Methods and compositions

Abstract

The present invention relates to an engineered microorganism for use in combination with a plant to increase the uptake of metals from a growth medium. In particular, engineering a microorganism to exploit plant hormonal synthesis, suppress stress response, and produce metal chelators is described.

Description

[0001] Methods and Compositions

[0002] Field

[0003] The present invention is in the field of bioremediation.

[0004] Background

[0005] Soil contamination poses a significant obstacle to land utilisation with common pollutants including petroleum hydrocarbons, agrochemicals, solvents, and heavy metals, the latter of which can render soil unsuitable for agriculture or habitation. Heavy metal accumulation is detrimental to human health with even low-level exposure capable of inducing organ damage. Heavy metal contamination can originate from industrial activity, mining, and agriculture. In contrast to organic contaminants, heavy metals are not broken down by either biotic or abiotic means and can persist in the soil. In the UK, the Environment agency has reported that the average concentration of Zinc and Nickel are 81.3 and 21.0 mg / kg respectively with sharp increases observed in urban areas.

[0006] Current methods of metal remediation include both physical and chemical approaches e.g. soil substitution and soil washing respectively. Substitution involves the excavation of the contaminated soil followed by replacement with new soil. Similarly, soil washing involves excavating the contaminated soil followed by extensive processing with one or more wash solutions containing small organic acids or chelators that remove soluble metal contaminants. Both methods are labour intensive, time consuming and the waste products usually require further specialised treatment to comply with environmental regulations. In the case of soil washing, high proportions of silt, clay or organic matter can render the process ineffective. The washing process also depletes the nutrient content of the soil and degrades soil structure.

[0007] Bioremediation represents an attractive alternative to chemical and physical soil remediation. Bioremediation is the deliberate use of a biological system to degrade, breakdown and remove environmental pollutants. Unlike traditional methods, bioremediation is less resource heavy and can be directly applied to polluted sites (in situ). Most bioremediation initiatives have focused on the use of plants, fungi, algae, or bacteria to degrade organic contaminants with less attention directed towards metal removal. Owing to toxicity, phytoremediation of heavy metals is frequently unsuccessful with common varieties unable to thrive in highly contaminated soil. Soil bioremediation using either bacteria or fungi can prove challenging as they must be introduced and removed using a liquid media and is frequently applied in ex situ processing.

[0008] The inventors have devised new solutions to address these problems.

[0009] Summary of invention

[0010] The inventors have surprisingly found that the application of engineered microorganisms to plants, such that a plant-endophytic relationship is formed between the plant and the microorganisms, enhances the ability of the plant to accumulate metals from the surrounding environment, for example from the soil, having clear uses in bioremediation. In addition, the invention can be applied to phytomining and represents a significant improvement compared to current approaches.

[0011] Detailed description of the invention

[0012] The invention provides various engineered microorganisms, methods, compositions and kits for putting the invention into effect.

[0013] The inventors have found that modulation of four pathways within a microorganism, either alone or in combination, produces a microorganism that, when associated with a partner plant in an endophytic relationship, increases the plants ability to take up and accumulate metals, without toxic effects on the plant. In some instances the ability to take up and accumulate metals is uncoupled to an effect on growth of the plant, i.e. the plant does not accumulate an increased amount of metal simply because it is growing faster.

[0014] The pathways are the auxin production pathway; pathways to reduce the production of ethylene; pathways to increase production of one or more metal chelators; and pathways to decrease the production of tryptophan.

[0015] Accordingly, the invention provides an engineered microorganism, wherein the microorganism has been engineered to:

[0016] (a) produce or overproduce auxin;

[0017] (b) reduce the production of ethylene;

[0018] (c) produce or overproduce one or more metal chelator molecules; and / or

[0019] (d) reduce or knockout the production of tryptophan.

[0020] Some microorganisms endogenously have one or more of these pathways. For example, although auxins are plant growth hormones, they are found in all kingdoms, with some bacteria also are able to produce auxins. Some bacteria such as Pseudomonas do not have a native or endogenous AUX1 and AUX2 gene.

[0021] Ethylene is another plant hormone, produced in response to stress (such as exposure to high levels of metals), and slows down plant growth.

[0022] The skilled person will appreciate that these pathways can be modulated by any means. For example in some embodiments small molecules can be used to effect modulation of the pathways. In other embodiments RIMA based methods such as RNAi may be used. In preferred embodiments the pathways are modulated by the expression of overexpression of one or more proteins or peptides.

[0023] Accordingly, in some embodiments:

[0024] (a) said production or overproduction of auxin is via the expression or overexpression of one or more proteins or peptides required for the production or auxin;

[0025] (b) said reduction of the production of ethylene is via the expression or overexpression of one or more proteins or peptides involved in the degradation or conversion of extracellular ethylene;

[0026] (c) said production or overproduction of one or more metal chelators is via the expression or overexpression of one or more proteins or peptides for the biosynthesis of one or more chelator molecules; and / or

[0027] (d) said reduction or knockout of the production of tryptophan is via one or more gene disruptions in the tryptophan biosynthesis pathway.

[0028] In some embodiments the microorganism may not endogenously express the one or more required proteins or peptides. In these instances it is appropriate to use the term "express", since there is no background level of expression of that specific protein or peptide. In other embodiments, the microorganism may already endogenously express the said protein or peptide. In these cases it is beneficial if the microorganism "overexpresses" the said protein or peptide, relative to the basal endogenous level of expression; or expresses a version of the protein or peptide that is not the endogenous version of the protein or peptide.

[0029] The term "overexpress" or "overexpression" can be used to describe modulating the transcription or translation of a gene of interest to a higher level than that of the endogenous or basal level of transcription or translation. The skilled person will appreciate that the typical means of overexpressing a protein or peptide is via the generation of an expression cassette, that comprises a promoter operably linked to the gene that encodes the protein or peptide. Accordingly in some embodiments the engineered microorganism has been engineered to comprise one or more expression cassettes.

[0030] In some embodiments one or more of the expression cassettes is genomically integrated into the host cell genome. In the same or different embodiment one or more of the expression cassettes is contained with an episomal vector, for example wherein the vector is selected from a plasmid, transposon, or bacteriophage.

[0031] Where the engineered microorganisms is engineered to express or overexpress more than one gene, the more than one gene may be co-located on the same polynucleotide molecule, or may be on separate polynucleotide molecules. For example a single vector such as a plasmid may comprise a promoter that drives expression of a single gene described herein, in other instances a single plasmid may comprise a first promoter that drives expression of a first gene described herein and a second promoter that drives expression of a second gene described here. In other instances a single vector such as a plasmid may have a first promoter that drives expression of a first and a second gene as set out herein -for example as an operon. Accordingly in some instances where the engineered microorganism expresses or overexpresses more than one gene described here, it may comprise at least two different episomally maintained vectors such as plasmid, or it may comprise a single episomally maintained vector such as a plasmid that comprises both genes, or it may comprise one or more genomically integrated expression cassettes.

[0032] The promoter driving expression of the one or more proteins or peptides may be any promoter. In some embodiments the promoter is selected from the group comprising or consisting of: a) a constitutive promoter; b) an inducible promoter; c) a BG17 promoter [SEQ ID NO: 9]; and / or d) a BG37 promoter [SEQ ID NO: 10].

[0033] In some embodiments the promoter is a synthetic promoter. In some embodiments the synthetic promoters are selected from BG17 and BG37, which have been characterised previously as suitable for constitutive heterologous gene expression in Pseudomonas putida (Zobel et al 2015 ACS Synthetic Biology 4: 1341-1351). In some embodiments the one or more proteins or peptides required for the production of auxin is AUXI, AUXII or both AUXI and AUXII.

[0034] In some embodiments the one or more proteins or peptides involved in the degradation of conversion of extracellular ethylene is ACC Deaminase (ACCD). ACCD refers to the protein, and acdS refers to the gene. Reference to overexpression of the acdS (or ACDS) gene and the ACCD protein are used interchangeably.

[0035] In some embodiments the one or more proteins or peptides for the biosynthesis of one or more chelator molecules is a sigma factor, or a protein or peptide that modulates the transcription of one or more proteins that is a chelator, for example a metal chelator. In some embodiments the sigma factor protein in PvdS.

[0036] In some embodiments the engineered microorganism has been engineered to express or overexpress: a) AUXI; b) AUXII; c) PvdS; d) ACDS; e) AUXI and AUXII; f) AUXI and PvdS; g) AUXI and ACDS; h) AUXII and PvdS; i) AUXII and ACDS; j) PvdS and ACDS; k) AUXI, AUXII and PvdS; l) AUXI, AUXII and ACDS; m) AUXI, PvdS and ACDS; n) AUXII, PvdS and ACDS; or o) AUXI, AUXII, PvdS and ACDS.

[0037] In some preferred embodiments the engineered microbe has been engineered to express or overexpress AUXI and AUXII.

[0038] In some embodiments the engineered microorganism has been engineered to express or overexpress all of AUXI, AUXII, ACCD and PvdS. As mentioned above, the promoter may in some instances be selected from BG17 or BG37, or the engineered microbe may be engineered to express genes from both the BG17 and BG37 promoters. For example in some instances the engineered microorganism may be engineered to express at least the following gene combinations from the specified promoters: a) BG17::AUXI b) BG37::AUXI c) BG17::AUXII d) BG37::AUXII e) BG17::PvdS f) BG37::PvdS g) BG17::ACDS h) BG37::ACDS i) BG17::AUXI + BG17::AUXII j) BG17::AUXI + BG37::AUXII k) BG37::AUXI + BG17::AUXII l) BG37::AUXI + BG37::AUXII m) BG17::AUXI + BG17::PvdS n) BG17::AUXI + BG37::PvdS o) BG37::AUXI + BG17::PvdS p) BG37::AUXI + BG37::PvdS q) BG17::AUXI + BG17::ACDS r) BG17::AUXI + BG37::ACDS s) BG37::AUXI + BG17::ACDS t) BG37::AUXI + BG37::ACDS u) BG17::AUXII + BG17::PvdS v) BG17::AUXII + BG37::PvdS w) BG37::AUXII + BG17::PvdS x) BG37::AUXII + BG37::PvdS y) BG17::AUXII + BG17::ACDS z) BG17::AUXII + BG37::ACDS aa) BG37::AUXII + BG17::ACDS ab) BG37::AUXII + BG37::ACDS ac) BG17::PvdS + BG17::ACDS ad) BG17::PvdS + BG37::ACDS ae) BG37::PvdS + BG17::ACDS af) BG37::PvdS + BG37::ACDS ag) BG17::AUXI + BG17::AUXII + BG17::PvdS ah) BG17::AUXI + BG17::AUXII + BG37::PvdS ai) BG17::AUXI + BG37::AUXII + BG17::PvdS aj) BG17::AUXI + BG37::AUXII + BG37::PvdS ak) BG37::AUXI + BG17::AUXII + BG17::PvdS al) BG37::AUXI + BG17::AUXII + BG37::PvdS am) BG37::AUXI + BG37::AUXII + BG17::PvdS an) BG37::AUXI + BG37::AUXII + BG37::PvdS ao) BG17::AUXI + BG17::AUXII + BG17::ACDS ap) BG17::AUXI + BG17::AUXII + BG37::ACDS aq) BG17::AUXI + BG37::AUXII + BG17::ACDS ar) BG17::AUXI + BG37::AUXII + BG37::ACDS as) BG37::AUXI + BG17::AUXII + BG17::ACDS at) BG37::AUXI + BG17::AUXII + BG37::ACDS au) BG37::AUXI + BG37::AUXII + BG17::ACDS av) BG37::AUXI + BG37::AUXII + BG37::ACDS aw) BG17::AUXI + BG17::PvdS + BG17::ACDS ax) BG17::AUXI + BG17::PvdS + BG37::ACDS ay) BG17::AUXI + BG37::PvdS + BG17::ACDS az) BG17::AUXI + BG37::PvdS + BG37::ACDS ba) BG37::AUXI + BG17::PvdS + BG17::ACDS bb) BG37::AUXI + BG17::PvdS + BG37::ACDS be) BG37::AUXI + BG37::PvdS + BG17::ACDS bd) BG37::AUXI + BG37::PvdS + BG37::ACDS be) BG17::AUXII + BG17::PvdS + BG17::ACDS bf) BG17::AUXII + BG17::PvdS + BG37::ACDS bg) BG17::AUXII + BG37::PvdS + BG17::ACDS bh) BG17::AUXII + BG37::PvdS + BG37::ACDS bi) BG37::AUXII + BG17::PvdS + BG17::ACDS bj) BG37::AUXII + BG17::PvdS + BG37::ACDS bk) BG37::AUXII + BG37::PvdS + BG17::ACDS bl) BG37::AUXII + BG37::PvdS + BG37::ACDS bm) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bn) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS bo) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS bp) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS bq) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS br) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS bs) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS bt) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS bu) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bv) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS bw) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS bx) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS by) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS bz) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS ca) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS cb) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS

