Pseudomonas cells and methods for valorisation of cyanide

Abstract

The present invention relates to bacterial cells and methods for valorisation of cyanide. In particular, the invention relates to engineered, or non-natural Pseudomonas cells capable of assimilating cyanide as carbon and nitrogen source, as well as methods for producing value-added compounds from cyanide using such cells.

Description

[0001] P7355PC00

[0002] Pseudomonas cells and methods for valorisation of cyanide

[0003] Technical field

[0004] The present invention relates to bacterial cells and methods for valorisation of cyanide. In particular, the invention relates to engineered, or non-natural Pseudomonas cells capable of assimilating cyanide as carbon and nitrogen source, as well as methods for producing value-added compounds from cyanide using such cells.

[0005] Background

[0006] Cyanide is a powerful toxic chemical that impacts the terminal cytochrome oxidase of most aerobic organisms, leading to a halt in the electron transport chain and ultimately cell death. Among the most toxic cyanides are hydrogen cyanide (HCN), sodium cyanide (NaCN), potassium cyanide (KCN), and calcium cyanide [Ca(CN)2]. These compounds are extremely poisonous and require careful handling to avoid severe health risks. The mining industry is nowadays responsible for most of its demand. In this case, cyanide is mainly produced for the mining of silver and gold, as it helps dissolve these metals allowing separation from other solids. The steel, plastic, fishing and chemical industries also use cyanides extensively. Even though considerable research has been directed to the quest for more environmentally friendly alternatives, cyanide is still widely used as leach reagent to extract heavy metals for its high purity recovery, robustness and relatively low costs. The resulting waste products, containing not only highly toxic cyanides but also other valuable resources, are generally discarded due to the lack of technically and economically feasible detoxification treatments. This possesses a clear environmental threat with a severe potential leakage to the natural surroundings and water sources.

[0007] Cyanide-containing wastes pose a serious environmental threat to natural surroundings and public health when not treated and disposed properly. Several physical, chemical and electrochemical detoxification systems have been employed, but these are not efficient or cost-effective while generating toxic by-products (Martinkova etal. 2024).

[0008] Summary

[0009] The invention is as defined in the claims.P7355PC00

[0010] This disclosure relates to Pseudomonas cells capable of assimilating cyanide and preferably valorising it into one or more metabolites or value-added compounds. In other words, provided herein are methods of incorporating cyanide into the biomass and / or other compounds, such as metabolites or value-added compounds, produced by a Pseudomonas cell capable of assimilating cyanide. Cyanide is a toxic one-carbon (Cl)-compound and a byproduct of several industrial processes, as described herein above. Thus, the present disclosure provides Pseudomonas cells for transformation of cyanide (literally and figuratively), thereby both reducing its toxicity by degradation and utilising of its degradation products for cellular growth and / or production of other compounds. Thus, provided herein are Pseudomonas cells and methods for efficient utilization of cyanide and C1 -feedstock.

[0011] Provided herein are Pseudomonas cells comprising a cyanidase (EC 3.5.5.1). Also provided herein is Pseudomonas cells capable of using cyanide as carbon- and nitrogen-source, said cells comprising a cyanidase capable of catalysing conversion of cyanide into formate and ammonia / ammonium. Further provided herein is Pseudomonas cells capable of converting cyanide to pyruvate via formation of formate and serine, said cells comprising a cyanidase capable of catalysing conversion of cyanide into formate and ammonia / ammonium.

[0012] Provided herein are also Pseudomonas cells comprising a nucleic acid encoding a cyanidase (EC 3.5.5.1) capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+), and one or more of:

[0013] a heterologous nucleic acid encoding a formate-THF ligase (FtfL; EC 6.3.4.3), a heterologous nucleic acid encoding a methenyl-THF cyclohydrolase (Fch; EC 3.5.4.9), and / or

[0014] a heterologous nucleic acid encoding a methylene-THF dehydrogenase (MdtA; EC 1.5.1.5),

[0015] wherein said cell is capable of converting said formate into 5,10-methylene-THF, and wherein said Pseudomonas cell further comprises a modification resulting in partial or total inactivation of an endogenous glycine cleavage system (GCS) and a modification resulting in partial or total inactivation of an endogenous SerABC pathway. In preferred embodiments, said Pseudomonas cell is capable of producing a compound, such as a pyruvate-derived compound.P7355PC00

[0016] Provided herein are also methods of producing a compound, such as a pyruvatederived compound, comprising:

[0017] i. providing a Pseudomonas cell described herein;

[0018] ii. cultivating and optionally propagating said cell in a cultivation medium, said cultivation medium comprising cyanide,

[0019] thereby obtaining a fermentation broth and / or cell comprising said compound; iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;

[0020] whereby said compound and / or said fermentation broth comprising said compound is produced.

[0021] Also provided are methods of detoxifying a waste product comprising cyanide, comprising:

[0022] i. providing a Pseudomonas cell described herein;

[0023] ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide,

[0024] thereby degrading said cyanide, and optionally obtaining cell biomass;

[0025] iii. optionally recovering said cell biomass;

[0026] whereby said waste product is detoxified.

[0027] Further provided herein are methods of valorising of a waste product comprising cyanide into a compound, comprising:

[0028] i. providing a Pseudomonas cell described herein;

[0029] ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide,

[0030] thereby obtaining a fermentation broth and / or cell comprising said compound; iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;

[0031] whereby said waste product comprising cyanide is valorised by production of said compound and / or said fermentation broth comprising said compound.P7355PC00

[0032] Also provided herein are methods of propagating a Pseudomonas cell in the presence of cyanide, comprising:

[0033] i. providing a Pseudomonas cell described herein;

[0034] ii. propagating said cell in a cultivation medium, said cultivation medium comprising cyanide,

[0035] thereby obtaining a fermentation broth comprising said cell and / or biomass of said cell;

[0036] iii. optionally recovering said fermentation broth and / or said biomass.

[0037] Further provided herein are methods of engineering a Pseudomonas cell described herein, comprising:

[0038] i. providing a Pseudomonas cell, optionally a P. putida cell, such as a non- pathogenic Pseudomonas cell, for example a P. putida KT2440 cell or a P. putida SEM11 cell;

[0039] ii. providing one or more nucleic acids described herein;

[0040] iii. modifying the cell of step i. with said one or more of the nucleic acids of step ii.; thereby obtaining said Pseudomonas cell described herein.

[0041] Description of Figures

[0042] Figure 1. CynD heterologous production in P. putida assayed via SDS-PAGE (asterisk representing CynD size at 37.5 kDa). Negative control harbouring an empty vector (eV) is also included.

[0043] Figure 2. P. putida can detoxify cyanide when expressing heterologous cyanide dihydratases, (a) Cyanide dihydratase reaction performed by CynD. (b) Plasmid map of pSEVA221 harbouring CynD of P. stutzeri or B. pumilus (cynDstut and cynDpum, respectively), (c) P. putida SEM11 grown at increasing concentrations of potassium cyanide (KCN) while overexpressing cynDs in DBM medium supplemented with 20 mM glucose (d) or said minimal medium without nitrogen (-N).

[0044] Figure 3. P. putida can detoxify cyanide and assimilate its product formate by expressing cyanide dihydratases, (a) Metabolic map of the growth-coupled selection based on serine auxotrophy (ASGG-Ci) (b) Growth profiles of ASGG-C1 grown in de Bont minimal medium, 20 mM glucose, 10 mM glycine with the supplementation of serine or formate (positive controls) or increasing concentrations of potassium cyanideP7355PC00

[0045] (KCN) while overexpressing cynD from P. stutzeri (cynDstut) or B. pumilus (cynDpum) or el / as a negative control, (c) Growth rates (in h-1) in (b) are given for each condition. Both (b) cell densities (estimated as the optical density measured at 600 nm, ODeoo) and (c) specific growth rates (., h-1) are indicated as average values ± standard deviation of four biological replicates. Individual data points are shown whenever relevant.

[0046] Figure 4. P. putida can detoxify cyanide and assimilate its subproducts formate and ammonia simultaneously, (a) Metabolic map of the growth-coupled selection based on serine auxotrophy (ASGG-Ci). (b) Strain ASGG-C1 was grown in DBM medium without nitrogen, 20 mM glucose, 10 mM glycine and increasing concentrations of potassium cyanide (KCN) while overexpressing cynD from P. stutzeri (cynDstut) or B. pumilus (cynDpum) or eV as a negative control in pSEVA221 vector, (c) Specific growth rates (in h-1) are given for each condition.

[0047] Figure 5. Glycine production from the central carbon metabolism serve as a substrate for cyanide-dependent formate assimilation, (a) Metabolic map of the growth-coupled selection based on serine auxotrophy (ASGG-Ci) overexpressing ItaE. (b) Genetic engineering of the promoter and RBS combination is depicted for ItaE overexpression, (c) Strains ASGG-C1 overexpressing LtaE were grown in DBM medium without nitrogen, 20 mM glucose with the supplementation of serine or formate (positive controls) or increasing concentrations of potassium cyanide (KCN) while overexpressing cynD from P. stutzeri (cynDstut) or B. pumilus (cynDpum) or eV as a negative control, (d) Specific growth rates (in h-1) are given for each condition.

[0048] Figure 6. A combination of endogenous and heterologous activities activates the serine threonine cycle (STC) and a novel architecture for Ci assimilation, the enhanced STC. (a) A serine and glycine auxotrophic strain with further modifications (ASGG-C1) to overexpress the endogenous ItaE and yiaY. LtaE overproduction yields glycine from central metabolism whereas YiaY promotes methanol oxidation by derepressing the activity of endogenous PQQ-dependent alcohol dehydrogenases. This P. putida strain was termed STC1. (b) Genomic constitutive overexpression of the endogenous ItaE and yiaY via promoter exchange with the strong, constitutive Prrcand P14G promoters, respectively, (c) Growth profile of strain STC1 in de Bont minimal medium with 20 mM glucose and 10 nM LaCh supplemented with 10 mM glycine, serine (positive control),P7355PC00

[0049] or 2 mM homoserine. When needed, 60 mM formate or 500 mM methanol were also supplemented, (d) Specific growth rates corresponding to the growth profiles shown in panel (c). (e) Metabolic map for strain STC1 harbouring the plasmids indicated in panel (f), encoding Mi of the HSC. Increasing the assimilation of Ci compounds directly via formaldehyde leads to a new synthetic metabolism, termed enhanced STC (eSTC). (f) Structure of plasmids used to overexpressEcltaE and the new variantEcltaE* (encodingEcLtaE with a C188Y mutation) under the constitutive expression of Ptlcand the canonical RBS. (g) Growth profiles of strain STC1 harbouring pS621EcltaE IEcltaE*in de Bont minimal medium with 20 mM glucose, 10 nM LaCh, and 500 mM methanol. The strain transformed with an empty vector (eV) is also plotted as a control, (h) Specific growth rates corresponding to the experiments shown in panel (g). Average values for bacterial growth (estimated as the optical density measured at 600 nm, ODeoo), and specific growth rate (p, in h-1) ± standard deviation of three biological replicates are represented in all cases. Individual data points are shown. Error bars in panel (h) correspond to 95% confidence intervals (Cl), with P-values < 0.05 for all pairwise comparisons where error bars do not overlap.

[0050] Figure 7. The enhanced serine-threonine cycle (eSTC), using the native PQQ-dependent methanol dehydrogenases, enables methanol assimilation at low substrate concentrations, (a) Metabolic map of the serine and glycine auxotrophic strain (termed in MrSTC: ASGG-C1) with further modifications as indicated, overexpressing the endogenous ItaE and yiaY genes. LtaE overproduction yields glycine from central carbon metabolism, whereas YiaY enables constitutive activity of endogenous PQQ-dependent alcohol dehydrogenases for methanol oxidation. This engineered P. putida strain was termed STC1. (b) Growth profiles of strain STC1 harbouring pS621EcltaE IEcltaE* in de Bont minimal medium with 20 mM glucose, 10 nM LaCh, and varying methanol concentrations. The strain carrying the empty vector (eV) is also plotted as a control, (c) Specific growth rates corresponding to the growth profiles of panel (b). Average values for the cell density (optical density measured at 600 nm, ODeoo) ± standard deviation of three biological replicates are represented. Individual data points are shown. Error rates in panel (h) are plotted as specific growth rate (p, in h-1) ± 95% confidence intervals; P-values were < 0.05 when error bars do not overlap

[0051] Figure 8. The enhanced serine threonine cycle (eSTC) also poses an advantage versus the STC when grown with formate as a co-substrate. (a) Metabolic map of theP7355PC00

[0052] STCO strain (STC1 without YiaY overexpression) showcasing the entry points of the different auxiliary carbon sources used, (b) Structure of plasmids used to overexpressEcltaE* under the constitutive expression compared to an empty vector, (c) Growth profiles of strain STCO harbouring pS621Ec / faE* in de Bont minimal medium with noted carbon sources (in equimolar carbon concentrations) and 30 mM formate (absence of formate as a negative control). The strain transformed with an empty vector (eV) is also plotted as a STC control, (d) Specific growth rates corresponding to the experiments shown in panel (c). Average values for bacterial growth (estimated as the optical density measured at 600 nm, ODeoo), and specific growth rate (p, in h-1) ± standard deviation of three biological replicates are represented in all cases. Individual data points are shown. Error bars in panel (d) correspond to 95% confidence intervals (Cl), with P-values < 0.05 for all pairwise comparisons where error bars do not overlap.

[0053] Figure 9. The enhanced serine threonine cycle (eSTC) promotes better growth parameters than its native counterpart (STC) when using potassium cyanide (KCN) as a Ci source, (a) Metabolic map of the STCO (AlldD-dld2) strain showcasing partial contributions to biomass of the carbon and nitrogen elements of cyanide, (b) Structure of plasmid used to overexpressEcltaE* (leading to eSTC; compared to an empty vector, eV; i.e. STC) and genomic random integration of cynDstut with tn5 transposition, (c) Growth profiles and growth rates of the noted strain harbouring pS621Ec / faE* in de Bont minimal medium with 20 mM glucose and increasing concentrations of KCN (absence of KCN as a negative control). The strain transformed with an empty vector (eV) is also plotted as a STC control. Left: Growth profile of 5 mM KCN compared to absence of C1 donor for eSTC and STC strains. Final cell density (middle) and specific growth rates (right) are given for each tested concentration of KCN. Average values for bacterial growth (estimated as the optical density measured at 600 nm, OD600), and specific growth rate (μ, in h-1) ± standard deviation of three biological replicates are represented in c-middle. Error bars in panels c-middle and c-right correspond to 95% confidence intervals (Cl), with P-values < 0.05 for all pairwise comparisons where error bars do not overlap.

[0054] Figure 10. L-lactate can be efficiently produced from cyanide detoxification by-product formate via the serine threonine cycle (STC). (a) Metabolic map of the STCO AlldD-dld2 strain showcasing the production of L-lactate by LdhA from pyruvate (and thus, L-serine), product of the assimilation of formate via the STC. (b) Structure of plasmidP7355PC00

[0055] used for production of L-lactate by expression of IdhA N109G (i.e. IdhA* encoding BtLdhA(N108G)) derived from B. taurus IdhA. IdhA expression is tightly linked to a neo gene, (c) Growth and compound assimilation of secretion profiles. STCO AlldD-dld2 strain harbouring the IdhA* plasmid was grown in de Bont minimal medium with glucose or acetate and formate. L-lactate production and secretion was observed. Growth rates, uptake rates for carbon sources, as well as L-lactate secretion rates were calculated for all conditions.

[0056] Figure 11. Revalorization of potassium cyanide (KCN) into added-value product L-lactate using the serine threonine cycle (STC). (a) Metabolic map of the STCO AlldD-dld2 strain showcasing the production of L-lactate by LdhA from pyruvate (and thus, L-serine), product of the assimilation of KCN via the STC. (b) Structure of the pBAMD1-4 plasmid cloned for random integration via Tn5 transposition of cynDstut in the genome of STCO AlldD-dld2 strain (noted as gcynDstut) (Same plasmid used in Figure 9). (c) L-lactate production using nitrogen-free de Bont minimal medium supplemented with 20 mM glucose and 2.5 mM KCN (or 2.5 mM formate as a control) was monitored using an in vitro indirect system based on the production of H2O2 from commercial lactate oxidase (LOX). Afterwards, H2O2 is used to reduce 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) via the horseradish peroxidase (HRP), state in which reduced ABTS+strongly absorbs at 734 nm. Absorbance and L-lactate are correlated in a concentration-dependent manner, (d) Growth profile of the previously noted strain using de Bont minimal medium supplemented with 20 mM glucose and nitrogen (noted as “+N”) and 2.5 mM KCN or 2.5 mM formate (control), as well as its L-lactate production and secretion profile for both formate and KCN conditions over 72 h. Average values for bacterial growth (estimated as the optical density measured at 600 nm, OD600), and L-lactate production and secretion (in mM) ± standard deviation of three biological replicates are represented, (e) Growth profile of the previously noted strain in nitrogen-free (noted as “-N”) de Bont minimal medium supplemented with 20 mM glucose and 2.5 mM KCN, as well as its L-lactate production and secretion profile for both formate and KCN conditions over 78 h. Controls contained nitrogen in the de Bont medium (noted as “+N”), either in combination with 2.5 mM formate or 2.5 mM KCN. Average values for bacterial growth (estimated as the optical density measured at 600 nm, OD600), and L-lactate production and secretion (in mM) ± standard deviation of three biological replicates are represented.P7355PC00

[0057] Detailed description

[0058] Definitions

[0059] The term homologue herein refers to variant of a nucleic acid encoding the same polypeptide or a functional variant of said polypeptide. The skilled person knows that different codons may encode the same amino acid, and it therefore follows that two different nucleic acid sequences may encode the same polypeptide. The skilled person knows how to derive one or more distinct DNA sequences from an amino acid sequence. The skilled person also knows how to derive an amino acid sequence from a DNA sequence. Software for performing reverse translations from an amino acid sequence to DNA sequences and translations from DNA sequences to amino acid sequences are available for performing this task.

[0060] The term functional variant herein refers to a functional variant of a protein or polypeptide, for example a functional variant of an enzyme. A functional variant of a given enzyme may have a different amino acid sequence than the given enzyme, but is able to perform the same function, either partially or totally. In other words, a functional variant of a given enzyme has the same function as the given enzyme, albeit the extent of the function may be different: the functional variant may be less active than the given enzyme, or the functional variant may be more active than the given enzyme. Other features of the functional variant may differ from the given enzyme, e.g. half-life. The functional variant retains at least some of the activity exerted by the given enzyme.

[0061] A functional variant of a given protein such as a given enzyme can be a variant having an amino acid sequence at least 70% identical to the given protein, or it can be a protein having a similar structure to the given protein. In the field of bioinformatics, this can be calculated using a "template modelling score" (TM-score), which is a recognized metric used to assess the similarity between two protein structures. The TM-score quantitatively measures this similarity on a scale from 0 to 1. Typically, scores below 0.20 indicate randomly chosen, unrelated proteins, whereas scores above 0.5 suggest that the structures share roughly the same fold, i.e. the same structure, and can thus be expected to share the same activity. A detailed methodology for calculating the TM-score is provided in the publication " Zhang Y and Skolnick J (2004). Scoring function for automated assessment of protein structure template quality. Proteins. 57 (4): 702-710."P7355PC00

[0062] The TM-score can be calculated, for example, by uploading two three-dimensional structures in PDB format to the online resource available at https: / / zhanggroup.org / TM-score / . In situations where a pair of three-dimensional structures for comparison is not available, established methods for predicting the three-dimensional structure of a polypeptide are well known to those skilled in the art. For instance, a neural network trained for this specific task, such as AlphaFold, can be employed.

[0063] 3D structures of proteins and / or domains thereof are available through different sources. Thus, 3D structures to be used with the present invention may be available through various databases or can be predicted structures. AlphaFold3, referred to herein as AlphaFold, represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication: Jumper, J., Evans, R., Pritzel, A. et al. " Highly accurate protein structure prediction with AlphaFold." Nature 596, 583-589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2.

