinyl.

Exposing and Developing Western Blots

In a dark room, the autoradiography cassette was opened and the straight edge of the light sensitive film was aligned vertically and placed on the cassette. The polyvinyl wrapped membrane was pressed down on the light sensitive film and the autoradiography cassette was closed. The membrane was exposed to the film for less than 1 minute for pulldowns and for 30 minutes to 1 hour for protein-protein interactions. The light sensitive film was developed using standard techniques.

Claims

What is claimed is:

1. A method of improving the ability of a microorganism to produce a target compound encoded by a target compound exogenous gene comprising genetically modifying the microorganism to include an exogenous efgA gene and expressing the exogenous efgA gene.

2. The method of claim 1 wherein the microorganism is a bacterium.

3. The method of claim 1 wherein the microorganism expresses endogenous def.

4. The method of claim 1 wherein microorganism is a non-methylotropic bacterium engineered to produce a desired commodity product using methanol or methane as a carbon source for growth.

5. The method of claim 1 wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO: 1 across the length of SEQ ID NO: 1.

6. The method of claim 1 wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO: 1.

7. The method of claim 1 wherein the exogenous efgA gene provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock.

8. The method of claim 1, wherein the microorganism that has been genetically modified has an increased ability to produce the target compound as compared to a wild-type microorganism not expressing the exogenous efgA gene.

9. The method of claim 1, wherein the target compound is butanol or a carotenoid.

10. The method of claim 1, wherein the microorganism uses methane or methanol as a source for growth.

11. A method of genetically altering a microorganism to express a polypeptide which senses formaldehyde production by the microorganism comprising genetically modifying the microorganism to include an exogenous efgA gene and expressing the exogenous efgA gene.

12. The method of claim 11 wherein the microorganism is a bacterium.

13. The method of claim 11 wherein the microorganism expresses endogenous def.

14. The method of claim 11 wherein microorganism is a non -methylotropic bacterium engineered to produce a desired commodity product using methanol or methane as a carbon source for growth.

15. The method of claim 11 wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO: 1 across the length of SEQ ID NO: 1.

16. The method of claim 11 wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO: 1.

17. The method of claim 11 wherein the exogenous efgA gene provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock.

18. A recombinant microorganism expressing an exogenous efgA gene.

19. The recombinant microorganism of claim 18, in which the recombinant microorganism is a bacterium.

20. The recombinant microorganism of claim 19, in which the bacterium is a non- methylotropic bacterium engineered to produce a desired commodity product using methanol or methane as a carbon source for growth.

21. The recombinant microorganism of claim 18, wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO: 1 across the length of SEQ ID NO: 1.

22. The recombinant microorganism of claim 18, wherein the exogenous efgA gene has at least 90% sequence identity to the nucleic acid of SEQ ID NO:l, that is at least 391 consecutive nucleic acids in length.

23. The recombinant microorganism of claim 18, wherein the exogenous efgA gene provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock.

24. The recombinant microorganism of claim 18, wherein the recombinant microorganism has an increased ability to produce a desired commodity product as compared to a wild-type microorganism not expressing the exogenous efgA gene.

25. The recombinant microorganism of claim 24, wherein the desired commodity product is butanol or a carotenoid.

26. The recombinant microorganism of claim 18, in which the recombinant microorganism can use formaldehyde as a carbon source for growth.

27. A recombinant microorganism expressing an exogenous efgA gene to produce EfgA polypeptide, and endogenous def gene and an exogenous gene pathway for the production of a target compound using methane or methanol as a carbon source for growth.

28. The recombinant microorganism of claim 27, in which the recombinant microorganism is a bacterium.

29. The recombinant microorganism of claim 28, in which the bacterium is a non- methylotropic bacterium.

30. The recombinant microorganism of claim 27, wherein the EfgA polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 across the length of SEQ ID NO:2.

31. The recombinant microorganism of claim 27, wherein the EfgA polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, that is at least 130 consecutive amino acids in length.

32. The recombinant microorganism of claim 27, wherein the EfgA polypeptide provides one or more EfgA activities selected from the group consisting of increased ability to survive, increased ability to grow, increased ability to divide, and decreased sensitivity to formaldehyde shock.

33. A recombinant non-methylotrophic bacterium expressing an exogenous efgA gene, wherein the efgA gene encodes a polypeptide that acts as a formaldehyde sensor.