[0039] In some instances the engineered microorganism has been engineered to express or overexpress only those proteins specified in any of clauses (a) - (cb) above.

[0040] As set out elsewhere herein, each gene may be operably linked to its own promoter, or may be linked a promoter that also drives expression of a second or further gene, for example a single promoter may drive expression of an operon comprising a number of genes.

[0041] The AUXI, AUXII, ACCD and PvdS can be any AUXI, AUXII, ACCD and PvdS. For example the genes may be endogenous to the microorganism, or may be heterologous to the microorganism, i.e. genes taken from a different organism or microorganism and expressed in the engineered microorganism of the invention.

[0042] In some embodiments the:

[0043] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1;

[0044] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2;

[0045] ACCD has an amino acid sequence of SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; and / or

[0046] PvdS has an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. It will be apparent that where the above proteins have less than 100% sequence identity with the stated sequences, they should retain their core biological activity. The activity of each of the four proteins is set out below, along with standard tests that the skilled person can perform to ensure that any variant of the above specified sequences retains the required activity.

[0047] AUXI (Auxin Biosynthesis Gene I) encodes enzymes involved in the indole-3-acetic acid (IAA) biosynthesis pathway. The activity of AUXI can be confirmed using the Salkowski Reagent Assay, which is a colorimetric test where culture supernatant from cells expressing AUXI turns pink / red in the presence of IAA. Other methods include LC-MS / MS Quantification. AUXII (Auxin Biosynthesis Gene II) is also part of the IAA synthesis pathway. Activity can be confirmed using the Salkowski assay above of LC-MS / MS quantification. ACCD (1-Aminocyclopropane-l-Carboxylate (ACC) Deaminase) breaks down ACC, a precursor of ethylene in plants, and so reduces ethylene levels, promoting root growth especially under stress. Activity of ACCD can be determined using the ACC Deaminase Activity Assay which involves measuring a-ketobutyrate formation from ACC using colorimetric detection (ninhydrin or DNPH-based assay). PvdS (Pyoverdine Sigma Factor) is an alternative sigma factor regulating the synthesis of pyoverdine, a fluorescent siderophore involved in iron acquisition in Pseudomonas spp. Activity of PvdS can be determined using the Pyoverdine Fluorescence Assay, which involves monitoring the fluorescence of culture supernatant at excitation 400 nm I emission 460 nm.

[0048] Accordingly in some embodiments the:

[0049] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA;

[0050] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA;

[0051] ACCD has an amino acid sequence of SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 provided that the ACCD retains the ability to breakdown ACC; and / or

[0052] PvdS has an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 provided that the PvdS retains the ability to regulate the synthesis of pyoverdine. In some instances the engineered microorgani may be engineered to express at least the following gene combinations from the sped promoters, or engineered to express or overexpress only the following gene combinatk from the specified promoters: a) BG17::AUXI b) BG37::AUXI c) BG17::AUXII d) BG37::AUXII e) BG17::PvdS f) BG37::PvdS g) BG17::ACDS h) BG37::ACDS i) BG17::AUXI + BG17::AUXII j) BG17::AUXI + BG37::AUXII k) BG37::AUXI + BG17::AUXII l) BG37::AUXI + BG37::AUXII m) BG17::AUXI + BG17::PvdS n) BG17::AUXI + BG37::PvdS o) BG37::AUXI + BG17::PvdS p) BG37::AUXI + BG37::PvdS q) BG17::AUXI + BG17::ACDS r) BG17::AUXI + BG37::ACDS s) BG37::AUXI + BG17::ACDS t) BG37::AUXI + BG37::ACDS u) BG17::AUXII + BG17::PvdS v) BG17::AUXII + BG37::PvdS w) BG37::AUXII + BG17::PvdS x) BG37::AUXII + BG37::PvdS y) BG17::AUXII + BG17::ACDS z) BG17::AUXII + BG37::ACDS aa) BG37::AUXII + BG17::ACDS ab) BG37::AUXII + BG37::ACDS ac) BG17::PvdS + BG17::ACDS ad) BG17::PvdS + BG37::ACDS ae) BG37::PvdS + BG17::ACDS af) BG37::PvdS + BG37::ACDS ag) BG17::AUXI + BG17::AUXII + BG17::Pvd< ah) BG17::AUXI + BG17::AUXII + BG37::Pvd< ai) BG17::AUXI + BG37::AUXII + BG17 PvdS aj) BG17::AUXI + BG37::AUXII + BG37::PvdS ak) BG37::AUXI + BG17::AUXII + BG17::PvdS al) BG37::AUXI + BG17::AUXII + BG37::PvdS am) BG37::AUXI + BG37::AUXII + BG17::PvdS ay) az) ba) bb) be) bd) be) BG17::AUXII + BG17::PvdS + BG17 :ACDS bf) BG17::AUXII + BG17::PvdS + BG37 ACDS bg) BG17::AUXII + BG37::PvdS + BG17 :ACDS bh) BG17::AUXII + BG37::PvdS + BG37 :ACDS bi) BG37::AUXII + BG17::PvdS + BG17: ACDS bj) BG37::AUXII + BG17::PvdS + BG37 ACDS bk) BG37::AUXII + BG37::PvdS + BG17 :ACDS bl) BG37::AUXII + BG37::PvdS + BG37: ACDS bm) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bn) bo) bp) bq) br) bs) bt) bu) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bv) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS bw) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS bx) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS by) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS bz) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS ca) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS cb) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS where:

[0053] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA;

[0054] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA;

[0055] ACCD has an amino acid sequence of SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 provided that the ACCD retains the ability to breakdown ACC; and / or

[0056] PvdS has an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

[0057] In some instances the engineered microorganism may be engineered to express at least the following gene combinations from the specified promoters, or engineered to express or overexpress only the following gene combinations from the specified promoters: a) BG17::AUXI + BG17::AUXII b) BG17::AUXI + BG37::AUXII c) BG37::AUXI + BG17::AUXII d) BG37::AUXI + BG37::AUXII where:

[0058] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / or

[0059] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

[0060] In some instances the engineered microorganism may be engineered to express at least the following gene combinations from the specified promoters, or engineered to express or overexpress only the following gene combinations from the specified promoters:

[0061] BG37: :AUXI + BG37: :AUXII where:

[0062] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / or

[0063] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

[0064] As described above, the modulation of one of the pathways results in the increased production of one or more auxins. The skilled person will appreciate that there are various auxins. In some embodiments the engineered microorganism has been engineered to as to overproduce an auxin selected from the group comprising or consisting of: indole-3- acetic acid, 4-chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid.

[0065] In preferred embodiments, the engineered microorganism is capable of endophytic symbiosis or plant colonisation of a partner plant or plant part. Preferably the engineered microorganism is able to colonise the root of a partner plant.

[0066] In some embodiments the engineered microorganism is naturally able to colonise a given partner plant or plant part, for example is naturally able to colonise the root of a partner plant - i.e. the engineered microorganism has not been engineered so as to have the ability to colonise the partner plant or plant part.

[0067] In other embodiments, the engineered microorganism is not naturally able to colonise a particular partner plant or plant part, and is instead engineered so as to enable to microorganism to colonise the partner plant or plant part.

[0068] In some embodiments the microorganism is a bacteria, fungi, or algae. In preferred embodiments the microorganism is a bacteria.

[0069] In some embodiments the microorganism is a bacteria selected from the group comprising or consisting of: a) a gram negative bacteria for example a bacteria belonging to the Pseudomonas genus; optionally Pseudomonas fluorescens; or b) a gram positive bacteria, optionally Bacillus sp.

[0070] In some embodiments the bacteria is of any one or more of the following: a Pseudomonas sp.; a Pantoea sp.; an Enterobacter sp.; a Ralstonia sp.; or a a Microbacterium sp.

[0071] In some embodiments the bacteria is any one or more of

[0072] Pseudomonas fluorescens; Pantoea ananatis; Pantoea stewartia; Enterobacter ludwigii; Pantoea agglomerans; Pseudomonas thivervalensis; Pantoea ananatis; Pseudomonas putida.

[0073] In some particular embodiments the bacteria is any one or more of the strains described in Zhang et al 2011 Chemosphere 83: 57-62 or Card et al 2015 Biological Control 102- 112, for example is, in some particular embodiments, any one or more of the following strains: strain Gl-21-2 of Pseudomonas sp.; Jl-7-5 of Pantoea ananatis; Jl-13-7 of Pantoea stewartia; Jl-17-2 of Enterobacter ludwigii; Jl-22-2 of Ralstonia sp.; JP3-3 of Pantoea agglomerans; Yl-3-9 of Pseudomonas thivervalensis; Yl-15-5 of Pantoea ananatis; G16 of Microbacterium sp.

[0074] In some particular embodiments the microorganism is Pseudomonas fluorescens.

