US20200277592A1 - Genetically optimised microorganism for producing molecules of interest - Google Patents

Genetically optimised microorganism for producing molecules of interest Download PDF

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US20200277592A1
US20200277592A1 US16/480,569 US201816480569A US2020277592A1 US 20200277592 A1 US20200277592 A1 US 20200277592A1 US 201816480569 A US201816480569 A US 201816480569A US 2020277592 A1 US2020277592 A1 US 2020277592A1
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microorganism
gene
genetically modified
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molecule
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Cedric Boisart
Nicolas Morin
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Enobraq
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    • C12Y401/01039—Ribulose-bisphosphate carboxylase (4.1.1.39)

Definitions

  • the invention concerns a genetically modified microorganism, capable of using carbon dioxide as an at least partial carbon source, for the production of molecules of interest. More specifically, the invention relates to a microorganism in which at least the glycolysis pathway is at least partially inhibited. The invention also relates to processes for the production of at least one molecule of interest using such a microorganism.
  • fermentation processes are used to produce molecules by a microorganism from a fermentable carbon source, such as glucose.
  • Bioconversion processes have also been developed to allow a microorganism to convert a co-substrate, not assimilable by said microorganism, into a molecule of interest.
  • a carbon source is required, not for the actual production of the molecule of interest, but for the production of cofactors, and more particularly NADPH, that may be necessary for bioconversion.
  • the production yield of such microbiological processes is low, mainly due to the need for cofactors and the difficulty of balancing redox metabolic reactions.
  • a source of carbon assimilable by the microorganism is still necessary.
  • it is necessary to provide a molecule (glucose, or other), certainly of lower industrial value, but which is sufficient to make the production of certain molecules not economically attractive.
  • CO 2 carbon dioxide
  • microorganisms genetically modified to capture CO 2 and use it as the main carbon source, in the same way as plants and photosynthetic microorganisms, has already been demonstrated.
  • microorganisms modified to express a functional RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase—EC 4.1.1.39) and a functional PRK (phosphoribulokinase—EC 2.7.1.19) to reproduce a partial Calvin cycle and convert ribulose-5-phosphate into two 3-phosphoglycerate molecules by capturing a carbon dioxide molecule have been developed.
  • the inventors By working on the solutions provided by the Calvin cycle to produce molecules of interest using CO 2 as carbon source, the inventors discovered that it is possible to increase the production yield of molecules of interest by coupling part of the Calvin cycle (PRK/RuBisCO) to at least partial inhibition of glycolysis. The inventors have also discovered that it is possible to increase the consumption of exogenous CO 2 during the production of molecules of interest, by also at least partially inhibiting the oxidative branch of the pentose phosphate pathway.
  • the microorganisms thus developed make it possible to produce on a large scale and with an industrially attractive yield a large number of molecules of interest, such as amino acids, organic acids, terpenes, terpenoids, peptides, fatty acids, polyols, etc.
  • the invention thus relates to a genetically modified microorganism expressing a functional RuBisCO enzyme and a functional phosphoribulokinase (PRK), and in which the glycolysis pathway is at least partially inhibited, said microorganism being genetically modified so as to produce an exogenous molecule of interest and/or to overproduce an endogenous molecule of interest, other than a RuBisCO or phosphoribulokinase enzyme.
  • PRK functional phosphoribulokinase
  • the genetically modified microorganism has an oxidative branch of the pentose phosphate pathway that is also at least partially inhibited.
  • the invention also concerns the use of a genetically modified microorganism according to the invention, for the production or overproduction of a molecule of interest, preferentially selected from amino acids, peptides, proteins, vitamins, sterols, flavonoids, terpenes, terpenoids, fatty acids, polyols and organic acids.
  • the present invention also concerns a biotechnological process for producing or overproducing at least one molecule of interest, characterized in that it comprises a step of culturing a genetically modified microorganism according to the invention, under conditions allowing the synthesis or bioconversion, by said microorganism, of said molecule of interest, and optionally a step of recovering and/or purifying said molecule of interest.
  • It also concerns a process for producing a molecule of interest comprising (i) inserting at least one sequence encoding an enzyme involved in the synthesis or bioconversion of said molecule of interest into a recombinant microorganism according to the invention, (ii) culturing said microorganism under conditions allowing the expression of said enzyme and optionally (iii) recovering and/or purifying said molecule of interest.
  • FIG. 1 General diagram of glycolysis, the pentose phosphate pathway and the Entner-Doudoroff pathway;
  • FIG. 2 Schematic representation of inhibition of the glycolysis pathway, according to the invention.
  • FIG. 3 Schematic representation of inhibition of the glycolysis pathway, combined with inhibition of the oxidative branch of the pentose phosphate pathway, according to the invention.
  • recombinant microorganism modified microorganism
  • recombinant host cell refers to microorganisms that have been genetically modified to express or overexpress endogenous nucleotide sequences, to express heterologous nucleotide sequences, or that have an altered expression of an endogenous gene.
  • “Alteration” means that the expression of the gene, or level of an RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits is regulated, so that the expression, the level or the activity is higher or lower than that observed in the absence of modification.
  • recombinant microorganism refers not only to the particular recombinant microorganism but to the progeny or the potential progeny of such a microorganism. As some modifications may occur in subsequent generations, due to mutation or environmental influences, these offspring may not be identical to the mother cell, but they are still understood within the scope of the term as used here.
  • an at least partially “inhibited” or “inactivated” metabolic pathway refers to an altered metabolic pathway that can no longer function properly in the microorganism considered, compared with the same wild-type microorganism (not genetically modified to inhibit said metabolic pathway).
  • the metabolic pathway may be interrupted, leading to the accumulation of an intermediate metabolite. Such an interruption may be achieved, for example, by inhibiting the enzyme necessary for the degradation of an intermediate metabolite of the metabolic pathway considered and/or by inhibiting the expression of the gene encoding that enzyme.
  • the metabolic pathway may also be attenuated, i.e. slowed down.
  • Such attenuation may be achieved, for example, by partially inhibiting one or more enzymes involved in the metabolic pathway considered and/or partially inhibiting the expression of a gene encoding at least one of these enzymes and/or by exploiting the cofactors required for certain reactions.
  • the expression “at least partially inhibited metabolic pathway” means that the level of the metabolic pathway considered is reduced by at least 20%, more preferentially at least 30%, 40%, 50%, or more, compared with the level in a wild-type microorganism. The reduction may be greater, and in particular be at least greater than 60%, 70%, 80%, 90%. According to the invention, inhibition may be total, in the sense that the metabolic pathway considered is no longer used at all by said microorganism. According to the invention, such inhibition may be temporary or permanent.
  • inhibition of gene expression means that the gene is no longer expressed in the microorganism considered or that its expression is reduced, compared with wild-type microorganisms (not genetically modified to inhibit gene expression), leading to the absence of production of the corresponding protein or to a significant decrease in its production, and in particular to a decrease of more than 20%, more preferentially 30%, 40%, 50%, 60%, 70%, 80%, 90%.
  • inhibition can be total, i.e. the protein encoded by said gene is no longer produced at all.
  • Inhibition of gene expression can be achieved by deletion, mutation, insertion and/or substitution of one or more nucleotides in the gene considered.
  • inhibition of gene expression is achieved by total deletion of the corresponding nucleotide sequence.
  • any method of gene inhibition known per se by the skilled person and applicable to a microorganism, may be used.
  • inhibition of gene expression can be achieved by homologous recombination (Datsenko et al., Proc Natl Acad Sci USA. 2000; 97:6640-5; Lodish et al., Molecular Cell Biology 4 th ed. 2000. W. H. Freeman and Company. ISBN 0-7167-3136-3); random or directed mutagenesis to modify gene expression and/or encoded protein activity (Thomas et al., Cell.
  • interfering RNA refers to any iRNA molecule (for example single-stranded RNA or double-stranded RNA) that can block the expression of a target gene and/or facilitate the degradation of the corresponding mRNA.
  • Gene inhibition can also be achieved by genome editing methods that allow direct genetic modification of a given genome, through the use of zinc finger nucleases (Kim et al., PNAS; 93: 1156-1160), transcription activator-like effector nucleases, or “TALEN” (Ousterout et al., Methods Mol Biol. 2016; 1338:27-42.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • meganucleases Daboussi et al., Nucleic Acids Res. 2012. 40:6367-79. Inhibition of gene expression can also be achieved by inactivating the protein encoded by said gene.
  • NADPH-dependent or “NADPH-consuming” biosynthesis or bioconversion means all biosynthesis or bioconversion pathways in which one or more enzymes require the concomitant supply of electrons obtained by the oxidation of an NADPH cofactor.
  • “NADPH-dependent” biosynthesis or bioconversion pathways notably concern the synthesis of amino acids (e.g. arginine, lysine, methionine, threonine, proline, glutamate, homoserine, isoleucine, valine) ⁇ -aminobutyric acid, terpenoids and terpenes (e.g. farnesene), vitamins and precursors (e.g.
  • sterols e.g. squalene, cholesterol, testosterone, progesterone, cortisone
  • flavonoids e.g. fr
  • exogenous refers to molecules that are not normally or naturally found in and/or produced by the microorganism considered.
  • endogenous or native refers to various molecules (nucleotide sequences, peptides, enzymes, etc.), designating molecules that are normally or naturally found in and/or produced by the microorganism considered.
  • the invention proposes genetically modified microorganisms for the production of a molecule of interest, endogenous or exogenous.
  • “Genetically modified” microorganism means that the genome of the microorganism has been modified to incorporate a nucleic sequence encoding an enzyme involved in the biosynthesis or bioconversion pathway of a molecule of interest, or encoding a biologically active fragment thereof. Said nucleic sequence may have been introduced into the genome of said microorganism or one of its ancestors, by any suitable molecular cloning method. In the context of the invention, the genome of the microorganism refers to all genetic material contained in the microorganism, including extrachromosomal genetic material contained, for example, in plasmids, episomes, synthetic chromosomes, etc.
  • the introduced nucleic sequence may be a heterologous sequence, i.e.
  • a transcriptional unit with the nucleic sequence of interest is introduced into the genome of the microorganism, under the control of one or more promoters.
  • Such a transcriptional unit also includes, advantageously, the usual sequences such as transcriptional terminators, and, if necessary, other transcription regulatory elements.
  • Promoters usable in the present invention include constitutive promoters, i.e. promoters that are active in most cellular states and environmental conditions, as well as inducible promoters that are activated or suppressed by exogenous physical or chemical stimuli, and therefore induce a variable state of expression depending on the presence or absence of these stimuli.
  • constitutive promoters i.e. promoters that are active in most cellular states and environmental conditions
  • inducible promoters that are activated or suppressed by exogenous physical or chemical stimuli, and therefore induce a variable state of expression depending on the presence or absence of these stimuli.
  • the microorganism is a yeast
  • inducible promoters that can be used in yeast are tetO-2, GAL10, GAL10-CYC1, PHO5.
  • the genetically modified microorganism according to the invention has the following features:
  • any microorganism can be used.
  • the microorganism is a eukaryotic cell, preferentially selected from yeasts, fungi, microalgae or a prokaryotic cell, preferentially a bacterium or cyanobacterium.
  • the genetically modified microorganism according to the invention is a yeast, preferentially selected from among the ascomycetes (Spermophthoraceae and Saccharomycetaceae), basidiomycetes ( Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium , and Filobasidiella ) and deuteromycetes yeasts belonging to Fungi imperfecti (Sporobolomycetaceae, and Cryptococcaceae).