[0064] AlphaFold DB is an online database which hosts over 200 million entries, encompassing the human proteome as well as the proteomes of 47 other key organisms relevant to research and global health. These entries are freely accessible at https: / / alphafold.ebi.ac.uk / . The (predicted) structure of a presumed functional variant of any of the proteins described herein as being expressed by the present Pseudomonas cells can be retrieved by inputting appropriate identifiers.

[0065] Structures of polypeptides not included in the AlphaFold DB may for example be predicted using the source code available at https: / / github.com / google-deepmind / alphafold, and / or a Colab notebook accessible at https: / / colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / Alp haFold.ipynb. To generate a 3D structure using the Colab notebook, the amino acid sequence of any protein of interest, in particular the proteins expressed by the Pseudomonas cell described herein, can be inserted.

[0066] Thus, any functional variant of a given enzyme described herein may refer to a protein having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, atP7355PC00

[0067] least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of the given enzyme.

[0068] The term identity with respect to a polynucleotide or polypeptide is defined herein as the percentage of nucleic acids or amino acids in the candidate sequence that are identical to the residues of a corresponding native nucleic acid or amino acid, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and considering any conservative substitutions according to the NCIIIB rules (http: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-llIB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Throughout the present disclosure, the term “at least 70%” when referring to a percentage of sequence homology of sequence identity shall refer to a sequence identity of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0069] The terms nucleic acid and polynucleotide are used interchangeably herein. A coding sequence (CDS), open-reading frame (ORF) and gene are all examples of nucleic acids. A cluster of genes and an operon are other examples of nucleic acids. The terms polypeptide and protein are used interchangeably herein. An enzyme is thus both a polypeptide and a protein, whereas a protein is not necessarily an enzyme. Enzymes are proteins capable of catalysing one or more chemical reactions. This is known to the skilled person.

[0070] The term assimilation with respect to a carbon or nitrogen source herein refers its incorporation into a cell’s metabolism. Once incorporated into a cell’s metabolism, said assimilated carbon or nitrogen may contribute to the propagation of the cell, forP7355PC00

[0071] example to formation of cellular biomass, and / or provide carbon or nitrogen for metabolite and / or product formation.

[0072] Where the L or D form (optical isomers) has not been specified it is to be understood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so that the peptides formed may be constituted of amino acids of L form, D form, or a sequence of mixed L forms and D forms.

[0073] The term heterologous when referring to a polypeptide, such as a protein or an enzyme, or to a polynucleotide, such as a gene or a nucleic acid, shall herein be construed to refer to a polypeptide or a polynucleotide which is not naturally present in a wild type cell. For example, the term “heterologous cyanidase” when applied to a Pseudomonas cell refers to a cyanidase which is not naturally present in a wild type Pseudomonas cell, e.g. a cyanidase derived from Pseudomonas stutzeri AK61 of Bacillus pumilus.

[0074] The terms endogenous, and native with respect to a polypeptides or polynucleotide, such as a gene, s gene cluster or an operon, refers herein to a polypeptide or polypeptide that is naturally found in the cell, i.e. are not heterologous. The terms endogenous, native to, innate, intrinsic, and inherent may be used interchangeably herein.

[0075] The term waste product herein refers to by-product or side-stream of a specific production process, such as of an industrial process. A byproduct or side-stream that is considered waste from carrying out one process, may the starting material of another process. Similar terms, which may be used interchangeably, are industrial byproduct or industrial side-stream.

[0076] The term mutation with respect to nucleic acids, such as genes, herein refers to substitutions, insertions, deletions, and indels. A mutation in a gene refers to a mutation in the coding sequence, in the promoter of the gene, or in the terminator of the gene. The term mutation with respect to a polypeptide herein refers to substitutions, insertions, deletions, and indels.P7355PC00

[0077] Lactate and / or lactic acid as terms herein also comprise any salt thereof. Lactic acid has a pKaof 3.86 and therefore exists in an equilibrium with lactate. The person skilled in the art will therefore know that removal of either the dissociated or non-dissociated form from a liquid solution containing both, will influence the equilibrium and pH of said solution. In the context of the present disclosure the terms lactate and lactic acid are used interchangeably. The skilled person is well capable of calculating the amount of either lactate and / or lactic acid at a defined pH. Similarly, herein the terms formate and / or formic acid may also comprise any salt thereof. Formic acid has a pKaof 3.75, and therefore exists in an equilibrium with formate, as described above for lactic acid and lactate. The terms formic acid and formate are used interchangeably herein.

[0078] Similarly, herein the terms acetate and / or acetic acid may comprise any salt thereof. Acetic acid has a pKaof 4.76 and therefore exists in an equilibrium with acetate, as described above for lactic acid and lactate. The terms acetate and acetic acid are used interchangeably herein. Similarly, ammonium has a pKaof 9.25, and exists in an equilibrium with ammonia, as described above for lactic acid and lactate. The terms ammonium and ammonia are used interchangeably herein.

[0079] The term cultivation herein refers to maintaining cells in culture conditions that enable survival of said cells. Preferably said culture conditions allow expression of enzyme(s) encoded by gene(s) contained in said cells.

[0080] The term propagation herein refers to maintaining cells in culture conditions that enable growth, multiplication and / or accumulation of biomass of said cells. Preferably said culture conditions also allow expression of the enzyme(s) encoded by gene(s) contained in said cells.

[0081] The term “in the presence of” herein refers to the proximity of a cell to a compound. When a cell is in the presence of a compound, the cell is in sufficiently close proximity to said compound as to be able to use said compound for the cell metabolism. Hence, for example, a cell may be in the presence of a compound when said compound is present in the cell’s immediate extracellular environment, such as present in the medium used to cultivate said cell; alternatively in the presence of also encompasses that said compound may be or have been produced by said host cell and either is present intracellularly within the cell or secreted by the cell.P7355PC00

[0082] “Partial inactivation” of a protein such as an enzyme refers to any genetic modification that reduces the enzymatic activity of said protein relative to the corresponding wildtype, unmodified protein, but does not abolish it completely. “Partial inactivation” encompasses, but is not limited to, point mutations, deletions, insertions, promoter modifications, gene truncations and expression-reducing mutations that lead to measurable but non-zero residual activity of the protein, in particular modifications in the sequence encoding the protein. Partial inactivation can also be achieved by modifying a regulator, such as a transcription factor binding to the sequence encoding the protein, modifying the activity of a protein involved in post-translation modification of the protein to be partially inactivated, and the like, as is known to the skilled person. For example, “partial inactivation” may be a measurable reduction in enzymatic activity relative to the wild-type enzyme, preferably by at least 10%, 20%, 30%, 50%, 70%, or up to 90%. “Total inactivation” refers to >95% or preferably >99% reduction in activity.” Similarly, partial inactivation of an endogenous pathway, cycle or system of a cell refers to any modification that reduces the activity of the endogenous pathway, cycle or system relative to the corresponding wild-type, unmodified endogenous pathway, cycle or system, but does not abolish the activity completely. Partial inactivation of an endogenous pathway, cycle or system of a cell may be obtained by partially inactivating a protein such as an enzyme involved in the endogenous pathway, cycle or system, as described above. For example, partial inactivation of a pathway, cycle or system may be a measurable reduction in the flux through the pathway, cycle or system, preferably by at least 10%, 20%, 30%, 50%, 70%, or up to 90%, or it may be a measurable reduction in the amount of metabolites normally produced by the endogenous pathway, cycle or system.

[0083] Pseudomonas cell

[0084] In a main aspect, the present disclosure relates to a Pseudomonas cell comprising a cyanidase and / or a nucleic acid encoding a cyanidase. In some embodiments, the Pseudomonas cell is an engineered cell, a recombinant cell, a non-natural cell, which does not occur in nature.

[0085] In a further main aspect, this disclosure concerns a Pseudomonas cell capable of using cyanide as a carbon- and nitrogen-source, said cell comprises a nucleic acid encoding a cyanidase capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+).P7355PC00

[0086] In another main aspect, this disclosure relates to a Pseudomonas cell capable of converting cyanide to pyruvate via formation of formate and serine, said cell comprising a nucleic acid encoding a cyanidase capable of catalysing conversion of cyanide into formate and ammonia / ammonium.

[0087] The cyanidase may be a heterologous cyanidase. In other words, in some embodiments, the cyanidase is not a native cyanidase of the Pseudomonas cell, but is sourced from another organism. The term “heterologous cyanidase” refers to a cyanidase which is not naturally expressed by the organism, such as by the Pseudomonas cell.

[0088] Skilled person is capable of engineering, such as genetically modifying, the cell, for example (over-)expressing one or more genes or proteins, deleting one or more genes or otherwise modifying the genome of the cell.

[0089] With respect to any of the nucleic acids disclosed herein, they may be genomically integrated in the genome of the Pseudomonas cell and / or be present on one or more vectors within said cell, such as on one or more plasmids. Suitable vectors and plasmids are known in the art and readily available to the skilled person.

[0090] Any of the Pseudomonas cells disclosed herein, when comprising a nucleic acid encoding a protein, such as an enzyme, may express, such as overexpress the protein encoded by said nucleic acid. Thus, any one or more of the nucleic acids disclosed herein may comprise one or more inducible promoters and / or constitutive promoters. Such promoters are known in the art and readily available to the skilled person. In some embodiments of the present disclosure, the nucleic acid is codon-optimised, such as codon-optimised for P. putida.

[0091] As is known to the skilled person, a Pseudomonas cell may naturally comprise one or more homologues of a certain nucleic acid, such as of a gene, or functional variants a polypeptide, such as an enzyme. Such natural homologues and functional variants may have arisen from a multiplication of one or more nucleic acids, such as arisen from a duplication event. With respect to functional variants of a polypeptides, such variants may have arisen from diversification of the homologue nucleotides. Within the context of the present disclosure, it is to be understood that in the context of modifications andP7355PC00

[0092] mutations resulting in partial or total inactivation or deletion of either a nucleic acid or a polypeptide.

[0093] Parental Pseudomonas cell

[0094] In some embodiments, the Pseudomonas cell is an isolated cell. The cell preferably has a status of being GRAS (Generally Regarded as Safe) and / or is classified as HV1 certified. In other words, preferably the Pseudomonas cell is a non-pathogenic Pseudomonas cell.

[0095] In preferred embodiments, the Pseudomonas cell is a Pseudomonas putida cell.

[0096] In other preferred embodiments, the Pseudomonas cell is a Pseudomonas putida KT2440 cell or a P. putida SEM11 cell, or a derivative of any of the two. Strain derivatives may be further engineered and / or evolved, such as harbouring one or more modifications, for example mutations, deletions, insertions etc.

[0097] SEM11 is a genome-reduced derivative of P. putida KT2440 and EM42. In some embodiments, the Pseudomonas cell is derived from a genome-reduced P. putida KT2440 cell, for example the cell is derived from a P. putida EM42 cell. P. putida EM42 is a genome-reduced derivative of P. putida KT2440, harbouring the following deletions: Aprophagel, Aprophage4, Aprophage3 Aprophage2, ATn7, AendA-1, AendA-2, AhsdRMS, Aflagellum, and ATn4652.

[0098] The skilled person is well-capable of obtaining strain P. putida EM42, P. putida KT2440 and P. putida SEM11. P. putida EM42 is known in the field and described, see for example Martinez-Garcia et al., 2014. P. putida SEM11 is known in the field and described, see for example Wirth et al., 2023. P. putida KT2440 is known in the field and described, see for example Bagdasarian etal., 1981.

[0099] It is preferred, that the cell comprises a cytochrome bd oxidase that is insensitive to cyanide (a cyanide-insensitive terminal oxidase), enabling the cell to respire and generate energy in the presence of cyanide. Said cyanide insensitive cytochrome bd oxidase may be a ubiquinol oxidase, such as CioAB encoded by cioAB (PP_4650-PP_4651) of P. putida. CioAB is an ubiquinol oxidase comprising subunit I and subunit II (Uniprot accession numbers Q88E17 and Q88E18). The terms cytochrome bdP7355PC00

[0100] oxidase and ubiquinol oxidase are equivalent, and may be used interchangeably herein.

[0101]

[0102] The inventors have found that a Pseudomonas cell comprising a cyanidase can propagate in the presence of cyanide.

[0103] The term cyanidase herein refers to an enzyme which is capable of hydrolysing cyanide to formate and ammonia (NH3) / ammonium (NH4+). The cyanidase of the present disclosure are capable of catalysing the reaction:

[0104] HCN + H2O = HCOOH + NH3

[0105] The EC number for this reaction is EC 3.5.5.1. The term cyanidase may also be referred to as cyanide dihydratase and the terms are interchangeable and may be used as such herein. The Pseudomonas cell, when comprising or expressing a cyanidase is this capable of converting cyanide into formate and ammonia / ammonium.

[0106] In some embodiments, the cyanidase is a cyanidase from Stutzerimonas stutzeri, previously known as Pseudomonas stutzeri. In some embodiments, the cyanidase is a cyanidase from Pseudomonas stutzeri, such as from Pseudomonas stutzeri AK61, for example CynDstut (SEQ ID NO: 1, GenBank ID D82961.1) or a functional variant thereof having at least 70% identity thereto, such as at least 75% identity, such as at least 80% identity, such as at least 81% identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity thereto. In other embodiments, the cyanidase is a cyanidase from Bacillus pumilus, such as CynDpum (SEQ ID NO: 2, GenBank ID AF492815.1), or a functional variant thereof having at least 70% identity thereto, such as at least 75% identity, such as at least 80% identity, such as at least 81% identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as atP7355PC00

[0107] least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity thereto.

[0108] The cyanidase may be expressed in the Pseudomonas cell by introduction of a nucleic acid, which encodes said cyanidase. In some embodiments, the nucleic acid encoding CynDstut (SEQ ID NO: 1) comprises or consists of cynDstut (SEQ ID NO: 3) or a homologue thereof having at least 70% identity thereto. In other embodiments, the nucleic acid encoding CynDpum (SEQ ID NO: 2) comprises or consists of cynDpum (SEQ ID NO: 4) or a homologue thereof having at least 70% identity thereto.

[0109] CynDstut and cynDstut may also be referred to as CynDstut and cynDstut, respectively, herein. CynDpum and cynDpum may also be referred to as CynDpumand cynDpum, respectively, herein.

[0110] The cell described in this section can be used in any one of the methods described herein, for example in the methods for valorisation of cyanide. Cyanide, an otherwise toxic compound, is, thus, degraded and further incorporated into the metabolism of the Pseudomonas cells as disclosed herein.

[0111] Formate assimilation

[0112] In addition to comprising a cyanidase and / or a nucleic acid encoding a cyanidase, the Pseudomonas cell may further be or have been engineered to produce 5,10-methylene-tetrahydrofolate (5,10-methylene-THF) from formate. In other words, the Pseudomonas cell may be capable of assimilating formate into 5,10-methylene-THF.

[0113] Preferably, the Pseudomonas cells of the present disclosure are formatotrophic.

[0114] Formatrophy may be established by genetically modifying the Pseudomonas cells so that they are capable of assimilating formate into the metabolism. In preferred embodiments, formate is converted into 5,10-methylene-THF by the cell as described herein.

[0115] Thus, in some embodiments, the Pseudomonas cell comprises, such as expresses:P7355PC00

[0116] i. a nucleic acid encoding a formate tetrahydrofolate ligase (formate-THF ligase; FtfL);

[0117] ii. a nucleic acid encoding a methenyltetrahydrofolate cyclohydrolase (methenyl-THF cyclohydrolase; Fch); and / or

[0118] iii. a nucleic acid encoding a methylene-tetrahydrofolate synthase (methylene-THF dehydrogenase; MdtA),

[0119] whereby said Pseudomonas cell is capable of converting formate into 5,10-methylene-THF.

[0120] FtfL has the EC no. EC 6.3.4.3 and is capable of catalysing the reaction:

[0121] ATP + formate + tetrahydrofolate <=> ADP + orthophosphate + 10-formyltetrahydrofolate

[0122] Fch has the EC no. EC 3.5.4.9 and is capable of catalysing the reaction:

[0123] 10-formyltetrahydrofolate <=> 5,10-methenyltetrahydrofolate + H2O

[0124] MtdA has the EC no. EC 1.5.1.5 and is capable of catalysing the reaction:

[0125] NADPH + 5,10-methenyltetrahydrofolate <=> NADP + 5,10-methylenetetrahydrofolate

[0126] Thus, in some embodiments, the Pseudomonas cell comprises, such as expresses, FtfL, Fch and MdtA.

[0127] Each of FtfL, Fch and MdtA may be from a methylotrophic and / or a formatotrophic microorganism, such as from a bacterium, for example from a bacterium of the species Methylobacterium extorquens, such as M. extorquens AM1.

[0128] Thus, in some embodiments, the Pseudomonas cell comprises MeFtfL (SEQ ID NO: 8), MeFch (SEQ ID NO: 9), and / or MeMdtA (SEQ ID NO: 10), or functional variants thereof having at least 70 % identity thereto, such as at least 75% identity, such as at least 80% identity, such as at least 81% identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as atP7355PC00

[0129] least 97% identity, such as at least 98% identity, such as at least 99% identity thereto. In preferred embodiments, the cell comprises MeFtfL (SEQ ID NO: 8), MeFch (SEQ ID NO: 9), and MeMdtA (SEQ ID NO: 10), or functional variants thereof having at least 70 % identity thereto, whereby said Pseudomonas cell is capable of converting formate into 5,10-methylene-THF.

[0130] In some embodiments, the Pseudomonas cell comprises:

[0131] i. a nucleic acid encoding a formate-THF ligase (FtfL; EC 6.3.4.3), such as MeFtfL (SEQ ID NO: 8);

[0132] ii. a nucleic acid encoding a methenyl-THF cyclohydrolase (Fch), such as MeFch (SEQ ID NO: 9; EC 3.5.4.9); and / or

[0133] iii. a nucleic acid encoding a methylene-THF dehydrogenase (MdtA), such as MeMdtA (SEQ ID NO: 10; EC 1.5.1.5),

[0134] or functional variants thereof having at least 70 % identity thereto,

[0135] whereby said Pseudomonas cell is capable of converting formate into 5,10-methylene-THF.

[0136] In other embodiments, the Pseudomonas cell comprises a cyanidase and further comprises MeFtfL (SEQ ID NO: 8), MeFch (SEQ ID NO: 9), and MeMdtA (SEQ ID NO: 10), or functional variants thereof having at least 70 % identity thereto. Thereby said Pseudomonas cell is capable of converting cyanide into 5,10-methylene-THF and ammonia.

[0137] 5,10-methylene-THF / glycine and serine auxotrophy

[0138] In addition to comprising a cyanidase or at least comprising a nucleic acid encoding a cyanidase, as well as being capable of converting formate into 5,10-methylene-THF, the Pseudomonas cell of the present disclosure may further be or have been engineered, so that it cannot synthesize 5,10-methylene-THF via the glycine cleavage system (GCS) and serine via the SerABC pathway (which is a de novo serine pathway), thereby coupling cell growth to the assimilation of cyanide to replenish the methylated THF pool and to synthesise the essential amino acid serine. In other words, the Pseudomonas cell may be engineered so that it cannot produce either 5,10-methylene-THF, glycine and serine, unless it assimilates cyanide into 5,10-methylene-THF, which is converted into serine over glycine (see Figure 3A). Hydroxymethylation of glycine and 5,10-methylene-THF to form serine may be catalysed by an L-serineP7355PC00

[0139] hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) and / or GlyA-ll (SEQ ID NO: 23), or functional variants thereof.

[0140] Thus, the Pseudomonas cell engineered according to the present disclosure, which can synthesise serine from cyanide (via 5,10-methylene-THF) and cannot produce serine via the SerABC pathway, is not a serine auxotroph. Similarly, the Pseudomonas cell engineered according to the present disclosure, in which the GCS system is inactivated, but which can synthesise 5,10-methylene-THF and glycine from cyanide (via formate), is not a 5,10-methylene-THF auxotroph or a glycine auxotroph. 5,10-methylene-THF is a native metabolite of the Pseudomonas cell.