[0075] In some instances the engineered microorganism is Pseudomonas fluorescens and is engineered to express at least the following gene combinations from the specified promoters, or engineered to express or overexpress only the following gene combinations from the specified promoters: a) BG17: :AUXI + BG17: :AUXII b) BG17: :AUXI + BG37: :AUXII c) BG37: :AUXI + BG17: :AUXII d) BG37: :AUXI + BG37: :AUXII where:

[0076] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / or

[0077] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

[0078] In some embodiments the microorganism is an endophyte of the Brassica genus, for example of Brassica napus.

[0079] Once the engineered microorganism of the invention has colonised a partner plant or plant part, for example a plant root, in some embodiments the microorganism allows the partner plant to accumulate higher levels of metals than the plant can accumulate in the absence of the engineered microorganism. In some embodiments then the engineered microorganism of the invention is able to colonise a partner plant, for example is able to colonise the root of a partner plant and enables accumulation in the partner plant of one or more metals from the surrounding media.

[0080] The metals may be selected from the group comprising or consisting of: transition and post transition metals; one or more of Zinc, Strontium, Nickel, Iron, Mercury, Cadmium, Lead, Copper, Chromium, Aluminium, Cobalt, Manganese, Silver, and Gold, optionally Zinc, Strontium and / or Nickel.

[0081] In some embodiments the engineered microorganism enables accumulation of metals in the partner plant to a higher concentration than is accumulated in the absence of the engineered microorganism. In some embodiments the engineered microorganism enables accumulation of metals in the partner plan to a higher concentration than is accumulated in the presence of a corresponding microorganism that has not been engineered, for example that has not been engineered so as to:

[0082] (a) produce or overproduce auxin;

[0083] (b) reduce the production of ethylene;

[0084] (c) produce or overproduce one or more metal chelator molecules; and / or

[0085] (d) reduce or knockout the production of tryptophan as described elsewhere herein.

[0086] Without wishing to be bound by any theory, one mechanism by which the engineered microorganism of the invention is considered to allow the plant to accumulate higher levels of metals is by chelation of the metals within the xylem and phloem of the plant, sequestering the metals in such a way so as to prevent them from entering the plant cells and so reducing the toxicity associated with metal accumulation. Accordingly in some embodiments when the engineered microorganism has been applied to a plant or plant part, the one or more metals are prevented from entering plant cells, optionally wherein the one or more metals are chelated within the plant root and prevented from entering plant cells.

[0087] In addition to providing the engineered microorganism as described herein, the invention also provides the various nucleic acids that are required to engineer the microorganism.

[0088] For example the invention provides an expression cassette comprising a promoter operably linked to any one or more of the following genes: AUXI, AUXII, PvdS and / or ACDS. As set out above in some embodiments: a) AUXI has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; b) AUXII has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA; c) ACCD has the protein sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 provided that the ACCD retains the ability to breakdown ACC; and / or d) PvdS has the protein sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

[0089] In some embodiments the promoter of the cassette may be the BG17 or BG37 promoter.

[0090] For example the invention provides an expression cassette comprising: a) the BG37 promoter operably linked to an polynucleotide that encodes an AUXI protein with a sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; b) the BG17 promoter operably linked to an polynucleotide that encodes an AUXI protein with a sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; c) the BG37 promoter operably linked to an polynucleotide that encodes an AUXII protein with a sequence of SEQ ID NO: 2 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA; d) the BG17 promoter operably linked to an polynucleotide that encodes an AUXII protein with a sequence of SEQ ID NO: 2 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA; e) the BG37 promoter operably linked to an polynucleotide that encodes an ACCD protein with a sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 provided that the ACCD retains the ability to breakdown ACC; f) the BG17 promoter operably linked to an polynucleotide that encodes an ACCD protein with a sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 provided that the ACCD retains the ability to breakdown ACC; g) the BG37 promoter operably linked to an polynucleotide that encodes a PvdS protein with a sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 provided that the PvdS retains the ability to regulate the synthesis of pyoverdine; h) the BG17 promoter operably linked to an polynucleotide that encodes a PvdS protein with a sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

[0091] The invention also provides an expression vector comprising one or more expression cassettes of the invention.

[0092] The invention also provides various methods of using the engineered microorganism of the invention, for example methods of preparing a plant, and methods of bioremediation and methods of extracting metals from a media.

[0093] Accordingly in one embodiment the invention provides a method of preparing a plant with an increased ability to accumulate metals wherein the method comprising contacting the plant or plant part with the engineered microorganism of the invention. It will be appreciated by the skilled person that in some embodiments the engineered microorganism is allowed to colonise the partner plant when the partner plant is very young, or pregermination, so as to ensure maximal colonisation of plant tissue. Accordingly in some embodiments the method comprises contacting one or more seeds or tubers or bulbs of said plant with one or more engineered microorganisms of the invention.

[0094] In some embodiments the plant or plant part is a seedling oor part of a seedling. In other embodiments the plant or plant part is a mature plant or part of a mature plant

[0095] In some embodiments the seed, tuber or bulb is coated with the one or more engineered microorganisms of the invention.

[0096] In some embodiments, the plant part, for example one or more seeds, tubers or bulbs are contacted (or coated) with at least 2, 3 or at least 4 different engineered microorganisms of the invention. In some embodiments each of the different engineered microorganisms has been engineered so as to express or overexpress either:

[0097] (a) a different gene, for example to express or overexpress a different gene selected from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS; and / or

[0098] (b) two or more different genes, for example to express or overexpress two or more different genes from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS. For example in some embodiments the plant part, for example one or more seeds, tubers or bulbs are contacted (or coated) with at least two, three or four or more different engineered microbes, where the engineered microbes have been engineered to express or overexpress the following proteins from the specified promoters: a) BG17: :AUXI b) BG37: :AUXI c) BG17: :AUXII d) BG37: :AUXII e) BG17: :PvdS f) BG37: :PvdS g) BG17: :ACDS h) BG37: :ACDS

[0099] In preferred embodiments the engineered microorganism is Pseudomonas fluorescens. Preferences for the sequences of AUXI, AUXII, PvdS and ACDS are as described elsewhere herein.

[0100] In some embodiments the plant part, for example one or more seeds, tubers or bulbs are contacted (or coated) with at least a first and second different engineered microbe, wherein: a) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; b) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; c) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG17 promoter; or d) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered microorganism has been engineered to express AUXII from the BG17 promoter.

[0101] The engineered microorganism of the invention may be applied to the plant or plant part at any stage, for example may be applied to the seed, tuber or bulb prior to germination; may be applied to the plant or a plant part during germination or early growth; or may be applied to an established and mature plant or plant part.

[0102] In some embodiments then the method comprises applying said one or more engineered microorganisms to a media into which the plant is subsequently planted. By media we include the meaning of any substrate in which a plant can grow. For example by media we include soil, water, and gravel for instance. In these embodiments the engineered bacteria is applied to the media, for example to metal contaminated land or water, and the relevant partner plant is subsequently introduced, for example a seed, tuber or bulb may be planted into the metal contaminated land or water that already contains the engineered microorganism.

[0103] On the other hand, the engineered microorganism may be applied to a media in which the plant is already growing. For example in some embodiments the media may be a metal contaminated soil or water in which plants are already established. The engineered microorganism(s) of the invention may be applied directly to the established plants. For example they may be applied to the soil or water in which the plant is growing in, for example to allow colonisation of the plant root.

[0104] As set out above, in any instance, the plant or plant part may be contacted with any number of different engineered microorganisms of the invention.

[0105] The partner plant which the engineered microorganisms is to colonise may be any plant, for example any plant able to be colonised by a microorganism.

[0106] The skilled person would be able to identify the most suitable plant and microorganism combination or combinations, for example for the type of media in which the plant is to be grown. For example certain microorganisms are known to colonise certain plant species.

[0107] In some embodiments the plant is: a) a Brassica species, for example B. napus; b) a root vegetable; c) a plant that grows from tubers d) an aquatic plant; e) a plant that grows in water, optionally watercress In some particular embodiments the invention provides a method of preparing a Brassica species plant, for example a B. napus plant with an increased ability to accumulate heavy metals, wherein said method comprises contacting seeds or seedlings of said plant with one or more engineered microorganisms of the invention.

[0108] The invention also provides the various plants that have been prepared by any of the methods of the invention, for example a plant colonised with an engineered microorganism of the invention.

[0109] Accordingly the invention provides a plant or plant part prepared according to any one of the methods of the invention, for example where the plant or plant part is a seed, tuber, bulb or seedling.

[0110] The invention also provides a plant of plant part that is colonised with one or more engineered microorganisms of the invention.

[0111] It will be clear to the skilled person that the applications of the engineered microorganisms and the colonised plants of the invention include bioremediation and biomining.

[0112] Accordingly the invention provides a method for removing metals from a media wherein said method comprises cultivating a plant or plant part of the invention in said media. For example cultivating a plant or plant part that has been colonised with an engineered microorganism of the invention.

[0113] Preferences for the microorganism, media, engineered genes and other features are as described elsewhere herein.

[0114] In some embodiments the method further comprises the step of harvesting the plants so that the plants are removed from the media. In this way, the metals that are accumulated in the plant biomass are removed from the media, reducing the metal content of the media.

[0115] In some embodiments it is considered to be important to monitor the metal content of the media, to determine the effect that cultivating the plants of the invention has on the metal content of the media.

[0116] Accordingly in some embodiments the method comprises: a) Determining the concentration of one or more metals in the media prior cultivating the plant in the media; and b) determining the concentration of the same one or more metals in the media i) during cultivation of the plant; and / or ii) after harvesting the plants.

[0117] The skilled person will appreciate that in some instances one growth cycle of the plant of the invention in the media is sufficient to reduce the metal content of the media to acceptable levels, or the "mine" and capture the amount of metal required.

[0118] However, in other embodiments it will be appreciated that repeated cycles of growth and harvest of the plants of the invention are required, with an amount of metal being removed from the media at each harvest. Accordingly in some embodiments the method comprises repeating following steps: a) cultivating the plant or plant part of the invention in the media; and b) harvesting the plants so that the plants are removed from the media.

[0119] In some embodiments, for example where the aim is to remove metals from the media so that the amount of metals falls below a particular level, step (a) and step (b) are repeated until the metal content of the media is below the predetermined threshold.

[0120] In some embodiments step (a) and step (b) are repeated until one or more of the following criteria are met:

[0121] (i) the content of Nickel in the media is at least lower than 1800 mg / kg; preferably wherein the content is at least lower than 230 mg / kg; most preferably wherein the content is at least lower than 75 mg / kg;

[0122] (ii) the content of Zinc in the media is at least lower than 900 mg / kg; preferably wherein the content is at least lower than 300 mg / kg; most preferably wherein the content is at least lower than 50 mg / kg; and / or

[0123] (iii) the content of Strontium in the media is at least lower than 1370 mg / kg; preferably wherein the content is at least lower than 121 mg / kg; most preferably wherein the content is at least lower than 65 mg / kg.

[0124] In some embodiments step (a) and step (b) are repeated until the metal content of the media is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% of the starting metal content. As mentioned elsewhere here, in addition to the engineered microorganisms and plants of the invention being used in bioremediation, they may also be used in methods of biomining, i.e. as a means of extracting metals from a media to obtain the metals, rather than to decontaminate land.