  • ascomycetes Spermophthoraceae and Saccharomycetaceae
  • basidiomycetes Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium , and Filobasidiella
  • deuteromycetes yeasts belonging to Fungi imperfecti Sporobolomycetaceae, and Cryptococcaceae
  • the genetically modified yeast according to the invention belongs to the genus Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Candida, Lipomyces, Rhodotorula, Rhodosporidium, Yarrowia , or Debaryomyces .
  • the genetically modified yeast according to the invention is selected from Pichia pastoris, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Schizosaccharomyces pombe, Candida albicans, Candida tropicalis, Rhodotorula glutinis, Rhodosporidium toruloides, Yarrowia lipolytica, Debaryomyces hansenii and Lipomyces starkeyi.
  • the genetically modified microorganism according to the invention is a fungus, and more particularly a “filamentous” fungus.
  • “filamentous fungi” refers to all filamentous forms of subdivision Eumycotina .
  • the genetically modified fungus according to the invention belongs to the genus Aspergillus, Trichoderma, Neurospora, Podospora, Endothia, Mucor, Cochliobolus or Pyricularia .
  • the genetically modified fungus according to the invention is selected from Aspergillus nidulans, Aspergillus niger, Aspergillus awomari, Aspergillus oryzae, Aspergillus terreus, Neurospora crassa, Trichoderma reesei , and Trichoderma viride.
  • the genetically modified microorganism according to the invention is a microalga.
  • microalga refers to all eukaryotic microscopic algae, preferentially belonging to the classes or superclasses Chlorophyceae, Chrysophyceae, Prymnesiophyceae, Diatomae or Bacillariophyta, Euglenophyceae, Rhodophyceae, or Trebouxiophyceae.
  • the genetically modified microalgae according to the invention are selected from Nannochloropsis sp. (e.g.
  • Nannochloropsis oculata Nannochloropsis gaditana, Nannochloropsis salina
  • Tetraselmis sp. e.g. Tetraselmis suecica, Tetraselmis chuii
  • Chlorella sp. e.g. Chlorella salina, Chlorella protothecoides, Chlorella ellipsoidea, Chlorella emersonii, Chlorella minutissima, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris
  • Chlamydomonas sp. e.g. Chlamydomonas reinhardtii
  • the genetically modified microorganism according to the invention is a bacterium, preferentially selected from phyla Acidobacteria, Actinobacteria, Aquificae, Bacterioidetes, Chlamydia, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfobacteria, Thermomicrobia, Thermotogae, or Verrucomicrobia.
  • the genetically modified bacterium according to the invention belongs to the genus Acaryochloris, Acetobacter, Actinobacillus, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Anaerobiospirillum, Aquifex, Arthrobacter, Arthrospira, Azobacter, Bacillus, Brevibacterium, Burkholderia, Chlorobium, Chromatium, Chlorobaculum, Clostridium, Corynebacterium, Cupriavidus, Cyanothece, Enterobacter, Deinococcus, Erwinia, Escherichia, Geobacter, Gloeobacter, Gluconobacter, Hydrogenobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Mesorhizobium, Methylobacterium, Microbacterium, Microcystis, Nitrobacter, Nitrosomonas, Nitrospina, Nitrospira, Nostoc,
  • the genetically modified bacterium according to the invention is selected from the species Agrobacterium tumefaciens, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Aquifex aeolicus, Aquifex pyrophilus, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium acetobutylicum, Clostridium beigerinckii, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Enterobacter sakazakii, Escherichia coli, Gluconobacter oxydans, Hydrogenobacter thermophilus, Klebsiella oxytoca, Lactococcus lactis, Lactobacillus plantarum, Mann
  • the microorganism can naturally express a functional RuBisCO and a functional PRK. This is the case, for example, for photosynthetic microorganisms such as microalgae and cyanobacteria.
  • RuBisCO there are several forms of RuBisCO in nature (Tabita et al., J Exp Bot. 2008; 59(7):1515-24. doi: 10.1093/jxb/erm361).
  • Forms I, II and III catalyze the carboxylation and oxygenation reactions of ribulose-1,5-biphosphate.
  • Form I is present in eukaryotes and bacteria. It consists of two types of subunits: large subunits (RbcL) and small subunits (RbcS).
  • the functional enzyme complex is a hexadecamer consisting of eight L subunits and eight S subunits.
  • RbcX (Liu et al., Nature. 2010 Jan. 14; 463(7278):197-202. doi: 10.1038/nature08651).
  • Form II is mainly found in proteobacteria, archaea (Archaea or archaebacteria) and dinoflagellate algae. Its structure is much simpler: it is a homodimer (formed by two identical RbcL subunits).
  • the genes encoding a type I RuBisCO may be called rbcL/rbcS (for example Synechococcus elongatus ), or cbxLC/cbxSC, cfxLC/cfxSC, cbbL/cbbS (for example Cupriavidus necator ).
  • the genes encoding a type II RuBisCO are generally called cbbM (for example Rhodospirillum rubrum ).
  • Form III is present in the archaea. It is generally found in the form of dimers of the RbcL subunit, or in pentamers of dimers.
  • rbcL for example Thermococcus kodakarensis
  • cbbL for example Haloferax sp.
  • PRKs Two classes of PRKs are known: class I enzymes found in proteobacteria are octamers, while class II enzymes found in cyanobacteria and plants are tetramers or dimers.
  • the genes encoding a PRK may be called prk (for example Synechococcus elongatus ), prkA (for example Chlamydomonas reinhardtii ), prkB (for example Escherichia coli ), prk1, prk2 (for example Leptolyngbya sp.), cbbP (for example Nitrobacter vulgaris ) or cfxP (for example Cupriavidus necator ).
  • the microorganism used does not naturally express a functional RuBisCO and a functional PRK
  • said microorganism is genetically modified to express heterologous RuBisCO and PRK.
  • the microorganism is transformed so as to integrate into its genome one or more expression cassettes integrating the sequences encoding said proteins, and advantageously the appropriate transcription factors.
  • the microorganism is genetically modified to express a type I RuBisCO. In another embodiment, the microorganism is genetically modified to express a type II RuBisCO. In another embodiment, the microorganism is genetically modified to express a type III RuBisCO.
  • the glycolysis pathway is at least partially inhibited, so that the microorganism is no longer able to use this metabolic pathway normally ( FIG. 1 —glycolysis).
  • the microorganism no longer has the ability to assimilate glucose in a similar way to a wild-type microorganism, in which the glycolysis pathway has not been inhibited (independently of any other genetic modification).
  • the microorganism is genetically modified to inhibit, totally or partially, glycolysis downstream of the production of glyceraldehyde-3-phosphate (G3P).
  • G3P glyceraldehyde-3-phosphate
  • glycolysis is inhibited upstream of the production of 1,3-biphospho-D-glycerate (1,3-BPG) or upstream of the production of 3-phosphoglycerate (3PG).
  • glyceraldehyde-3-phosphate (G3P) and 3-phosphoglycerate (3PG) can be managed (i) by two enzymes acting concomitantly, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12, abbreviated GAPDH or more rarely G3PDH) and phosphoglycerate kinase (E.C. 2.7.2.3, abbreviated PGK), or (ii) by a single non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase enzyme (EC 1.2.1.9, abbreviated GAPN).
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • PGK phosphoglycerate kinase
  • Glyceraldehyde-3-phosphate dehydrogenase catalyzes the reversible conversion of G3P to 1,3-biphospho-D-glycerate (1,3-BPG), using the pair NAD + /NADH as electron donor/acceptor in the direction of the reaction.
  • the genes encoding GAPDH may be called gapA, gapB, gapC (e.g. Escherichia coli, Arabidopsis thaliana ), GAPDH, GAPD, G3PD, GAPDHS (e.g. Homo sapiens ), TDH1, TDH2, TDH3 (e.g. Saccharomyces cerevisiae ), gap, gap2, gap3 (e.g. Mycobacterium sp., Nostoc sp.).
  • Phosphoglycerate kinase catalyzes the reversible conversion of 1,3-BPG to 3PG, using the pair ATP/ADP as cofactor.
  • the genes encoding PGK may be called PGK, PGK1, PGK1, PGK2, PGK3, pgkA, PGKB, PGKC, cbbK, cbbKC, cbbKP (e.g. Cupriavidus necator ).
  • Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzes the conversion of G3P to 3PG, without going through 1,3-BPG. This reaction is catalyzed in the presence of the cofactor pair NADP + /NADPH, which acts as an electron acceptor.
  • GAPN e.g. Bacillus sp., Streptococcus sp.
  • GAPN1 e.g. Chlamydomonas sp.
  • the microorganism is genetically modified so that the expression of the gene encoding glyceraldehyde 3-phosphate dehydrogenase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • the expression of the gene encoding phosphoglycerate kinase may also be at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • the microorganism is genetically modified so that the expression of the gene encoding non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • Tables 3, 4 and 5 below list, as examples, the sequences encoding a glyceraldehyde 3-phosphate dehydrogenase, a phosphoglycerate kinase and a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that can be inhibited depending on the target microorganism.
  • the skilled person knows which gene corresponds to the enzyme of interest to be inhibited depending on the microorganism.
  • 3-phosphoglycerate (3PG) is no longer possible through glycolysis, or at least significantly reduced, in the genetically modified microorganism according to the invention.
  • the microorganism is a yeast of the genus Saccharomyces cerevisiae in which the expression of the TDH1 (Gene ID: 853395), TDH2 (Gene ID: 853465) and/or TDH3 gene (Gene ID: 853106) is at least partially inhibited.
  • the microorganism is a yeast of the genus Saccharomyces cerevisiae in which the expression of the PGK1 gene (Gene ID: 5230) is at least partially inhibited.
  • the microorganism is a yeast of the genus Saccharomyces cerevisiae in which the expression of the PGK1 gene (Gene ID: 5230), the expression of the TDH1 gene (Gene ID: 853395), TDH2 (Gene ID: 853465) and/or the expression of the TDH3 gene (Gene ID: 853106) are at least partially inhibited.
  • the microorganism is an Escherichia coli bacterium in which the expression of the gapA gene (Gene ID: 947679) is at least partially inhibited.
  • the microorganism is an Escherichia coli bacterium in which the expression of the pgk gene (Gene ID: 947414) is at least partially inhibited.
  • the microorganism is an E. coli bacterium in which the expression of the pgk gene (Gene ID: 947414), and/or the expression of the gapA gene (Gene ID: 947679) are at least partially inhibited.
  • the genetically modified microorganism which expresses a functional RuBisCO and a functional PRK, is on the other hand capable of producing 3PG by capturing CO 2 from ribulose-5-phosphate produced by the pentose phosphate pathway ( FIG. 2 ).
  • microorganism Since the enzymes necessary for the metabolism of 3PG to pyruvate are not inhibited in the microorganism, said microorganism can then metabolize 3PG to produce pyruvate and ATP.
  • the genetically modified microorganism is able to produce pyruvate and NADPH cofactors using CO 2 as complementary carbon source.
  • “complementary” carbon source means that the microorganism uses CO 2 as a partial carbon source, in addition to the carbon atoms provided by fermentable sugars (glucose, galactose, sucrose, fructose, etc.), which constitute the majority or main carbon source for pyruvate production.
