Coding

Part:BBa_K2586019

Designed by: Robert Warneke, Janek Meißner   Group: iGEM18_Goettingen   (2018-09-11)

GAT: Glyphosate N-Acetyltransferase


This part encodes for an enzyme that n-acetylates the herbicide and antibiotic glyphosate. The GAT-enzyme is able to confer glyphosate resistance to organisms such as Escherichia coli, Arabidopsis, tobacco and maize. The acetylation of glyphosate may provide a new and alternative strategy in glyphosate support on crop plants. We integrated this part into soil bacterium Bacillus subtilis to enable the bacteria to inactivate glyphosate, which would otherwise be deadly to them.

Protein structure of Glyphosate N-acetyltransferase bound to acetyl COA and 3-phosphoglycerate





Molecular weight 17 kDa
Protein length 146 aa
Gene length 483 bp
Function mediates glyphosate tolerance
Product N-acetyl glyphosate
Ideal pH 6.8
Essential no




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Engineering bacteria to disarm glyphosate

Many years ago it has been found that glyphosate can be inactivated by acetylation yielding N-acetylglyphosate ( Castle et al.,2004). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. The glyphosate N-acetyltransferase GAT from the Gram-positive soil bacterium Bacillus licheniformis transfers the acetyl group from acetyl-CoA onto the amino group of the weedkiller ( Castle et al.,2004).

Interestingly, B. subtilis possesses a putative glyphosate N-acetyltransferase, which is designated as YitI and shares 59% overall amino acid identity with GAT of B. licheniformis. However, the native GAT enzymes are very poor catalysts for N-acetylation of glyphosate ( Castle et al.,2004). Moreover, despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (Siehl et al., 2007 JBC). As an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to N-acetylglyphosate the GAT was also subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide ( Castle et al.,2004). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate ( Castle et al.,2004). Therefore, we expect that bacteria like B. subtilis expressing the optimized GAT should tolerate high amounts of glyphosate even though it is taken up from the environment via the high-affinity glyphosate transporter GltT (see above). To evaluate the potential of the GAT to allow B. subtilis in the presence of glyphosate concentrations that are toxic for the wild type bacteria, we wanted to express the gat gene in B. subtilis (Figure 1).


Fig. 1. A. Sequence alignment showing the similarities of the wild type (wt) gat alleles from Bacillus licheniformis and Bacillus subtilis as well as the gat alleles R7 and R11 from B. licheniformis that were obtained by 11 rounds of gene shuffling. The red arrows indicate the 31 amino acid exchanges in the glyphosate N-acetyltransferase (Gat) variant R11. The optimized Gat variants shown an up to a 4,500-fold increase in catalytic efficiency (kcat/Km) relative to the native enzyme. The GenBank accession numbers for the sequences: B. licheniformis ST401 GAT, AX543338; R7 GAT, AY597417; R11 GAT, AY597418; B. subtilis YitI, CAA70664. B. Structure of the Gat R7 variant (PDBid: 2JDD) in ternary complex with acetyl-CoA and the competitive inhibitor 3-phosphoglycerate (3PG). The four active site residues (Arg-21, Arg-73, Arg-111, and His-138), which are labelled in black color, contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. C. Top view on the active site of Gat R7. D. Construction of a plasmid for the expression of the gat R11 variant in B. subtilis . Expression is driven by the constitutively active σA-dependent Palf4 promoter The plasmid pAC7::gat R11 integrates into the amyE locus of the B. subtilis chromosome in single copy. lacZ, beta-galactosidase; amyE-5' and amyE-3', fragments of the B. subtilis amyE for homologous recombination; amp, ampicillin resistance gene for the selection in E. coli; kan, kanamycin resistance gene for the selection in B. subtilis ; ori pBR322, origin of replication for E. coli.

Fig. 2. A. Growth of the wild type and ΔgltT mutant and of the isogenic strains overexpressing the gat R11 and aroA* genes on CS-Glc minimal medium in the presence of different amounts of glyphosate. B. Growth of the strains described in A on CS-Glc minimal medium in the presence of different amounts of Roundup® Alphee.

GAT confers tolerance to glyphosate

We ordered the gat gene as a g-Block from Integrated DNA Technologies, which was free of charge because the company supports the iGEM competition. Next, we amplified the gat gene by PCR using the g-Block as template DNA. An artificial promoter and a ribosome binding site was attached during the PCR (Figure 1). The promoter-gene fusion was cloned and introduced in single copy into the genomes of the B. subtilis wild type strain and the gltT mutant strain to further increase its glyphosate tolerance (Figure 2). To assess the potential of the GAT to disarm glyphosate in the different B. subtilis strains, we propagated the bacteria on CS-Glc minimal medium agar plates that were supplemented with different amounts of glyphosate. As shown in Figure 2, the strains synthesizing the GAT tolerated significantly more glyphosate than the wild type strain. Moreover, the inactivation of the gltT glyphosate transporter gene and the overexpression of the gat gene confers high-level glyphosate tolerance to the bacteria. Thus, when introduced into B. subtilis , the optimized gat gene confers indeed robust tolerance to glyphosate. To conclude, the engineered bacteria possess two interesting properties: (i) B. subtilis promotes plant growth by protecting plants against pathogens and (ii), the bacteria take up glyphosate from the environment and inactivate the weedkiller!