[0141] The glycine cleavage system (GCS) is well known in the art. It is also known as the glycine decarboxylase complex or GDC. The system is a series of enzymes that are triggered in response to high concentrations of the amino acid glycine. The same set of enzymes is sometimes referred to as glycine synthase when it runs in the reverse direction to form glycine. The GCS is composed of four proteins: the T-protein, P-protein, L-protein, and H-protein. The H-protein is responsible for interacting with the three other proteins and acts as a shuttle for some of the intermediate products in glycine decarboxylation.

[0142] L-serine hydroxymethyltransferase

[0143] In some embodiments, the Pseudomonas cell comprises a cyanidase and an L-serine hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) and / or GlyA-ll (SEQ ID NO: 23), or functional variants thereof having at least 70% identity thereto. Said cyanidase may be any one or both of CynDstut (SEQ ID NO: 1) and CynDpum (SEQ ID NO: 2), or functional variants thereof. In some embodiments, the Pseudomonas cell comprises:

[0144] - CynDstut (SEQ ID NO: 1) and GlyA-l (SEQ ID NO: 22);

[0145] - CynDstut (SEQ ID NO: 1) and GlyA-ll (SEQ ID NO: 23);

[0146] - CynDpum (SEQ ID NO: 2) and GlyA-l (SEQ ID NO: 22);

[0147] - CynDpum (SEQ ID NO: 2) and GlyA-ll (SEQ ID NO: 23);

[0148] - CynDstut (SEQ ID NO: 1), GlyA-l (SEQ ID NO: 22) and GlyA-ll (SEQ ID NO:

[0149] 23); and / or

[0150] - CynDpum (SEQ ID NO: 2), GlyA-l (SEQ ID NO: 22) and GlyA-ll (SEQ ID NO:

[0151] 23);P7355PC00

[0152] or functional variants thereof having at least 70% identity thereto.

[0153] Starting from a wildtype Pseudomonas cell, which is capable of de novo synthesis of 5,10-methylene-THF and serine, a 5,10-methylene-THF and serine auxotroph Pseudomonas cell may be constructed by inactivating the endogenous glycine cleavage system(s) (AgcvTHP-\ / \\) as well as inactivating one or more of the later reactions of the endogenous serine biosynthesis, for example one or more of an endogenous D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95), phosphoserine aminotransferase (EC 2.6.1.52) and phosphoserine phosphatase (EC 3.1.3.3).

[0154] Inactivation of the endogenous glycine cleavage system (GCS), such as inactivation of the GcvTHP-l / ll operons, results in a Pseudomonas cell that is incapable of de novo synthesis of both 5,10-methylene-THF and glycine (see Figure 3A), unless their synthesis is restored by other enzymatic activities. Where a 5,10-methylene-THF auxotrophic Pseudomonas cell results from the inactivation of said endogenous GCS, the Pseudomonas cell may also be referred to as a glycine auxotrophic cell. Thus, the terms 5,10-methylene-THF auxotroph and glycine auxotroph may be used interchangeably herein, provided the auxotrophy is the result of inactivation of the endogenous GCS.

[0155]

[0156] de novo serine

[0157] Thus, de novo serine synthesis of a Pseudomonas cell, such as a P. putida cell, may be disrupted by inactivation one or more of SerA, SerB and SerC (SerABC or SerABC pathway), and / or one or more of the nucleic acids encoding said enzyme(s). The SerABC pathway of bacteria, including Pseudomonads, is well known in the art (Pizer, Lewis I., 1963., Zhang et al., 2017).

[0158] In preferred embodiments, the Pseudomonas cell comprises an endogenous D-3- phosphoglycerate dehydrogenase (EC 1.1.1.95), and said cell comprises a modification resulting in partial or total inactivation of said endogenous D-3- phosphoglycerate dehydrogenase. Thus, in some embodiments, the Pseudomonas cell comprises modification resulting in partial or total inactivation of the D-3- phosphoglycerate dehydrogenase SerA (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity thereto. SerA (SEQ ID NO: 15) may also be referred to as a bifunctional D-3-phosphoglycerate dehydrogenase / a-ketoglutarate reductase.P7355PC00

[0159] SerA (SEQ ID NO: 15) or functional variants thereof, may be encoded by serA (SEQ ID NO: 14) or homologues thereof. Therefore, a modification resulting in partial or total inactivation of SerA may be a mutation within or a deletion of at least a part of serA itself. However, it may also be a mutation in a cis-regulatory element of serA, such as a mutation in a promoter of serA; a mutation in a terminator of serA; or a mutation in a trans-regulatory element of serA, such as in a nucleic acid encoding a transcription factor regulating its transcription. Thus, in some embodiments, said modification resulting in partial or total inactivation of SerA (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity thereto, comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerA or a functional variant thereof having at least 70% identity thereto, such as serA (SEQ ID NO: 14) or a homologue thereof having at least 70% identity thereto.

[0160] In other embodiments, the Pseudomonas cell comprises an endogenous phosphoserine aminotransferase (EC 2.6.1.52), and said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine aminotransferase. Thus, in some embodiments, the Pseudomonas cell comprises a modification resulting in partial or total inactivation of the phosphoserine aminotransferase SerC (SEQ ID NO: 68) or a functional variant thereof having at least 70% identity thereto. SerC (SEQ ID NO: 68) or functional variants thereof, may be encoded by serC (SEQ ID NO: 67) or homologues thereof. Thus, in some embodiments, the modification resulting in partial or total inactivation of SerC (SEQ ID NO: 68) or a functional variant thereof having at least 70% identity thereto, comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerC or a functional variant thereof having at least 70% identity thereto, such as serC (SEQ ID NO: 67) or a homologue thereof having at least 70% identity thereto.

[0161] In other embodiments, the Pseudomonas cell comprises an endogenous phosphoserine phosphatase (EC 3.1.3.3), and said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine phosphatase. Thus, in some embodiments, the Pseudomonas cell comprises a modification resulting in partial or total inactivation of the phosphoserine phosphatase SerB (SEQ ID NO: 66) or a functional variant thereof having at least 70% identity thereto.P7355PC00

[0162] SerB (SEQ ID NO: 66) or functional variants thereof, may be encoded by serB (SEQ ID NO: 65) or homologues thereof. Thus, in some embodiments, the modification resulting in partial or total inactivation of SerB (SEQ ID NO: 66) or a functional variant thereof having at least 70% identity thereto, comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerB or a functional variant thereof having at least 70% identity thereto, such as serB (SEQ ID NO: 65) or a homologue thereof having at least 70% identity thereto.

[0163] Similarly to serA, a modification resulting in partial or total inactivation of SerC or SerB may be a mutation within or a deletion of at least a part of serC or serB, respectively; in a cis-regulatory element of serC and / or serB, such as a promoter of serC and / or serB; in a terminator of serC and / or serB; or in a trans-regulatory element, such as in a nucleic acid encoding a transcription factor regulating either or both of their transcription.

[0164] Thus, the cell may harbour one or more modifications rendering its native / endogenous serine biosynthesis inactive and alternative engineering strategies to disrupting the expression or interfering with the activity of SerA, SerB and / or SerC may exist to achieve this.

[0165] In some embodiments, the Pseudomonas cell comprises a cyanidase, an L-serine hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) and / or GlyA-ll (SEQ ID NO: 23), and a modification resulting in partial or total inactivation of SerA (SEQ ID NO: 15), SerC (SEQ ID NO: 68) and / or SerB (SEQ ID NO: 66), or functional variants thereof having at least 70% identity thereto.

[0166] In some embodiments, the Pseudomonas cell comprises:

[0167] - a cyanidase, an L-serine hydroxymethyltransferase, and a modification resulting in partial or total inactivation of SerA (SEQ ID NO: 15);

[0168] - a cyanidase, an L-serine hydroxymethyltransferase, and a modification resulting in partial or total inactivation of SerB (SEQ ID NO: 66);

[0169] - a cyanidase, an L-serine hydroxymethyltransferase, and a modification resulting in partial or total inactivation of SerC (SEQ ID NO: 68);

[0170] - CynDstut (SEQ ID NO: 1) and a modification resulting in partial or total inactivation of SerA (SEQ ID NO: 15);P7355PC00

[0171] - CynDpum (SEQ ID NO: 2) and a modification resulting in partial or total inactivation of SerA (SEQ ID NO: 15);

[0172] or functional variants thereof having at least 70% identity thereto.

[0173] In addition to not being capable of de novo serine synthesis via the SerABC pathway, the Pseudomonas cell should also be modified, so that synthesis of 5,10-methylene-THF is dependent on the conversion of formate thereto. Conversion of formate into 5,10-methylene-THF may be as described herein above. In other words, the Pseudomonas cell should be modified not to escape the coupling of growth to the assimilation of 5,10-methylene-THF via hydroxymethylation of 5,10-methylene-THF and glycine to form the essential amino acid serine. This may be achieved by inactivating, if any, the endogenous GCS, for example as described in this section.

[0174] Glycine cleavage system (GCS)

[0175] As explained herein above, the glycine cleavage system (GCS) is well known in the art. The endogenous GCS of the Pseudomonas cell may comprise or consist of a dihydrolipoyl dehydrogenase (LpdG; EC 1.8.1.4); a glycine cleavage system protein H (GcvH); a glycine dehydrogenase (EC 1.4.4.2), which may also be referred to as glycine cleavage system protein P (GcvP); and a dihydrolipoyl dehydrogenase (EC 2.1.2.10), which may also be referred to as glycine cleavage system protein T (GcvT).

[0176] Wildtype Pseudomonas cell often comprises more than one copy of the genes encoding the GCS proteins. In other words, the cell may comprise multiple GCS gene homologues or multiple GCS nucleic acids. Genes or nucleic acids encoding, at least some of, the GCS proteins may be comprised within one or more operons. Thus, without being bound by theory, one or more of the GCS genes or nucleic acids may be located in one or more operons in the genome of a wildtype Pseudomonas cell. Such operon(s) may be referred to as GCS operon(s) herein. For example, wildtype P. putida comprises two operons comprising genes encoding proteins of the GCS. These two GCS operons may be referred to as Gcv-I, or gcvTHP-l, and Gcv-ll, or gcvTHP-ll, respectively. Importantly, if the Pseudomonas cell comprises multiple homologues of the genes encoding proteins of the GCS, each gene homologues should be inactivated. For example, inactivation of the GCS may be achieved by disruption of one or more genes and their homologues, encoding essential proteins for the GCS to function, so that the GCS of said Pseudomonas cell is rendered non-functional. AP7355PC00

[0177] skilled person is capable of identifying the GCS proteins and the genes encoding the GCS proteins, including their homologues.

[0178] Hence, in some embodiments, the Pseudomonas comprises at least one endogenous GCS operon, for example two endogenous GCS operons, wherein said cell comprises a modification resulting in partial or total inactivation of said at least one endogenous GCS operon. In other embodiments, the Pseudomonas comprises two endogenous GCS operons, and said cell comprises at least one modification, such as at least two modifications, resulting in partial or total inactivation of said two endogenous GCS operons.

[0179] The endogenous GCS operon(s), such as Gcv-I and Gcv-ll, may comprise or consist of one or more nucleic acids encoding a GCS protein H (GcvH), a glycine dehydrogenase (GcvP; EC 1.4.4.2) and an aminomethyltransferase (GcvT; EC 2.1.2.10). The skilled person will know that there are multiple routes / engineering strategies to achieve complete inactivation of a GCS operon of a Pseudomonas cell. Importantly, if the cell comprises more than one homologue of the genes encoding the proteins of the GCS, such as more than one GCS operon, the cell should be modified in a manner so that the inactivation of one gene homologue is not compensated by the existence of another functional homologue.

[0180] Hence, in some embodiments, the Pseudomonas cell comprises two endogenous GCS operons comprising or consisting of:

[0181] I. a first operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-l (SEQ ID NO: 16), GcvP-l (SEQ ID NO: 17) and GcvT-l (SEQ ID NO: 18); and / or

[0182] II. a second operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-ll (SEQ ID NO: 19), GcvP-ll (SEQ ID NO: 20) and GcvT-ll (SEQ ID NO: 21),

[0183] or functional variants thereof having at least 70% identity to thereto.

[0184] The Pseudomonas cell may comprise a modification resulting in partial or total inactivation of said at least one endogenous GCS operons, for example two endogenous GCS operons. Thus, in some embodiments, said modification resulting in partial or total inactivation of said at least one endogenous GCS operon comprises orP7355PC00

[0185] consists of a mutation in or a deletion of at least a part of gcvTHP-l and / or gcvTHP-ll. In some embodiments, the Pseudomonas cell comprises two endogenous GCS operons and said modification comprises or consists of a mutation in or a deletion of at least a part of said two endogenous GCS operons. In some embodiments, the Pseudomonas cell comprises gcvTHP-l and gcvTHP-ll, and said modification comprises or consists of a mutation in or a deletion of at least a part of gcvTHP-l and gcvTHP-ll. In some embodiments, the modification resulting in partial or total inactivation of said two endogenous GCS operons comprises or consists of a mutation in or a deletion of at least a part of a first nucleic acid encoding GcvH-l, GcvP-l and GcvT-l, or functional variants thereof having at least 70% identity to thereto, and a mutation in or a deletion of at least a corresponding part of a second nucleic acid encoding GcvH-ll, GcvP-ll and GcvT-ll, or functional variants thereof having at least 70% identity to thereto. For example, if the part of a first nucleic acid encoding GcvH-l is mutated or deleted, a mutation in or deletion of the part of a second nucleic acid encoding GcvH-ll, and resulting in the same effect as for the first nucleic acid, is considered “a mutation in or deletion of the corresponding part in a second nucleic acid”.

[0186] The same logic applies mutatis mutandis if the Pseudomonas cell comprises more than two GCS operons. Skilled person is capable of partially or totally inactivating the GCS of the Pseudomonas.

[0187] Serine-Threonine-Cycle (STC)

[0188] In addition to comprising a cyanidase or at least comprising a nucleic acid encoding a cyanidase, being capable of converting formate into 5,10-methylene-THF, and modifications for inactivation of de novo 5,10-methylene-THF and serine production and coupling their biosynthesis to assimilation of cyanide, the Pseudomonas cell of the present disclosure may further comprise the enzyme activities depicted in Table 1.

[0189] The enzymatic reactions listed in Table 1 represents a cycle, which may be referred to as the Serine-Threonine Cycle (STC). The reactions of the STC may be catalysed by heterologous enzymes, which the Pseudomonas cell have been genetically modified to express, and / or endogenous enzymes, i.e. enzyme activities that are native to the Pseudomonas cell.

[0190] Table 1: Characteristics of Serine-Threonine Cycle (STC) enzymesP7355PC00

[0191] AbbreEC no.

[0192] Enzyme name Reaction

[0193] viation

[0194] Serine hydroxy2.1.2.1 Glycine + 5,10-Methylene- GlyA

[0195] methyltransferase tetrahydrofolate + H2O <=> Serine (-I to -II)

[0196] (SHMT) + Tetra hydrofol ate TdcG Serine 4.3.1.17

[0197] Serine <=> NH3 + Pyruvate (-I to -III) dehydratase

[0198] Pyruvate 6.4.1.1 ATP + Pyruvate + CO2 <=> ADP + PycAB

[0199] carboxylase Orthophosphate + Oxalacetate 2.7.1.40 ATP + Pyruvate + H2O <=> PhosphoenolPykA Orthophosphate + AMP + pyruvate synthetase

[0200] Phosphoenolpyruvate Phosphoeno4.1.1.31 CO2 + Phosphoenolpyruvate + Ppc lpyruvate H2O <=> Orthophosphate + carboxylase Oxaloacetate Aspartate 2.6.1.1 Oxalacetate + Glutamate <=> L- AspC

[0201] aminotransferase Aspartate + 2-Ketoglutarate 2.7.2.4 ATP + L-Aspartate <=> ADP + 4- LysC Aspartate kinase

[0202] phospho-L-aspartate Aspartate1.2.1.11 4-phospho-L-aspartate + NADPH Asd semialdehyde <=> L-Aspartate 4-semialdehyde + dehydrogenase phosphate + NADP 1.1.1.3 L-aspartate 4-semialdehyde + Hom or Homoserine

[0203] NAD(P)H <=> L-homoserine + PP_0664 dehydrogenase

[0204] NAD(P) 2.7.1.39 Homoserine + ATP <=> 0- ThrB Homoserine kinase

[0205] phospho-homoserine + ADP 4.2.3.1 O-phospho-homoserine + H2O ThrC Threonine synthase

[0206] <=> Threonine + POT 4.1.2.5 / Threonine <=> L-Glycine + LtaE L-threonine aldolase

[0207] 4.1.2.48 Acetaldehyde

[0208]

[0209] In instances, where one or more of the enzymatic reactions listed in Table 1 is native to the Pseudomonas cell, said reactions may be catalysed by multiple enzymes, which may be functional variants of each other and encoded by different gene homologues.P7355PC00

[0210] As a non-limiting example, the serine hydroxymethyltransferase (SHMT) GlyA is found in two variants in P. putida, that is GlyA-l and GlyA-ll, which is encoded by glyA-l and glyA-ll, respectively.

[0211] Hence, in some embodiments, the Pseudomonas cell is capable of catalysing:

[0212] I. hydroxymethylation of glycine with 5,10-methylene-THF into serine by a L-serine hydroxymethyltransferase such as GlyA-l (SEQ ID NO: 22) or GlyA-ll (SEQ ID NO: 23);

[0213] II. deamination of said serine into pyruvate by a serine dehydratase / serine deaminase (EC 4.3.1.17), such as TdcG-l (SEQ ID NO: 26), TdcG-ll (SEQ ID NO: 27) orTdcG-lll (SEQ ID NO: 28);

[0214] III. carboxylation of said pyruvate into oxaloacetate, such as;

[0215] i. by a pyruvate carboxylase (EC 6.4.1.1), such as PycAB comprising or consisting of PycA (SEQ ID NO: 32) and PycB (SEQ ID NO: 33); and / or ii. via conversion of said pyruvate to phosphoenolpyruvate by phosphoenolpyruvate synthetase (EC 2.7.1.40), such as PykA (SEQ ID NO: 36), and conversion of said phosphoenolpyruvate into said oxaloacetate by a phosphoenolpyruvate carboxylase (EC 4.1.1.31), such as Ppc (SEQ ID NO: 38);

[0216] IV. transamination of said oxaloacetate into aspartate by an aspartate aminotransferase (EC 2.6.1.1), such as AspC (SEQ ID NO: 40);

[0217] V. conversion of said aspartate into homoserine, such as via:

[0218] iii. conversion of aspartate into 4-phospho-L-aspartate by an aspartate kinase (EC 2.7.2.4), such as LysC (SEQ ID NO: 42);

[0219] iv. conversion of said 4-phospho-L-aspartate into L-aspartate 4-semialdehyde by an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11), such as Asd (SEQ ID NO: 44); and

[0220] v. conversion of said L-aspartate 4-semialdehyde into homoserine by a homoserine dehydrogenase (EC 1.1.1.3), such as Hom (SEQ ID NO: 46) or PP_0664 (SEQ ID NO: 47);

[0221] VI. conversion of said homoserine into threonine, such as via:

[0222] vi. conversion of homoserine into O-phospho-homoserine by a homoserine kinase (EC 2.7.1.39), such as ThrB (SEQ ID NO: 50); and

[0223] vii. conversion of said O-phospho-homoserine into threonine by a threonine synthase (EC 4.2.3.1), such as ThrC (SEQ ID NO: 52);P7355PC00

[0224] and / or

[0225] VII. conversion of said threonine into glycine and acetaldehyde by L-threonine aldolase (EC 4.1.2.5 / 4.1.2.48), such as an L-threonine aldolase from P. putida, for example LtaE (SEQ ID NO: 5),EcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7),

[0226] or functional variants thereof having at least 70 % identity thereto.