  • the genetically modified microorganism according to the invention makes it possible to increase carbon yield, by fixing and using the CO 2 normally lost during glucose metabolism via the pentose phosphate pathway, for the production of pyruvate (and subsequently molecules of interest).
  • the genetically modified microorganism according to the invention is also modified in such a way that the oxidative branch of the pentose phosphate pathway is also at least partially inhibited.
  • the microorganism is genetically modified to inhibit the oxidative branch of the pentose phosphate pathway upstream of ribulose-5-phosphate production ( FIG. 1 —pentose phosphate pathway).
  • the interruption of the oxidative branch of the pentose phosphate pathway upstream of ribulose-5-phosphate (Ru5P) production specifically targets one or more reactions in the Ru5P synthesis process from glucose-6-phosphate (G6P).
  • This synthesis is generally catalyzed by the successive actions of three enzymes: (i) glucose-6-phosphate dehydrogenase (EC. 1.1.1.49, abbreviated G6PDH), (ii) 6-phosphogluconolactonase (E.C. 3.1.1.31, abbreviated PGL), and (iii) 6-phosphogluconate dehydrogenase (EC 1.1.1.44, abbreviated PGD).
  • G6PDH Glucose-6-phosphate dehydrogenase catalyzes the first reaction of the pentose phosphate pathway, i.e. the oxidation of glucose-6-phosphate to 6-phosphogluconolactone (6PGL), with concomitant reduction of one molecule of NADP to NADPH.
  • the genes encoding G6PDH may be called G6PD (for example in Homo sapiens ), G6pdx (for example in Musculus ), gsdA (for example in Aspergillus nidulans ), zwf (for example in Escherichia coli ), or ZWF1 (for example in Saccharomyces cerevisiae ).
  • 6-Phosphogluconolactonase is a hydrolase that catalyzes the synthesis of 6-phosphogluconate (6PGA) from 6PGL.
  • the genes encoding PGL may be called pgl (for example in Escherichia coli, Synechocystis sp.) pgls (for example in Rhodobacteraceae bacterium ), or SOL (for example in Saccharomyces cerevisiae ).
  • 6-Phosphogluconate dehydrogenase is an oxidoreductase that catalyzes the synthesis of Ru5P from 6PGA, with concomitant reduction of an NADP molecule to NADPH and emission of a CO 2 molecule.
  • the genes encoding PGD may be called gnd (for example in Escherichia coli, Saccharomyces cerevisiae ), PGD (for example in Homo sapiens ), gntZ (for example in Bacillus subtilis ), or 6-PGDH (for example in Lactobacillus paracollinoides ).
  • the microorganism is genetically modified so that the expression of the gene encoding glucose-6-phosphate dehydrogenase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • the microorganism is genetically modified so that the expression of the gene encoding 6-phosphogluconolactonase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • the microorganism is genetically modified so that the expression of the gene encoding 6-phosphogluconate dehydrogenase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • Tables 6, 7 and 8 below list, as examples, the sequences encoding a glucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase and a 6-phosphogluconate dehydrogenase that can be inhibited depending on the target microorganism.
  • the skilled person knows which gene corresponds to the enzyme of interest to be inhibited depending on the microorganism.
  • ribulose-5-phosphate (Ru5P) is no longer possible through the pentose phosphate pathway, or at least significantly reduced, in the genetically modified microorganism according to the invention.
  • the microorganism is a yeast of the genus Saccharomyces cerevisiae in which the expression of the ZWF1 gene is at least partially inhibited.
  • the yeast of the genus Saccharomyces cerevisiae is genetically modified so that the expression of the TDH1, TDH2, TDH3 and/or PGK1 genes, and the expression of the ZWF1 gene are at least partially inhibited.
  • the microorganism is a bacterium of the genus Escherichia coli in which the expression of the zwf gene is at least partially inhibited.
  • the bacterium of the genus Escherichia coli is genetically modified so that the expression of the gapA and/or pgk genes, and the expression of the zwf gene are at least partially inhibited.
  • the microorganism is a filamentous fungus of the genus Aspergillus , such as Aspergillus niger or Aspergillus terreus , genetically modified so that the expression of the pgk and gsdA genes is partially inhibited.
  • the genetically modified microorganism which expresses a functional RuBisCO and a functional PRK, and whose glycolysis pathway and oxidative branch of the pentose phosphate pathway are at least partially inhibited, is no longer capable of producing 3PG via the glycolysis pathway or Ru5P via the oxidative branch of the pentose phosphate pathway.
  • it is capable of producing Ru5P by diverting the production of fructose-6-phosphate (F6P) and/or glyceraldehyde-3-phosphate (G3P), produced at the beginning of glycolysis (upstream of inhibition).
  • transketolase EC 2.2.1.1
  • transaldolase EC 2.2.1.2
  • ribose-5-phosphate isomerase EC 5.3.1.6
  • ribulose-5-phosphate epimerase EC 5.1.3.1
  • microorganism Since the enzymes necessary for the metabolism of 3PG to pyruvate are not inhibited in the microorganism according to the invention, said microorganism can then metabolize 3PG to produce pyruvate and ATP.
  • the genetically modified microorganism is able to produce pyruvate by using exogenous CO 2 as complementary carbon source.
  • the genetically modified microorganism according to the invention makes it possible to increase the carbon yield, by fixing and using exogenous CO 2 , for the production of pyruvate (and subsequently molecules of interest). Here again, there is an increase in carbon yield.
  • the genetically modified microorganism according to the invention has an Entner-Doudoroff pathway, and this is at least partially inhibited.
  • This pathway mainly found in bacteria (especially Gram-negative bacteria), is an alternative to glycolysis and the pentose pathway for the production of pyruvate from glucose. More precisely, this pathway connects to the pentose phosphate pathway at P-gluconate to feed glycolysis, particularly at pyruvate.
  • the microorganism is genetically modified to inhibit Entner-Doudoroff pathway reactions downstream of 6-phosphogluconate production. This inhibition eliminates a possible competing pathway, and ensures the availability of 6-phosphogluconate as a substrate for PRK/RuBisCO engineering.
  • the interruption of the Entner-Doudoroff pathway downstream of 6-phosphogluconate production specifically targets one or more reactions in the pyruvate synthesis process from 6-phosphogluconate.
  • This synthesis is initiated by the successive actions of two enzymes: (i) 6-phosphogluconate dehydratase (“EDD”—EC. 4.2.1.12), and (ii) 2-dehydro-3-deoxy-phosphogluconate aldolase (“EDA”—E.C. 4.1.2.14).
  • 6-Phosphogluconate dehydratase catalyzes the dehydration of 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate.
  • the genes encoding 6-phosphogluconate dehydratase may be called edd (GenBank NP_416365, for example, in Escherichia coli ), or ilvD (for example, in Mycobacterium sp.).
  • 2-Dehydro-3-deoxy-phosphogluconate aldolase catalyzes the synthesis of a pyruvate molecule and a glyceraldehyde-3-phosphate molecule from the 2-keto-3-deoxy-6-phosphogluconate produced by 6-phosphogluconate dehydratase.
  • the genes encoding 2-dehydro-3-deoxy-phosphogluconate aldolase may be called eda (GenBank NP_416364, for example, in Escherichia coli ), or kdgA (for example in Thermoproteus tenax ), or dgaF (for example in Salmonella typhimurium ).
  • the microorganism is genetically modified so that the expression of the gene encoding 6-phosphogluconate dehydratase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • the microorganism is genetically modified so that the expression of the gene encoding 2-dehydro-3-deoxy-phosphogluconate aldolase is at least partially inhibited. Preferentially, gene expression is completely inhibited.
  • Tables 9 and 10 below list, as examples, the sequences encoding a 6-phosphogluconate dehydratase and a 2-dehydro-3-deoxy-phosphogluconate aldolase that can be inhibited depending on the target microorganism.
  • the skilled person knows which gene corresponds to the enzyme of interest to be inhibited depending on the microorganism.
  • pyruvate production is no longer possible via the Entner-Doudoroff pathway, or at least significantly reduced.
  • the microorganism is a bacterium of the genus Escherichia coli in which the expression of the edd gene is at least partially inhibited.
  • the bacterium of the genus Escherichia coli is genetically modified so that the expression of the gapA, and edd genes are at least partially inhibited.
  • the genetically modified microorganism which expresses a functional RuBisCO and a functional PRK, and whose glycolysis pathway and Entner-Doudoroff pathway are at least partially inhibited, is no longer capable of producing 3PG by glycolysis or pyruvate by the Entner-Doudoroff pathway.
  • the carbon flow from glucose is therefore preferably directed towards PRK/RuBisCO engineering.
  • the genetically modified microorganism is transformed so as to produce an exogenous molecule of interest and/or to overproduce an endogenous molecule of interest.
  • molecule of interest preferentially refers to a small organic molecule with a molecular mass less than or equal to 0.8 kDa.
  • “improved” yield refers to the quantity of the finished product.
  • the carbon yield corresponds to the ratio of quantity of finished product to quantity of fermentable sugar, particularly by weight.
  • the carbon yield is increased in the genetically modified microorganisms according to the invention, compared with wild-type microorganisms, placed under identical culture conditions.
  • the carbon yield is increased by 2%, 5%, 10%, 15%, 18%, 20%, or more.
  • the genetically modified microorganism according to the invention may produce a larger quantity of molecules of interest (finished product) than heterologous molecules produced by a genetically modified microorganism simply to produce or overproduce that molecule.
  • the genetically microorganism may also overproduce an endogenous molecule compared with the wild-type microorganism.
  • the overproduction of an endogenous molecule is mainly understood in terms of quantities.
  • the genetically modified microorganism produces at least 20%, 30%, 40%, 50%, or more by weight of the endogenous molecule than the wild-type microorganism.
  • the microorganism according to the invention is genetically modified so as to produce or overproduce at least one molecule among amino acids, terpenoids, terpenes, vitamins and/or vitamin precursors, sterols, flavonoids, organic acids, polyols, polyamines, aromatic molecules obtained from stereospecific hydroxylation, via an NADP-dependent cytochrome p450, etc.
  • the microorganism is genetically modified to overproduce at least one amino acid, preferentially selected from arginine, lysine, methionine, threonine, proline, glutamate, homoserine, isoleucine, valine, and ⁇ -aminobutyric acid.
  • the microorganism is genetically modified to produce or overproduce molecules from the terpenoid pathway, such as farnesene, and from the terpene pathway.
  • the microorganism is genetically modified to produce or overproduce a vitamin or precursor, preferentially selected from pantoate, pantothenate, transneurosporene, phylloquinone and tocopherols.
  • the microorganism is genetically modified to produce or overproduce a sterol, preferentially selected from squalene, cholesterol, testosterone, progesterone and cortisone.
  • the microorganism is genetically modified to produce or overproduce a flavonoid, preferentially selected from frambinone and vestinone.
  • the microorganism is genetically modified to produce or overproduce an organic acid, preferentially selected from coumaric acid, 3-hydroxypropionic acid, citric acid, oxalic acid, succinic acid, and itaconic acid.
  • an organic acid preferentially selected from coumaric acid, 3-hydroxypropionic acid, citric acid, oxalic acid, succinic acid, and itaconic acid.
  • the microorganism is genetically modified to produce or overproduce a polyol, preferentially selected from sorbitol, xylitol and glycerol.
  • the microorganism is genetically modified to produce or overproduce a polyamine, preferentially spermidine.