[0125] Accordingly the invention also provides a method of recovering metals from a media wherein the method comprises the method for removing metals from a media of the invention, and further comprises processing the harvested plants so as to extract the metal content.

[0126] As demonstrated by the inventors, the majority of the accumulated metals is concentrated in the root of the plant. Accordingly in preferred embodiments it is the root part of the plant is processed so as to extract the metal content.

[0127] Since metal accumulation was shown to be restricted to the root of a plant, the invention also provides a means to grow a food crop in metal contaminated media, for example where the method comprises:

[0128] (a) cultivating a plant or plant part of the invention in the metal contaminated media;

[0129] (b) harvesting the plants so that the plants are removed from the growth media; and

[0130] (c) removing and discarding the root portion of the plant.

[0131] In this way, the metal contaminated part of the plant is removed, leaving the non-metal contaminated remainder for food use.

[0132] The invention also provides a seed, tuber or bulb coated with one or more engineered microorganisms of the invention.

[0133] The invention also provides Pseudomonas fluorescens for use in a method of bioremediation.

[0134] The invention also provides a method of bioremediation wherein said method comprises applying Pseudomonas fluorescens to a plant or plant part.

[0135] Sequences:

[0136] Brief description of the figures

[0137] Fig 1: (A-D) Zinc accumulation in wildtype B. napus in the presence and absence of P. fluorescens expressing either AuxI, AuxII, ACCD, and PvdS. The effect of each gene was measured under the control of BG17 and BG37 promoters. All experiments were conducted in triplicate, (n = 3).

[0138] Fig 2: Quantification of Zinc accumulation within different tissue types of WT B. napus in the presence of P. fluorescens expressing either AuxI, AuxII and ACCD. Zinc concentration (mg / kg) of the substrate soil given in the figure legend.

[0139] Fig 3: Nickel (Right) and Strontium (Left) accumulation in WT B. napus in the presence and absence of WT P. fluorescens. Fig 4: Representative microscopy images showing the examination of WT P. fluorescens (A) and P. fluorescens expressing E2 Crimson red (B) using brightfield and fluorescent imaging (pictured left and right respectively). Fluorescent images were taken with an excitation wavelength of 633 nm and emission wavelength of 650-800 nm.

[0140] Fig 5: Representative microscopy images showing the colonisation of B. napus by P. fluorescens. Panels A and B show brightfield (left) and fluorescent (right) microscopy images of shoot structures containing WT P. fluorescens and WT P. fluorescens expressing E2 Crimson red respectively. Panels C and D show the same microscopy procedure for the root structures.

[0141] Fig 6: Experiments conducted P. fluorescens expressing AuxI and AuxII with promoters BG17 and BG37 grown in Zinc. Zn accumulation was analysed after one month of growth.

[0142] The invention also provides the following numbered embodiment paragraphs:

[0143] 1. An engineered microorganism, wherein the microorganism has been engineered to:

[0144] (a) produce or overproduce auxin;

[0145] (b) reduce the production of ethylene;

[0146] (c) produce or overproduce one or more metal chelator molecules; and / or

[0147] (d) reduce or knockout the production of tryptophan.

[0148] 2. The engineered microorganism of paragraph 1 wherein :

[0149] (a) said production or overproduction of auxin is via the expression or overexpression of one or more proteins or peptides required for the production or auxin;

[0150] (b) said reduction of the production of ethylene is via the expression or overexpression of one or more proteins or peptides involved in the degradation or conversion of extracellular ethylene;

[0151] (c) said production or overproduction of one or more metal chelators is via the expression or overexpression of one or more proteins or peptides for the biosynthesis of one or more chelator molecules; and / or

[0152] (d) said reduction or knockout of the production of tryptophan is via one or more gene disruptions in the tryptophan biosynthesis pathway.

[0153] 3. The engineered microorganism of paragraph 2 wherein : (a) said one or more proteins or peptides required for the production of auxin is AUXI, AUXII or both AUXI and AUXII;

[0154] (b) said one or more proteins or peptides involved in the degradation of conversion of extracellular ethylene is ACC Deaminase (ACCD); and / or

[0155] (c) said one or more proteins or peptides for the biosynthesis of one or more chelator molecules is a sigma factor, optionally PvdS.

[0156] 4. The engineered microorganism of paragraph 2 or 3 wherein the engineered microorganism has been engineered to express or overexpress: a) AUXI; b) AUXII; c) PvdS; d) ACDS; e) AUXI and AUXII; f) AUXI and PvdS; g) AUXI and ACDS; h) AUXII and PvdS; i) AUXII and ACDS; j) PvdS and ACDS; k) AUXI, AUXII and PvdS; l) AUXI, AUXII and ACDS; m) AUXI, PvdS and ACDS; n) AUXII, PvdS and ACDS; or o) AUXI, AUXII, PvdS and ACDS.

[0157] 5. The engineered microorganism of any one of paragraphs 1-3 wherein the engineered microorganism has been engineered to express or overexpress AUXI, AUXII, ACCD and PvdS.

[0158] 6. The engineered microorganism of any one of paragraphs 3-5 wherein:

[0159] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, optionally provided that the AUXI retains the ability to produce IAA;

[0160] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 optionally provided that the AUXII retains the ability to produce IAA;

[0161] ACCD has an amino acid sequence of SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 optionally provided that the ACCD retains the ability to breakdown ACC;

[0162] PvdS has an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 optionally provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

[0163] 7. The engineered microorganism of any of the preceding paragraphs wherein the auxin is selected from the group comprising or consisting of: indole-3-acetic acid, 4- chloroindole-3-acetic acid, phenylacetic acid, indole-3-butyric acid, and indole-3-propionic acid.

[0164] 8. The engineered microorganism of any of paragraphs 1-7 wherein the engineered microorganism comprises a nucleotide sequence encoding any one or more of AUXI, AUXII, PvdS and / or ACDS or any combination thereof operably linked to a promoter sequence thus forming one or more expression cassettes.

[0165] 9. The engineered microorganism of paragraph 8 wherein the promoter is selected from the group comprising or consisting of: a) a constitutive promoter; b) an inducible promoter; c) a BG17 promoter [SEQ ID NO: 9]; and / or d) a BG37 promoter [SEQ ID NO: 10].

[0166] 10. The engineered microorganism of any of the preceding paragraphs wherein the microorganism has been engineered to express at least the following gene combinations from the specified promoters, or engineered to express only the following gene combinations from the specified promoters: a) BG17: :AUXI b) BG37: :AUXI c) BG17: :AUXII d) BG37: :AUXII e) BG17: :PvdS f) BG37::PvdS g) BG17::ACDS h) BG37::ACDS i) BG17::AUXI + BG17::AUXII j) BG17::AUXI + BG37::AUXII k) BG37::AUXI + BG17::AUXII l) BG37::AUXI + BG37::AUXII m) BG17::AUXI + BG17::PvdS n) BG17::AUXI + BG37::PvdS o) BG37::AUXI + BG17::PvdS p) BG37::AUXI + BG37::PvdS q) BG17::AUXI + BG17::ACDS r) BG17::AUXI + BG37::ACDS s) BG37::AUXI + BG17::ACDS t) BG37::AUXI + BG37::ACDS u) BG17::AUXII + BG17::PvdS v) BG17::AUXII + BG37::PvdS w) BG37::AUXII + BG17::PvdS x) BG37::AUXII + BG37::PvdS y) BG17::AUXII + BG17::ACDS z) BG17::AUXII + BG37::ACDS aa) BG37::AUXII + BG17::ACDS ab) BG37::AUXII + BG37::ACDS ac) BG17::PvdS + BG17::ACDS ad) BG17::PvdS + BG37::ACDS ae) BG37::PvdS + BG17::ACDS af) BG37::PvdS + BG37::ACDS ag) BG17::AUXI + BG17::AUXII + BG17::PvdS ah) BG17::AUXI + BG17::AUXII + BG37::PvdS ai) BG17::AUXI + BG37::AUXII + BG17::PvdS aj) BG17::AUXI + BG37::AUXII + BG37::PvdS ak) BG37::AUXI + BG17::AUXII + BG17::PvdS al) BG37::AUXI + BG17::AUXII + BG37::PvdS am) BG37::AUXI + BG37::AUXII + BG17::PvdS an) BG37::AUXI + BG37::AUXII + BG37::PvdS ao) BG17::AUXI + BG17::AUXII + BG17::ACDS ap) BG17::AUXI + BG17::AUXII + BG37::ACDS aq) BG17::AUXI + BG37::AUXII + BG17::ACDS ar) BG17::AUXI + BG37::AUXII + BG37 ACDS as) BG37::AUXI + BG17::AUXII + BG17 ACDS at) BG37::AUXI + BG17::AUXII + BG37 ACDS au) BG37::AUXI + BG37::AUXII + BG17 :ACDS av) BG37::AUXI + BG37::AUXII + BG37 :ACDS aw) BG17::AUXI + BG17::PvdS + BG17 ACDS ax) ay) az) ba) bb) be) bd) be) BG17::AUXII + BG17::PvdS + BG17 :ACDS bf) BG17::AUXII + BG17::PvdS + BG37 ACDS bg) BG17::AUXII + BG37::PvdS + BG17 :ACDS bh) BG17::AUXII + BG37::PvdS + BG37 :ACDS bi) BG37::AUXII + BG17::PvdS + BG17 ACDS bj) BG37::AUXII + BG17::PvdS + BG37 ACDS bk) BG37::AUXII + BG37::PvdS + BG17 :ACDS bl) BG37::AUXII + BG37::PvdS + BG37 ACDS bm) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bn) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS bo) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS bp) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS bq) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS br) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS bs) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS bt) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS bu) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS bv) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS bw) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS bx) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS by) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS bz) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS ca) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS cb) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS. 11. The engineered microorganism of any of the preceding paragraphs wherein the microorganism has been engineered to express at least the following gene combinations from the specified promoters, or engineered to express only the following gene combinations from the specified promoters: a) BG17: :AUXI + BG17: :AUXII b) BG17: :AUXI + BG37: :AUXII c) BG37: :AUXI + BG17: :AUXII d) BG37: :AUXI + BG37: :AUXII where:

[0167] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / or

[0168] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

[0169] 12. The engineered microorganism of any one of paragraphs 8-11 wherein one or more of the expression cassettes is genomically integrated into the host cell genome.

[0170] 13. The engineered microorganism of any one of paragraphs 8-12 wherein one or more of the expression cassettes is contained with an episomal vector, optionally wherein the vector is selected from a plasmid, transposon, or bacteriophage.

[0171] 14. The engineered microorganism of any of paragraphs 1-13 wherein the engineered microorganism is capable of endophytic symbiosis or plant colonisation, optionally is able to colonise the root of a partner plant.

[0172] 15. The engineered microorganism of any of paragraphs 1-14 wherein the microorganism is a bacteria, fungi, or algae.