[0227] The acetaldehyde produced by reaction VII above may be further converted into acetate by an aldehyde dehydrogenase, such as AldB, which may in turn be converted into acetyl-CoA by an acetyl-coenzyme A synthetase, such as AcsA, by the Pseudomonas cell. Thus, in some embodiments, the Pseudomonas cell is further capable of catalysing the conversion of acetaldehyde into acetate by an aldehyde dehydrogenase, such as AldB. In some embodiments, the Pseudomonas cell is capable of catalysing the conversion of acetate into acetyl-CoA by an acetyl-coenzyme A synthetase, such as AcsA.

[0228] In some embodiments, the Pseudomonas cell comprises one or more of:

[0229] i. a nucleic acid encoding a L-serine hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) or GlyA-ll (SEQ ID NO: 23), for example glyA-l (SEQ ID NO: 24) or glyA-ll (SEQ ID NO: 25);

[0230] ii. a nucleic acid encoding a serine dehydratase / serine deaminase (EC 4.3.1.17), such as TdcG-l (SEQ ID NO: 26), TdcG-ll (SEQ ID NO: 27) or TdcG-lll (SEQ ID NO: 28), for example tdcG-\ (SEQ ID NO: 29), tdcG-ll (SEQ ID NO: 30) or tdcG-lll (SEQ ID NO: 31);

[0231] iii. a nucleic acid encoding a pyruvate carboxylase (EC 6.4.1.1), such as PycAB comprising or consisting of PycA (SEQ ID NO: 32) and PycB (SEQ ID NO: 33), for example pycA (SEQ ID NO: 34) and / or pycB (SEQ ID NO: 35);

[0232] iv. a nucleic acid encoding a phosphoenolpyruvate synthetase (EC 2.7.1.40), such as PykA (SEQ ID NO: 36), for example pykA (SEQ ID NO: 37);

[0233] v. a nucleic acid encoding a phosphoenolpyruvate carboxylase (EC 4.1.1.31), such as Ppc (SEQ ID NO: 38), for example ppc (SEQ ID NO: 39);

[0234] vi. a nucleic acid encoding an aspartate aminotransferase (EC 2.6.1.1), such as AspC (SEQ ID NO: 40), for example aspc (SEQ ID NO: 41);

[0235] vii. a nucleic acid encoding an aspartate kinase (EC 2.7.2.4), such as LysC (SEQ ID NO: 42), for example lysC (SEQ ID NO: 43);P7355PC00

[0236] viii. a nucleic acid encoding an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11), such as Asd (SEQ ID NO: 44), such as asd (SEQ ID NO: 45);

[0237] ix. a nucleic acid encoding a homoserine dehydrogenase (EC 1.1.1.3), such as Hom (SEQ ID NO: 46) or PP_0664 (SEQ ID NO: 47), for example horn () (SEQ ID NO: 48) or PP_0664 (SEQ ID NO: 49);

[0238] x. a nucleic acid encoding a homoserine kinase (EC 2.7.1.39), such as ThrB (SEQ ID NO: 50), such as thrB (SEQ ID NO: 51);

[0239] xi. a nucleic acid encoding a threonine synthase (EC 4.2.3.1), such as ThrC (SEQ ID NO: 52), such as thrC (SEQ ID NO: 53); and / or

[0240] xii. a nucleic acid encoding an L-threonine aldolase (EC 4.1.2.5 / 4.1.2.48) from P. putida, such as LtaE (SEQ ID NO: 5),EcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), for example I

[0241]

[0242] taE (SEQ ID NO: 71),EcltaE (SEQ ID NO: 69) orEcltaE* (SEQ ID NO: 70), respectively;

[0243] or functional variants or homologues thereof having at least 70 % identity thereto.

[0244] As is understood from this section, the STC cycle can efficiently provide metabolites which are required or useful for growth of the Pseudomonas cell and thus the production of cell biomass.

[0245] The data as presented herein confirm that the disclosed strain design(s), i.e. the proposed pathway(s) and / or pathway module(s), provided by the inventors allow for efficient assimilation of cyanide (via formate), and thus, efficient valorisation of this, otherwise toxic, C1-feedstock into biomass or various compounds.

[0246] In some embodiments, the Pseudomonas cell comprises a nucleic acid encoding a cyanidase (EC 3.5.5.1) capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+), and a heterologous nucleic acid encoding a formate-THF ligase (FtfL; EC 6.3.4.3), a heterologous nucleic acid encoding a methenyl-THF cyclohydrolase (Fch; EC 3.5.4.9), and a heterologous nucleic acid encoding a methylene-THF dehydrogenase (MdtA; EC 1.5.1.5), wherein said cell is capable of converting said formate into 5,10-methylene-THF, and wherein said Pseudomonas cell further comprises a modification resulting in partial or total inactivation of an endogenous GCS and a modification resulting in partial or total inactivation of an endogenous SerABC pathway.P7355PC00

[0247] eSTC

[0248] To further improve the assimilation of carbon derived from cyanide, the Pseudomonas cell may comprise a L-serine aldolase which may aid in the carbon assimilation via formaldehyde. This may be achieved by expression of a heterologous L-serine aldolase that can catalyse the condensation of formaldehyde and glycine into serine.

[0249] The heterologous L-serine aldolase may be an L-serine aldolase from Escherichia, such as from Escherichia coli, for exampleEcLtaE (SEQ ID NO: 6), orEcLtaE* (SEQ ID NO: 7), or functional variants thereof having at least 70% identity thereto, such as at least 75% identity, such as at least 80% identity, such as at least 81% identity, such as at least 82% identity, such as at least 83% identity, such as at least 84% identity, such as at least 85% identity, such as at least 86% identity, such as at least 87% identity, such as at least 88% identity, such as at least 89% identity, such as at least 90% identity, such as at least 91% identity, such as at least 92% identity, such as at least 93% identity, such as at least 94% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity thereto.EcLtaE* is identical toEcLtaE with the exception of the mutation C188Y.

[0250] Hence, in some embodiments, the Pseudomonas cell comprises, preferably overexpresses, a nucleic acid encoding a heterologous L-serine aldolase capable of converting glycine and formaldehyde into serine, such asEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto.

[0251] The heterologous L-serine aldolase may be expressed in the Pseudomonas cell by introduction of a nucleic acid that encodes the aldolase. In some embodiments, the nucleic acid encodingEcLtaE (SEQ ID NO: 6) comprises or consists ofEcltaE (SEQ ID NO: 69) or a homologue thereof having at least 70% identity thereto. In other embodiments, the nucleic acid encodingEcLtaE* (SEQ ID NO: 7) comprises or consists ofEcltaE* (SEQ ID NO: 70) or a homologue thereof having at least 70% identity thereto.

[0252] Thus, in some embodiments, the Pseudomonas cell of the present disclosures comprises:P7355PC00

[0253] i. a nucleic acid encoding CynDstut (SEQ ID NO: 1) and a nucleic acid encodingEcLtaE (SEQ ID NO: 6)

[0254] ii. a nucleic acid encoding CynDpum (SEQ ID NO: 2) and a nucleic acid encodingEcLtaE (SEQ ID NO: 6);

[0255] iii. a nucleic acid encoding CynDstut (SEQ ID NO: 1) and a nucleic acid encodingEcLtaE* (SEQ ID NO: 7); or

[0256] iv. a nucleic acid encoding CynDpum (SEQ ID NO: 2) and a nucleic acid encodingEcLtaE* (SEQ ID NO: 7);

[0257] or functional variants thereof having at least 70% identity thereto.

[0258] In another aspect, the present disclosure relates to a Pseudomonas cell comprising a nucleic acid encoding a heterologous L-serine aldolase capable of converting glycine and formaldehyde into serine. Such Pseudomonas cells may be useful for assimilation of not only cyanide, but also other C1-compounds / feedstocks, including methanol and formate. A skilled person knows, that C1 -compounds, such as methanol and formate, can be converted into formaldehyde by various enzymatic reactions. As demonstrated by the data herein, Pseudomonas cells in the form of P. putida cells have been successfully engineered by the present inventors express a heterologous L-serine aldolase for efficient assimilation of a C1 -compound via formaldehyde.

[0259] Further modifications

[0260] The efficiency of the conversion of cyanide into either cellular biomass or a compound of interest may be increased by one or more further modifications of the Pseudomonas cell in combination with modifications described herein above.

[0261] For example, it may be beneficial that any endogenous synthesis of medium-chain length polyhydroxyalkanoates (mcl-PHAs) by the Pseudomonas cell is either partially or totally inactivated. In other words, it may further enhance the assimilation of cyanide, that the cell is not capable of producing mcl-PHAs as they may function as a “metabolic sink” or so-called bypass pathway. Thus, in some embodiments, the Pseudomonas cell is not capable of producing polyhydroxyalkanoates (PHAs) having a medium-length carbon chain, i.e. mcl-PHAs. Hence, in some embodiments the Pseudomonas cell comprises an endogenous pha cluster and said cell comprises a modification resulting in partial or total inactivation of said endogenous pha cluster.P7355PC00

[0262] The endogenous pha cluster of said Pseudomonas cell may comprise or consist of one or more nucleic acids encoding PhaC1 (SEQ ID NO: 54), PhaZ (SEQ ID NO: 55), PhaC2 (SEQ ID NO: 56), PhaD (SEQ ID NO: 57), PhaF (SEQ ID NO: 58) and Phal (SEQ ID NO: 59), or functional variants thereof having at least 70% identity thereto. These nucleic acids may be comprised within one or more operons. Thus, in some embodiments, the endogenous pha cluster comprises or consists of:

[0263] i. a first operon comprising or consisting of one or more nucleic acids encoding PhaC1, PhaZ and PhaD; and

[0264] ii. a second operon comprising or consisting of one or more nucleic acids encoding PhaF and Phal;

[0265] or functional variants thereof having at least 70% identity thereto.

[0266] In some embodiments of the present disclosure, the endogenous pha cluster in said cell is partially or totally inactivated, wherein the endogenous pha cluster consists of: i. a first operon comprising nucleic acids encoding PhaC1 (SEQ ID NO: 54), PhaC2 (SEQ ID NO: 56), PhaZ (SEQ ID NO: 55), and PhaD (SEQ ID NO: 57); and ii. a second operon comprising nucleic acids encoding PhaF (SEQ ID NO: 58), and Phal (SEQ ID NO: 59),

[0267] or functional variants thereof having at least 70% identity thereto.

[0268] The inactivation of the endogenous pha cluster may comprise or consist of a mutation or a deletion of all or part of the endogenous pha cluster. Preferably, the inactivation consists of a mutation or deletion of at least one of the first and second operon, such as a mutation or deletion of the first operon, a mutation or deletion of the second operon, or a mutation or deletion of both the first and the second operons. The same logic applies mutatis mutandis if the Pseudomonas cell comprises more than two operons. Skilled person is capable of partially or totally inactivating the pha cluster of the cell to either partially or completely abolish mcl-PHA synthesis by the cell.

[0269] The endogenous pha cluster may be partially or totally inactivated, if one or more of PhaC1 (SEQ ID NO: 54), PhaC2 (SEQ ID NO: 56), PhaZ (SEQ ID NO: 55), PhaD (SEQ ID NO: 57), PhaF (SEQ ID NO: 58) and Phal (SEQ ID NO: 59), or a functional variant thereof having at least 70% identity thereto, are partially or totally inactivated, so that the cell is not capable of producing mcl-PHAs. Thus, in further embodiments, the partial or total inactivation comprises or consists of a mutation in or a deletion of atP7355PC00

[0270] least one or more of a nucleic acid encoding PhaC1, PhaC2, PhaZ and PhaD; a nucleic acid encoding PhaF and Phal, or a functional variant thereof having at least 70% identity to PhaC1, PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively.

[0271] To further improve assimilation and in particular biomass formation, it may be beneficial to inactivate any endogenous FAD-dependent glycine / D-amino acid oxidase (EC 1.4.3.19). FAD-dependent glycine / D-amino acid oxidase may produce glyoxylate from glycine and thus compete with the biomass formation, wherefore glycine is needed for production of the essential amino acid serine. Thus, in some embodiments, the Pseudomonas cell comprises an endogenous FAD-dependent glycine / D-amino acid oxidase, and said cell comprises a modification resulting in partial or total inactivation of said endogenous FAD-dependent glycine / D-amino acid oxidase. In some embodiments, said FAD-dependent glycine / D-amino acid oxidase is ThiO (SEQ ID NO: 73) or a functional variant thereof having at least 70% identity thereto. Thus, in some embodiments, said modification resulting in partial or total inactivation of ThiO (SEQ ID NO: 73) or a functional variant thereof having at least 70% identity thereto, comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding ThiO or a functional variant thereof having at least 70% identity thereto, such as thiO (SEQ ID NO: 72) or a homologue thereof having at least 70% identity thereto.

[0272] In addition to the above-described modifications, it may be advantageous to also delete or inactivate the Pseudomonas cells’ ability to create biofilm-producing adhesins. P. putida comprises one or more endogenous biofilm-producing adhesins, including LapA (SEQ ID NO: 74) and LapF (SEQ ID NO: 76). Inactivation of biofilm-producing adhesins, such as LapA and LapF or functional variants thereof, is within reach of the skilled person, and can be achieved for example by deleting part or all of one or more of lapA (SEQ ID NO: 75) and lapF (SEQ ID NO: 77), or homologues thereof having at least 70% identity thereto. Thus, in some embodiments the cell comprises a modification resulting in partial or total inactivation of said one or more endogenous biofilm-producing adhesins, for example the cell comprises a modification resulting in partial or total inactivation of LapA (SEQ ID NO: 74) and / or LapF (SEQ ID NO: 76), or functional variants thereof having at least 70% identity thereto. In other embodiments, the cell comprises a modification comprising or consisting of a mutation in or a deletion of at least a part of a nucleic acid encoding LapA and / or LapF or functional variants thereof having at least 70% identity thereto. Thus, in some embodiments, the cellP7355PC00

[0273] comprises a modification comprising or consisting of a mutation in or a deletion of at least a part of lapA (SEQ ID NO: 75) and / or lapF (SEQ ID NO: 77), or homologues thereof having at least 70% identity thereto.

[0274] Growth on cyanide

[0275] An advantage of the Pseudomonas cells disclosed herein is their ability to tolerate, including propagate, grow and / or respire, in the presence of cyanide, and in particular their ability to valorise cyanide, including using cyanide as a nitrogen- and / or carbon-source for growth and / or production of bulk or value-added compounds, such as pyruvate-derived compounds.

[0276] Thus, in preferred embodiments, the Pseudomonas cell comprising a cyanidase and / or a nucleic acid encoding a cyanidase is capable of utilizing cyanide as sole nitrogen source for growth. In other words, preferably the Pseudomonas cell expressing a cyanidase is able to use cyanide as a source of nitrogen to sustain its propagation. In some embodiments, the Pseudomonas cell is capable of assimilating at least 20 % of the nitrogen provided in cyanide, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or more.

[0277] Preferably, the Pseudomonas cell is also capable of using cyanide as a carbon source. In some embodiments, the Pseudomonas cell is capable of assimilating at least 20 % of the carbon provided in cyanide, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or more.

[0278] Thus, in other embodiments, the Pseudomonas cell is capable of utilizing cyanide as sole nitrogen source and as a carbon source. For example, in some embodiments, the Pseudomonas cell is able to utilize cyanide as a carbon source for the generation of pyruvate via formation of formate and serine.P7355PC00

[0279] Valorisation of cyanide

[0280] The Pseudomonas cell disclosed herein may be used for conversion of cyanide into more useful compounds, and hence valorise cyanide. In particular, the cells disclosed herein may be used for production of pyruvate-derived compounds, including but not limited to lactate. Valorisation also encompasses cell growth, i.e. that the cell is able to use cyanide as a substrate for growth.

[0281] Methods

[0282] Described herein are methods relating various applications of the Pseudomonas cells of the present disclosure, including methods for producing value-added compounds from cyanide. The methods described herein may be particularly useful for producing bulk and value-added compounds by assimilation of cyanide. Provided herein are also methods of detoxifying a waste product comprising cyanide, methods of valorising a waste product comprising cyanide and methods of propagating a Pseudomonas cell in the presence of cyanide. Methods of engineering a Pseudomonas cell are also provided.

[0283] Thus, it is a main aspect of the present disclosure to provide a method of propagating a Pseudomonas cell in the presence of cyanide, comprising:

[0284] i. providing a Pseudomonas cell as described elsewhere herein;

[0285] ii. propagating said cell in a cultivation medium, said cultivation medium comprising cyanide, thereby obtaining a fermentation broth comprising said cell and / or biomass of said cell;

[0286] iii. optionally recovering said fermentation broth and / or said biomass.

[0287] In another main aspect, the present disclosure relates to a method of producing a compound, such as a pyruvate-derived compound, comprising:

[0288] i. providing a Pseudomonas cell as described elsewhere herein;

[0289] ii. cultivating and optionally propagating said cell in a cultivation medium, said cultivation medium comprising cyanide, thereby obtaining a fermentation broth and / or cell comprising said compound;

[0290] iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;

[0291] whereby said compound and / or said fermentation broth comprising said compound is produced.P7355PC00

[0292] In some embodiments, the Pseudomonas cell is capable of producing pyruvate or a compound derived from pyruvate.

[0293] In some embodiments, the Pseudomonas cell is capable of producing lactate. Thus, in some embodiments, said method of producing a compound is a method of producing lactate. The cell being capable of producing or producing lactate may comprise a lactate dehydrogenase or a nucleic acid encoding a lactate dehydrogenase (LDH; EC 1.1.1.28). Said lactate dehydrogenase may for example be from Bos taurus, such as LdhA from B. taurus or a functional variant thereof, for example BtLdhA(N108G) (SEQ ID NO: 60) or a functional variant thereof having at least 70% identity thereto. In some embodiments, the cell further comprises at least one modification resulting a partial or total inactivation of an endogenous lactate dehydrogenase, such as an L-lactate dehydrogenase (EC 1.1.2.3) and / or a D-lactate dehydrogenase 2 (EC 1.1.5.12). The modification may for example comprise or consist of a mutation in or a deletion of at least a part of a nucleic acid encoding said endogenous lactate dehydrogenase. Thus, for example the Pseudomonas cell may further comprise at least one modification resulting in partial or total inactivation of an L-lactate dehydrogenase (EC 1.1.2.3), such as LldD (SEQ ID NO: 61), and a D-lactate dehydrogenase 2 (EC 1.1.5.12), such as Dld2 (SEQ ID NO: 62), or functional variants thereof having at least 70% identity thereto. In some embodiments, said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding LldD and Dld2 or functional variants thereof having at least 70% identity thereto, such as IldD (SEQ ID NO: 63) and dld2 (SEQ ID NO: 64) or homologues thereof having at least 70% identity thereto.

[0294] In some embodiments, said compound, such as said pyruvate-derived compound, is a mixture of pyruvate or lactate. In some embodiments, said compound, such as said pyruvate-derived compound, is pyruvate. In some embodiments, said compound, such as said pyruvate-derived compound, is lactate.

[0295] The cells of the present disclosure may be useful for detoxifying a waste product comprising cyanide. Said detoxification of a waste product may comprise assimilation of said cyanide from the waste product by a cell as described in the present disclosure. Said assimilation of cyanide from said waste product comprising cyanide, by a cell of the present disclosure or by a method described herein, may result in the detoxificationP7355PC00

[0296] of said waste product via the partial or complete reduction of cyanide from or in said waste product. Thus, herein provided is a method of detoxifying a waste product comprising cyanide, comprising:

[0297] i. providing a Pseudomonas cell as described elsewhere herein;

[0298] ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide, thereby degrading said cyanide and optionally obtaining cell biomass;

[0299] iii. optionally recovering said cell biomass;

[0300] whereby said waste product is detoxified.

[0301] Provided herein is a method of valorising of a waste product comprising cyanide into a compound, comprising:

[0302] i. providing a Pseudomonas cell as described elsewhere herein;

[0303] ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide, thereby obtaining a fermentation broth and / or cell comprising said compound;

[0304] iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;

[0305] whereby said waste product comprising cyanide is valorised by production of said compound and / or said fermentation broth comprising said compound. Valorisation of a waste product comprising cyanide may entail assimilation and conversion of said cyanide, comprised within the waste product, to a compound. Said compound may be a bulk or value-added compound. As an example, said compound may be a pyruvate derived compound, such as pyruvate or lactate.