  • the microorganism is genetically modified to produce or overproduce an aromatic molecule from a stereospecific hydroxylation, via an NADP-dependent cytochrome p450, preferentially selected from phenylpropanoids, terpenes, lipids, tannins, fragrances, hormones.
  • the genetically modified microorganism is advantageously cultured in a culture medium including the substrate to be converted.
  • the production or overproduction of a molecule of interest by a genetically modified microorganism according to the invention is obtained by culturing said microorganism in an appropriate culture medium known to the skilled person.
  • appropriate culture medium generally refers to a sterile culture medium providing essential or beneficial nutrients for the maintenance and/or growth of said microorganism, such as carbon sources; nitrogen sources such as ammonium sulfate; sources of phosphors, for example, potassium phosphate monobasic; trace elements, for example, salts of copper, iodide, iron, magnesium, zinc or molybdate; vitamins and other growth factors such as amino acids or other growth promoters.
  • An antifoam agent can be added as needed.
  • this appropriate culture medium may be chemically defined or complex.
  • the culture medium may thus be identical or similar in composition to a synthetic medium, as defined by Verduyn et al. (Yeast. 1992. 8:501-17), adapted by Visser et al. (Biotechnology and bioengineering. 2002. 79:674-81), or commercially available such as yeast nitrogen base (YNB) medium (MP Biomedicals or Sigma-Aldrich).
  • the culture medium may include a simple carbon source, such as glucose, galactose, sucrose, molasses, or the by-products of these sugars, optionally supplemented with CO 2 as carbon co-substrate.
  • a simple carbon source such as glucose, galactose, sucrose, molasses, or the by-products of these sugars, optionally supplemented with CO 2 as carbon co-substrate.
  • the simple carbon source must allow the normal growth of the microorganism of interest. It is also possible, in some cases, to use a complex carbon source, such as lignocellulosic biomass, rice straw, or starch. The use of a complex carbon source usually requires pretreatment before use.
  • the culture medium contains at least one carbon source among monosaccharides such as glucose, xylose or arabinose, disaccharides such as sucrose, organic acids such as acetate, butyrate, propionate or valerate to promote different kinds of polyhydroxyalkanoate (PHA), treated or untreated glycerol.
  • monosaccharides such as glucose, xylose or arabinose
  • disaccharides such as sucrose
  • organic acids such as acetate, butyrate, propionate or valerate to promote different kinds of polyhydroxyalkanoate (PHA), treated or untreated glycerol.
  • PHA polyhydroxyalkanoate
  • any culture method allowing the production on an industrial scale of molecules of interest can be considered.
  • the culture is done in bioreactors, especially in batch, fed-batch and/or continuous culture mode.
  • the culture associated with the production of the molecule of interest is in fed-batch mode corresponding to a controlled supply of one or more substrates, for example by adding a concentrated glucose solution whose concentration can be between 200 g/L and 700 g/L.
  • a controlled supply of vitamins during the process can also be beneficial to productivity (Alfenore et al., Appl Microbiol Biotechnol. 2002. 60:67-72). It is also possible to add an ammonium salt solution to limit the nitrogen supply.
  • Fermentation is generally carried out in bioreactors, with possible steps of solid and/or liquid precultures in Erlenmeyer flasks, with an appropriate culture medium containing at least a simple carbon source and/or an exogenous CO 2 supply, necessary for the production of the molecule of interest.
  • the culture conditions of the microorganisms according to the invention are easily adaptable by the skilled person, depending on the microorganism and/or the molecule to be produced/overproduced.
  • the culture temperature is between 20° C. and 40° C. for yeasts, preferably between 28° C. and 35° C., and more particularly around 30° C., for S. cerevisiae .
  • the culture temperature is between 25° C. and 35° C., preferably 30° C., for Cupriavidus necator.
  • the invention therefore also relates to the use a genetically modified microorganism according to the invention, for the production or overproduction of a molecule of interest, preferentially selected from amino acids, peptides, proteins, vitamins, sterols, flavonoids, terpenes, terpenoids, fatty acids, polyols and organic acids.
  • the invention also relates to a biotechnological process for producing at least one molecule of interest, characterized in that it comprises a step of culturing a genetically modified microorganism according to the invention, under conditions allowing the synthesis or bioconversion, by said microorganism, of said molecule of interest, and optionally a step of recovering and/or purifying said molecule of interest.
  • the microorganism is genetically modified to express at least one enzyme involved in the synthesis of said molecule of interest.
  • the microorganism is genetically modified to express at least one enzyme involved in the bioconversion of said molecule of interest.
  • the invention also relates to a process for producing a molecule of interest comprising (i) inserting at least one sequence encoding an enzyme involved in the synthesis or bioconversion of said molecule of interest into a recombinant microorganism according to the invention, (ii) culturing said microorganism under conditions allowing the expression of said enzyme and optionally (iii) recovering and/or purifying said molecule of interest.
  • a fungus particularly a filamentous fungus, such as Aspergillus niger , genetically modified to express a functional PRK and a functional type I or II RuBisCO, and in which the expression of the pgk (Gene ID: 4982539) and gsdA (Gene ID: 497979751) genes is at least partially inhibited.
  • fungus particularly a filamentous fungus, such as Aspergillus terreus or Aspergillus niger , genetically modified to express a functional PRK and a functional type I or II RuBisCO, and in which the expression of the pgk (Gene ID: 4354973) and gsdA (Gene ID: 4316232) genes is at least partially inhibited.
  • a yeast such as a yeast of the genus Saccharomyces cerevisiae genetically modified to express a functional PRK and a functional type I or II RuBisCO, a farnesene synthase and in which the expression of a PGK1 gene (Gene ID: 5230) is at least partially inhibited.
  • a bacterium such as a bacterium of the genus Escherichia coli , genetically modified to express a functional PRK and a functional type I or II RuBisCO, and in which the expression of the gapA gene (Gene ID: 947679) is at least partially inhibited.
  • This overproduction can also occur in a strain where at least partial inhibition of the gapA gene is combined with at least partial inhibition of the zwf gene (Gene ID: 946370).
  • a bacterium such as a bacterium of the genus Escherichia coli , genetically modified to express a functional PRK and a functional type I or II RuBisCO, as well as a glutamate decarboxylase gadB (Gene ID: 946058), and in which the expression of the gapA gene (Gene ID: 947679) is at least partially inhibited.
  • This overproduction can also occur in a strain where at least partial inhibition of the gapA gene is combined with at least partial inhibition of the zwf gene (Gene ID: 946370).
  • a bacterium such as a bacterium of the genus Escherichia coli , genetically modified to express a functional PRK and a functional type I or II RuBisCO, as well as an enzymatic activity allowing the oxidation of glyoxylate to oxalate, preferentially a glyoxylate dehydrogenase FPGLOXDH1 (mRNA: BAH29964.1), a glyoxylate oxidase GLO (mRNA: AOW73106.1), or a lactate dehydrogenase LDHA (Gene ID: 3939), and in which the expression of the gapA (Gene ID: 947679) and zwf (Gene ID: 946370) genes is at least partially inhibited.
  • PRK-RuBisCO engineering i) in a strain inhibited for glycolysis on the yield of a NADPH-dependent biosynthetic pathway (for example farnesene synthesis), and (ii) in a strain inhibited for glycolysis and for the oxidative branch of the pentose phosphate pathway on the yield of a biosynthetic pathway of interest (for example citrate synthesis).
  • the maximum theoretical yield of pyruvate production by the pentose phosphate pathway is therefore 0.82 g pyruvate /g glucose (g of synthesized pyruvate, per g of glucose consumed), while it is 0.98 g pyruvate /g glucose by the glycolysis pathway.
  • the carbon fixation flux is redirected to the oxidative branch of the pentose phosphate pathway and then to PRK/RuBisCO engineering (see FIG. 2 ).
  • This flux is related to the end of the glycolysis pathway, at the level of 3-phosphoglycerate (3PG) formation, with the following yield:
  • the integration of the modifications according to the invention into a microorganism makes it possible to recover the carbon molecule otherwise lost by decarboxylation in the pentose pathway.
  • the maximum theoretical carbon fixation yield is therefore 0.98 g pyruvate /g glucose , which improves by 20.5% the yield obtained by the production of pyruvate by the pentose phosphate pathway, while producing NADPH.
  • PRK/RuBisCO engineering is integrated into a strain that is both inhibited for glycolysis (for example ⁇ PGK1 in the case of S. cerevisiae yeast) and for the oxidative branch of the pentose phosphate pathway (for example ⁇ ZWF1 in the case of S. cerevisiae yeast).
  • ⁇ PGK1 in the case of S. cerevisiae yeast
  • ⁇ ZWF1 oxidative branch of the pentose phosphate pathway
  • the calculation is applied to citrate production in S. cerevisiae yeast, in a wild-type strain and in a modified strain modified according to the invention incorporating PRK/RuBisCO engineering and deleted for the PGK1 gene so as to inhibit the glycolysis pathway, and for the ZWF1 gene to inhibit the oxidative branch of the pentose pathway.
  • the corresponding g citrate /g glucose yield is 1.28, a maximum theoretical increase of about 20% compared with the yield of the wild-type strain.
  • FBAs flux balance analyses
  • FBAs are based on mathematical models that simulate metabolic networks at the genome scale (Orth et al., Nat Biotechnol. 2010; 28: 245-248). Reconstructed networks contain the known metabolic reactions of a given organism and integrate the needs of the cell, in particular to ensure cell maintenance or growth. FBAs make it possible to calculate the flow of metabolites through these networks, making it possible to predict theoretical growth rates as well as metabolite production yields.
  • the reactions necessary to simulate the production of molecules through heterologous pathways have also been added to the model.
  • a farnesene synthase reaction (EC 4.2.3.46 or EC 4.2.3.47) has been added for the heterologous production of farnesene.
  • acetoacetyl-CoA reductase (EC 1.1.1.36) and poly-hydroxybutyrate synthase (EC 2.3.1.B2 or 2.3.1.B5) reactions were added to the model to simulate a heterologous production pathway of ⁇ -hydroxybutyrate, the monomer of polyhydroxybutyrate.
  • a glutamate decarboxylase reaction (EC 4.1.1.15) was added for the heterologous production of ⁇ -aminobutyric acid.
  • an aconitate decarboxylase reaction (EC 4.1.1.6) was added for the heterologous production of itaconic acid.
  • a lactate dehydrogenase reaction (EC 1.1.1.27) was added for the heterologous production of oxalate
  • the simulations were carried out by applying to the model a set of constraints reproducible by the skilled person, aimed at simulating the in vivo culture conditions of a strain of S. cerevisiae under the conditions described according to the invention (for example presence of unrestricted glucose in the medium, aerobic culture condition).
  • simulations are performed by virtually inactivating the reactions of the enzymes PGK1 (for example glutamate, ⁇ -hydroxybutyric acid, farnesene) and ZWF1 (for example citrate production), in order to simulate the decreases in glycolysis activity and the pentose phosphate pathway, described according to the invention.
  • PGK1 for example glutamate, ⁇ -hydroxybutyric acid, farnesene
  • ZWF1 for example citrate production
  • Simulations are carried out in parallel on an unmodified “wild-type strain” model in order to evaluate the impact of the improvements described according to the invention on the production yield of the biosynthetic pathways tested.
  • CEN.PK 1605 (Mat a HIS3 leu2-3.112 trp1-289 ura3-52 MAL.28c) derived from the commercial strain CEN.PK 113-7D (GenBank: JRIV00000000 is engineered to produce NADPH without CO 2 loss and thus allow the improvement of alpha-farnesene production from glucose.