[0173] 16. The engineered microorganism of any one of the preceding paragraphs wherein the microorganism is an endophyte of the Brassica genus, optionally Brassica napus. 17. The engineered microorganism of any of the preceding paragraphs wherein the microorganism is a bacteria selected from the group comprising or consisting of: a) a gram negative bacteria optionally a bacteria belonging to the Pseudomonas genus; optionally Pseudomonas fluorescens; b) a gram positive bacteria, optionally Bacillus sp; c) any one or more of the following: a Pseudomonas sp.; a Pantoea sp.; an Enterobacter sp.; a Ralstonia sp.; or a a Microbacterium sp.; d) any one or more of Pseudomonas fluorescens; Pantoea ananatis; Pantoea stewartia; Enterobacter ludwigii; Pantoea agglomerans; Pseudomonas thivervalensis; Pantoea ananatis; Pseudomonas putida; or e) any one or more of the following strains from Zhang et al 2011 Chemosphere 83: 57-62: strain Gl-21-2 of Pseudomonas sp.; Jl-7-5 of Pantoea ananatis; Jl-13-7 of Pantoea stewartia; Jl-17-2 of Enterobacter ludwigii; Jl-22-2 of Ralstonia sp.; JP3-3 of Pantoea agglomerans; Yl-3-9 of Pseudomonas thivervalensis; Yl-15-5 of Pantoea ananatis; G16 of Microbacterium sp.

[0174] 18. The engineered microorganism of any of the preceding paragraphs wherein the microorganism is Pseudomonas fluorescens.

[0175] 19. The engineered microorganism of any of the preceding paragraphs wherein the microorganism is Pseudomonas fluorescens and is engineered to express at least the following gene combinations from the specified promoters, or engineered to express or overexpress only the following gene combinations from the specified promoters: a) BG17: :AUXI + BG17: :AUXII b) BG17: :AUXI + BG37: :AUXII c) BG37: :AUXI + BG17: :AUXII d) BG37: :AUXI + BG37: :AUXII where:

[0176] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / or

[0177] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

[0178] 20. The engineered microorganism of any one of the preceding paragraphs wherein the engineered microorganism is able to colonise a partner plant, optionally able to colonise the root of a partner plant and enables accumulation in the partner plant of one or more metals from the surrounding media, optionally wherein the metals are selected from the group comprising or consisting of: transition and post transition metals; optionally one or more of Zinc, Strontium, Nickel, Iron, Mercury, Cadmium, Lead, Copper, Chromium, Aluminium, Cobalt, Manganese, Silver, and Gold, optionally Zinc, Strontium and / or Nickel.

[0179] 21. The engineered microorganism of paragraph 20 wherein the engineered microorganism enables accumulation of metals in the partner plant to a higher concentration than is accumulated in the absence of the engineered microorganism.

[0180] 22. The engineered microorganism of paragraph 20 or 21 wherein the engineered microorganism enables accumulation of metals in the partner plan to a higher concentration than is accumulated in the presence of a corresponding microorganism that has not been engineered according to any of the preceding paragraphs.

[0181] 23. The engineered microorganism of any of paragraphs 20-22 wherein the metals accumulate within the xylem and / or phloem of the recipient plant.

[0182] 24. The engineered microorganism of any of paragraphs 20-23 wherein the one or more metals are prevented from entering plant cells, optionally wherein the one or more metals are chelated within the plant root and prevented from entering plant cells.

[0183] 25. An expression cassette comprising a promoter operably linked to any one or more of the following genes: AUXI, AUXII, PvdS and / or ACDS optionally wherein a) AUXI has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 optionally provided that the AUXI retains the ability to produce IAA; ; b) AUXII has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 optionally provided that the AUXII retains the ability to produce IAA;; c) ACCD has the protein sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 optoinally provided that the ACCD retains the ability to breakdown ACC; and / or d) PvdS has the protein sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 optionally provided that the PvdS retains the ability to regulate the synthesis of pyoverdine;

[0184] And / or b) the promoter is the BG17 or BG37 promoter.

[0185] 26. An expression vector comprising one or more expression cassettes of paragraph 25.

[0186] 27. A method of preparing a plant with an increased ability to accumulate metals, wherein said method comprises contacting one or more plant parts, optionally one or more seeds or tubers or bulbs of said plant with one or more engineered microorganisms of any of the preceding paragraphs.

[0187] 28. The method of paragraph 27 wherein said one or more plant parts, optionally one or more seeds, tubers or bulbs are contacted with at least 2, 3 or at least 4 different engineered microorganisms, optionally wherein each microorganism has been engineered so as to express or overexpress either:

[0188] (a) a different gene, optionally to express or overexpress a different gene selected from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0189] (b) two or more different genes, optionally to express or overexpress two or more different genes from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0190] 29. The method of paragraph wherein the one or more plant part, optionally one or more seeds, tubers or bulbs are contacted (or coated) with at least two, three or four or more different engineered microbes, where the engineered microbes have been engineered to express or overexpress the following proteins from the specified promoters: a) BG17: :AUXI b) BG37: :AUXI c) BG17: :AUXII d) BG37: :AUXII e) BG17: :PvdS f) BG37: :PvdS g) BG17: :ACDS h) BG37: :ACDS.

[0191] 30. The method of paragraph wherein the one or more plant part, optionally one or more seeds, tubers or bulbs are contacted (or coated) with at least a first and second different engineered microbe, wherein : a) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; b) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; c) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG17 promoter; or d) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered microorganism has been engineered to express AUXII from the BG17 promoter.

[0192] 31. The method of any of paragraphs 29 or 30 wherein the seed, tuber or bulb is coated with the one or more engineered microorganisms of any of the preceding paragraphs.

[0193] 32. A method of preparing a plant with an increased ability to accumulate metals, wherein said method comprises contacting one or more parts of said plant with one or more engineered microorganisms of any of the preceding paragraphs, optionally wherein one or more parts of said plant is a seed, tuber or bulb.

[0194] 33. The method of paragraph 32 wherein the plant is a seedling.

[0195] 34. The method of paragraph 32 wherein the plant is a mature plant. 35. The method of any of paragraphs 32-34 wherein said contacting comprises applying said one or more engineered microorganisms to a media into which the said plant is subsequently planted.

[0196] 36. The method of any of paragraphs 32-34 wherein contacting occurs by applying the one or more engineered microorganisms to the media in which the plant is already growing in.

[0197] 37. The method of any of paragraphs 32-34 wherein said one or more parts of said plant is contacted with the one or more engineered microorganisms at the same time as the plant is planted into a media.

[0198] 38. The method of any of paragraphs 32-37 wherein said one or more engineered microorganisms is at least 2, 3 or at least 4 different engineered microorganisms, optionally wherein each microorganism has been engineered so as to express or overexpress either:

[0199] (a) a different gene, optionally to express or overexpress a different gene selected from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0200] (b) two or more different genes, optionally to express or overexpress two or more different genes from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0201] 39. The method of any of paragraphs 32-38 wherein the plant is: a) a Brassica species, optionally B. napus; b) a root vegetable; c) a plant that grows from tubers d) an aquatic plant; e) a plant that grows in water, optionally watercress.

[0202] 40. A method of preparing a Brassica species plant, optionally B. napus plant with an increased ability to accumulate heavy metals, wherein said method comprises contacting seeds or seedlings of said plant with one or more engineered microorganisms of any of the preceding paragraphs, wherein said engineered microorganism is P. fluorescens engineered so as to overexpress any one or more of AUXI, AUXII, PvdS and ACDS.

[0203] 41. A plant or plant part prepared according to any one of the methods of any of the preceding paragraphs , optionally wherein said plant or plant part is a seed, tuber, bulb or seedling. 42. A method for removing metals from a media wherein said method comprises cultivating a plant or plant part according to paragraph 41 in said media.

[0204] 43. The method of paragraph 42 wherein the media is soil or water.

[0205] 44. The method of paragraph 42 or 43 wherein the method further comprises: harvesting the plants so that the plants are removed from the growth media.

[0206] 45. The method of paragraph 44 wherein the method comprises: a) Determining the concentration of one or more metals in the media prior cultivating the plant in the media; and b) determining the concentration of the same one or more metals in the media i) during cultivation of the plant; and / or ii) after harvesting the plants.

[0207] 46. The method of paragraph 42-45 wherein the method comprises repeating steps a) cultivating the plant or plant part of paragraph 36 in the media; and b) harvesting the plants so that the plants are removed from the media.

[0208] 47. The method of paragraph 46 wherein step (a) and step (b) are repeated until the metal content of the media is below a predetermined threshold.

[0209] 48. The method of paragraph 46 wherein step (a) and step (b) are repeated until the metal content of the media is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% of the starting metal content.

[0210] 49. The method of paragraph 46 wherein step (a) and step (b) are repeated until one or more of the following criteria are met:

[0211] (i) the content of Nickel in the media is at least lower than 1800 mg / kg; preferably wherein the content is at least lower than 230 mg / kg; most preferably wherein the content is at least lower than 75 mg / kg;

[0212] (ii) the content of Zinc in the media is at least lower than 900 mg / kg; preferably wherein the content is at least lower than 300 mg / kg; most preferably wherein the content is at least lower than 50 mg / kg; and / or

[0213] (iii) the content of Strontium in the media is at least lower than 1370 mg / kg; preferably wherein the content is at least lower than 121 mg / kg; most preferably wherein the content is at least lower than 65 mg / kg. 50. A method of recovering metals from a media wherein the method comprises the method for removing metals from a plant growth media according to any one of the preceding paragraphs, and further comprises processing the harvested plants so as to extract the metal content.

[0214] 51. The method paragraph 50 wherein the root part of the plant is processed so as to extract the metal content.

[0215] 52. A method of producing a food crop from a metal contaminated media wherein the method comprises:

[0216] (a) cultivating a plant or plant part according to paragraph 36 in the metal contaminated media;

[0217] (b) harvesting the plants so that the plants are removed from the growth media; and

[0218] (c) removing and discarding the root portion of the plant.

[0219] 53. A seed, tuber or bulb coated with one or more engineered microorganisms of any of the preceding paragraphs.

[0220] 54. Pseudomonas fluorescens for use in a method of bioremediation.

[0221] 55. A method of bioremediation wherein said method comprises applying Pseudomonas fluorescens to a plant or plant part.

[0222] The invention also provides the further numbered embodiments:

[0223] 1. An engineered microorganism, wherein the microorganism has been engineered to:

[0224] (a) produce or overproduce auxin;

[0225] (b) reduce the production of ethylene;

[0226] (c) produce or overproduce one or more metal chelator molecules; and / or

[0227] (d) reduce or knockout the production of tryptophan.

[0228] 2. The engineered microorganism of embodiment 1 wherein : (a) said one or more proteins or peptides required for the production of auxin is AUXI, AUXII or both AUXI and AUXII;

[0229] (b) said one or more proteins or peptides involved in the degradation of conversion of extracellular ethylene is ACC Deaminase (ACCD); and / or

[0230] (c) said one or more proteins or peptides for the biosynthesis of one or more chelator molecules is a sigma factor, optionally PvdS.