[0306] The produced compound may for example be a pyruvate-derived compound. In some embodiments said compound is lactate. In some embodiments, said compound is pyruvate. In some embodiments said cell is capable of assimilating at least 20 % of the carbon provided in cyanide, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or more.P7355PC00

[0307] Also provided herein is a method of engineering a Pseudomonas cell as described herein, comprising:

[0308] i. providing a Pseudomonas cell, optionally a P. putida cell, such as a non- pathogenic Pseudomonas cell, for example a P. putida KT2440 cell or a P. putida SEM11 cell;

[0309] ii. providing one or more nucleic acids as described herein;

[0310] iii. modifying the cell of step i. with said one or more of the nucleic acids of step ii.; thereby obtaining said Pseudomonas cell as described herein.

[0311] The Pseudomonas cell of the present disclosure is capable of degrading cyanide, assimilating cyanide, and propagating in the presence of cyanide, but it may also be capable of degrading, assimilating and propagating in the presence of other compounds, in particular compounds derivable from cyanide, such as other C1 compounds, for example formate as disclosed herein. It follows, that if the cultivation medium and / or waste product comprises other carbon- and / or nitrogen-containing compounds in addition to cyanide, including cyanide degradation products, said compounds may also be used as substrates for cell growth or enzymatic reactions of the cell.

[0312] £H

[0313] It may be preferable to adjust the pH of the cultivation medium to improve cyanide assimilation. The present inventors have realized that cyanide assimilation may be improved by cultivating and propagating the Pseudomonas cell in cultivation medium with a pH that is above neutral.

[0314] In some embodiments, the pH of said cultivation medium is maintained at and / or is at least 7, such as at least 7.5, such as at least 8, such as at least 8.5, such as at least 9, such as at least 9.5, such as at least 10, such as at least 10.5, such as at least 11, such as at least 11.5, or more. In other embodiments, the pH of said fermentation broth is maintained at and / or is at least 7, such as at least 7.5, such as at least 8, such as at least 8.5, such as at least 9, such as at least 9.5, such as at least 10, such as at least 10.5, such as at least 11, such as at least 11.5, or more.P7355PC00

[0315] Cultivation medium and co-feeding

[0316] The cultivation medium used in the methods described herein may be any cultivation medium allowing the Pseudomonas cell to grow in the presence of cyanide and in particular to assimilate cyanide as sole nitrogen and / or carbon source.

[0317] The concentration of cyanide in the cultivation medium may be at least 2.5 mM, for example between 2.5 mM and 10 mM. In some embodiments, said cultivation medium comprises at least 2.5 mM cyanide, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM cyanide, or more.

[0318] In preferred embodiments, the cultivation medium does not comprise serine and / or a source of serine enabling the Pseudomonas cell to escape the engineered coupling of serine production with assimilation of cyanide. Not having serine in the cultivation medium and thus not supplying the cell with this essential amino acid forces the cell to produce serine via conversion of 5,10-methylene-THF to serine over glycine. 5,10-methylene-THF may be produced by the cell upon assimilating cyanide, such as upon conversion of cyanide into formate and ammonia and further converting said formate into 5,10-methylene-THF, as explained herein above. The coupling of serine synthesis to synthesis of 5,10-methylene-THF, forces the assimilation of formate and thus cyanide, since this assimilation is the only way for the cell to acquire both 5,10-methylene-THF and the essential amino acid serine, when cultivated in a serine-free cultivation medium.

[0319] With respect to the methods described herein, for example herein above, co-feeding of one or more substrates may be useful. For example, co-feeding of cyanide with one or more additional carbon and / or nitrogen sources may increase biomass accumulation relative to feeding of cyanide alone. Co-feeding of cyanide with one or more additional carbon and / or nitrogen sources may increase product formation relative to feeding of cyanide alone.

[0320] In some embodiments, said cultivation medium comprises cyanide and at least one other co-substrate, such as glucose, methanol, acetate, pyruvate or glycerol. Thus, in some embodiments, the cultivation medium may comprise cyanide and glucose. In further other embodiments, the cultivation medium may comprise cyanide andP7355PC00

[0321] methanol. In other embodiments the cultivation medium may comprise cyanide and acetate. In further other embodiments, the cultivation medium may comprise cyanide and pyruvate. In further some embodiments, the cultivation medium may comprise cyanide and glycerol. In some embodiments, the cultivation medium may comprise cyanide and formate. The cultivation medium may comprise cyanide and more than one additional co-substrate. As an example, the cultivation medium may comprise cyanide, glucose and glycerol. As an alternative example, the cultivation medium may comprise cyanide, acetate and glycerol.

[0322] In some embodiments, said cultivation medium comprises another nitrogen source than cyanide. In other embodiments, said cultivation medium comprises cyanide and at least one more nitrogen source. In other words, the cultivation medium may comprise two or more nitrogen sources, wherein at least one of said nitrogen sources is cyanide. Examples of nitrogen sources include ammonia / ammonium, such as a salt thereof.

[0323] Items

[0324] 1. A Pseudomonas cell comprising a nucleic acid encoding a cyanidase (EC 3.5.5.1).

[0325] 2. A Pseudomonas cell expressing a heterologous cyanidase (EC 3.5.5.1).

[0326] 3. A Pseudomonas cell capable of using cyanide as a carbon- and nitrogensource, said cell comprising a nucleic acid encoding a cyanidase (EC 3.5.5.1) capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+).

[0327] 4. A Pseudomonas cell capable of converting cyanide to pyruvate via formation of formate and serine, said cell comprising a nucleic acid encoding a cyanidase (EC 3.5.5.1) capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+).

[0328] 5. A Pseudomonas cell comprising a nucleic acid encoding a heterologous L- serine aldolase capable of converting glycine and formaldehyde into serine, such as an L-serine aldolase from Escherichia, such as from Escherichia coli,P7355PC00

[0329] for exampleEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), or functional variants thereof having at least 70% identity thereto.

[0330] 6. The Pseudomonas cell according to any one of the preceding items, wherein said cell is able to utilize cyanide as sole nitrogen source for growth.

[0331] 7. The Pseudomonas cell according to any one of the preceding items, wherein said cell is able to utilize cyanide as a carbon source for the generation of pyruvate via formation of formate and serine.

[0332] 8. The Pseudomonas cell according to any one of the preceding items, wherein said cyanidase is a heterologous cyanidase.

[0333] 9. The Pseudomonas cell according to any one of the preceding items, wherein said cyanidase is a cyanidase from Stutzerimonas stutzeri (formerly known as Pseudomonas stutzeri), such as from Stutzerimonas stutzeri AK61 (formerly known as Pseudomonas stutzeri AK61), for example CynDstut (SEQ ID NO: 1), or a cyanidase from Bacillus pumilus, such as CynDpum (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity to SEQ ID NO: 1 or SEQ ID NO: 2, respectively, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of the cyanidase as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0334] 10. The Pseudomonas cell according to any one of the preceding items, wherein said cell is an isolated cell.

[0335] 11. The Pseudomonas cell according to any one of the preceding items, wherein said Pseudomonas cell is a P. putida cell, such as a non-pathogenic Pseudomonas cell, for example a P. putida KT2440 cell or a P. putida SEM11 cell.P7355PC00

[0336] 12. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of assimilating at least 20 % of the carbon provided in cyanide, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or more.

[0337] 13. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of assimilating at least 20 % of the nitrogen provided in cyanide, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or more.

[0338] 14. The Pseudomonas cell according to any one of the preceding items, wherein said cell expresses, such as overexpresses, the protein encoded by said nucleic acid.

[0339] 15. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises a modification, such as a mutation, rendering said cell unable to synthesize serine by one or more endogenous enzymes.

[0340] 16. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises a modification resulting in partial or total inactivation of an endogenous SerABC pathway.

[0341] 17. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95),

[0342] wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous D-3-phosphoglycerate dehydrogenase, optionally wherein said D-3-phosphoglycerate dehydrogenase is SerA (SEQ ID NO: 15), or a functional variant thereof having at least 70% identity thereto, or aP7355PC00

[0343] functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of SerA as set forth in SEQ ID NO: 15,

[0344] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerA or a functional variant thereof having at least 70% identity thereto, such as serA (SEQ ID NO: 14), or a homologue thereof having at least 70% identity thereto.

[0345] 18. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous phosphoserine phosphatase (EC 3.1.3.3) wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine phosphatase, optionally wherein said phosphoserine phosphatase is SerB (SEQ ID NO: 66), or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of SerB as set forth in SEQ ID NO: 66,

[0346] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerB or a functional variant thereof having at least 70% identity thereto, such as serB (SEQ ID NO: 65) or a homologue thereof having at least 70% identity thereto.

[0347] 19. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous phosphoserine aminotransferase (EC 2.6.1.52) wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine aminotransferase, optionally wherein said phosphoserine aminotransferase is SerC (SEQ ID NO: 68) or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50,P7355PC00

[0348] such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of SerC as set forth in SEQ ID NO: 68,

[0349] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerC or a functional variant thereof having at least 70% identity thereto, such as serC (SEQ ID NO: 67) or a homologue thereof having at least 70% identity thereto.

[0350] 20. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous glycine cleavage system (GCS), wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous GCS.

[0351] 21. The Pseudomonas cell according to any one of the preceding items, wherein said endogenous GCS comprises or consists of a dihydrolipoyl dehydrogenase (LpdG; EC 1.8.1.4), a GCS protein H (GcvH), a glycine dehydrogenase (GcvP; EC 1.4.4.2) and an aminomethyltransferase (GcvT; EC 2.1.2.10).

[0352] 22. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises at least one endogenous GCS operon comprises or consists of one or more nucleic acids encoding a GCS protein H (GcvH), a glycine dehydrogenase (GcvP; EC 1.4.4.2) and an aminomethyltransferase (GcvT; EC 2.1.2.10).

[0353] 23. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises two endogenous GCS operons comprising or consisting of: i. a first operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-l (SEQ ID NO: 16), GcvP-l (SEQ ID NO: 17) and GcvT-l (SEQ ID NO: 18); and / or

[0354] ii. a second operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-ll (SEQ ID NO: 19), GcvP-ll (SEQ ID NO: 20) and GcvT-ll (SEQ ID NO: 21),P7355PC00

[0355] or functional variants thereof having at least 70% identity to thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of GcvH-l (SEQ ID NO: 16), GcvP-l (SEQ ID NO: 17) and GcvT-l (SEQ ID NO: 18); GcvH-ll (SEQ ID NO: 19), GcvP-ll (SEQ ID NO: 20) orGcvT-ll (SEQ ID NO: 21).

[0356] 24. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises a modification resulting in partial or total inactivation of said at least one endogenous GCS operon, for example said two endogenous GCS operons,

[0357] optionally wherein said modification comprises or consists of a mutation in or a deletion of at least a part of gcvTHP-l and / or gcvTHP-ll,

[0358] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of gcvTHP-l and gcvTHP-ll.

[0359] 25. The Pseudomonas cell according to any one of the preceding items, wherein the modification resulting in partial or total inactivation of said at least one endogenous GCS operon comprises or consists of a mutation in or a deletion of at least a part of a first nucleic acid encoding GcvH-l, GcvP-l and GcvT-l, or functional variants thereof having at least 70% identity to thereto,

[0360] and at least a corresponding part of a second nucleic acid encoding GcvH-ll, GcvP-ll and GcvT-ll, or functional variants thereof having at least 70% identity to thereto.

[0361] 26. The Pseudomonas cell according to any one of the preceding items, wherein said cell is formatotrophic.

[0362] 27. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of converting formate into 5,10-methylene-THF.P7355PC00

[0363] 28. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises:

[0364] i. a nucleic acid encoding a formate-THF ligase (FtfL; EC 6.3.4.3), such as MeFtfL (SEQ ID NO: 8);

[0365] ii. a nucleic acid encoding a methenyl-THF cyclohydrolase (Fch; EC 3.5.4.9), such as MeFch (SEQ ID NO: 9); and / or

[0366] iii. a nucleic acid encoding a methylene-THF dehydrogenase (MdtA; EC 1.5.1.5), such as MeMdtA (SEQ ID NO: 10),

[0367] or functional variants thereof having at least 70 % identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of MeFtfL (SEQ ID NO: 8), MeFch (SEQ ID NO: 9) or MeMdtA (SEQ ID NO: 10), whereby said Pseudomonas cell is capable of converting formate into 5,10- methylene-THF.

[0368] 29. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of catalysing:

[0369] i. hydroxymethylation of glycine with 5,10-methylene-THF into serine by a L- serine hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) or GlyA-ll (SEQ ID NO: 23);

[0370] ii. deamination of said serine into pyruvate by a serine dehydratase / serine deaminase (EC 4.3.1.17), such as TdcG-l (SEQ ID NO: 26), TdcG-ll (SEQ ID NO: 27) orTdcG-lll (SEQ ID NO: 28);

[0371] iii. carboxylation of said pyruvate into oxaloacetate, such as;

[0372] i. by a pyruvate carboxylase (EC 6.4.1.1), such as PycAB comprising or consisting of PycA (SEQ ID NO: 32) and PycB (SEQ ID NO: 33); and / or ii. via conversion of said pyruvate to phosphoenolpyruvate by phosphoenolpyruvate synthetase (EC 2.7.1.40), such as PykA (SEQ ID NO: 36), and conversion of said phosphoenolpyruvate into said oxaloacetate by a phosphoenolpyruvate carboxylase (EC 4.1.1.31), such as Ppc (SEQ ID NO: 38);P7355PC00

[0373] iv. transamination of said oxaloacetate into aspartate by an aspartate aminotransferase (EC 2.6.1.1), such as AspC (SEQ ID NO: 40);

[0374] v. conversion of said aspartate into homoserine, such as via:

[0375] i. conversion of aspartate into 4-phospho-L-aspartate by an aspartate kinase (EC 27.2.4), such as LysC (SEQ ID NO: 42);

[0376] ii. conversion of said 4-phospho-L-aspartate into L-aspartate 4- semialdehyde by an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11), such as Asd (SEQ ID NO: 44); and

[0377] iii. conversion of said L-aspartate 4-semialdehyde into homoserine by a homoserine dehydrogenase (EC 1.1.1.3), such as Hom (SEQ ID NO: 46) or PP_0664 (SEQ ID NO: 47);

[0378] vi. conversion of said homoserine into threonine, such as via:

[0379] i. conversion of homoserine into O-phospho-homoserine by a homoserine kinase (EC 2.7.1.39), such as ThrB (SEQ ID NO: 50); and ii. conversion of said O-phospho-homoserine into threonine by a threonine synthase (EC 4.2.3.1), such as ThrC (SEQ ID NO: 52); and / or

[0380] vii. conversion of said threonine into glycine and acetaldehyde by L-threonine aldolase (EC 4.1.2.5 / 4.1.2.48), such as an L-threonine aldolase from P. putida, for example LtaE (SEQ ID NO: 5),EcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7),

[0381] or functional variants thereof having at least 70 % identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof.

[0382] 30. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises one or more of:

[0383] i. a nucleic acid encoding a L-serine hydroxymethyltransferase (EC 2.1.2.1), such as GlyA-l (SEQ ID NO: 22) or GlyA-ll (SEQ ID NO: 23), for example glyA-l (SEQ ID NO: 24) or glyA-ll (SEQ ID NO: 25);P7355PC00

[0384] ii. a nucleic acid encoding a serine dehydratase / serine deaminase (EC 4.3.1.17), such as TdcG-l (SEQ ID NO: 26), TdcG-ll (SEQ ID NO: 27) or TdcG-lll (SEQ ID NO: 28), for example tdcG-\ (SEQ ID NO: 29), tdcG-ll (SEQ ID NO: 30) or tdcG-lll (SEQ ID NO: 31);

[0385] iii. a nucleic acid encoding a pyruvate carboxylase (EC 6.4.1.1), such as PycAB comprising or consisting of PycA (SEQ ID NO: 32) and PycB (SEQ ID NO: 33), for example pycA (SEQ ID NO: 34) and / or pycB (SEQ ID NO: 35);

[0386] iv. a nucleic acid encoding a phosphoenolpyruvate synthetase (EC 2.7.1.40), such as PykA (SEQ ID NO: 36), for example pykA (SEQ ID NO: 37);

[0387] v. a nucleic acid encoding a phosphoenolpyruvate carboxylase (EC 4.1.1.31), such as Ppc (SEQ ID NO: 38), for example ppc (SEQ ID NO: 39);

[0388] vi. a nucleic acid encoding an aspartate aminotransferase (EC 2.6.1.1), such as AspC (SEQ ID NO: 40), for example aspc (SEQ ID NO: 41); vii. a nucleic acid encoding an aspartate kinase (EC 2.7.2.4), such as LysC (SEQ ID NO: 42), for example lysC (SEQ ID NO: 43);

[0389] viii. a nucleic acid encoding an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11), such as Asd (SEQ ID NO: 44), such as asd (SEQ ID NO: 45); ix. a nucleic acid encoding a homoserine dehydrogenase (EC 1.1.1.3), such as Hom (SEQ ID NO: 46) or PP_0664 (SEQ ID NO: 47), for example hom () (SEQ ID NO: 48) or PP_0664 (SEQ ID NO: 49); x. a nucleic acid encoding a homoserine kinase (EC 2.7.1.39), such as ThrB (SEQ ID NO: 50), such as thrB (SEQ ID NO: 51);

[0390] xi. a nucleic acid encoding a threonine synthase (EC 4.2.3.1), such as ThrC (SEQ ID NO: 52), such as thrC (SEQ ID NO: 53); and / or xii. a nucleic acid encoding an L-threonine aldolase (EC 4.1.2.5 / 4.1.2.48) from P. putida, such as LtaE (SEQ ID NO: 5),EcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), for example ItaE (SEQ ID NO: 71),EcltaE (SEQ ID NO: 69) orEcltaE* (SEQ ID NO: 70), respectively;

[0391] or functional variants or homologues thereof having at least 70 % identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at leastP7355PC00

[0392] 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof.

[0393] 31. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous FAD-dependent glycine / D-amino acid oxidase (EC 1.4.3.19) wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous FAD-dependent glycine / D-amino acid oxidase, optionally wherein said FAD-dependent glycine / D-amino acid oxidase is ThiO (SEQ ID NO: 73) or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least O.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of ThiO (SEQ ID NO: 73),

[0394] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding ThiO or a functional variant thereof having at least 70% identity thereto, such as thiO (SEQ ID NO: 72) or a homologue thereof having at least 70% identity thereto.

[0395] 32. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises, such as overexpresses, a nucleic acid encoding an L- threonine aldolase (EC 4.1.2.5 / 4.1.2.48), such as an L-threonine aldolase from P. putida, for example LtaE (SEQ ID NO: 5), capable of converting threonine into glycine and acetaldehyde and / or serine into glycine and formaldehyde.

[0396] 33. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises, preferably overexpresses, a nucleic acid encoding a heterologous L-serine aldolase capable of converting glycine and formaldehyde into serine, such asEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93,P7355PC00

[0397] at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure ofEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7).

[0398] 34. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises one or more endogenous biofilm-producing adhesins, wherein said cell comprises a modification resulting in partial or total inactivation of said one or more endogenous biofilm-producing adhesins, optionally wherein said one or more endogenous biofilm-producing adhesins is LapA (SEQ ID NO: 74) and / or LapF (SEQ ID NO: 76), or functional variants thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of LapA (SEQ ID NO: 74) or LapF (SEQ ID NO: 76),

[0399] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding LapA and / or LapF or functional variants thereof having at least 70% identity thereto, such as lapA (SEQ ID NO: 75) and / or lapF (SEQ ID NO: 77), or homologues thereof having at least 70% identity thereto.