  • the glycolysis pathway was inactivated by deletion of the PGK1 gene. Once glycolysis is inhibited, the resulting yeast strain is no longer able to use glucose as a source of carbon and energy. It is therefore necessary to supply the biomass synthesis pathways with glycerol and the energy pathways with ethanol.
  • the strains in which PGK1 is deleted are grown on YPGE (yeast extract peptone glycerol ethanol) medium.
  • the deletion of the PGK1 gene was obtained as follows:
  • the coding phase of the G418 resistance gene derived from the KanMX cassette contained on plasmid pUG6 (P30114—Euroscarf), was amplified with the oligonucleotides CB101 (SEQ ID NO: 1) and CB102 (SEQ ID NO: 2):
  • SEQ ID NO: 1 CB101 (forward): 5′-ACAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAAT ATAAAACA ATGGGTAAGGAAAAGACTCACGTTTC -3′
  • SEQ ID NO: 2 CB102 (reverse): 5′-GGGAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAATTC AATTCAAT TTAGAAAAACTCATCGAGCATCAAATGAAAC -3′
  • the underlined portion of the oligonucleotides is perfectly homologous to the Kan sequence and the rest of the sequence corresponds to the regions adjacent to the coding phase of the PGK1 gene on the Saccharomyces cerevisiae genome so as to generate a PCR amplicon containing at its ends homologous recombination sequences of the PGK1 gene locus.
  • strain CEN.PK 1605 was grown in a volume of 50 mL of complex rich medium YPD (yeast extract peptone dextrose) at 30° C. to an optical density at 600 nm of 0.8. The cells were centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • YPD yeast extract peptone dextrose
  • the supernatant was removed and the cells were resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells were resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix was prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 5 or 10 ⁇ L of purified PCR reaction (deletion cassette) and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) were added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath.
  • the tube was centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant was discarded.
  • the cells were resuspended in 2 mL of YPGE (yeast extract peptone glycerol ethanol) medium, transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells were then centrifuged for 1 minute at 5,000 rpm at room temperature. The supernatant was removed and the cells were resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread over 180 ⁇ g/mL YPGE+G418.
  • YPGE yeast extract peptone glycerol ethanol
  • the yeast lacks the alpha-farnesene synthase gene (AFS1; SEQ ID NO: 71; GenBank accession number AY182241).
  • the seven genes required for PRK-RuBisCO engineering were cloned on four plasmid vectors capable of autonomous replication, with compatible origins of replication and each carrying a different gene for complementation of auxotrophy or of antibiotic resistance, allowing the selection of strains containing the three or four plasmid constructs.
  • Two of these plasmids are single-copy, with an Ars/CEN origin of replication and the third is multicopy with a 2 ⁇ origin.
  • strain EQ-0134 was grown in a volume of 50 mL of complex rich medium YPGE (yeast extract peptone glycerol ethanol) at 30° C. The cells are centrifuged for 5 minutes at 2,500 rpm at room temperature. The supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • YPGE yeast extract peptone glycerol ethanol
  • the following transformation mix is prepared: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L (3 ⁇ g of one of the following combinations, pFPP45+pFPP56+pFPP20 or pL4+pFPP45+pFPP56+pFPP20) and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) were added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube was centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant was discarded. The cells were resuspended in 2 mL YNB (yeast nitrogen base including ammonium sulfate) with glycerol and ethanol, transferred to a 14 mL tube and incubated for 2 hours at 30° C. under atmosphere enriched with 10% CO 2 .
  • YNB yeast nitrogen base including ammonium sulfate
  • the final mix is spread on YNB agar medium including ammonium sulfate+CSM without LUW (leucine uracil, tryptophan)+nourseothricin if applicable, with glycerol and ethanol as carbon sources.
  • LUW leucine uracil, tryptophan
  • strain CEN.PK 1605 is transformed with the following plasmid combination: pL4+pFL36+pCM185+pV51TEF.
  • the clones obtained were genotyped for all engineering genes and then adapted on liquid medium YNB ammonium sulfate and glucose.
  • cultures with a volume greater than or equal to 50 mL in Erlenmeyer flasks of at least 250 mL are inoculated in order to adapt the strain to the use of the PRK/RuBisCO engineering.
  • This adaptation is carried out on YNB+CSM-LUW culture medium with 20 g/L glucose, in the presence of nourseothricin if necessary and an exogenous CO 2 supply as described above.
  • the strains are adapted to a minimum mineral medium free of the amino acids and nitrogenous bases included in the CSM-LUW, i.e. only YNB with 20 g/L glucose, nourseothricin if necessary and an exogenous CO 2 supply as described above.
  • Saccharomyces cerevisiae strain EQ-0253 with a deletion in the glycolytic pathway at the PGK1 gene, is grown to produce farnesene while overproducing NADPH without CO 2 loss, using a PRK and a RuBisCO.
  • This strain of interest is compared with a reference strain EQ-0353 producing farnesene following the introduction of a heterologous alpha-farnesene synthase, without deletion of PGK1 or addition of PRK and RuBisCO.
  • Strains EQ-0253 (CEN.PK1605 ⁇ pgk1::kan) (pL4+pFPP56+pFPP20+pFPP45) and EQ-0353 (CEN.PK1605) (pL4+pFL36+pCM185+pV51TEF) were grown in a YNB medium with 20 g/L D-glucose, to which 100 ⁇ g/L nourseothricin was added.
  • a pre-culture containing 20 mL of culture medium was inoculated at 0.05 OD 600 nm into a 250 mL baffled Erlenmeyer flask, shaken at 120 rpm for 24 h at 30° C.
  • the culture also conducted in Erlenmeyer flasks (500 mL, baffled) from the second pre-culture, was inoculated at 0.05 OD 600 nm into 100 mL of the same culture medium, to which 50 ⁇ g/mL ampicillin, 10 ⁇ L antifoam (Antifoam 204, Sigma, A6426) and 10% (v/v) dodecane were added (Tippman et al., Talanta (2016), 146: 100-106). The cultures were shaken at 120 rpm at 30° C. in the presence of 10% CO 2 . Growth was monitored by measuring turbidity at 600 nm.
  • the transfer line and source temperatures were 250° C. and 200° C. respectively.
  • An external calibration including seven points was performed using the farnesene isomer mix (Sigma, W383902) for the quantification of ⁇ -farnesene produced by the strains.
  • a Rezex ROA-Organic Acid H + column (8%) 150 ⁇ 7 8 mm, 8 ⁇ m particle size (Phenomenex, 00H-0138-KO) was used with a Carbo-H pre-column 4 ⁇ 3.0 mm.
  • the temperature of the column was 35° C. and the flow rate was set at 0.5 mL/min.
  • Isocratic elution was performed with an aqueous mobile phase at 5 mM H 2 SO 4 and lasted 30 min A volume of 20 ⁇ L was injected for each sample.
  • the identification of compounds was based on the comparison of retention times with standards.
  • the external calibration includes 10 points of variable glucose concentration (0-20 g/L).
  • the carbon yield Y ⁇ -farnesene/Glc is calculated in grams of farnesene produced per gram of glucose consumed for both strains EQ-0253 and EQ-0353,
  • Y ⁇ - farnesene / Glc farnesene ⁇ ⁇ ( mg ⁇ / ⁇ L ⁇ ⁇ aqueous ) glucose ⁇ ⁇ ( mg ⁇ / ⁇ L ⁇ ⁇ aqueous ) .
  • the coding phase of the hygromycin B resistance gene derived from the hphMX cassette (loxP-pAgTEF1-hph-tAgTEF1-loxP) and contained on plasmid pUG75 (P30671)—Euroscarf), is amplified with the oligonucleotides Sdzwf1 and Rdzwf1 (Table 14). This makes it possible to generate a ⁇ zwf1 PCR amplicon containing at its ends homologous recombination sequences of the glucose-6-phosphate dehydrogenase ZWF1 gene locus.
  • the underlined portion of the oligonucleotides corresponds to the portion perfectly homologous to the sequence of the selection gene, the rest of the sequence corresponding to the regions adjacent to the coding phase of the target gene to be deleted on the Saccharomyces cerevisiae genome.
  • strain CEN.PK 1605 (Mat a HISS leu2-3.112 trp1-289 ura3-52 MAL.28c) derived from the commercial strain CEN.PK 113-7D (GenBank: JRIV00000000 ) is transformed with the ⁇ zwf1 PCR fragment described above.
  • strain CEN.PK 1605 is grown in a volume of 50 mL of complex rich medium YPD (yeast extract peptone dextrose, here 20 g/L glucose) at 30° C. to an optical density at 600 nm of 0.8.
  • the cells are centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • the supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix is prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L of purified PCR reaction (deletion cassette) and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) are added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube is centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant is discarded. The cells are resuspended in 2 mL of YPD (yeast extract peptone dextrose) medium, transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at room temperature.
  • YPD yeast extract peptone dextrose
  • the supernatant is removed and the cells are resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread on YPD+HygromycinB (200 ⁇ g/mL).
  • the colonies obtained were genotyped for the validation of the deletion of the ZWF1 gene and referenced EQSC-002 (CEN.PK 1605 ⁇ zwf1::hph).
  • the coding phase of the nourseothricin resistance gene derived from the natMX cassette (loxP-pAgTEF1-nat-tAgTEF1-loxP) contained on the plasmid (pUG74 (P30670)—Euroscarf) is amplified with the oligonucleotides Sdidh1 and Rdidh1 (Table 13). This makes it possible to generate a ⁇ idh1 PCR amplicon containing at its ends homologous recombination sequences of the isocitrate dehydrogenase IDH1 subunit gene locus.
  • strains EQSC-002 and CEN.PK1605 are grown in a volume of 50 mL of complex rich medium YPD (yeast extract peptone dextrose, here 20 g/L glucose) at 30° C. to an optical density at 600 nm of 0.8.
  • the cells are centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • the supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix is prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L of purified PCR reaction (deletion cassette) and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) are added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube is centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant is discarded. The cells are resuspended in 2 mL of YPD (yeast extract peptone dextrose), transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at room temperature.
  • YPD yeast extract peptone dextrose
  • the supernatant is removed and the cells are resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread on YPD+HygromycinB 200 ⁇ g/mL, 50 nourseothricin.
  • the colonies obtained were genotyped for the validation of the deletion of the IDH1 gene and are called EQSC-003 (CEN.PK 1605 ⁇ zwf1::hph, ⁇ idh1::nat) and EQSC-005 (CEN.PK 1605 ⁇ idh1::nat)
  • Strain CEN.PK 1606 (Mat alpha HIS3 leu2-3.112 trp1-289 ura3-52 MAL.28c) derived from the commercial strain CEN.PK 113-7D (GenBank: JRIV00000000) is transformed with the PCR fragment for inactivation of the PGK1 gene.
  • strain CEN.PK 1606 is grown in a volume of 50 mL of complex rich medium YPD (yeast extract peptone dextrose, here 20 g/L glucose) at 30° C. to an optical density at 600 nm of 0.8.
  • the cells are centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • the supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix is prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L of purified PCR reaction (deletion cassette) and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) are added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube is centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant is discarded. The cells are resuspended in 2 mL of YPGE (yeast extract peptone 20 g/L glycerol, 30 g/L ethanol), transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at room temperature.
