Patent Application
20220340915
The amino acid sequence of a representative tryptophan halogenase, PyrH, is shown in sequence listing.
According to the World Health Organization, the number of hungry people in the world reached 821 million in 2017, meaning that one in every nine people globally is suffering from food insecurity. Population increase and climate change will make the situation even more challenging. On top of that issue, the current agricultural practices, including the application of chemical fertilizers and pesticides, are environmentally unsustainable. Therefore, there is an urgent need to develop novel agricultural technologies to enhance global food production and lower the food costs in a sustainable way.
Soil bacteria are known to benefit crop growth in a variety of ways, such as producing indole-3-acetic acid (IAA), a native auxin that can stimulate plant growth and enhance crop yield/quality. The chlorinated derivatives of IAA, such as 4-chloro-IAA (4-CI-IAA) and 5-chloro-IAA (5-CI-IAA), have been reported to be much more active and thus are better plant growth promoters than IAA. However, chlorinated auxins are only present in very few plant species and no soil bacteria can produce these powerful plant growth stimulator, which has limited their efficiency and utility in agricultural applications.
In this invention, a novel method was developed to enable soil bacteria to produce powerful chlorinated auxins such as 5-CI-IAA. A heterologous halogenase, such as L-tryptophan 5-halogenase, can be introduced into soil bacteria, including but not limited to Pseudomonas. The resulting strains can acquire the ability to produce chlorinated IAAs, which can be used in agriculture to stimulate crop growth and enhance food production. Therefore, this invention represents a novel and sustainable agricultural biotechnology that can increase food production without the need of chemical fertilizers, herbicides and pesticides that are harmful to the environment and human health.
A novel method was established to engineer soil bacteria for the ability to produce chlorinated auxins, which have strong plant growth-promoting ability and are not naturally produced by soil bacteria. Different L-tryptophan halogenases can be selected and introduced to soil bacteria for the production of specific chlorinated auxins. This method can be applied to a variety of soil microbes fit for different crops and environmental conditions, yielding a wide arsenal of chlorinated auxins-producing soil microbes for various crops and regions.
The present disclosure covers methods for constructing engineered strains of soil bacteria for the capability to produce chlorinated auxins. In the following description, numerous specific details are provided for a thorough understanding of specific preferred embodiments. However, those skilled in the art will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In some cases, well-known structures, materials, or operations are not shown or described in detail in order to avoid obscuring aspects of the preferred embodiments. Furthermore, the described features, genes, structures, strains, or characteristics may be combined in any suitable manner in a variety of alternative embodiments. Thus, the following more detailed description of the embodiments of the present invention, as illustrated in some aspects in the drawings, is not intended to limit the scope of the invention, but is merely representative of the various embodiments of the invention.
In this specification and the claims that follow, singular forms such as “a,” “an,” and “the” include plural forms unless the content clearly dictates otherwise. All ranges disclosed herein include, unless specifically indicated, all endpoints and intermediate values. In addition, “optional” or “optionally” refer, for example, to instances in which subsequently described circumstance may or may not occur, and include instances in which the circumstance occurs and instances in which the circumstance does not occur. The terms “one or more” and “at least one” refer, for example, to instances in which one of the subsequently described circumstances occurs, and to instances in which more than one of the subsequently described circumstances occurs.
In one embodiment, the present disclosure provides methods for engineering soil bacteria to produce chlorinated auxins such as 5-CI-IAA. By way of example, the present disclosure provides for introducing a microbial L-tryptophan 5-halogenase (PyrH) from Streptomyces rugosporus into two Pseudomonas strains to produce 5-CI-IAA. The methods described herein generally provide for a method to enable soil bacteria to produce chlorinated auxins that can better promote crop growth. Preferably, this method allows soil bacteria to produce chlorinated IAAs by incorporating a foreign halogenase, which will make soil bacteria more beneficial to crops and thus a useful agricultural biotechnology to enhance crop production. The details for the procedure are provided.