[0400] 35. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises an endogenous pha cluster,

[0401] wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous pha cluster,

[0402] optionally wherein the endogenous pha cluster comprises or consists of one or more nucleic acids encoding PhaC1 (SEQ ID NO: 54), PhaZ (SEQ ID NO: 55), PhaC2 (SEQ ID NO: 56), PhaD (SEQ ID NO: 57), PhaF (SEQ ID NO: 58) and Phal (SEQ ID NO: 59), or functional variants thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at leastP7355PC00

[0403] 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof.

[0404] 36. The Pseudomonas cell according to any one of the preceding items, wherein said endogenous pha cluster comprises or consists of:

[0405] i. a first operon comprising or consisting of one or more nucleic acids encoding PhaC1, PhaZ and PhaD; and

[0406] ii. a second operon comprising or consisting of one or more nucleic acids encoding PhaF and Phal,

[0407] or functional variants thereof having at least 70% identity thereto or functional variants thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of PhaC1, PhaZ, PhaD, PhaF or Phal.

[0408] 37. The Pseudomonas cell according to any one of the preceding items, wherein said endogenous pha cluster is partially or totally inactivated, such as wherein one or more of PhaC1, PhaC2, PhaZ, PhaD, PhaF and Phal, or a functional variant thereof having at least 70% identity thereto are partially or totally inactivated, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of PhaC1, PhaC2, PhaZ, PhaD, PhaF and Phal.

[0409] 38. The Pseudomonas cell according to any one of the preceding items, wherein said modification resulting in partial or total inactivation of said endogenous pha cluster comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding one or more of PhaC1, PhaC2, PhaZ, PhaD, PhaF and Phal, preferably a mutation or a deletion of a nucleic acid encoding PhaC1, PhaC2, PhaZ, PhaD, and / or a nucleic acid encoding PhaF and Phal, or functional variants thereof having at least 70% identity thereto, or a functionalP7355PC00

[0410] variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of PhaC1, PhaC2, PhaZ, PhaD, PhaF and Phal.

[0411] 39. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of producing a compound, such as a pyruvate-derived compound, optionally wherein said compound is pyruvate or lactate, or a mixture of pyruvate and lactate.

[0412] 40. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of producing pyruvate.

[0413] 41. The Pseudomonas cell according to any one of the preceding items, wherein said cell is capable of producing lactate,

[0414] optionally wherein said cell comprises, such as overexpresses, a nucleic acid encoding a lactate dehydrogenase (LDH; EC 1.1.1.28), such as BtLdhA(N108G) (SEQ ID NO: 60) or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of BtLdhA(N108G) (SEQ ID NO: 60).

[0415] 42. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises at least one modification resulting in partial or total inactivation of an endogenous lactate dehydrogenase, preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding said lactate dehydrogenase.

[0416] 43. The Pseudomonas cell according to any one of the preceding items, wherein said cell comprises at least one modification resulting in partial or totalP7355PC00

[0417] inactivation of a L-lactate dehydrogenase (EC 1.1.2.3), such as LldD (SEQ ID NO: 61), and a D-lactate dehydrogenase 2 (EC 1.1.5.12), such as Dld2 (SEQ ID NO: 62), or functional variants thereof having at least 70% identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof,

[0418] preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding LldD and Dld2 or functional variants thereof having at least 70% identity thereto, such as IldD (SEQ ID NO: 63) and dld2 (SEQ ID NO: 64) or homologues thereof having at least 70% identity thereto.

[0419] 44. The Pseudomonas cell according to any one of the preceding items, wherein said nucleic acid is genomically integrated in the genome of said cell or is present on a vector within said cell, such as on a plasmid.

[0420] 45. The Pseudomonas cell according to any one of the preceding items, wherein said nucleic acid comprises an inducible promoter and / or constitutive promoter.

[0421] 46. The Pseudomonas cell according to any one of the preceding items, wherein said nucleic acid is codon-optimised, such as codon-optimised for P. putida.

[0422] 47. The Pseudomonas cell according to any one of the preceding items, wherein said nucleic acid encoding:

[0423] i. CynDstut (SEQ ID NO: 1) comprises or consists of cynDstut (SEQ ID NO: 3); and / or

[0424] ii. CynDpum (SEQ ID NO: 2) comprises or consists of cynDpum (SEQ ID NO: 4);

[0425] or homologues thereof having at least 70% identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93,P7355PC00

[0426] at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of thereof.

[0427] 48. The Pseudomonas cell according to any one of the preceding items, wherein the cell is a recombinant cell.

[0428] 49. A method of producing a compound, such as a pyruvate-derived compound, comprising:

[0429] i. providing a Pseudomonas cell as defined in any one of items 1 to 48; ii. cultivating and optionally propagating said cell in a cultivation medium, said cultivation medium comprising cyanide,

[0430] thereby obtaining a fermentation broth and / or cell comprising said compound;

[0431] iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;

[0432] whereby said compound and / or said fermentation broth comprising said compound is produced.

[0433] 50. The method according to any one of the preceding items, wherein said compound, such as said pyruvate-derived compound, is lactate, optionally wherein said cell comprises, such as overexpresses, a nucleic acid encoding a lactate dehydrogenase (LDH; EC 1.1.1.28), such as BtLdhA(N108G)( (SEQ ID NO: 60) or a functional variant thereof having at least 70% identity thereto, or a functional variant having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof.

[0434] 51. The method according to any one of the preceding items, wherein said compound, such as said pyruvate-derived compound, is lactate, wherein said cell comprises at least one modification resulting in partial or total inactivation of a L-lactate dehydrogenase (EC 1.1.2.3), such as LldD (SEQ ID NO: 61), and a D-lactate dehydrogenase 2 (EC 1.1.5.12), such as Dld2 (SEQ ID NO: 62), orP7355PC00

[0435] functional variants thereof having at least 70% identity thereto, or functional variants having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure thereof, preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding LldD and Dld2 or functional variants thereof having at least 70% identity thereto, such as IldD (SEQ ID NO: 63) and dld2 (SEQ ID NO: 64) or homologues thereof having at least 70% identity thereto.

[0436] 52. The method according to any one of the preceding items, wherein said compound, such as said pyruvate-derived compound, is pyruvate or lactate.

[0437] 53. A method of detoxifying a waste product comprising cyanide, comprising:

[0438] i. providing a Pseudomonas cell as defined in any one of items 1 to 48; ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide,

[0439] thereby degrading said cyanide and optionally obtaining cell biomass; iii. optionally recovering said cell biomass;

[0440] whereby said waste product is detoxified.

[0441] 54. A method of valorising of a waste product comprising cyanide into a compound, comprising:

[0442] i. providing a Pseudomonas cell as defined in any one of items 1 to 48; ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide,

[0443] thereby obtaining a fermentation broth and / or cell comprising said compound;

[0444] iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;P7355PC00

[0445] whereby said waste product comprising cyanide is valorised by production of said compound and / or said fermentation broth comprising said compound.

[0446] 55. The method according to any one of the preceding items, wherein said compound is lactate and said cell is as defined in any one of items 1 to 48 or wherein said compound is pyruvate.

[0447] 56. A method of engineering a Pseudomonas cell as defined in any one of items 1 to 48, comprising:

[0448] i. providing a Pseudomonas cell, optionally a P. putida cell, such as a non- pathogenic Pseudomonas cell, for example a P. putida KT2440 cell or a P. putida SEM11 cell;

[0449] ii. providing one or more nucleic acids as defined in any one of items 1 to 48;

[0450] iii. modifying the cell of step i. with said one or more of the nucleic acids of step ii.;

[0451] thereby obtaining said Pseudomonas cell as defined in any one of items 1 to 48.

[0452] 57. A method of propagating a Pseudomonas cell in the presence of cyanide, comprising:

[0453] i. providing a Pseudomonas cell as defined in any one of items 1 to 48; ii. propagating said cell in a cultivation medium, said cultivation medium comprising cyanide,

[0454] thereby obtaining a fermentation broth comprising said cell and / or biomass of said cell;

[0455] iii. optionally recovering said fermentation broth and / or said biomass.

[0456] 58. The method according to any one of the preceding items, wherein said cultivation medium comprises at least 2.5 mM cyanide, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM cyanide, or more.P7355PC00

[0457] 59. The method according to any one of the preceding items, wherein the pH of said cultivation medium and / or fermentation broth is maintained at or is at least 7, such as at least 7.5, such as at least 8, such as at least 8.5, such as at least 9, such as at least 9.5, such as at least 10, such as at least 10.5, such as at least 11, such as at least 11.5, or more.

[0458] 60. The method according to any one of the preceding items, wherein said cultivation medium comprises cyanide and at least one other co-substrate, such as glucose, methanol, acetate, formate, pyruvate or glycerol.

[0459] 61. The method according to any one of the preceding items, wherein said cultivation medium does not comprise serine.

[0460] 62. The method according to any one of the preceding items, wherein said cultivation medium comprises another nitrogen source than cyanide.

[0461] 63. The method according to any one of the preceding items, wherein said cultivation medium comprises cyanide and at least one more nitrogen source.

[0462] Examples

[0463] Example 1: Materials and methods

[0464] Bacterial strains, medium composition and culture conditions. All bacterial strains and plasmids are listed in Table 2 and Table 3, respectively. E. coli DH5a p / r (Platt et al. 2000) was used as cloning host, while the reduced-genome P. putida strain SEM11 (Platt et al. 2000) was selected for quantitative physiology and engineering purposes unless indicated otherwise. Lysogeny broth (LB) complex medium (containing 10 g L"1tryptone, 5 g L"1yeast extract, and 10 g L"1NaCI) and de Bont minimal (DBM) medium were used for all cultivations (Hartmans et al. 1989). DBM medium contained 3.88 g L"1K2HPO4, 1.63 g L“1NaH2PO4, 2 g L“1(NH4)2SO4, and 0.1 g L“1MgCl2·6H2O with the initial pH adjusted at 7.0 and supplemented with a trace elements solution [10 mg L"1ethylenediaminetetraacetic acid (EDTA), 2 mg L"1ZnSO4·7H2O, 1 mg L"1CaCl2·2H2O, 5 mg L“1FeSO4·7H2O, 0.2 mg L“1Na2MoO4·2H2O, 0.2 mg L“1CuSO4·5H2O, 0.4 mg L“1CoCl2·6H2O, and 1 mg L"1MnCl2·2H2O] (Nikel et al. 2016). When needed, kanamycin (Km), gentamicin (Gm), streptomycin (Sm) and ampicilin (Amp) were supplied at 50 pg mL-1, 10 pug mL-1, 100 pg mL-1and 100 µg mL-1respectively.P7355PC00

[0465] Overnight cultures in LB medium were diluted 1 / 100 to inoculate a 5-mL preculture of DBM medium with 20 mM glucose in a 50-mL culture tube and incubated at 30°C and 250 rpm for ca. 18 h. This overnight culture was washed with DBM medium without any carbon source prior to the inoculation of the main culture in 96-well microtiter plates with the appropriate carbon source(s) as described in the text. All growth assays were performed in the presence of antibiotics, unless the strain did not harbor any plasmid. For cultivations in 96-well microtiter plates, 150 pL of a cell suspension at an ODeoo of 0.05 were incubated in an Epoch2 microtiter plate reader (BioTek Instruments Inc.; Winooski, VT, USA) with 50 pL of mineral oil to prevent evaporation. Measurements obtained with microtiter plate readers were calibrated against a tabletop spectrophotometer. The specific growth rate (p) and, when relevant, the extension of the lag phase ( ) were calculated using QurvE (www.qurveanalysis.com) by performing a smooth spline fit on the growth data (Wirth et al. 2023). For production experiments, baffled 250 mL Erlenmeyer flasks were inoculated with 50 mL at initial ODeoo 0.05, and cultivated at 30°C and 200 rpm.

[0466] Construction of (deletion) plasmids. The suicide plasmids and overexpression plasmids noted in Table 3 were constructed using USER cloning. For deletion plasmids, DNA fragments, consisting of ca. 500-bp upstream and downstream regions around the locus to be eliminated, were amplified with Phusion U Hot Start™ DNA polymerase (ThermoFisher Scientific Co.) using uracil-containing primers. The pGNW2 backbone (Wirth et al. 2020) was digested with Dpn\ prior to mixing 1 pL of Dpn\-treated vector with 100 ng of each PCR fragment and 1 pL of USER™ enzyme (New England BioLabs) in a final volume of 10 pL. The reaction was incubated for 30 min at 37°C, followed by a temperature decrease over 3 min (from 28°C to 20°C, 1°C per step) and a final incubation step at 10°C for at least 10 min. Finally, chemically-competent E. coli DH5a p / r cells were transformed via heat shock with 5 pL of the USER mix; upon recovery, the cell suspension was plated onto selective LB medium agar plates containing the corresponding antibiotic.

[0467] Two homologues of cyanide dihydratases (cynD) from Bacillus pumilus (cynDpum', AF492815.1) and Pseudomonas stutzeri AK61 (cynDstut; AIY29195.1) were codon optimized for Pseudomonas putida (Benchling. 2018. Biology software. Benchling, San Francisco, CA.) and synthetized by Twist Bioscience (San Francisco, California, USA)P7355PC00

[0468] (see Table 3). These were cloned in overexpression plasmids. Construction of overexpression plasmids were cloned in a similar fashion using the same cloning method.

[0469] Table 3. Plasmids.

[0470] Reference Plasmid Relevant characteristics3

[0471] or source Suicide vector used for deletions in Gram- Wirth et al. pGNW2 negative bacteria; oriT, traJ, lacZo., conditional

[0472] 2020

[0473] RK6 replication origin, PEM7^msfGFP\ KmR

[0474]

[0475] Derivative of vector pGNW2 carrying homology Turlin et al. pGNW2 AserA

[0476] regions to delete serA (PP_5155) KmR2022 Derivative of vector pGNW2 carrying homology

[0477] regions to integrate P4*^mtdA-fch-ftfL module pGNW2 p / 7a:: P4*- from M. extorquens AM1 into the native deleting This work mtdA-fch-ftfL

[0478] pha (PP_5002-8) locus based on a previous work’s vector harboring a P4 promoter; KmRpGNW2- AgcvTHP- Derivative of vector pGNW2 carrying homology Turlin et al. I regions to delete gcvTHP-l (PP_0986-9) KmR2022 pGNW2 Agcv77- / P- Derivative of vector pGNW2 carrying homology Turlin et al. II regions to delete gcvTHP-ll (PP_5192-4, KmR2022

[0479] Derivative of vector pGNW2 carrying homology Turlin et al. pGNW2 Af / 7 / O

[0480] regions to delete thiO (PP_0612) KmR2022 Derivative of vector pGNW2 carrying homology Calero et al. pGNW2 A / apA

[0481] regions to delete lapA (PP_0168, KmR2022 Derivative of vector pGNW2 carrying homology

[0482] pGNW2 A / apF This work regions to delete lapF (PP_0806 KmR

[0483] Derivative of vector pGNW2 carrying homology

[0484] pGNW2- regions to insert promoter P14G with BCD10

[0485] This work P / rae:: Pi4G(BCD10) instead of endogenous promoter of ItaE

[0486] (PP_023i KmR

[0487] Derivative of vector pGNW2 carrying homology

[0488] regions to insert promoter Ptrc with SEVA

[0489] pGNW2- canonical RBS (AGGAGGAAAAACAT) instead This work P / faE:: Ptrc(RBS)

[0490] of endogenous promoter of ItaE (PP_023i

[0491] KmRP7355PC00

[0492] Derivative of vector pGNW2 carrying homology

[0493] pGNW2- regions to insert promoter P14G with BCD10

[0494] This work Py / ay:: Pi4G(BCD10) instead of endogenous promoter of yiaY

[0495] (PP_2682)', KmR

[0496] Derivative of vector pGNW2 carrying homology

[0497] pGNW2 A / - regions to delete IldD (PP 4736) and dld2

[0498] dld2

[0499] (PP_4737)\ KmR

[0500]

[0501] Helper plasmid for gene deletions;

[0502] Volke et al. pQURE6 H conditionally-replicating vector carrying

[0503] 2020 XylS / Pm— > / -Sce / and Pi4g(BCD2)^mRFP; GmR

[0504]

[0505] Control vector derivative of pSEVA221

[0506] harboring a P G promoter and the translational

[0507] pS221 eV This work coupler BCD10 followed by a start and stop codon (ATGTAA); oriT oriV RK2); KmR

[0508] Derivative vector of pSEVA221, harboring a

[0509] Pi4G promoter and a bicistronic design RBS pS221 ■ cynDpum This work (BCD10) expressing cynD from Bacillus

[0510] pumilus C1 (cynDpum ', oriT oriV(RK2); KmR

[0511] Derivative vector of pSEVA221, harboring a

[0512] Pi4g promoter and a bicistronic design RBS pS221 ■ cynDstut This work (BCD10) expressing cynD from Pseudomonas

[0513] stutzeri AK61 (cynDstut)', oriT oriV(RK2); KmR

[0514] Control vector derivative of pSEVA621

[0025] harboring a Ptrcpromoter and the canonical

[0515] Turlin etal. pS621 eV SEVA ribosome binding site (RBS) followed by

[0516] 2023 a start and stop codon (ATGTAA); oriT

[0517] or / V(RK2); GmR

[0518] Derivative of vector pSEVA621 harboring a Ptlc

[0519] promoter and the canonical SEVA ribosome pS621 ■EcltaE This work binding site (RBSHEc / taE; oriT oriV( RK2);

[0520] GmR

[0521] Derivative of vector pSEVA621 harboring a Ptlc

[0522] pS621 ■EcltaE* promoter and the canonical SEVA ribosome

[0523] This work (C188Y) binding site (RBS)->EcltaE* (C188Y); oriT

[0524] or / V(RK2); G

[0525]

[0526] mRP7355PC00

[0527] Martinez- Tn5 delivery plasmid; conditional RK6

[0528] pBAMD1-4 Garcia eta / .

[0529] replication origin; SmR, AmpR

[0530] 2014 pBAMD1-4-Pi4G Derivative of vector pBAMD1-4 carrying a

[0531] (BCD10)^ PI4G(BCD10) — > cynDpummodule from B. This work cynDpum pumilus', SmR, AmpR

[0532] pBAMD1-4-Pi4G Derivative of vector pBAMD1-4 carrying a

[0533] (BCD10)^ PI4G(BCD10) — > cynDstut module from P. stutzeri This work cynDstut AK61; SmR, AmpR

[0534] Derivative of vector pSEVA331 harboring a constitutive expression of engineered IdhA pS331e7dM*

[0535] (N109G) from Bos taurus (encoding

[0536] BtLdhA(N108G) of SEQ ID NO: 60); KmR

[0537] aAntibiotic markers: Gm, gentamicin; Km, kanamycin; Sm, streptomycin; Amp, ampicilin.

[0538] Construction of mutant P. putida strains. The corresponding suicide pGNW2-derivative plasmid was delivered into the cells by triparental conjugation with the corresponding DH5a " / .pir harboring the specific suicide plasmid, the P. putida strain of interest, and the E. coli helper strain E. coli HB101 carrying plasmid pRK2013 (Figurski et al. 1979). The three strains were incubated in LB plates for over 5 h at 30°C and subsequently plated in LB plates supplemented with the antibiotic of interest and Irgasan. Positive co-integration events were further transformed with pQURE6 H (Table 3), a conditionally-replicative plasmid bearing the meganuclease gene / -Sce / (Volke et al. 2021). I-Scel cuts pGNW2 co-integrants within the chromosome, thus forcing a second homologous recombination event. This was performed by electroporating 50 ng of plasmid DNA into 50 pL of freshly-prepared electrocompetent P. putida cells, previously washed three times with 300 mM sucrose. Electroporation was performed with a Gene Pulser XCell (Bio-Rad) set to 2.5 kV, 25 F capacitance and 200 Q resistance in a 2-mm gap cuvette. Cells were recovered in 1 mL of LB medium supplemented with 2 mM of 3-methylbenzoate (3-mBz) for at least 3 h at 30°C and plated onto LB medium agar containing the corresponding antibiotic(s) and 1 mM 3-mBz to induce both plasmid replication and l-Scel expression. Positive clones were identified by colony PCR, verified by DNA sequencing, and cured from the resolving plasmid by serial dilution under non-selective conditions.P7355PC00

[0539] Table 2. Bacterial strains.