  • YPGE yeast extract peptone 20 g/L glycerol, 30 g/L ethanol
  • the supernatant is removed and the cells are resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread over YPGE+150 ⁇ g/mL G418.
  • the colonies obtained were genotyped for the validation of the deletion of the PGK1 gene and referenced EQSC-008 (CEN.PK 1605, ⁇ pgk1::kan).
  • the haploid strains of opposite mating types EQSC-003 (CEN.PK 1605 ⁇ zwf1::hph, ⁇ idh1::nat) and EQSC-008 (CEN.PK 1606 ⁇ pgk1::kan) are grown overnight on agar medium:YPD (yeast extract peptone dextrose) for strain EQSC-008 and YPGE (yeast extract peptone glycerol ethanol) for strain EQSC003, at 30° C.
  • YPD yeast extract peptone dextrose
  • YPGE yeast extract peptone glycerol ethanol
  • YPGE yeast extract peptone glycerol ethanol
  • agar medium +150 ⁇ g/mL G418+200 ⁇ g/mL hygromycin B.
  • the G418 and hygromycin B double selection eliminates the two parental strains, only the MAT a/MAT alpha, ZWF1/ ⁇ zwf1::hph, IDH1/ ⁇ idh1::nat, PGK1/ ⁇ pgk1::kan diploid strains grow on this medium. An isolated diploid clone from this crossing is collected.
  • the presence of the three cassettes ⁇ zwf1::hph, ⁇ idh1::nat, ⁇ pgk1::kan is validated by growth tests on YPGE (yeast extract peptone glycerol ethanol) agar medium supplemented with 150 ⁇ g/mL G418 or 200 ⁇ g/mL hygromycin B or 50 ⁇ g/mL nourseothricin.
  • the strain obtained is referenced EQSC-009 (CEN.PK 1607, MAT a/MAT alpha, ZWF1/ ⁇ zwf1::hph, IDH1/ ⁇ idh1::nat, PGK1/ ⁇ pgk1::kan).
  • strain EQSC-009 (CEN.PK 1607, MAT a/MAT alpha, ZWF1/ ⁇ zwf1::hph, IDH1/ ⁇ idh1::nat, PGK1/ ⁇ pgk1::kan) is grown on YPGE (yeast extract peptone glycerol ethanol) agar medium overnight at 30° C. The cells are then placed in liquid culture in a deficient medium (Sporulation Medium, 1% potassium acetate+leucine+uracil+tryptophan) to induce meiosis of the diploid cells and thus lead to the formation of tetrads containing four haploid spores.
  • YPGE yeast extract peptone glycerol ethanol
  • the tetrads are spread on YNB.GE medium (yeast nitrogen base, glycerol, ethanol)+leucine+uracil+tryptophan+1 g/L glutamic acid+20 mg/L methionine+40 mg/L cysteine and immediately dissected (using a microdissector) to isolate the spores on the same medium.
  • the spores are germinated for several days at 30° C.
  • the genetic content of the haploid cells thus obtained is tested by growth on selective media: YPGE (yeast extract peptone glycerol ethanol) supplemented with 150 ⁇ g/mL G418 or 200 ⁇ g/mL hygromycin B or 50 ⁇ g/mL nourseothricin and their mating type is tested by crossing with two tectrix strains of mating type MAT a or MAT alpha.
  • the colonies obtained are genotyped for the validation of the deletion of the PGK1, IDH1, ZWF1 genes and the absence of transcripts corresponding to these genes is validated by real-time PCR after reverse transcription of ribonucleic acids.
  • One of the strains obtained is referenced EQSC-004 (CEN.PK 1606 MAT alpha ⁇ zwf1::hph, ⁇ idh1::nat, ⁇ pgk1::kan)
  • the six genes required for PRK-RuBisCO engineering are cloned on three plasmid vectors capable of autonomous replication, with compatible origins of replication and each carrying a different auxotrophic complementation gene, allowing the selection of strains containing the three plasmid constructs (see WO 2015107496). Two of these plasmids are single-copy with an ARS/CEN origin of replication and the third is multicopy with a 2 ⁇ origin.
  • strain EQSC-004 (CEN.PK 1606 ⁇ zwf1::hph, ⁇ idh1::nat, ⁇ pgk1::kan) was grown in a volume of 50 mL of complex rich medium YPGE (yeast extract peptone glycerol ethanol) at 30° C. to an optical density at 600 nm of 0.8.
  • the cells are centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • the supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix is prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L (3 ⁇ g) of a combination of pFPP45+pFPP56+pFPP20 and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) are added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube is centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant is discarded. The cells are resuspended in 2 mL of YPGE (yeast extract peptone glycerol ethanol)+2 mg/L doxycycline, transferred into a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at room temperature.
  • YPGE yeast extract peptone glycerol ethanol
  • the supernatant is removed and the cells are resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread over YNB.GE (yeast nitrogen base, glycerol, ethanol)+1 g/L glutamic acid+20 mg/L methionine+40 mg/L cysteine+2 mg/L doxycycline.
  • the strain obtained is referenced: EQSC-006 (CEN.PK 1606 ⁇ zwf1::hph, ⁇ idh1::nat, ⁇ pgk1::kan) (pFPP45+pFPP56+pFPP20).
  • strain EQSC-005 (CEN.PK 1605 ⁇ idh1::nat) was grown in a volume of 50 mL of complex rich medium YPGE (yeast extract peptone glycerol ethanol) at 30° C. to an optical density at 600 nm of 0.8.
  • the cells are centrifuged for 5 minutes at 2,500 rpm at room temperature.
  • the supernatant is removed and the cells are resuspended in 25 mL of sterile water and centrifuged again for 5 minutes at 2,500 rpm at room temperature. After removing the supernatant, the cells are resuspended in 400 ⁇ L of 100 mM sterile lithium acetate.
  • a transformation mix is prepared in a 2 mL tube as follows: 250 ⁇ L of 50% PEG, 10 ⁇ L of “carrier” DNA at 5 mg/mL, 36 ⁇ L of 1 M lithium acetate, 10 ⁇ L (3 ⁇ g) of a combination of pV51TEF+pFL36+pCM185 and 350 ⁇ L of water.
  • the resuspended cells (50 ⁇ L) are added to the transformation mixture and incubated at 42° C. for 40 minutes in a water bath. After incubation, the tube is centrifuged for 1 minute at 5,000 rpm at room temperature and the supernatant is discarded. The cells are resuspended in 2 mL of YPD (yeast extract peptone dextrose), transferred to a 14 mL tube and incubated for 2 hours at 30° C. at 200 rpm. The cells are then centrifuged for 1 minute at 5,000 rpm at room temperature.
  • YPD yeast extract peptone dextrose
  • the supernatant is removed and the cells are resuspended in 1 mL of sterile water and centrifuged again for 1 minute and resuspended in 100 ⁇ L of sterile water and spread on YNBD (yeast nitrogen base dextrose)+2 mg/L doxycycline.
  • the strain obtained is referenced: EQSC-007 (CEN.PK 1605 ⁇ idh1::nat) (pV51TEF+pFL36+pCM185).
  • strains EQSC-006 and EQSC-007 to growth on YNB (yeast nitrogen base) liquid medium with glucose and CO 2 .
  • Batch-mode cultures in Erlenmeyer flasks are carried out with the appropriate culture medium and a 10% exogenous CO 2 supply, in a shaking incubator (120 rpm, 30° C.), with inoculation at 0.05 OD 600 nm measured using an EON spectrophotometer (BioTek Instruments).
  • the strain of interest is grown on YNB+CSM-LUW medium with 10 g/L glycerol and 7.5 g/L ethanol, +50 mg/L glutamate under conditions where PRK expression is not induced.
  • cultures with a volume greater than or equal to 50 mL in Erlenmeyer flasks of at least 250 mL are inoculated in order to adapt the strain to the use of the PRK/RuBisCO engineering.
  • This adaptation is carried out on YNB+CSM-LUW culture medium with 20 g/L glucose, 50 mg/L glutamate and an exogenous CO 2 supply as described above.
  • the strains are adapted to a minimum mineral medium free of all amino acids except those indicated below, and nitrogenous bases included in the CSM-LUW, i.e. only YNB with, in final concentrations, 20 g/L glucose, 1 g/L glutamate, 40 mg/L L-cysteine and 20 mg/L L-methionine and an exogenous CO 2 supply as described above.
  • Saccharomyces cerevisiae strain EQSC-006 with a deletion in the glycolytic pathway at the PGK1 gene, in the oxidative part of the pentose phosphate pathway and in the Krebs cycle, is grown to produce citrate without CO 2 loss, using PRK and RuBisCO.
  • This strain of interest is compared with a reference strain EQSC-007 producing citrate following inactivation of the IDH1 gene, without deletion of PGK1 or ZWF1 or addition of PRK and RuBisCO.
  • EQSC-006 CEN.PK 1605 ⁇ zwf1::hph, ⁇ idh1::nat, ⁇ pgk1::kan, pFPP45+pFPP56+pFPP20
  • EQSC-007 CEN.PK 1605 ⁇ idh1::nat, pV51TEF+pFL36+pCM185
  • yeast nitrogen base (YNB) medium supplemented with 20 g/L D-glucose (YNB D20).
  • a pre-culture containing 20 mL of culture medium was inoculated at 0.05 OD 600 nm into a 250 mL baffled Erlenmeyer flask, shaken at 120 rpm at 30° C.
  • 50 mL of medium was inoculated at 0.05 OD 600 nm into a 250 mL Erlenmeyer flask and shaken at 120 rpm, at 30° C.
  • the culture was carried out in Erlenmeyer flasks (500 mL, baffled) from the second pre-culture, inoculated at 0.05 OD 600 nm into 100 mL of the same medium, at 30° C., 120 rpm. Growth was monitored by measuring turbidity at 600 nm.
  • An isocratic elution at a flow rate of 0.5 mL/min was carried out with an aqueous solution of 0.037% formic acid (v/v) whose pH was adjusted to 4.5 with ammonium hydroxide.
  • the column oven temperature was 65° C.
  • the mass spectrometry analytical conditions were: negative electrospray mode, source temperature 450° C., needle voltage 3 kV, cone voltage 50 V.
  • a seven-point external calibration was performed using a commercial sodium citrate solution.
  • a Rezex ROA-Organic Acid H + column (8%) 150 ⁇ 7.8 mm, 8 ⁇ m particle size (Phenomenex, 00H-0138-KO) was used with a Carbo-H 4 ⁇ 3.0 mm pre-column.
  • the column oven temperature was 35° C. and the flow rate was set at 0.5 mL/min
  • a 30 min isocratic elution was performed with an aqueous mobile phase at 5 mM H 2 SO 4 .
  • a volume of 20 ⁇ L was injected for each sample. The identification of the compounds was based on the comparison of retention times with standards.
  • the external calibration included 10 points of variable glucose concentration (0 to 20 g/L).
  • the Y citrate/Glc mass yield was calculated in grams of citrate produced per gram of glucose consumed for both strains EQSC-006 and EQSC-007,
  • Escherichia coli strain K12 MG1655 with a deleted sucA gene was used.
  • This strain is derived from a gene deletion bank (Baba et al. Mol Syst Biol. 2006; 2:2006.0008) in Escherichia coli and supplied by the coli Genetic Stock Center under the name JW0715-2 and with reference 8786. (JW0715-2: MG1655 ⁇ sucA::Kan)
  • the selection cassette was deleted using a recombinase.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC002: MG1655 ⁇ sucA
  • the deletion of the edd-eda operon is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • the deletion of the gapA gene is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • ribosome binding sequences presented in Table 19 below, with variable translation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun. 21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic Acids Res. 2013 May; 41(9):e98) are inserted between the coding phase for each gene.