The present disclosure also provides for the methods to express useful enzymes in soil bacteria.
The genes encoding a L-tryptophan halogenase such as PyrH can be directly amplified from the genome or cDNA of a related microbial strain or chemically synthesized. The genes may be modified for improved activity or expression.
Expression of a L-tryptophan halogenase can be achieved in a variety of hosts such as Escherichia coli, Pseudomonas putida or other microbial strains. Any suitable bacterial strain, vector or culture condition may be used for the expression of L-tryptophan halogenases and production of chlorinated auxins. By way of example, suitable bacterial strains include Escherichia coli and two Pseudomonas strains. Alternatively, any species or strain of Pseudomonas may be used. Broadly, a suitable microbial strain is any soil bacterium strain capable of expressing a L-tryptophan halogenase. In some embodiments, chlorinated auxins may be generated by a microbial strains harboring a vector or vectors that encode for a L-tryptophan halogenase. The vector or vectors may be plasmids.
Chlorinated auxins may be produced by soil bacteria harboring a L-tryptophan halogenase from exogenously supplied substrates such as L-tryptophan and IAA. Chlorinated auxins may also be generated by soil bacteria harboring a L-tryptophan halogenase directly without supply of any said substrates.
The engineered microbial strains that harbor a L-tryptophan halogenase are grown in an appropriate medium. If there is an inducible promoter in the vector, a specific inducer will be added into the culture to induce protein expression. If a constitutive promoter is used, no inducer is needed. To produce a chlorinated auxin, such as 5-CI-IAA, substrates such as IAA will be added to the culture if the engineered strains don't naturally produce the substrate. No substrates are needed if the original soil bacteria have the ability to produce IAA.
The following examples are illustrative only and are not intended to limit the disclosure in any way. One skilled in the art would recognize various known methods and conditions for cloning or synthesizing a halogenase gene, expressing a halogenase in a soil bacterium, and analyzing the production of chlorinated IAAs. Each of these various embodiments are within the scope of the invention.
The following materials and methods may be used in carrying out the various embodiments of the invention.
Streptomyces rugosporus NRRL 21084 was obtained from USDA Agricultural Research Service Culture Collection. It was grown at 30° C. in YEME medium for the preparation of genomic DNA. Escherichia coli XL1-Blue and BL21(DE3) was purchased from Agilent. Pseudomonas putida KT2440 (ATCC 47054) was obtained from the American Type Culture Collection and Pseudomonas chlororaphis O6 was acquired from Dr. David Britt at Utah State University. Both Pseudomonas strains were routinely grown at 30° C. in LB medium.
Escherichia coli XL1-Blue was used for DNA cloning and amplification. Escherichia coli BL21(DE3) and pET28a (Novagen) were used for protein expression in Escherichia coli. Escherichia coli cells were grown in Luria-Bertani (LB) medium. pMiSI was used as the expression vector in Pseudomonas. When necessary, kanamycin was added into the culture medium at 50 μg/mL.
The genomic DNA of Streptomyces rugosporus was isolated using standard methods. Plasmids in Escherichia coli were extracted using a GeneJET™ Plasmid Miniprep Kit (Fermentas).
The pyrH gene (GenBank accession number AFV71318) was amplified from the genome of Streptomyces rugosporus via PCR using a set of specific primers, including 5′-AATTCATATGATTCGCAGCGTTGTGATTGTTG-3′ and 5′-AATTAAGCTTTTATTGAATACTGGCCAGGTATTC-3′. The Phusion high-fidelity DNA polymerase from Thermo Fisher Scientific was used. The PCR program used for amplification of the pyrH gene consisted of an initial denaturation at 98° C. for 5 minutes, 20 cycles of touchdown program (98° C. for 30 seconds, annealing at 70° C. for 40 seconds, decreasing 0.5° C. per cycle, and extension at 72° C. for 90 seconds), 20 cycles of regular program (98° C. for 30 seconds, annealing at 60° C. for 40 seconds, and extension at 72° C. for 90 seconds), followed by a final extension at 72° C. for 10 minutes.