[0540] Reference Strain Relevant characteristics

[0541] or source Escherichia coli

[0542] Cloning host; F“ A-endA1 glnX44(AS) thiE1

[0543] recA 1 relA 1 spoT 1 gyrA96(NalR) rfbC1 deoR Platt et al. DH5a Ap / r

[0544] nupG < 80(lacZAM15) A(argF-lac)U169 2000 hsdR17(n<~ mF), Ap / rlysogen

[0545]

[0546] Figurski Conjugative helper strain; hsdR-M+, proA2,

[0547] and HB101 pRK2013 leuB6, thi-1, recA harbouring plasmid

[0548] Helinski PRK2013, KanR

[0549] 1979 Blattner et K-12 MG1655 Wild-type strain; F A rph-1

[0550] al. 1997 Pseudomonas putida

[0551] Reduced-genome derivative of P. putida

[0552] Wirth et al. SEM11 KT2440 (Nikel et al. 2016) and

[0553] 2023 EM42(Martinez-Garcia et al. 2014).

[0554] Derivative of P. putida SEM11 AserA,

[0555] AgcvTHP-l (PP_0986-9) AgcvTHP-ll

[0556] ASGG-Ci This work (PP_5192-4) pha(PP_5002-8) P4*^mtdA- fch-ftfL (Turlin et al. 2022)

[0557] ASGG-Ci Derivative of P. putida ASGG-Ci,

[0558] This work P / rae:: PI4G(BCD 10) PI4G(BCD 10)^ltaE

[0559]

[0560] ASGG-Ci Derivative of P. putida ASGG-Ci,

[0561] This work P / raE:: Pfrc(RBS) Pfrc(RBS)^ / taE

[0562]

[0563] Derivative of P. putida ASGG- Ci, P / faE:: Pfrc

[0564] STC0 (SEVA RBS) AthiO (PP_0612) AiapA This work (PP_0168) AiapF (PP_0806)

[0565] Derivative of P. putida STC0, AlidD

[0566] STC0 AlldD-dld2 This work (PP_4736) Adld2 (PP_4737)P7355PC00

[0567] Derivative of P. putida STCO, MldD-dld2, with

[0568] STCO lldD-dld2

[0569] cynDstut from Pseudomonas stutzeri AK61 This work g CynDstut

[0570] randomly integrated using pBAMD1-4 system

[0571] Derivative of P. putida STCO,

[0572] STC1 This work Py / a / : PI4G(BCD10)

[0573]

[0574] Chemicals and reagents. Chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise indicated, and oligonucleotides were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA). DNA sequencing was performed at Eurofins Genomics (Ebersberg, Germany). All primers were designed by standard methods. PCR reactions were performed using Phusion U Hot Start™ DNA polymerase, purchased from ThermoFisher Scientific Co. (Waltham, MA, USA). The commercial One Taq™ master mix from New England BioLabs (Ipswich, MA, USA) was used for colony PCRs. 4-Hydroxy-2-oxobutanoate (HOB, in its lactone form) was purchased from Synthenova (France).

[0575] Random Tn5-genomic integration. Plasmid pBAMD1-4-Pi4c(BCD10)^cynD were constructed using USER protocol as previously described. The plasmid was inserted into P. putida STCO AlldD-dld2 by tri-parental mating. The recipient strain, helper strain E. coli HB101 harbouring pRK600 and the E. coli donor strains were mixed and plated on a warm LB plate and incubated at 30°C overnight. The next day, the biomass was streaked on a DBM medium plate containing 20 mM glucose, 5 mM potassium cyanide (KCN) and streptomycin and incubated at 30°C to select for P. putida strains able to detoxify KCN, produce formate and subsequently complement the L-serine auxotrophy. The growth phenotype of individual colonies was confirmed by performing another passage in selective condition.

[0576] Analytical procedures. Extracellular concentrations of formate, lactate, glucose and acetate were measured in culture supernatants. Supernatants were analyzed in a high-performance liquid chromatography instrument (UltiMate™ 3000 Basic Automated System, ThermoFisher Scientific Co.) equipped with an Aminex HPX-87P column (BioRad) and a refractive index detector SHodex RI-101 (Showa Denko America Inc., NY, USA). Specific consumption rates for acetate (qA), formate (qp) and glucose (qc), as well as lactate secretion rate (q were determined with the following equation:

[0577] I AS

[0578] q

[0579]

[0580] s =X~biP7355PC00

[0581] Where qs corresponds to the biomass-specific substrate consumption rate (mmol gcDw’1h-1), Xis the average biomass concentration between two sampling time points (gcDw L"1), Z1S is the difference in substrate concentration between two sampling time points (mM) and At refers to the time between two sampling points (h). The rate values reported are an average of the values determined individually for three biological replicates. Cell-dry-weight (CDW) values were obtained from ODeoo measurements by applying a correlation factor reported previously to be 0.533*OD600 gCDW L⁻1(Rodriguez et al. 2018).

[0582] Indirect In vitro detection of L-lactate via ABTS and lactate oxidase. The lactate oxidase (LOX) from Aerococcus viridans was purchased from Sigma-Aldrich (cat. # L9795). LOX activity on lactate was assayed coupling the reaction with the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) catalysed by a commercial type II peroxidase from horseradish (HRP) purchased from Sigma-Aldrich. The assay was prepared by adding 10 pL of culture supernatant (diluted at least 1 / 1000), 2 mM ABTS, 50 mM sodium phosphate buffer (pH = 7.0), 0.2 U mL-1 LOX and 1 U mL-1 peroxidase in a final volume of 200 pL. The corresponding blanks were prepared by omitting either LOX or sample from the mixture, and a positive control was included replacing the culture supernatant with commercial L-lactate (2 mM final concentration). The samples were incubated at room temperature and the feasibility of the reaction was confirmed qualitatively observing a shift of colour from transparent to dark green. The absorbance at 734 nm (A734) was monitored in 95 s-intervals at 30°C in an EPOCH2 microplate reader (BioTek Instruments, Winooski, VT, USA). The L-lactate concentrations were identified by determining the linear range concentrationspecific using commercial lactate and decreasing concentrations from 2 mM to 0.04 mM (final concentration). Calibrations curves were identified and used for the samples, which were diluted at least 1 / 1000 times.

[0583] Statistical analysis. Data analysis was performed using Prism 9.0.2 (GraphPad Software Inc.; San Diego, CA, USA). All reported values are indicated as averages ± standard deviation of at least three independent biological replicates as specified in the legend of the corresponding figures. Considering that most negative controls (i.e., strains transformed with empty vectors) did not show any growth, enzyme variants that restored methylotrophic growth were considered to be statistically significant (Ferreira et al. 2007).P7355PC00

[0584] Example 2: Cyanide detoxification via cyanide dihydratases in P. putida

[0585] In order to permit P. putida to detoxify cyanide, we expressed heterologously two cyanide dihydratases from Pseudomonas stutzeri AK61 (GenBank ID D82961.1) and Bacillus pumilus (GenBank ID AF492815.1), hereafter cynDstut (CynDstut; SEQ ID NO: 1 encoded by SEQ ID NO: 3) and cynDpum, (CynDpum; SEQ ID NO: 2 encoded by SEQ ID NO: 4), respectively (Figure 1). These cyanide dihydratases have been previously reported to break cyanide into formate and ammonia (Figure 2A). We overexpressed constitutively cynDstut and cynDpum, from plasmid pSEVA221 and transformed them into strain SEM11, with an empty vector (eV) as a negative control (Figure 2B). When we tested these strains in DBM medium supplemented with 20 mM glucose and increasing concentration of potassium cyanide (KCN), we observed that the expression of this detoxification system permitted growth up to 5 mM KCN (Figure 2C). The negative control harbouring an empty vector plasmid did not grow at any concentrations tested (KCN > 1.25 mM).

[0586] To test if the limited toxicity is caused by the detoxification of cyanide, we tested the same conditions in a DBM nitrogen-free medium (termed -N). Thus, growth can only be promoted when degradation of cyanide to formate and ammonia occurs. The latter contributes to growth, thereby coupling nitrogen assimilation via NH3 to growth. We tested concentrations from 0.16 mM to 10 mM KCN in DBM nitrogen-free medium and 20 mM glucose (Fig. 2D). As expected, the detoxification of cyanide led to growth in a concentration-dependent manner. The absence of KCN did not result in increased cell density. Thus, heterologous expression of cyanide dihydratases leads to cyanide detoxification, which permits growth at higher KCN concentrations but also, enables growth-coupling via the assimilation of its degradation products, ammonia and formate.

[0587] Example 3: Cyanide-derived formate assimilation via tetrahydrofolate in P. putida To enable growth on formate, the second product of cyanide detoxification, via cyanide and CynDs, we constructed a growth-coupled selection strain that requires its assimilation to replenish the methylated tetrahydrofolate (THF) pool and the essential amino acid serine. In methylotrophic bacteria, such as Methylobacterium extorquens AM1, carbon can be assimilated through 5,10-methylene-THF. In this case, carbon is assimilated into glycine, forming serine. These activities are catalysed by FtfL (formate-THF ligase; SEQ ID NO: 11), Fch (methenyl-THF cyclohydrolase; SEQ ID NO: 12), and MtdA (5,10-methylene-THF dehydrogenase; SEQ ID NO: 13), as well as endogenousP7355PC00

[0588] GlyA (serine hydroxymethyltransferase; SEQ ID NOs: 22 and 23) (Claassens et al. 2020). Thus, an operon encoding Fch, FtfL and MtdA from M. extorquens was constitutively overexpressed in the phaC1ZC2DFI locus (thereby resulting in deletion of the pha cluster APP_5003-8; SEQ ID NOs: 54 to 59). A 5,10-methylene-THF and serine auxotroph was constructed by deleting the glycine cleavage systems (AgcvTHP-l / ll; SEQ ID NOs: 16 to 21) and D-3-phosphoglycerate dehydrogenase (AserA; SEQ ID NO: 14) in strain SEM11. The resulting strain was termed ASGG-Ci (Figure 3A).

[0589] After transformation of each of the cynD-containing plasmids in ASGG-Ci, we tested growth of the resulting strains in the presence of KCN in DBM medium with 20 mM glucose and 10 mM glycine. Indeed, 2.5 and 5 mM KCN replenished the serine auxotrophy via its degradation product formate, unlike the negative control harbouring an empty vector plasmid (eV) (Figure 3B-C). Growth rates and maximum cell densities (ODeoo) were similar regardless of whether cyanide or formate (as positive control) were supplemented, regardless of whether the strain harboured CynDpumor CynDSfur (Figure 3B-C).

[0590] To further develop the cyanide assimilation of both carbon and nitrogen, we attempted the same experiment as showcased in Figure 3 but employing DBM medium nitrogen-free. Thus, the nitrogen required for cell growth was uniquely provided by cyanide detoxification or subsequently, serine deamination succeeding carbon assimilation into glycine (Figure 4A). Thus, strains ASGG-Ci harbouring the pS221 plasmids with cynDs were grown in DBM medium nitrogen-free (-N), 20 mM glucose, 10 mM glycine and 2.5 or 5 mM KCN. Formate was instead used as a control. When KCN was available, growth was recovered instantaneously, whereas with formate, growth seemed to be impaired by the limited amount of nitrogen present, which required to be transformed first into serine (Figure 4B-C). Negative control (eV) did not showcase auxotrophic recovery in the presence of cyanide (Figure 4B-C). Nonetheless, we aimed to have cyanide as an only nitrogen source, as well as its carbon replenishing 5,10-methylene-THF and subsequently, serine.

[0591] Example 4: Carbon and nitrogen assimilation from the detoxification of cyanide via the serine threonine cycle

[0592] In order to valorise cyanide for its carbon and nitrogen matter, it is required for glycine to be synthesised from the central carbon metabolism. Thus, we overexpressed theP7355PC00

[0593] native L-threonine aldolase encoding gene (ItaE, SEQ ID NO: 71 encoding SEQ ID NO: 5 (LtaE)) to drive flux out of the TCA cycle (i.e. from oxalacetate) through an energydemanding degradation pathway encompassing L-aspartate, homoserine, L-threonine and lastly, glycine (Figure 5A). LtaE (SEQ ID NO: 5) also yields acetaldehyde as coproduct, which can synthetise acetyl-CoA. The combination of modules (i.e. Ci assimilation and glycine regeneration) form the serine threonine cycle (STC). The STC supports mixotrophic growth of P. putida from methanol or formate (as presented in Example 4). Genomic overexpression of ItaE was carried out via a combination of two constitutive promoters and RBS (i.e. Pfrc(RBS) and PI4G(BCD10)) in ASGG-Ci strain (Figure 5A-B). The resulting strains were transformed with the pS221 cynD plasmids and grown in DBM medium without nitrogen, supplemented with 20 mM glucose and increasing concentrations of KCN. As controls, we also included formate and glycine, or formate in DBM medium with nitrogen. An empty vector control was also included (Figure 5C-D). Glycine could be synthesised from the TCA cycle and higher concentrations of KCN promoted higher cell densities with both cynD variants. Both promoter combinations yielded comparable results in terms of glycine generation.

[0594] Furthermore, all combinations of ItaE promoters, CynD variants and KCN concentrations led to comparable specific growth rates (p) of ~ 0.3 h-1(Figure 5D). Negative control harbouring the empty vector did not show growth except for the positive control. No strains were able to grow in glucose only showcasing that the growth-coupled selections are tight.

[0595] Example 5: Construction of a stable P. putida strain for cyanide-derived formate assimilation via the serine threonine cycle

[0596] To build a consolidated and stable P. putida strain, we engineered the relevant activities expressed from the genome of P. putida ASGG-Ci. In addition to the Ci assimilation module from methylotrophic bacteria, as well as the native ItaE to regenerate glycine, we also deleted native thiO (SEQ ID NO: 72) encoding a FAD-dependent glycine / D-amino acid oxidase (ThiO, SEQ ID NO: 73) that produces glyoxylate (Turlin et al. 2022) and could decrease flux through the STC competing with biomass formation. Finally, since we observed substantial biomass aggregation in media containing high one-carbon compound concentrations, we deleted the genes encoding the biofilm-producing adhesins LapA (SEQ ID NO: 74 encoded by SEQ ID NO: 75) and LapF (SEQ ID NO: 76 encoded by SEQ ID NO: 77). The resulting strain was termed P. putida STCO. Moreover, since methanol is a potentialP7355PC00

[0597] secondary / auxiliary carbon source for bioproduction, we also overexpressed yiaY (encoding YiaY; Uniprot accession no. Q88JG7) to upregulate endogenous PQQ-dependent MeDHs, terming the resulting strain STC1.

[0598] Initially, we tested if P. putida STC1 can assimilate methanol into l-serine and regenerate glycine from pyruvate or oxaloacetate (Figure 6a). When strain STC1 was incubated in DBM medium with 20 mM glucose and 10 nM LaCh, both formate and methanol were efficiently assimilated and promoted growth, with p ~ 0.3 and 0.28 h-1and maximum optical density (ODeoo) ~ 2.4 and 2.1, respectively (Figure 6c-d). Thus, the full STC was realized in P. putida via overproduction of the endogenous PQQ-MeDH and l-threonine aldolase as well as heterologous Ci-assimilation via THF.

[0599] Example 6: An enhanced serine-threonine cycle, encompassing a serine aldolase activity, outperforms the parental pathway for carbon assimilation

[0600] Feedstock oxidation to CO2 cannot be completely abolished in an aerobic heterotroph, and we hypothesized that adding extra entry points for Ci moieties into central carbon metabolism could improve the ability of the engineered strain to utilize Ci substrates (Figure 6e). Formaldehyde is a toxic compound produced by different native and heterologous pathways. An aldolase from E. coli (i.e.EcltaE) catalyses the condensation of formaldehyde and glycine, yielding serine (L-serine aldolase reaction). Thus, we hypothesized that including theEcltaE in the STC architecture could enable additional carbon assimilation at the formaldehyde level. To test this scenario, we transformed P. putida STC1, where all relevant STC genes are stably integrated in the chromosome, with plasmid pSEVA621Ec / faE (Figure 6f). We also testedEcltaE*, a variant of theEcLtaE carrying a C188Y point mutation described elsewhere to display faster kinetics (Schann et al. 2024).

[0601] When the fully engineered strain was incubated in DBM medium with 20 mM glucose, 10 nM LaCh, and 500 mM methanol, all growth parameters improved compared to P. putida STC1. A decreased lag phase was observed, with faster growth (p ~ 0.21 h-1forEcltaE*), and increased maximum cell density (ODeoo ~ 3, Figure 6g-h). The wild-typeEcltaE supported a similar growth profile asEcltaE*, albeit the growth rate was equivalent to the STC1 strain (Figure 6g-h). We named the new cycle enhanced serine-threonine cycle (eSTC). A calibration assay was performed to identify the concentration range at which methanol is assimilated (Figure 7). We tested the STC1P7355PC00

[0602] strain harboring eitherEcltaE orEcltaE* in DBM medium with 20 mM glucose and 10 nM LaCh at different methanol concentrations (Figure 7). No growth was observed for any of the variants when methanol was omitted, highlighting the need for a Ci substrate. Importantly, PedH (Uniprot accession no. Q88JH0), the native PQQ-MeDH overexpressed by YiaY and lanthanum chloride, supported synthetic methylotrophy even at low (7.8 mM) methanol concentrations. Hence, both eSTC variants (with eitherEcltaE orEcltaE*) outperformed the parental STC in terms of maximum cell growth (ODeoo) and lag phase reduction, exhibiting more consistent growth profiles at lower methanol concentrations. Since specific growth rates and maximum cell densities were not substantially affected by the methanol concentration, further engineering efforts could be used to overcome other potential bottlenecks (e.g., glycine regeneration, Figure 7b-c).

[0603] Example 7: The enhanced serine-threonine cycle outperforms the parental variant with formate as a co-substrate

[0604] We used strain STCO and tested whether the eSTC promotes increased growth with formate (as a proxy for cyanide and initially as a co-substrate). Formaldehyde can be produced from serine, by native L-threonine aldolase (LtaE) in P. putida. The L-serine aldolase from E. coli (EcLtaE, SEQ ID NO: 6 encoded by SEQ ID NO: 69), which can catalyse the condensation of formaldehyde and glycine, yielding serine (L-serine aldolase reaction), has the potential to restore part of the formaldehyde produced by LtaE, creating a balance between formaldehyde production and assimilation by the native and heterologous LtaEs (Figure 8A).

[0605] We testedEcLtaE* (SEQ ID NO: 7 encoded by SEQ ID NO: 70), the variant of the L-serine aldolaseEcLtaE carrying the C188Y mutation. We transformed STCO with plasmid pSEVA621 ■EcltaE* and an empty vector (eV) as a parental control (Figure 8B). Apart from glucose, we also tested acetate, glycine, pyruvate and glycerol as additional carbon sources (in equimolar carbon concentrations), in the presence of 30 mM formate (Figure 8C). Again, formate was used as a proxy for cyanide. As a negative control, absence of formate did not showcase any growth. Comparisons between pSEVA621 ■EcltaE* and pSEVA621 e\ / showcased statistically-significant differences in all conditions in terms of growth rates and maximal cell densities - except for those where glycine was involved (as then, replenishment of the serine auxotrophy from glycine becomes trivial) (Figure 8C-D). From those, acetate showcased the highestP7355PC00

[0606] growth rate (µ > 0.4 h-1), and glycerol resulted in the highest cell densities when overexpressing the heterologous L-serine aldolase activity (ODeoo ~ 2.5) (Figure 8C-D). Cells did not grow when glycine was the only primary carbon source.

[0607] Therefore, we showcase that the enhanced variant of the STC (eSTC) promotes increased growth (either growth rates or max. cell density) for both methanol and formate as a source of carbon.