  • the succession of each coding phase interspersed by an RBS sequence is constructed by successive insertions into a pZA11 vector (Expressys) that contains a PLtetO-1 promoter, a p15A origin of replication and an ampicillin resistance gene.
  • RBS intercistronic sequences Name RBS sequences A (SEQ ID NO: 9) AGGAGGTTTGGA B (SEQ ID NO: 10) AACAAAATGAGGAGGTACTGAG C (SEQ ID NO: 11) AAGTTAAGAGGCAAGA D (SEQ ID NO: 12) TTCGCAGGGGGAAG E (SEQ ID NO: 13) TAAGCAGGACCGGCGGCG F (SEQ ID NO: 14) CACCATACACTG
  • EQ.EC 006 ⁇ (EQ.EC 004+pEQEC005): MG1655 ⁇ sucA ⁇ edd-eda ⁇ gapA::kan (RuBisCO)
  • Clones are selected on LB medium supplemented with 2 g/L glycerol and 5 g/L pyruvate and with 100 mg/L ampicillin. After obtaining a sufficient quantity of biomass, cultures with a volume greater than or equal to 50 mL in a minimum 250 mL Erlenmeyer flask are inoculated in order to adapt the strain to the use of the PRK/RuBisCO engineering. This adaptation is carried out on LB culture medium with 2 g/L glucose, and an exogenous CO 2 supply at 37° C. as described above.
  • cells from 500 mL of LB culture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere CO 2 .
  • MS medium 40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere
  • Residual glutamate and glucose are measured with a bioanalyzer (Sakura Seiki).
  • the carbon yield Y p/s is calculated in grams of glutamate produced per gram of glucose consumed.
  • This step is performed in the same way as example 4A] above.
  • the strain obtained is called EQ.EC002: MG1655 ⁇ sucA
  • the deletion of the zwf gene (GeneID: 946370) is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol, as detailed in Example 4A].
  • the strain obtained is called EQ.EC010: MG1655 ⁇ sucA ⁇ zwf
  • the deletion of the gapA gene in the Escherichia coli K-12 strain EQ.EC010 is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol, as detailed in Example 4A].
  • ribosome binding sequences presented in Table 17 (see Example 4A]), with variable translation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun. 21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic Acids Res. 2013 May; 41(9):e98) are inserted between the coding phase of each gene.
  • the succession of each coding phase interspersed by an RBS sequence is constructed by successive insertions into a pZA11 vector (Expressys) that contains a PLtetO-1 promoter, a p15A origin of replication and an ampicillin resistance gene.
  • icfA carbonic anhydrase
  • EQ.EC 014 ⁇ (EQ.EC 011+pEQEC006): MG1655 ⁇ sucA ⁇ zwf ⁇ gapA (RuBisCO+PRK)
  • EQ.EC 015 ⁇ (EQ.EC 011+pEQEC007): MG1655 ⁇ sucA ⁇ zwf ⁇ gapA (RuBisCO+PRK+carbonic anhydrase)
  • clones are selected on LB glycerol, pyruvate medium supplemented with 100 mg/L ampicillin.
  • An adaptation and evolution phase of the strains with PRK and RuBisCO engineering is performed as described in Example 4A].
  • cells from 500 mL of LB culture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH4) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere CO 2 .
  • MS medium 40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH4) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere CO 2 .
  • Residual glutamate and glucose are measured with a bioanalyzer (YSI Inc.).
  • the carbon yield Y p/s is calculated in grams of glutamate produced per gram of glucose consumed.
  • This step is performed in the same way as example 4A] above.
  • the strain obtained is called EQ.EC002: MG1655 ⁇ sucA
  • the deletion of the zwf gene (GeneID: 946370) is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol, as detailed in Example 4A].
  • the strain obtained is called EQ.EC010: MG1655 ⁇ sucA ⁇ zwf
  • the deletion of the gapA gene in the Escherichia coli K-12 strain EQ.EC010 is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol, as detailed in Example 4A]. Deletions are verified by genotyping and sequencing and the name of the strains obtained is:
  • ribosome binding sequences presented in Table 17 (see Example 4A]
  • RBS ribosome binding sequences
  • Table 17 The succession of each coding phase interspersed by an RBS sequence is constructed by successive insertions into a pZA11 vector (Expressys) that contains a PLtetO-1 promoter, a p15A origin of replication and an ampicillin resistance gene.
  • the addition of a glutamate dehydrogenase (gdhA) and a pyruvate carboxylase (pycA) allows a better production of glutamic acid.
  • pycA pyruvate carboxylase
  • CA carbonic anhydrase
  • CA also allows an interconversion of bicarbonate ions into available CO 2 molecules and improves the efficiency of RuBisCO.
  • clones are selected on LB glycerol, pyruvate medium supplemented with 100 mg/L ampicillin.
  • An adaptation and evolution phase of the strains with PRK and RuBisCO engineering is performed as described in Example 4A].
  • cells from 500 mL of LB culture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere CO 2 .
  • MS medium 40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin at a pressure of 0.1 atmosphere
  • Residual glutamate and glucose are measured with a bioanalyzer (YSI Inc.).
  • the carbon yield Y p/s is calculated in grams of glutamate produced per gram of glucose consumed.
  • the increase in reducing power obtained through the genetic modifications proposed according to the invention may also have a considerable gain over existing metabolic pathways.
  • This C. necator H16 strain has a megaplasmid pHG1 and two chromosomes.
  • the deletion of the gapA gene is performed by generating a vector containing the Bacillus subtilis suicide gene sacB for Gram-negative bacteria (Quandt et al., Gene. 1993 May 15; 127(1):15-21; Lindenkamp et al., Appl Environ Microbiol. 2010 August; 76(16):5373-82 and Appl Environ Microbiol. 2012 August; 78(15):5375-83).
  • PCR amplicons corresponding to adjacent regions of the edd and eda genes are cloned by restriction according to the procedure described in Srinivasan et al. (Appl Environ Microbiol. 2002 December; 68(12):5925-32), in plasmid pJQ200mp18Cm.
  • the modified plasmid pJQ200mp18Cm:: ⁇ edd-eda is then transformed into an E. coli strain S17-1 by the calcium chloride transformation method.
  • the transfer of genetic material into C. necator is done by conjugation by depositing on agar a spot of C. necator culture on a dish containing a cell monolayer of S17-1 bacteria. Selection is made on nutrient broth (NT) medium at 30° C. in the presence of 10% sucrose for purposes of selection (Hogrefe et al., J Bacteriol. 1984 April; 158(1):43-8) and validated on a mineral medium containing 50 ⁇ g/mL chloramphenicol.
  • NT nutrient broth
  • EQCN_002 The deletions are validated by genotyping and sequencing.
  • the resulting strain EQCN_002 therefore has deletions of the genes of the Entner-Doudoroff metabolic pathway edd-eda.
  • EQCN_002 H16 ⁇ edd-eda.
  • Two PCR amplicons corresponding to adjacent regions of the gapA gene are cloned by restriction according to the procedure described in Lindenkamp et al. 2012, in plasmid pjQ200mp18Tc.
  • the modified plasmid pjQ200mp18Tc:: ⁇ gapA is then transformed into an E. coli strain S17-1 by the calcium chloride transformation method.
  • the transfer of genetic material is done by conjugation by depositing on agar a spot of C. necator culture on a plate containing a cell monolayer of S17-1 bacteria. Selection is made on nutrient broth (NT) medium at 30° in the presence of 10% sucrose for purposes of selection (Hogrefe et al., J Bacteriol. 1984 April; 158(1):43-8) and validated on a mineral medium containing 25 ⁇ g/mL tetracycline.
  • NT nutrient broth
  • EQCN_003 H16 ⁇ edd-eda ⁇ gapA.
  • Strain EQCN_003 with a deletion in the glycolytic pathway at the gapA gene and in the Entner-Doudoroff pathway at the edd-eda genes, is grown to improve PHB production yield by fixing exogenous CO 2 via the use of the PRK and RuBisCO enzymes.
  • the inoculum from a frozen stock is spread on solid medium at a rate of 50 to 100 ⁇ L from a cryotube incubated at 30° C. for 48 to 96 h in the presence of fructose.
  • the expression of genes encoding RuBisCO and PRK are maintained in C. necator under heterotrophic aerobic conditions (Rie Shimizu et al., Sci Rep. 2015; 5: 11617. Published online 2015 Jul. 1).
  • strain of interest EQCN_003 improving PHB production yield is compared with a reference strain H16 naturally accumulating PHB under heterotrophic conditions in the presence of a nutritional limitation.
  • the productivity of the strains is compared in bioreactors.
  • Cultures carried out in bioreactors are seeded from solid and/or liquid amplification chains in Erlenmeyer flasks under the conditions described above.
  • the bioreactors of type My-control (Applikon Biotechnology, Delft, Netherlands) 750 mL or Biostat B (Sartorius Stedim, Göttingen, Germany) 2.5 L, are seeded at a density equivalent to 0.01 OD 620 nm .
  • the accumulation of PHB is decoupled from growth.
  • the culture is regulated at 30° C., aeration is between 0.1 VVM (gas volume/liquid volume/min) and 1 VVM in order to maintain a minimum dissolved oxygen concentration above 20% (30° C., 1 bar), shaking is adapted according to the scale of the bioreactor used.
  • the inlet gas flow consists of air optionally supplemented with CO 2 .
  • CO 2 supplementation is between 1% and 10%.
  • the pH is adjusted to 7 with a 14% or 7% ammonia solution.
  • the fed-batch culture method allows a supply of non-limiting carbon substrate combined with a limitation of phosphorus or nitrogen, while maintaining a constant carbon/phosphorus or carbon/nitrogen ratio.
  • PHB extraction and quantification are performed according to the method of Brandl et al. (Appl Environ Microbiol. 2013 July; 79(14):4433-9).
  • the protocol consists in adding 1 mL of chloroform to 10 mg of lyophilized cells, followed by 850 ⁇ L of methanol and 150 ⁇ L of sulfuric acid. The mixture is heated for 2.5 h at 100° C., cooled and 500 ⁇ L of water is added. The two phases are separated by centrifugation and the organic phase is dried by adding sodium sulfate The samples are filtered and analyzed as described by Müller et al. (Appl Environ Microbiol. 2013 July; 79(14):4433-9).
  • H16 ⁇ edd-eda ⁇ gapA shows a 5% increase in carbon yield, corresponding here to the ratio grams of PHB per gram of fructose consumed.
  • An Escherichia coli K-12 strain genetically modified to increase the yield of its glutamate production according to example 4B] can also be modified to allow the constitutive expression of a glutamate decarboxylase gadB (Gene ID: 946058) and thus increase the production yield of ⁇ -aminobutyric acid.
  • Overexpression of the gadB gene is subcloned into a bacterial expression vector pZE21MCS (EXPRESSYS).
  • This vector has a ColE1 origin of replication and a kanamycin antibiotic resistance gene.