[0231] 3. The engineered microorganism of any one of embodiments 1 or 2 wherein the engineered microorganism has been engineered to express or overexpress AUXI, AUXII, ACCD and PvdS.

[0232] 4. The engineered microorganism of any one of embodiments 2 or 3 wherein:

[0233] AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1;

[0234] AUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2;

[0235] ACCD has an amino acid sequence of SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3;

[0236] PvdS has an amino acid sequence of SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4.

[0237] 5. The engineered microorganism of any of embodiments 1-4 wherein the engineered microorganism comprises a nucleotide sequence encoding any one or more of AUXI, AUXII, PvdS and / or ACDS or any combination thereof operably linked to a promoter sequence thus forming one or more expression cassettes and wherein promoter is selected from the group comprising or consisting of: a) a BG17 promoter [SEQ ID NO: 9]; b) a BG37 promoter [SEQ ID NO: 10]; c) a constitutive promoter; and / or d) an inducible promoter. 6. The engineered microorganism of any of embodiments 1-5 wherein the engineered microorganism is capable of endophytic symbiosis or plant colonisation, optionally is able to colonise the root of a partner plant.

[0238] 7. The engineered microorganism of any one of the preceding embodiments wherein the microorganism is an endophyte of the Brassica genus, optionally Brassica napus.

[0239] 8. The engineered microorganism of any of the preceding embodiments wherein the microorganism is selected from any one or more of: a) Pseudomonas fluorescens b) a gram negative bacteria optionally a bacteria belonging to the Pseudomonas genus; optionally Pseudomonas fluorescens; c) a gram positive bacteria, optionally Bacillus sp; d) any one or more of the following: a Pseudomonas sp.; a Pantoea sp.; an Enterobacter sp.; a Ralstonia sp.; or a a Microbacterium sp.; e) any one or more of Pseudomonas fluorescens; Pantoea ananatis; Pantoea stewartia; Enterobacter ludwigii; Pantoea agglomerans; Pseudomonas thivervalensis; Pantoea ananatis; Pseudomonas putida; or f) any one or more of the following strains from Zhang et al 2011 Chemosphere 83: 57-62: strain Gl-21-2 of Pseudomonas sp.; Jl-7-5 of Pantoea ananatis; Jl-13-7 of Pantoea stewartia; Jl-17-2 of Enterobacter ludwigii; Jl-22-2 of Ralstonia sp.; JP3-3 of Pantoea agglomerans; Yl-3-9 of Pseudomonas thivervalensis; Yl-15-5 of Pantoea ananatis; G16 of Microbacterium sp.

[0240] 9. The engineered microorganism of any of the preceding embodiments wherein when the engineered microorganism has colonised a partner plant or plant part, enables accumulation of metals in the partner plant to a higher concentration than is accumulated: a) in the absence of the engineered microorganism; and / or b) in the presence of a corresponding microorganism that has not been engineered according to any of the preceding embodiments.

[0241] 10. A method of preparing a plant with an increased ability to accumulate metals, wherein said method comprises contacting one or more seeds or tubers or bulbs of said plant with one or more engineered microorganisms of any of the preceding embodiments. 11. The method of embodiment 10 wherein said one or more seeds, tubers or bulbs are contacted with at least 2, 3 or at least 4 different engineered microorganisms, wherein each microorganism has been engineered so as to express or overexpress either:

[0242] (a) a different gene, optionally to express or overexpress a different gene selected from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0243] (b) two or more different genes, optionally to express or overexpress two or more different genes from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

[0244] 12. The method of any of embodiments 10 or 11 wherein the seed, tuber or bulb is coated with the one or more engineered microorganisms of any of the preceding embodiments.

[0245] 13. A method of preparing a plant with an increased ability to accumulate metals, wherein said method comprises contacting one or more parts of said plant with one or more engineered microorganisms of any of the preceding embodiments.

[0246] 14. The method of embodiment 13 wherein the plant is a seedling or a mature plant.

[0247] 15. The method of any of embodiments 10-14 wherein the plant is: a) a Brassica species, optionally B. napus; b) a root vegetable; c) a plant that grows from tubers d) an aquatic plant; e) a plant that grows in water, optionally watercress.

[0248] 16. A method of preparing a Brassica species plant, optionally B. napus plant with an increased ability to accumulate heavy metals, wherein said method comprises contacting seeds or seedlings of said plant with one or more engineered microorganisms of any of the preceding embodiments, wherein said engineered microorganism is P. fluorescens engineered so as to overexpress any one or more of AUXI, AUXII, PvdS and ACDS.

[0249] 17. A plant or plant part prepared according to any one of the methods of any of embodiments 10-16, optionally wherein said plant or plant part is a seed, tuber, bulb or seedling.

[0250] 18. A plant or plant part that has been colonised by one or more engineered microorganisms according to any of the preceding embodiments. 19. A method for removing metals from a media wherein said method comprises cultivating a plant or plant part according to embodiment 17 or 18 in said media, optionally wherein the media is soil or water.

[0251] 20. The method of embodiment 19 wherein the method further comprises: harvesting the plants so that the plants are removed from the growth media.

[0252] 21. The method of embodiment 20 wherein the method comprises repeating steps a) cultivating the plant or plant part of embodiment 17 or 18 in the media; and b) harvesting the plants so that the plants are removed from the media.

[0253] 22. A method of recovering metals from a media wherein the method comprises the method for removing metals from a plant growth media according to any one of embodiments 19-21, and further comprises processing the harvested plants so as to extract the metal content.

[0254] 23. The method embodiment 22 wherein only the root part of the plant is processed so as to extract the metal content.

[0255] 24. A seed, tuber or bulb coated with one or more engineered microorganisms of any of the preceding embodiments.

[0256] 25. A method of bioremediation wherein said method comprises applying Pseudomonas fluorescens to a plant or plant part.

[0257] Examples

[0258] The invention will now be illustrated by the following non-limiting example.

[0259] Example 1: Experimental procedures to enable the modification of Pseudomonas fluorescens and colonisation of Brassica napus for the purpose of improving metal uptake from soil.

[0260] 1.1 Preparation and transformation of an expression vector into P. fluorescens

[0261] 1.1.1 Molecular biology approaches Points 1.1.1.1 to 1.1.1.11 illustrate some of the exemplary molecular biology methods that can be used to prepare a vector to express genes of interest in P. fluorescens.

[0262] 1.1.1.1 Preparation of Miller Lysogeny Broth (LB) Media for the culturing of E. coli The medium was prepared with 1% (w / v) Peptone au casein (Merck, Nottingham, UK), 0.5% (w / v) Yeast Extract (Merck, Nottingham, UK), 1% (w / v) NaCI (+1.6% (w / v) agar included for plates). The mixture was then autoclaved at 121°C for 20 minutes.

[0263] 1.1.1.2 Preparation of Murashige and Skoog medium for the culturing of P. fluorescens

[0264] The medium was prepared by dissolving MS basal salts (Millipore Sigma) in distilled water at a concentration of 4.43 g / L. Sucrose was added to the medium at a final concentration of 30 g / L as a carbon source. The pH of the medium was adjusted to 5.8 using 1 M NaOH or 1 M HCI before autoclaving at 121°C for 20 minutes. After autoclaving, the medium was poured into culture dishes and stored at 4°C.

[0265] 1.1.1.3 Plasmid DNA Isolation

[0266] Plasmid DNA was extracted from overnight cultures of E. coli using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol.

[0267] 1.1.1.4 DNA Quantification

[0268] DNA solution concentrations were determined by measuring absorbance at 260 nm with the BioDrop pLITE (BioDrop Ltd., Cambridge, UK), following the manufacturers' instructions.

[0269] 1.1.1.5 DNA Recovery, Purification, and Concentration

[0270] PCR products were recovered from an agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA), in accordance with the manufacturer's instructions. In some cases, DNA was concentrated using the Zymo Clean and Concentrate kit (Zymo Research) following the provided manufacturer's instructions.

[0271] 1.1.1.6 Restriction Enzymes

[0272] Following the selection of the enzymes, a reaction mix was prepared in a microcentrifuge tube that contained the DNA sample (usually between 0.5-1.0 pg), the specific buffer recommended for the enzyme, the restriction enzyme itself (typically 1-10 units per pg of DNA), and nuclease-free water to adjust the final volume. After gently mixing the components, the reaction mix was incubated at the optimal temperature for the chosen enzyme, typically around 37°C, for a period typically ranging from 1 to 2 hours. After digestion, the restriction enzyme was inactivated, by heating to 65-80°C for 10-20 minutes or as per the manufacturer's guidelines. The resulting DNA fragments were then analyzed to confirm digestion. This analysis was performed using agarose gel electrophoresis or other appropriate methods that allowed for the visualization of the cleaved DNA fragments. In some circumstances, the digested DNA fragments was purified using a DNA purification kit to remove any residual enzymes and buffer components.

[0273] 1.1.1.7 Ligation

[0274] Ligation was carried out using T4 DNA Ligase (NEB) according to the manufacturer's protocol. The plasmid to insert molar ratio was typically 3: 1 for successful ligation. The ligation mixture was generally incubated for 1 hour at room temperature or overnight at 4°C with 5 pL of the ligation reaction being used to transform NEB DH5a cells.

[0275] 1.1.1.8 DNA Sequence Verification

[0276] DNA plasmids were sequenced by Eurofins Genomics (Ebersberg, Germany). For submission, 15 pL plasmid DNA between 50-100 ng pL-1 was pre-mixed with 2 pL primer (starting concentration 10 pM).

[0277] 1.1.1.9 Transformation of competent E. coli cells

[0278] E. coli (NEB 5a) were subjected to chemical transformation following the supplier's guidelines. Initially, 50 pL aliquots of cells were thawed on ice and combined with 1-50 ng of DNA, followed by incubation on ice for 30 minutes. Subsequently, the cells underwent heat shock at 42°C for 30 seconds before being returned to ice for 2 minutes. To facilitate recovery, 950 pL of SOC medium was added to the cells, and the mixture was incubated with shaking at 37°C and 250 rpm for 1 hour. After incubation, the cells were centrifuged, resuspended in 100 pL of solution, and then plated onto LB agar plates containing selective antibiotics. The plates were incubated overnight at 37°C to allow for colony growth.

[0279] 1.1.1.10 Transformation of electrically competent P. fluorescens cells

[0280] Prior to transformation, electrocompetent P. fluorescens cells were prepared. An overnight culture of cells was grown in LB broth, followed by subsequent sub-culturing until the culture reached mid log phase. The cells were then harvested, washed, and resuspended in ice-cold sterile water or buffer to create a dense cell suspension suitable for electroporation.