[0608] Example 8: The eSTC architecture for cyanide detoxification and subsequent assimilation

[0609] Above, we have demonstrated the eSTC as an architecture that drives more formate assimilation for serine production. To prove that this enhancement of Ci assimilation also supports efficient cyanotrophy, we inserted the genes encoding cynDstut or cynDpum in the genome of STCO AIID-dld2 via Tn5 transposon integration, and identified the best-performing single clones that display efficient cyanide degradation (in DBM medium added with 20 mM glucose and 5 mM cyanide) (Figure 9A-B). A cyanotrophic clone harbouring the insertion of cynDstut was deemed the best candidate for further testing, thus it was transformed with the pSEVA621Ec / faE* plasmid, and growth profiles and cyanide assimilation were compared to a corresponding strain bearing an empty pSEVA621 vector as a control (Figure 9B-C). The eSTC performed better in terms of supporting growth (cell density and growth rate) when co-fed glucose together with cyanide, especially at higher concentrations of the latter (Figure 9C). No bacterial growth is detected in the absence of cyanide.

[0610] Example 9: L-Lactate production as a carbon sink for cyanide assimilation

[0611] In order to prove assimilation and also valorisation of cyanide, we decided to use pyruvate, an intermediate of the STC / eSTC, as a monomer for L-lactate production. L-lactate can be produced from pyruvate via its reduction by LdhA. In our case, we used the IdhA variant N109G of Bos taurus (codon optimized for P. putida KT2440, i.e.BtldhA* encoding BtLdhA(N108G) of SEQ ID NO: 60). Moreover, native D- and L-lactate dehydrogenases encoded by dld2 (SEQ ID NO: 64 encoding SEQ ID NO: 62) and IldD (SEQ ID NO: 63 encoding SEQ ID NO: 61), respectively, were deleted in P. putida strain STCO to prevent product re-assimilation.P7355PC00

[0612] As a proof-of-principle experiment, we again first used formate as a C1 -compound (and proxy for cyanide) to replenish serine auxotrophy (Figure 10A). Thus, we grew STCO AlldD-dld2 harbouring a pSEVA331ef / d / 7A plasmid (Figure 10B) in DBM medium and co-fed either glucose, glucose and glycine, acetate, or acetate and glycine (in equimolar carbon concentrations). All conditions were supplemented with 30 mM formate as a representation of cyanide detoxification. As observed in Figure 10C, acetate conditions barely produced L-lactate, whereas glucose conditions had a substantial product formation. This effect was also observed in the growth pattern, since acetate conditions promoted high cell densities ODeoo 1.7 albeit a longer lagphase. Cell incubated in glucose conditions grew poorly (ODeoo < 1) since most carbon was destined for product formation. Glucose consumption rates were similar, irrespective of the presence of glycine, similarly to formate (yet slightly higher in glucose and glycine compared to glucose only). Interestingly, formate was not fully consumed in the acetate conditions.

[0613] Example 10: L-Lactate production from cyanide via the (e)STC

[0614] Direct L-lactate production from cyanide was tested by using strain STCO AlldD-dld2 gcynDstut, in which we inserted the cynDstut gene in the chromosome using transposon insertion (as explained in Example 8). We transformed said strain with plasmid pSEVA231BtldhA for L-lactate production The cells were grown in nitrogen-free de Bont minimal medium with 20 mM glucose and 2.5 mM of potassium cyanide (KCN). Growth profiles and L-lactate production were compared to those in a strain supplemented with a nitrogen-containing controls with KCN and formate in two independent experiments. L-lactate production and secretion was assessed in vitro via an L-lactate oxidase, which produces hydrogen peroxide. The latter is coupled to a colorimetric reaction coupling the reaction to ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] catalysed by a type II horseradish peroxidase that can be measured by recording the absorbance at 734 nm (Figure 11C). As reported in Figure 11 D and 11 E, L-lactate bioproduction reached similar levels, though the presence of cyanide led to an increased lag phase, notably in the presence of nitrogen in the medium (Figure 11E). The results obtained through this indirect in vitro assessment complemented those for formate in Example 9.P7355PC00

[0615] Sequence overview

[0616] SEQ ID Characteristics [name, organism (or synthetic construct), type NO: (DNA / AA)]

[0617] 1 CynDstut, Pseudomonas stutzeri AK61, protein

[0618] 2 CynDpum, Bacillus pumilus, protein

[0619] 3 cynDstut, synthetic construct, DNA

[0620] 4 cynDpum, synthetic construct, DNA

[0621] 5 LtaE, P. putida, protein

[0622] 6EcLtaE, E. coli, protein

[0623] 7EcLtaE*, synthetic construct, protein

[0624] 8 MeftfL, Methylobacterium extorquens AM1, DNA

[0625] 9 Mefch, Methylobacterium extorquens AM1, DNA

[0626] 10 MemtdA, Methylobacterium extorquens AW\, DNA

[0627] 11 MeFtfL, Methylobacterium extorquens AM1, protein

[0628] 12 MeFch, Methylobacterium extorquens AM1, protein

[0629] 13 MeMtdA, Methylobacterium extorquens AM1, protein

[0630] 14 serA, P. putida, DNA

[0631] 15 SerA, P. putida, protein

[0632] 16 gcvH-\, P. putida, DNA

[0633] 17 gcvP-\, P. putida, DNA

[0634] 18 gcvT-\, P. putida, DNA

[0635] 19 gcvH-W, P. putida, DNA

[0636] 20 gcvP-W, P. putida, DNA

[0637] 21 gcvT-W, P. putida, DNA

[0638] 22 GlyA-l, P. putida, protein

[0639] 23 GlyA-ll, P. putida, protein

[0640] 24 glyA-\, P. putida, DNA

[0641] 25 glyA-\ I, P. putida, DNA

[0642] 26 TdcG-l, P. putida, protein

[0643] 27 TdcG-ll, P. putida, protein

[0644] 28 TdcG-ll I, P. putida, protein

[0645] 29 tdcG-\, P. putida, DNA

[0646] 30 tafcG-ll, P. putida, DNA

[0647] 31 tafcG-lll, P. putida, DNA

[0648]

[0649] P7355PC00

[0650] 32 PycA, P. putida, protein

[0651] 33 PycB, P. putida, protein

[0652] 34 pycA, P. putida, DNA

[0653] 35 pycB, P. putida, DNA

[0654] 36 PykA, P. putida, protein

[0655] 37 pykA, P. putida, DNA

[0656] 38 Ppc, P. putida, protein

[0657] 39 ppc, P. putida, DNA

[0658] 40 AspC, P. putida, protein

[0659] 41 aspC, P. putida, DNA

[0660] 42 LysC, P. putida, protein

[0661] 43 lysC, P. putida, DNA

[0662] 44 Asd, P. putida, protein

[0663] 45 asd, P. putida, DNA

[0664] 46 Hom, P. putida, protein

[0665] 47 PP_0664, P. putida, protein

[0666] 48 hom, P. putida, DNA

[0667] 49 PP_0664, P. putida, DNA

[0668] 50 ThrB, P. putida, protein

[0669] 51 thrB, P. putida, DNA

[0670] 52 ThrC, P. putida, protein

[0671] 53 thrC, P. putida, DNA

[0672] 54 PhaC1, P. putida, protein

[0673] 55 PhaZ, P. putida, protein

[0674] 56 PhaC2, P. putida, protein

[0675] 57 PhaD, P. putida, protein

[0676] 58 PhaF, P. putida, protein

[0677] 59 Phal, P. putida, protein

[0678] 60 BtLdhA (N108G), synthetic construct, protein 61 LldD, P. putida, protein

[0679] 62 Dld2, P. putida, protein

[0680] 63 IldD, P. putida, DNA

[0681] 64 dld2, P. putida, DNA

[0682] 65 serB, P. putida, DNA

[0683]

[0684] P7355PC00

[0685] 66 SerB, P. putida, protein

[0686] 67 serC, P. putida, DNA

[0687] 68 SerC, P. putida, protein

[0688] 69EcltaE, E. coli, DNA

[0689] 70EcltaE*, synthetic construct, DNA

[0690] 71 ItaE, P. putida, DNA

[0691] 72 thiO, P. putida, DNA

[0692] 73 ThiO, P. putida, protein

[0693] 74 LapA, P. putida, protein

[0694] 75 lapA, P. putida, DNA

[0695] 76 LapF, P. putida, protein

[0696] 77 lapF, P. putida, DNA

[0697]

[0698] References

[0699] Bagdasarian, etal. (1981). " Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas." Gene 16: 237-247.

[0700] Martinez-Garcia et al., 2014. Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb. Cell Fact. 13: 159.

[0701] Wirth et al., 2023. Recursive genome engineering decodes the evolutionary origin of an essential thymidylate kinase activity in Pseudomonas putida KT2440. mBio 2023, 14:e01081-01023

[0702] Claassens et al., 2020. 'Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator', Metab Eng, 62: 30-41.

[0703] Schann et al., 2024. 'Design, construction and optimization of formaldehyde growth biosensors with broad application in biotechnology', Microb. Biotechnol., 17: e14527. Turlin et al., 2022. 'Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation', Metab. Eng., 74: 191-205.

[0704] Platt et al., 2000. Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome. Plasmid, 43:12-23.

[0705] Hartmans et al., 1989. Metabolism of styrene oxide and 2-phenylethanol in the styrenedegrading Xanthobacter strain 124X. Appl. Environ. Microbiol. 55:2850-2855.

[0706] Nikel et al., 2016. Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegradation of aromatic compounds. Environ.

[0707] Microbiol. 18:3565-3582.

[0708] Wirth et al., 2023. QurvE: user-friendly software for the analysis of biological growth and fluorescence data. Nat. Protoc. 18:2401-2403.P7355PC00

[0709] Wirth et al., 2020. Accelerated genome engineering of Pseudomonas putida by I-Scel— mediated recombination and CRISPR-Cas9 counterselection. Microb.

[0710] Biotechnol. 13:233-249.

[0711] Calero et al., 2022. Role of the CrcB transporter of Pseudomonas putida in the multilevel stress response elicited by mineral fluoride. Environ Microbiol 24:5082-5104. Volke et al., 2020. Synthetic control of plasmid replication enables target- and selfcuring of vectors and expedites genome engineering of Pseudomonas putida. Metab Eng Commun 10:e00126.

[0712] Turlin et al., 2023 Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses. mSystems 8:e0000423.

[0713] Martinez-Garcia, et al., 2014. New transposon tools tailored for metabolic engineering of gram-negative microbial cell factories. Front Bioeng Biotechnol 2:46.

[0714] Figurski et al., 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652.

[0715] Volke et al., 2021. Rapid genome engineering of Pseudomonas assisted by fluorescent markers and tractable curing of plasmids. Bio-protocol 11:e3917.

[0716] Blattner et al., 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-1462.

[0717] Rodriguez et al., 2018. Behavior of several Pseudomonas putida strains growth under different agitation and oxygen supply conditions. Biotechnol Prog 34:900-909.

[0718] Ferreira, 2007. The Benjamini-Hochberg method in the case of discrete test statistics. Int. J. Biostat. 3:11.

[0719] Martinkova et al., 2024. Biotransformation of free cyanide to formic acid by a cyanide hydratase-formamidase cascade reaction. Process Biochem. 142: 62-67. hftp: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur J Biochem (1985) Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984)

[0720] Zhang et al., 2017. Coupling between d-3-phosphoglycerate dehydrogenase and d-2-hydroxyglutarate dehydrogenase drives bacterial l-serine synthesis. Proceedings of the National Academy of Sciences 114.36 (2017): E7574-E7582.

[0721] Pizer, Lewis I., 1963. The pathway and control of serine biosynthesis in Escherichia coli. Journal of Biological Chemistry 238.12: 3934-3944.

Claims

P7355PC00Claims1. A Pseudomonas cell comprising a nucleic acid encoding a cyanidase (EC 3.5.5.1) capable of catalysing conversion of cyanide into formate (HCOOH) and ammonia (NH3) / ammonium (NH4+), and one or more of:a heterologous nucleic acid encoding a formate-THF ligase (FtfL; EC 6.3.4.3), a heterologous nucleic acid encoding a methenyl-THF cyclohydrolase (Fch; EC 3.5.4.9), and / ora heterologous nucleic acid encoding a methylene-THF dehydrogenase (MdtA; EC 1.5.1.5),wherein said cell is capable of converting said formate into 5,10-methylene- THF, andwherein said Pseudomonas cell further comprises a modification resulting in partial or total inactivation of an endogenous glycine cleavage system (GCS) and a modification resulting in partial or total inactivation of an endogenous SerABC pathway,preferably wherein said cell is capable of producing a compound, such as a pyruvate-derived compound.

2. The Pseudomonas cell according to claim 1, wherein said cell is capable of: i. assimilating at least 20 % of the carbon provided in cyanide, such as at least 50%, such as at least 75%, such as at least 85%, such as at least 90%, such as at least 95%, or more; and / orii. assimilating at least 20 % of the nitrogen provided in cyanide, such as at least 50%, such as at least 75%, such as at least 85%, such as at least 90%, such as at least 95%, or more.

3. The Pseudomonas cell according to any one of the preceding claims, wherein said cyanidase is a heterologous cyanidase, preferably wherein said cyanidase is a cyanidase from Stutzerimonas stutzeri (formerly known as Pseudomonas stutzeri), such as from Stutzerimonas stutzeri AK61 (formerly known as Pseudomonas stutzeri AK61) or from Bacillus pumilus.

4. The Pseudomonas cell according to any one of the preceding claims, wherein said cyanidase is CynDstut (SEQ ID NO: 1), or CynDpum (SEQ ID NO: 2), or aP7355PC00functional variant thereof having at least 70% identity to SEQ ID NO: 1 or 2, respectively.

5. The Pseudomonas cell according to any one of the preceding claims, wherein said Pseudomonas cell is a P. putida cell, such as a non-pathogenic Pseudomonas cell, for example a P. putida KT2440 cell or a P. putida SEM11 cell.

6. The Pseudomonas cell according to any one of the preceding claims, wherein said cell comprises:i. an endogenous D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95), wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous D-3-phosphoglycerate dehydrogenase, optionally wherein said D-3-phosphoglycerate dehydrogenase is SerA (SEQ ID NO: 15) or a functional variant thereof having at least 70% identity thereto,preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerA or a functional variant thereof having at least 70% identity thereto, such as serA (SEQ ID NO: 14) or a homologue thereof having at least 70% identity thereto;ii. an endogenous phosphoserine phosphatase (EC 3.1.3.3) wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine phosphatase, optionally wherein said phosphoserine phosphatase is SerB (SEQ ID NO: 66) or a functional variant thereof having at least 70% identity thereto,preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerB or a functional variant thereof having at least 70% identity thereto, such as serB (SEQ ID NO: 65) or a homologue thereof having at least 70% identity thereto; and / oriii. an endogenous phosphoserine aminotransferase (EC 2.6.1.52) wherein said cell comprises a modification resulting in partial or total inactivation of said endogenous phosphoserine aminotransferase, optionallyP7355PC00wherein said phosphoserine aminotransferase is SerC (SEQ ID NO: 68) or a functional variant thereof having at least 70% identity thereto, preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding SerC or a functional variant thereof having at least 70% identity thereto, such as serC (SEQ ID NO: 67) or a homologue thereof having at least 70% identity thereto.

7. The Pseudomonas cell according to any one of the preceding claims, wherein said endogenous GCS comprises or consists of a dihydrolipoyl dehydrogenase (LpdG; EC 1.8.1.4), a GCS protein H (GcvH), a glycine dehydrogenase (GcvP; EC 1.4.4.2) and an aminomethyltransferase (GcvT; EC 2.1.2.10), further wherein said cell comprises two endogenous GCS operons comprising or consisting of:i. a first operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-l (SEQ ID NO: 16), GcvP-l (SEQ ID NO: 17) and GcvT-l (SEQ ID NO: 18); and / orii. a second operon (gcvTHP-l) comprising or consisting of one or more nucleic acids encoding GcvH-ll (SEQ ID NO: 19), GcvP-ll (SEQ ID NO: 20) and GcvT-ll (SEQ ID NO: 21),or functional variants thereof having at least 70% identity to thereto.

8. The Pseudomonas cell according to any one of the preceding claims, wherein said cell comprises a modification resulting in partial or total inactivation of said two endogenous GCS operons, optionally wherein said modification comprises or consists of a mutation in or a deletion of at least a part of gcvTHP-l and gcvTHP-ll, preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a first nucleic acid encoding GcvH- I, GcvP-l and GcvT-l, or functional variants thereof having at least 70% identity to thereto, and at least a corresponding part of a second nucleic acid encoding GcvH-ll, GcvP-ll and GcvT-ll, or functional variants thereof having at least 70% identity to thereto.

9. The Pseudomonas cell according to any one of the preceding claims, wherein:i. said formate-THF ligase (FtfL; EC 6.3.4.3) is MeFtfL (SEQ ID NO: 8);P7355PC00ii. said methenyl-THF cyclohydrolase (Fch; EC 3.5.4.9) is MeFch (SEQ ID NO: 9); and / oriii. said methylene-THF dehydrogenase (MdtA; EC 1.5.1.5) is MeMdtA (SEQ ID NO: 10),or functional variants thereof having at least 70 % identity thereto.

10. The Pseudomonas cell according to any one of the preceding claims, wherein said cell comprises a nucleic acid encoding a heterologous L-serine aldolase capable of converting glycine and formaldehyde into serine, such asEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto.

11. A Pseudomonas cell comprising a nucleic acid encoding a heterologous L- serine aldolase capable of converting glycine and formaldehyde into serine, such as an L-serine aldolase from Escherichia, such as from Escherichia coli, for exampleEcLtaE (SEQ ID NO: 6) orEcLtaE* (SEQ ID NO: 7), or functional variants thereof having at least 70% identity thereto.

12. A method of producing a compound, such as a pyruvate-derived compound, comprising:i. providing a Pseudomonas cell as defined in any one of claims 1 to 11; ii. cultivating and optionally propagating said cell in a cultivation medium, said cultivation medium comprising cyanide,thereby obtaining a fermentation broth and / or cell comprising said compound;iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;whereby said compound and / or said fermentation broth comprising said compound is produced.

13. A method of detoxifying a waste product comprising cyanide, comprising: i. providing a Pseudomonas cell as defined in any one of claims 1 to 11; ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivationP7355PC00medium comprising cyanide,thereby degrading said cyanide and optionally obtaining cell biomass; iii. optionally recovering said cell biomass;whereby said waste product is detoxified.

14. A method of valorising of a waste product comprising cyanide into a compound, comprising:i. providing a Pseudomonas cell as defined in any one of claims 1 to 11; ii. cultivating and optionally propagating said cell in a cultivation medium comprising said waste product or a part / fraction thereof, said cultivation medium comprising cyanide,thereby obtaining a fermentation broth and / or cell comprising said compound;iii. optionally recovering said fermentation broth, and / or said compound from said cell and / or fermentation broth;whereby said waste product comprising cyanide is valorised by production of said compound and / or said fermentation broth comprising said compound.

15. The cell or the method according to any one of the preceding claims, wherein said compound, such as said pyruvate-derived compound, is lactate, optionally wherein said cell comprises a nucleic acid encoding a lactate dehydrogenase (LDH; EC 1.1.1.28), such as BtLdhA(N108G) (SEQ ID NO: 60) or a functional variant thereof having at least 70% identity thereto.

16. The cell or the method according to any one of the preceding claims, wherein said compound, such as said pyruvate-derived compound, is lactate, wherein said cell comprises at least one modification resulting in partial or total inactivation of an L-lactate dehydrogenase (EC 1.1.2.3), such as LldD (SEQ ID NO: 61), and a D-lactate dehydrogenase 2 (EC 1.1.5.12), such as Dld2 (SEQ ID NO: 62), or functional variants thereof having at least 70% identity thereto, preferably wherein said modification comprises or consists of a mutation in or a deletion of at least a part of a nucleic acid encoding LldD and Dld2 or functional variants thereof having at least 70% identity thereto, such as IldD (SEQ ID NO: 63) and dld2 (SEQ ID NO: 64) or homologues thereof having at least 70% identity thereto.

Images