The PCR product was then digested with NdeI and HindIII at 37° C. for 2 hours, which was ligated into the pET28a expression vector between the NdeI and HindIII sites (
The ligation product was introduced into Escherichia coli XL1-Blue competent cells through chemical transformation. The transformants were grown on LB agar supplemented with 50 μg/mL kanamycin at 37° C. overnight.
Colonies were picked from the agar plate into 5 mL of LB broth with 50 μg/mL kanamycin at 37° C. and 250 rpm overnight. The plasmids were extracted and digested with NdeI and HindIII at 37° C. for 2 hours. The digestion of correct pET28a-pyrH is shown in
This new plasmid pET28a-pyrH was then introduced into Escherichia coli BL21(DE3) through chemical transformation. The transformants were grown on LB agar supplemented with 50 μg/mL kanamycin at 37° C. overnight.
A colony was picked from the agar plate into 5 mL of LB broth with 50 μg/mL kanamycin at 37° C. and 250 rpm. When the OD600 value reached 0.5, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added into the culture at a final concentration of 200 μM. The culture was maintained at 28° C. and 250 rpm for 16 hours.
The cells were harvested by centrifugation at 4,000 rpm for 7 minutes. The cells were resuspended in 3 mL of lysis buffer (20 mM Tris-Cl, 500 mM NaCl, pH 7.9). After 10 minutes of ultrasonication (18 W, 30 s of interval), the resultant lysates were centrifuged at 20,000 rpm for 10 minutes. Insoluble proteins were dissolved in 8 M urea.
Both soluble and insoluble fractions were analyzed by 12% SDS-PAGE. As shown in
To test whether the expressed PyrH has the function of L-tryptophan 5-halogenase in Escherichia coli BL21(DE3), L-tryptophan was fed at 100 mg/L into the IPTG-induced broth of in Escherichia coli BL21(DE3)/pET28a-pyrH. After 1 day, 1 mL of the broth was taken from the culture and centrifuged at 15,000 rpm for 7 minutes. The supernatant was analyzed on an Agilent 6130 single quadrupole LC-MS equipped with an Agilent Eclipse XDB-C18 column (5 μm, 250 mm×4.6 mm). The sample was eluted with a gradient mobile phase of methanol-water (containing 0.1% formic acid) from 30% to 90% over 20 minutes at a flow rate of 1 mL/min. As shown in
Pseudomonas represents a most common family of soil bacteria. pMiSI is a Pseudomonas expression vector that has been used previously for expression of carotenoid biosynthetic genes. The pyrH gene was amplified from the genome of Streptomyces rugosporus via PCR using a set of specific primers, including 5′-AATTGTTTAAACATGATTCGCAGCGTTGTGATTGTTG-3′ and 5′-AATTGAAGCTTTTATTGAATACTGGCCAGGTATTC-3′. The Phusion high-fidelity DNA polymerase from Thermo Fisher Scientific was used. The PCR program used for amplification of the pyrH gene consisted of an initial denaturation at 98° C. for 5 minutes, 20 cycles of touchdown program (98° C. for 30 seconds, annealing at 70° C. for 40 seconds, decreasing 0.5° C. per cycle, and extension at 72° C. for 90 seconds), 20 cycles of regular program (98° C. for 30 seconds, annealing at 60° C. for 40 seconds, and extension at 72° C. for 90 seconds), followed by a final extension at 72° C. for 10 minutes.
The PCR product was then digested with PmeI and HindIII at 37° C. for 2 hours, which was subsequently ligated into the pMiSI expression vector between the PmeI and HindIII sites (
The ligation product was introduced into Escherichia coli XL1-Blue competent cells through chemical transformation. The transformants were grown on LB agar supplemented with 50 μg/mL kanamycin at 37° C. overnight.