[0281] For the transformation step, a plasmid containing the gene of interest was mixed with the electrocompetent P. fluorescens cells in a pre-chilled electroporation cuvette. An electric pulse was applied using an electroporator under specific conditions suitable for Pseudomonas species. Following the pulse, a recovery medium such as SOC was added to the cuvette, and the cell suspension was transferred to a culture tube for incubation at 30°C. The cells were allowed to recover and express the transformed plasmid for 1-2 hours. After the recovery period, the transformed cells were plated on selective agar plates containing antibiotics for plasmid selection. The plates were then incubated at 30°C overnight, and the growth of transformed colonies was monitored. In some instances, adjustments to parameters such as DNA concentration, electroporation conditions, and recovery time were needed based on the specific P. fluorescens strain and plasmid used.

[0282] 1.1.1.11 Agarose Gel Electrophoresis

[0283] Agarose gel electrophoresis was employed to separate DNA fragments based on their sizes. Typically, PCR reaction products were subjected to electrophoresis on a 0.8% agarose gel. Agarose gels were prepared by dissolving agarose in lx TAE buffer (50x TAE Buffer; consisting of 242 g / L Tris base, 57.1 mL / L glacial acetic acid, and 100 mL / L 0.5 M EDTA, pH 8.0), which was then melted completely and allowed to cool to around 50°C. SYBR Safe (Invitrogen, Paisley, UK) was diluted 10,000 times in the agarose gel for staining purposes, depending on the type of DNA to be visualized (insert and vector amplification and colony PCR products, respectively). Once the gel was poured and solidified in a mold, it was removed and immersed in TAE buffer, then run in Bio-Rad Wide Mini-Sub Cell GT Tanks (Bio-Rad, Hemel Hempstead, UK) at a constant voltage of 100 V for 50 minutes using a Bio-Rad Power Pac Basic (Bio-Rad). Before loading onto the gel, DNA samples were mixed with 6x Gel Loading Dye, purple (NEB). A 5 pL portion of HyperLadder 1 kb (Bioline, Taunton, MA, USA) served as the marker for all gels. Visualization of DNA was achieved using an integrated UV transilluminator and camera system.

[0284] 1.2 Colonisation of B. napus with P. fluorescens and determining the efficacy metal uptake

[0285] Points 1.2.1 to 1.2.2 illustrate exemplary methods required to facilitate translocation of P. fluorescens into B. napus seeds.

[0286] 1.2.1 Sterilization of B. napus seeds

[0287] A 50% v / v bleach solution was prepared with 100 mL of autoclaved DI water, 100 mL of bleach, and 50 pL of Tween 20. The final solution was stored at room temperature for up to 30 days. Under a biological safety hood, 500 pL of the bleach solution was added to a tube of B. napus seeds and resuspended for about 5 minutes. After removing the bleach, the seeds were washed with autoclaved DI water 6 times. They were left to dry in the hood until plating. 1.2.2 Translocation of the bacteria into the plants

[0288] The translocation of P. fluorescens into the B. napus tissue was accomplished using culture plates made with MS medium. After autoclaving, the MS medium was poured into culture plates and left to cool. Bacterial cultures prepared the day before in LB media were taken and used to inoculate MS plates. B. napus seeds were then placed onto the MS medium plate, with the bacteria underneath them. They were then wrapped in aluminium foil and left to germinate in alternating 16 hour day and 8 hour night cycles conducted at 24°C and 20°C respectively. The seedlings were verified to contain the bacteria with fluorescence microscopy.

[0289] 1.3 Analysis of plant accumulation of heavy metal

[0290] Metal content analysis was performed using a three step procedure of acid digestion, filtration and mass spectroscopy. Briefly, 150 mg of plant material was digested using microwaves in a covered vessel with a 3: 1 mixture of concentrated nitric acid (HNO3) and hydrochloric acid (HCI). After the material had digested and cooled to room temperature, the sample was diluted with an acidic solution comprising 500 mL DI water, 10.75 mL concentrated HNO3 and 2.75 mL concentrated HCI. The sample was filtered to eliminate insoluble residues and analyzed to determine the elemental content using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

[0291] Example 2. B. napus colonised by P. fluorescens expressing multiple different genes shows a marked increases in metal accumulation.

[0292] This study aimed to measure the contribution of AuxI, AuxII, ACCD, and PvdS expressed in P. fluorescens in the uptake of metals by N. napus. P. fluorescens cells were transformed with expression plasmids containing either auxl, auxll, accd, and pvdS under the control of the BG37 or the BG17 promoters. B. napus seeds colonised by P. fluorescens, harbouring the vectors of interest, were germinated and planted in soils containing varying concentrations of Zinc (i.e. 1000 mg / kg of dry soil, 3000 mg / kg of dry soil, and 5000 mg / kg of dry soil). After maturation, plant samples were harvested from the pots, including their roots, and dried for 5 days. Dried plants were then weighed, and a portion of the plant, weighing ~150 mg, was digested using Nitric Acid and Hydrochloric Acid in a microwave digestor as set out above. The samples were processed through ICP-MS instrument to quantify metal accumulation. As shown in Fig. 1, a comparison of metal content in B. napus samples grown in combination with P. fluorescens, harbouring the aforementioned vectors, showed a significant accumulation of Zinc compared to B. napus alone. Expression of all four genes separately across all four selected Zinc concentrations showed higher accumulation of metal compared to B. napus grown in the absence of P. fluorescens. The data further showed that the degree of Zinc enrichment correlated with the concentration of Zinc added to the soil. Moreover, the data showed that the expression of auxl had the greatest impact on the accumulation of Zinc compared to the other tested genes.

[0293] To determine if the efficacy of the combination was limited to Zinc, a further experiment was conducted using soil supplemented with either Strontium or Nickel at varying concentrations. B. napus was grown in the presence and absence of wildtype P. fluorescens. As shown in Fig. 2, the presence of wildtype P. fluorescens gave rise to increased uptake of both Strontium and Nickel showing that a variety of metals could be enriched using this approach.

[0294] In a parallel study quantifying Zinc accumulation by tissue type, B. napus was grown in combination with P. fluorescens expressing either Auxl, AuxII or ACCD in soil supplemented with varying concentrations of Zinc. As shown in Fig. 3, dissection of individual plant structures followed by digestion and ICP-MS analysis showed a significant accumulation of Zinc in the plant's root structures.

[0295] To determine the prevalence of P. fluorescens across the B. napus plant tissue brightfield and fluorescence microscopy was conducted. To allow for detection, P. fluorescens was transformed with a plasmid harbouring E2 Crimson red. As shown in Fig. 4, the expression of the plasmid in P. fluorescens could be readily detected with bright far-red fluorescence microscopy. B. napus, colonised with either wild type or E2 Crimson red expressing P. fluorescens, was grown for 5 weeks. As shown in Fig. 5, subsequent examination of plant tissue structures showed P. fluorescens was able to colonise both shoot and root tissue.

[0296] Example 3. Expression of combinations of Auxl, AuxII, ACCD, and PvdS enhance metal uptake in plants, and is further enhanced by use of specific promoters

[0297] B. napus seeds were colonised by two strains of P. fluorescens, one that expressed AUXI and one that expressed AUXII. To optimise expression, different combinations of the BG17 and BG37 promoters were used. The seeds were germinated and planted in soils containing either no Zinc or 5000 mg Zinc per kg of dry soil. Plant samples were harvested after 1 month of growth from the pots, including their roots, and dried for 5 days. Dried plants were then weighed, and a portion of the plant, weighing ~150 mg, was digested using Nitric Acid and Hydrochloric Acid in a microwave digestor as set out above. The samples were processed through ICP-MS instrument to quantify metal accumulation. Data is presented in Fig 6.

[0298] The multiplex experiments demonstrate that combinations of genes, particularly AuxI and AuxII, significantly enhance zinc accumulation in Brassica napus when paired with Pseudomonas fluorescens. Specific multiplex configurations outperform individual gene constructs, particularly under BG37 promoter control.

Claims

Claims1. An engineered microorganism, wherein the microorganism has been engineered to express at least the following gene combinations from the specified promoters, or engineered to express only the following gene combinations from the specified promoters: a) BG37: :AUXI + BG37: :AUXII b) BG17: :AUXI + BG17: :AUXII c) BG17: :AUXI + BG37: :AUXII d) BG37: :AUXI + BG17: :AUXII.

2. The engineered microorganism of claim 1 wherein:AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, optionally provided that the AUXI retains the ability to produce IAA; andAUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 optionally provided that the AUXII retains the ability to produce IAA.

3. The engineered microorganism of any of the preceding claims wherein the microorganism is a bacteria selected from the group comprising or consisting of: a) a gram negative bacteria optionally a bacteria belonging to the Pseudomonas genus; optionally Pseudomonas fluorescens; b) a gram positive bacteria, optionally Bacillus sp; c) any one or more of the following: a Pseudomonas sp.; a Pantoea sp.; an Enterobacter sp.; a Ralstonia sp.; or a a Microbacterium sp.; d) any one or more of Pseudomonas fluorescens; Pantoea ananatis; Pantoea stewartia; Enterobacter ludwigii; Pantoea agglomerans; Pseudomonas thivervalensis; Pantoea ananatis; Pseudomonas putida; or e) any one or more of the following strains from Zhang et al 2011 Chemosphere 83: 57-62: strain Gl-21-2 of Pseudomonas sp.; Jl-7-5 of Pantoea ananatis; Jl-13-7 of Pantoea stewartia; Jl-17-2 of Enterobacter ludwigii; Jl-22-2 of Ralstonia sp.; JP3-3 of Pantoea agglomerans; Yl-3-9 of Pseudomonas thivervalensis; Yl-15-5 of Pantoea ananatis; G16 of Microbacterium sp.

4. The engineered microorganism of any of the preceding claims wherein the microorganism is Pseudomonas fluorescens.

5. The engineered microorganism of any of the preceding claims wherein the BG17 promoter has a sequence of [SEQ ID NO: 9]; and the BG37 promoter has a sequence of [SEQ ID NO: 10].

6. The engineered microorganism of any of the preceding claims wherein the microorganism has been further engineered to express or overexpress PvdS and / or ADDC.

7. The engineered microorganism of claim 6 wherein the promoter or promoters driving expression of PvdS and / or ADDC are selected from the group comprising or consisting of: a) a constitutive promoter; b) an inducible promoter; c) a BG17 promoter [SEQ ID NO: 9]; and / or d) a BG37 promoter [SEQ ID NO: 10].