Fig. 3. A. Growth of the B. subtilis wild type strain in presence of 0 - 3.5 mM Glyphosate. B. Growth of the ∆gltT B. subtilis strain with increasing glyphosate concentration (0-30 mM). C. Growth of the gat B. subtilis strain with increasing glyphosate concentration (0-30 mM. D.Growth of the aroA* B. subtilis strain with increasing glyphosate concentration (0-30 mM E. Growth of the aroA* B. subtilis strain with increasing glyphosate concentration (0-30 mM). Growth of the B. subtilis strain containing both ∆gltT and gat with increasing glyphosate concentration (0-30 mM). F. Growth of the B. subtilis strain containing both ∆gltT and aoA* with increasing glyphosate concentration (0-30 mM). Bacteria were cultivated in liquid Cs-Glu medium.

Characeterization of GAT conferred tolerance to glyphosate

Cultivation of the different strains in liquid media allows more detailed characterisation. We analysed growth of the bacteria in CS-Glu medium supplemented with increasing amounts of glyphosate, using a platereader. In general, the analysis supports previous results. The strain containing aroA* confers only a slight increase in resistance in comparison to the WT strain and hence a significantly lower resistance in comparison the ∆gltT strain (Figure 3 A, B and D ). However, for the strain combining aroA* and ∆gltT a 9-fold higher glyphosate concentration is needed to reduce the growth rate by 50% (Figure 3 F. Figure Y ).

Further characterisation of the Gat enzyme supports previous results and underline significant increased resistance in both strains conferring gat or a combination of gat and ∆gltT (Figure 3 C and E, Figure 4).

Fig. 4. The relationship between the growth rate (µ) and the glyphosate (GS) concentration for the B. subtilis wild type (WT) strain SP1, the ΔgltT mutant strain BP233, the gat B. subtilis strain, the aroA* B. subtilis strain, the B. subtilis strain containing both ∆gltT and aroA* and the the B. subtilis strain containing both ∆gltT and gat in CS-Glc minimal medium supplemented with increasing amounts of glyphosate (GS).


A novel nechanism conferring resistance to glyphosate

In the past years, the underlying molecular mechanisms conferring resistance to glyphosate have been intensively studied in bacteria and plants. In many cases glyphosate resistance is directly linked to the target of the herbicide. For instance, bacteria like S. aureus are a priori resistant to glyphosate because this organism synthesizes an insensitive EPSP synthase (13-16). Moreover, increased cellular levels of the EPSP synthase, which can be achieved either by overexpression of the coding gene or by gene amplification, can confer resistance to glyphosate (Figure 5) (9, 17-21). Due to the increased cellular levels of the EPSP synthase, the glyphosate is probably titrated away and sufficient amounts of the precursor for aromatic amino acid biosynthesis can be produced.

As mentioned in the design section, several glyphosate-insensitive EPSP synthase mutant variants have been isolated and engineered (11, 22-35). Often, a single amino acid exchange is sufficient to render the EPSP synthase insensitive to glyphosate. However, although glyphosate resistance is frequently linked to the target of the herbicide, resistance against the herbicide may occur by other means. Several studies have demonstrated that glyphosate can be detoxified by covalent modification. For instance, the Gat glyphosate N-acetyltransferase from Bacillus licheniformis, which was subjected to directed evolution for creating an enzyme with higher efficiency and increased specificity for the herbicide, converts glyphosate to N-acetylglyphosate, which is not herbicidal and is not an effective inhibitor of EPSP synthases (Figure 9) (36-39). Moreover, the hygromycin phosphotransferases Hph and GlpA from E. coli and Pseudomonas pseudomallei, respectively, phosphorylate glyphosate and thus confer tolerance to the herbicide (Fig. 3) (40,41). Interestingly, the gat gene has also been used as a selection marker for genetic engineering of bacteria (39). The enzymes that covalently modify glyphosate have been successfully introduced into crops to increase herbicide resistance (42).

Recently, it has been also demonstrated that enhanced export of glyphosate can reduce toxicity of the herbicide. For instance, the overexpression of the uncharacterized membrane proteins MFS40 and YhhS from Aspergillus oryzae and E. coli, respectively, with similarity to the major facilitator secondary transporter superfamily, enhance glyphosate tolerance of E. coli (Figure 5) (43,44). It will be interesting to test whether these transporters are suitable for engineering crops to enhance glyphosate tolerance. Proteins of unknown function can also increase glyphosate tolerance. For instance, the overexpression of the igrA gene product from the Pseudomonas sp. strain PG2982 increases glyphosate tolerance in transgenic rice (45-47). Finally, many bacteria can survive in the presence of glyphosate because they are able to degrade the herbicide (Figure 5) (48-57). To conclude, several mechanisms of glyphosate resistance have been described over the past years and novel mechanisms allowing survival with the herbicide are certainly identified in the future. We have observed that the deletion of the gltT gene in B. subtilis confers high-level glyphosate resistance to the bacteria. The B. subtilis gltT mutant strain could be a suitable host to identify glyphosate uptake systems from plants by expressing cDNA libraries and by screening for transformants that are unable to grow with glyphosate.


Fig. 5. Mechanisms conferring resistance to glyphosate. GltT and GltP, B. subtilis glutamate transporters; YhhS and MFS40, major facilitator secondary transporters from E. coli and A. oryzae; GAT, B. licheniformis glyphosate N-acetyltransferase; GlpA, P. pseudomallei hygromycin phosphotransferase; PEP, phosphoenolpyruvate; 3PS, 3-phosphoshikimate; EPSP, enolpyruvylshikimate-3-phosphate; EPSPS, enolpyruvylshikimate-3-phosphate synthase. Membrane topologies of the transporters were predicted using the web-based protein topology tool Protter (5).


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[edit]
Categories
//chassis/prokaryote/bsubtilis
Parameters
protein