Colonies were picked from the agar plate into 5 mL of LB broth with 50 μg/mL kanamycin at 37° C. and 250 rpm overnight. The plasmids were extracted and digested with PmeI and HindIII at 37° C. for 2 hours. The digestion of correct pMiSI-pyrH is shown in
To test whether PyrH can be expressed in Pseudomonas, pMiSI-pyrH was introduced into the model soil bacterium Pseudomonas putida KT2440 through electroporation. The transformants were grown on LB agar with 50 μg/mL kanamycin at 30° C. overnight. L-Rhamnose was added at a concentration of 0.2% (w/v) to induce protein expression. After 36 hours, the cells were harvested by centrifugation at 4,000 rpm for 10 minutes. The cells were lysed as described in Example 4 and protein expression was analyzed by 12% SDS-PAGE. As shown in
Although SDS-PAGE analysis indicated that PyrH was expressed in Pseudomonas putida KT2440, it is unclear whether the L-tryptophan 5-halogenase is functional in this soil bacterium. To test this, L-tryptophan was added into the fermentation broth of P. putida KT2440/pMiSI-pyrH at 100 mg/L.
After 36 hours, 1 mL of the broth was taken from the culture and centrifuged at 15,000 rpm for 7 minutes. The supernatant was analyzed on an Agilent 1200 HPLC equipped with an Agilent Eclipse XDB-018 column (5 μm, 250 mm×4.6 mm). The sample was eluted with a gradient mobile phase of methanol-water (containing 0.1% formic acid) from 30% to 90% over 20 minutes at a flow rate of 1 mL/min. The broth was analyzed by HPLC. As shown in
IAA is a most common and widely used plant auxin. It is known that chlorinated derivatives of IAA are much effective than IAA in promoting plant growth. Because IAA shares the same indole ring as L-tryptophan, it is of interest to test whether P. putida KT2440/pMiSI-pyrH has relaxed substrate specificity and can halogenate IAA to yield a more powerful auxin. To this end, I supplied IAA into the rhamnose-induced broth of this engineered strain.
As shown in
To confirm that the halogenation really occurred at C-5 of IAA, the product was purified from the broth and collected its 1D and 2D NMR spectra. The 1H, 13C and HMBC NMR spectra are shown in
Pseudomonas chlororaphis O6 is a crop-benefiting soil bacterium that was known to colonize the roots of crops such as wheat. It was found to produce IAA. To enable soil bacteria to directly produce 5-CI-IAA without exogenous supply of IAA, the pMiSI-pyrH plasmid was introduced into Pseudomonas chlororaphis O6 by electroporation. The correct transformant of Pseudomonas chlororaphis O6/pMiSI-pyrH was selected on LB agar with 50 μg/mL kanamycin.
Pseudomonas chlororaphis O6/pMiSI-pyrH was grown in 50 mL of LB broth with 50 μg/mL kanamycin at 30° C. and 250 rpm. L-Rhamnose was added at a final concentration of 0.2% (w/v) to induce protein expression. Wild type Pseudomonas chlororaphis O6 was grown in LB broth as the control. After 36 hours, 1 mL of broth was taken from these cultures and centrifuged at 15,000 rpm for 7 minutes. The supernatants were analyzed on an Agilent 1200 HPLC equipped with an Agilent Eclipse XDB-C18 column (5 μm, 250 mm×4.6 mm). The sample was eluted with a gradient mobile phase of methanol-water containing 0.1% formic acid from 30% to 90% over 20 minutes at a flow rate of 1 mL/min. the broth was analyzed by HPLC.
As shown in
It will be appreciated that various of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, and are also intended to be encompassed by the following claims.
A provisional patent application (U.S. 63/013,367) has been previously filed for this invention on Apr. 21, 2020.