8. The engineered microorganism of any of claims 6 or 7 wherein the microorganism has been engineered to express at least the following gene combinations from the specified promoters, or engineered to express only the following gene combinations from the specified promoters: a) BG17::AUXI + BG17::AUXII + BG17::PvdS b) BG17::AUXI + BG17::AUXII + BG37::PvdS c) BG17::AUXI + BG37::AUXII + BG17::PvdS d) BG17::AUXI + BG37::AUXII + BG37::PvdS e) BG37::AUXI + BG17::AUXII + BG17::PvdS f) BG37::AUXI + BG17::AUXII + BG37::PvdS g) BG37::AUXI + BG37::AUXII + BG17::PvdS h) BG37::AUXI + BG37::AUXII + BG37::PvdS i) BG17::AUXI + BG17::AUXII + BG17::ACDS j) BG17::AUXI + BG17::AUXII + BG37::ACDS k) BG17::AUXI + BG37::AUXII + BG17::ACDS l) BG17::AUXI + BG37::AUXII + BG37::ACDS m) BG37::AUXI + BG17::AUXII + BG17::ACDS n) BG37::AUXI + BG17::AUXII + BG37::ACDSo) BG37::AUXI + BG37::AUXII + BG17::ACDS p) BG37::AUXI + BG37::AUXII + BG37::ACDS q) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS r) BG17::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS s) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS t) BG17::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS u) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS v) BG17::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS w) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS x) BG17::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS y) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG17::ACDS z) BG37::AUXI + BG17::AUXII + BG17::PvdS + BG37::ACDS ab) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG17::ACDS ac) BG37::AUXI + BG17::AUXII + BG37::PvdS + BG37::ACDS ad) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG17::ACDS ae) BG37::AUXI + BG37::AUXII + BG17::PvdS + BG37::ACDS af) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG17::ACDS ag) BG37::AUXI + BG37::AUXII + BG37::PvdS + BG37::ACDS9. The engineered microorganism of the preceding claims wherein: a) AUXI has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 optionally provided that the AUXI retains the ability to produce IAA; b) AUXII has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 optionally provided that the AUXII retains the ability to produce IAA; c) ACCD has the protein sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 optionally provided that the ACCD retains the ability to breakdown ACC; and d) PvdS has the protein sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 optionally provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

10. The engineered microorganism of the preceding claims wherein one or more polynucleotides encoding one or more promoter: :gene combinations is genomically integrated into the host cell genome.

11. The engineered microorganism of the preceding claims wherein one or more polynucleotides encoding one or more promoter: :gene combinations is contained within and expressed from an episomal vector, optionally wherein the vector is selected from a plasmid, transposon, or bacteriophage.

12. The engineered microorganism of any of the preceding claims wherein the microorganism is Pseudomonas fluorescens and is engineered to express at least one of the following gene combinations from the specified promoters, or engineered to express or overexpress only the following gene combinations from the specified promoters: a) BG17: :AUXI + BG17: :AUXII b) BG17: :AUXI + BG37: :AUXII c) BG37: :AUXI + BG17: :AUXII d) BG37: :AUXI + BG37: :AUXII where:AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / orAUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

13. The engineered microorganism of any one of the preceding claims wherein the engineered microorganism is able to colonise a partner plant, optionally able to colonise the root of a partner plant and enables accumulation in the partner plant of one or more metals from the surrounding media, optionally wherein the metals are selected from the group comprising or consisting of: transition and post transition metals; optionally one or more of Zinc, Strontium, Nickel, Iron, Mercury, Cadmium, Lead, Copper, Chromium, Aluminium, Cobalt, Manganese, Silver, and Gold, optionally Zinc, Strontium and / or Nickel.

14. The engineered microorganism of claim 13 wherein the metal is zinc.

15. The engineered microorganism of claim 13 or 14 wherein the engineered microorganism enables accumulation of metals in the partner plant to a higher concentration than is accumulated in the absence of the engineered microorganism.

16. The engineered microorganism of any of claims 13-15 wherein the engineered microorganism enables accumulation of metals in the partner plan to a higher concentration than is accumulated in the presence of a corresponding microorganism that has not been engineered according to any of the preceding claims.

17. The engineered microorganism of any of claims 13-16 wherein the metals accumulate within the xylem and / or phloem of the recipient plant.

18. The engineered microorganism of any of claims 13-17 wherein the one or more metals are prevented from entering plant cells, optionally wherein the one or more metals are chelated within the plant root and prevented from entering plant cells.

19. An expression cassette comprising a BG17 or BG37 promoter operably linked to any one or more of the following genes: AUXI, AUXII, PvdS and / or ACDS.

20. The expression cassette of claim 19 wherein a) AUXI has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 optionally provided that the AUXI retains the ability to produce IAA; b) AUXII has the protein sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 optionally provided that the AUXII retains the ability to produce IAA; c) ACCD has the protein sequence of SEQ ID NO: 3 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 optionally provided that the ACCD retains the ability to breakdown ACC; and d) PvdS has the protein sequence of SEQ ID NO: 4 or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 optionally provided that the PvdS retains the ability to regulate the synthesis of pyoverdine.

21. An expression vector comprising one or more expression cassettes of claim 20.

22. A method of preparing a plant with an increased ability to accumulate metals, wherein said method comprises contacting one or more plant parts, optionally one or more seeds or tubers or bulbs of said plant with one or more engineered microorganisms of any of the preceding claims.

23. The method of claim 22 wherein said one or more plant parts, optionally one or more seeds, tubers or bulbs are contacted with at least 2, 3 or at least 4 different engineered microorganisms, optionally wherein each microorganism has been engineered so as to express or overexpress either:(a) a different gene, optionally to express or overexpress a different gene selected from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.(b) two or more different genes, optionally to express or overexpress two or more different genes from the group comprising or consisting of AUXI, AUXII, PvdS and ACDS.

24. The method of claim 22 or 23 wherein the one or more plant parts, optionally one or more seeds, tubers or bulbs are contacted with or coated with at least a first and second different engineered microbe, wherein : a) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; b) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered microorganism has been engineered to express AUXII from the BG37 promoter; c) the first engineered microorganism has been engineered to express AUXI from the BG37 promoter and the second engineered microorganism has been engineered to express AUXII from the BG17 promoter; or d) the first engineered microorganism has been engineered to express AUXI from the BG17 promoter and the second engineered.

25. The method of claim 22-24 wherein the one or more plant parts, optionally one or more seeds, tubers or bulbs are contacted with or coated with at least two, three or four or more different engineered microbes, where the engineered microbes have been engineered to express or overexpress the following proteins from the specified promoters: a) BG17: :AUXIb) BG37: :AUXI c) BG17: :AUXII d) BG37: :AUXII e) BG17: :PvdS f) BG37: :PvdS g) BG17: :ACDS h) BG37: :ACDS.

26. The method of any of claims 22-25 wherein the seed, tuber or bulb is coated with the one or more engineered microorganisms of any of the preceding claims.

27. The method of any of claims 22-25 wherein said contacting comprises applying said one or more engineered microorganisms to a media into which the said plant is subsequently planted.

28. The method of any of claims 22-25 wherein contacting occurs by applying the one or more engineered microorganisms to the media in which the plant is already growing in.

29. The method of any of claims 22-25 wherein said one or more parts of said plant is contacted with the one or more engineered microorganisms at the same time as the plant is planted into a media.

30. The method of claims 22-29 wherein the plant is a seed.

31. The method of claims 22-29 wherein the plant is a seedling.

32. The method of claims 22-29 wherein the plant is a mature plant.

33. The method of any of claims 22-32 wherein the plant is: a) a Brassica species, optionally B. napus; b) a root vegetable; c) a plant that grows from tubers d) an aquatic plant; e) a plant that grows in water, optionally watercress.

34. A method of preparing a Brassica species plant, optionally B. napus plant with an increased ability to accumulate heavy metals, wherein said method comprises contactingseeds or seedlings of said plant with one or more engineered microorganisms of any of the preceding claims, wherein said engineered microorganism is P. fluorescens engineered so as to overexpress the following genes from the specified promoters: a) BG17: :AUXI + BG17: :AUXII b) BG17: :AUXI + BG37: :AUXII c) BG37: :AUXI + BG17: :AUXII d) BG37: :AUXI + BG37: :AUXII where:AUXI has an amino acid sequence of SEQ ID NO: 1, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 provided that the AUXI retains the ability to produce IAA; and / orAUXII has an amino acid sequence of SEQ ID NO: 2, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2 provided that the AUXI retains the ability to produce IAA.

35. A method of preparing a Brassica species plant, optionally B. napus plant with an increased ability to accumulate heavy metals, wherein said method comprises contacting seeds or seedlings of said plant with one or more engineered microorganisms of any of the preceding claims, wherein said engineered microorganism is P. fluorescens engineered so as to overexpress any one or more of AUXI, AUXII, PvdS and ACDS.

36. A plant or plant part prepared according to any one of the methods of any of the preceding claims, optionally wherein said plant or plant part is a seed, tuber, bulb or seedling.

37. A method for removing metals from a media wherein said method comprises cultivating a plant or plant part according to claim 36 in said media.

38. The method of claim 37 wherein the media is soil or water.

39. The method of claim 37 wherein the media is soil or water in the environment rather than in a laboratory.

40. The method of claims 37-39 wherein the method further comprises harvesting the plants so that the plants are removed from the growth media.

41. The method of any of claims 37-40 wherein the method comprises: a) Determining the concentration of one or more metals in the media prior to cultivating the plant in the media; and b) determining the concentration of the same one or more metals in the media i) during cultivation of the plant; and / or ii) after harvesting the plants.

42. The method of any of claims 37-41 wherein the method comprises repeating steps a) cultivating the plant or plant part of claim 36 in the media; and b) harvesting the plants so that the plants are removed from the media.

43. The method of claim 42 wherein step (a) and step (b) are repeated until the metal content of the media is below a predetermined threshold.

44. The method of claim 42 or 43 wherein step (a) and step (b) are repeated until the metal content of the media is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or lower of the starting metal content.

45. The method of claim 42 or 43 wherein step (a) and step (b) are repeated until one or more of the following criteria are met:(i) the content of Zinc in the media is at least lower than 900 mg / kg; preferably wherein the content is at least lower than 300 mg / kg; most preferably wherein the content is at least lower than 50 mg / kg; and / or(ii) the content of Nickel in the media is at least lower than 1800 mg / kg; preferably wherein the content is at least lower than 230 mg / kg; most preferably wherein the content is at least lower than 75 mg / kg;(iii) the content of Strontium in the media is at least lower than 1370 mg / kg; preferably wherein the content is at least lower than 121 mg / kg; most preferably wherein the content is at least lower than 65 mg / kg.

46. The method of any of claims 37-45 wherein the concentration of zinc in the media prior to cultivating said plant or plant part is 1000 mg / Kg, 3000 mg / Kg, and 5000 mg / Kg.

47. A method of recovering metals from a media wherein the method comprises the method for removing metals from a plant growth media according to any one of the preceding claims, and further comprises processing the harvested plants so as to extract the metal content.

48. The method claim 47 wherein the root part of the plant is processed so as to extract the metal content.

49. A method of producing a food crop from a metal contaminated media wherein the method comprises:(a) cultivating a plant or plant part according to claim 36 in the metal contaminated media;(b) harvesting the plants so that the plants are removed from the growth media; and(c) removing and discarding the root portion of the plant.

50. A seed, tuber or bulb coated with one or more engineered microorganisms of any of the preceding claims.

51. Pseudomonas fluorescens for use in a method of bioremediation.

52. A method of bioremediation wherein said method comprises applying Pseudomonas fluorescens to a plant or plant part.60

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