  • the coding phase of the gadB gene (Gene ID: 946058) is amplified from the genome of strain MG1655 ⁇ sucA with primers homologous to the Escherichia coli K-12 genome covering positions 1570595 to 1570645 and 1572095 to 1572045. Each of these primers is coupled to floating sequences homologous over 18 nucleotides at the ends of the fragment obtained by amplifying vector pZE21MCS excluding the multiple cloning site. The two amplicons are combined according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech to form plasmid pEQEC030 allowing the constitutive overexpression of the gadB gene.
  • CDS The coding sequences (CDS) of the genes described in Table 24 are amplified and assembled into blocks according to the protocol provided with the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) so as to obtain three integration blocks described in Table 24. Each block is then amplified according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech to form the plasmids described below in Table 24.
  • ribosome binding sequences presented in Table 19 (Example 4B]), with variable translation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun. 21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic Acids Res. 2013 May; 41(9):e98) are inserted between the coding phase for each gene.
  • the succession of each coding phase interspersed by an RBS sequence is constructed by successive insertions into a pZA11 vector (Expressys) that contains a PLtetO-1 promoter, a p15A origin of replication and an ampicillin resistance gene.
  • the addition of a glutamate decarboxylase (gadB) also allows a conversion of glutamate to gamma-aminobutyrate (GABA).
  • clones are selected on LB glycerol, pyruvate medium supplemented with 100 mg/L ampicillin and 30 mg/L kanamycin.
  • An adaptation and evolution phase of the strains with PRK and RuBisCO engineering is performed as described in Example 4A].
  • cells from 500 mL of LB culture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin and 30 mg/L kanamycin at a pressure of 0.1 atmosphere CO 2 , at 30° C. at pH 3.5.
  • MS medium 40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L
  • the GABA concentration is measured by high-performance liquid chromatography (HPLC), using an OptimaPak C18 column (4.6 ⁇ 150 mm, RS Tech Corporation, Daejeon, Korea). The samples are centrifuged at 12,000 rpm for 5 minutes, 100 ⁇ L of the supernatant transferred into a new Eppendorf tube. The following reagents are added to these tubes: 200 ⁇ L of 1 M sodium bicarbonate buffer (pH 9.8), 100 ⁇ L of 80 g/L dansyl chloride in acetonitrile and 600 ⁇ L of double-distilled water. The mixture is incubated at 80° C. for 40 minutes. The reaction is stopped by adding 100 ⁇ L of 2% acetic acid.
  • HPLC high-performance liquid chromatography
  • the mixture is centrifuged at 12,000 rpm for 5 minutes.
  • the supernatant is then filtered through a 0.2 ⁇ m Millipore filter and analyzed by HPLC on an Agilent system using a UV detector.
  • Derivatized samples are separated using a binary non-linear gradient using eluent A [tetrahydrofuran/methanol/sodium acetate 50 mM at pH 6.2 (5: 75: 420, by volume)] and eluent B (methanol). Residual glucose is measured with a bioanalyzer (YSI Inc.).
  • the carbon yield Y p/s is calculated in grams of GABA produced per gram of glucose consumed.
  • isocitrate dehydrogenase (icd) expression allows the metabolic flux to be redirected to the glyoxylic shunt.
  • Inactivation of malate synthase (aceB) and succinate dehydrogenase (sdhA) prevents the glyoxylate and succinate, respectively, produced from being re-consumed.
  • Deletion of the succinate dehydrogenase gene increases succinate production under aerobic conditions (Yang et al., Microbiol res. 2014 May-June; 169(5-6):432-40). Deletion of the malate synthase gene allows the accumulation of glyoxylate which will be converted to oxalate by the constitutive expression of glyoxylate dehydrogenase.
  • an Escherichia coli K-12 strain MG1655 in which the sdhA gene has been deleted is used.
  • This strain is derived from a gene deletion bank (Baba et al. Mol Syst Biol. 2006; 2:2006.0008) in Escherichia coli K-12 and supplied by the coli Genetic Stock Center under the name JW0715-2 and with reference 8302. (JW0713-1: MG1655 ⁇ sdhA::Kan).
  • the selection cassette is deleted using a recombinase.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC040: MG1655 ⁇ sdhA
  • the deletion of the aceB gene (GeneID 948512) is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistance gene expression cassette and having a 5′ sequence homologous over 50 nucleotides to the adjacent regions of the deletion locus, i.e. at positions 4215428 to 4215478.and 4217129.to 4217079 on the chromosome thus generating recombination arms of the cassette on the bacterial genome on either side of the aceB gene coding sequence.
  • the Escherichia coli K-12 strain EQ.EC040 is transformed by electroporation with plasmid pRedET according to the kit protocol.
  • the colonies obtained are selected on rich complex medium LB agar with 0.2% glucose, 0.0003% tetracycline.
  • a second transformation of the deletion cassette is performed by electroporation in cells expressing RedET and the colonies are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC041: MG1655 ⁇ sdhA ⁇ aceB
  • the replacement of the native promoter of the icd gene (Gene ID: 945702) by a weaker promoter is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • the icd gene promoter is replaced by a cassette coupling the promoter P oxb1 , characterized as weak, and an antibiotic resistance gene cassette to allow the selection of the insertion of the P oxb1 cassette with an antibiotic resistance gene.
  • the amplification of a fusion fragment using the NEBuilder® HiFi DNA Assembly Master Mix Kit allows the replacement promoter to be combined with an antibiotic selection cassette.
  • the Escherichia coli K-12 strain EQ.EC041 is transformed by electroporation with plasmid pRedET according to the kit protocol.
  • the colonies obtained are selected on rich complex medium LB agar with 0.2% glucose, 0.0003% tetracycline.
  • a second transformation of the deletion cassette is performed by electroporation in cells expressing RedET and the colonies are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC042: MG1655 ⁇ sdhA ⁇ aceB P icd ::P oxb1
  • the deletion of the zwf gene (GeneID: 946370) is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistance gene expression cassette and having a 5′ sequence homologous over 50 nucleotides to the adjacent regions of the deletion locus, i.e. at positions 1934789 to 1934839 and 1936364 to 1936314 on the chromosome thus generating recombination arms of the cassette on the bacterial genome on either side of the entire operon.
  • the Escherichia coli K-12 strain EQ.EC042 is transformed by electroporation with plasmid pRedET according to the kit protocol.
  • the colonies obtained are selected on rich complex medium LB agar with 0.2% glucose, 0.0003% tetracycline.
  • a second transformation of the deletion cassette is performed by electroporation in cells expressing RedET and the colonies are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC043: MG1655 ⁇ sdhA ⁇ aceB P icd ::P oxb1 ⁇ zwf
  • the deletion of the gapA gene is performed by homologous recombination and the use of the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit (Gene Bridges) according to the supplier's protocol.
  • Oligonucleotides designed to amplify an FRT-PKG-gb2-neo-FRT resistance gene expression cassette and having a 5′ sequence homologous over 50 nucleotides to the adjacent regions of the deletion locus, i.e. the coding phase of the gene (gapA) (GenBank: X02662.1) thus generating recombination arms of the cassette on the bacterial genome.
  • the Escherichia coli K-12 strain EQ.EC043 is transformed by electroporation with plasmid pRedET according to the kit protocol.
  • the colonies obtained are selected on rich complex medium LB agar with 0.2% glucose, 0.0003% tetracycline.
  • Transformation of the amplicon obtained in the first step in the presence of RedET recombinase is induced by 0.3% arabinose in liquid LB for 1 h.
  • a second electroporation of the cells expressing RedET by the deletion cassette is performed and the colonies are selected on LB agar supplemented with 0.2% glycerol and 0.3% pyruvate, 0.0003% tetracycline and added with 0.3% L-arabinose and 0.0015% kanamycin.
  • Plasmid p707-Flpe (provided in the Quick & Easy E. coli Gene Deletion Red®/ET® Recombination Kit by Gene Bridges) is transformed by electroporation according to the kit protocol. The cells are selected on LB agar supplemented with 0.2% glucose, 0.0003% tetracycline and added with 0.3% L-arabinose. A counter-selection of the clones obtained is carried out by verifying that they are no longer able to grow on the same medium supplemented with 0.0015% kanamycin.
  • the strain obtained is called EQ.EC044: MG1655 ⁇ sdhA ⁇ aceB P icd ::P oxb1 ⁇ zwf ⁇ gapA
  • CDS The coding sequences (CDS) of the FPGLOXDH1 (Gene ID: 946058) and aceA (Gene ID: 948517) genes subcloned into a bacterial expression vector pZE21MCS (EXPRESSYS) as synthetic operons according to the structure described in Table 24.
  • This vector has a ColE1 origin of replication and a kanamycin antibiotic resistance gene.
  • Each of these primers is coupled to floating sequences homologous over 18 nucleotides at the ends of the fragment obtained by amplifying vector pZE21MCS excluding the multiple cloning site.
  • the two amplicons are combined according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech to form plasmid pEQEC035 allowing the constitutive overexpression of the FPGLOXDH1 and aceA genes.
  • CDS coding sequences of the genes described in the Table 2 are amplified and assembled into blocks according to the protocol provided with the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) to obtain three integration blocks described in Table 26. Each block is then amplified according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech to form the plasmids described below in Table 24.
  • ribosome binding sequences presented in Table 19 (Example 4B]), with variable translation efficiencies (Levin-Karp et al., ACS Synth Biol. 2013 Jun. 21; 2(6):327-36. doi: 10.1021/sb400002n; Zelcbuch et al., Nucleic Acids Res. 2013 May; 41(9):e98) are inserted between the coding phase for each gene.
  • the succession of each coding phase interspersed by an RBS sequence is constructed by successive insertions into a pZA11 vector (Expressys) that contains a PLtetO-1 promoter, a p15A origin of replication and an ampicillin resistance gene.
  • clones are selected on LB glycerol, pyruvate medium supplemented with 100 mg/L ampicillin and 30 mg/L kanamycin.
  • An adaptation and evolution phase of the strains with PRK and RuBisCO engineering is performed as described in Example 4A].
  • cells from 500 mL of LB culture are inoculated into 20 mL of MS medium (40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L CaCO 3 , 100 mg/L ampicillin and 30 mg/L kanamycin at a pressure of 0.1 atmosphere CO 2 , at 30° C. at pH 3.5.
  • MS medium 40 g/L glucose, 1 g/L MgSO 4 .7H 2 O, 20 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .7H 2 O, 2 g/L yeast extract, 30 g/L
  • the succinate concentration is measured by high-performance liquid chromatography (HPLC), culture samples are centrifuged at 12,000 g for 5 min.
  • the culture supernatant is filtered through a 0.2 ⁇ m Millipore filter and analyzed on an Agilent HPLC system (series 1100) equipped with a cation-exchange column.
  • Agilent HPLC system series 1100
  • a UV absorbance detector Agilent Technologies, G1315D
  • RI detector Agilent Technologies, HP1047A
  • the samples are separated on a 5 mM H 2 S0 4 mobile phase at a flow rate of 0.4 mL/min.
  • the column oven temperature is 65° C.
  • Residual glucose is measured with a bioanalyzer (Ysi Inc.) or by HPLC-refractometry with an Aminex HPX87-H column.
  • the carbon yield Y p/s is calculated in grams of succinate produced per gram of glucose consumed.
  • the pellets are washed twice with 10 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA and stored at ⁇ 20° C.
  • Samples (1 mL) are transferred into a tube pre-cooled with 0.75 g of glass beads (425-600 ⁇ m) and introduced into a Fast Prep homogenizer (Thermo Scientific, Erembodegem, Netherlands) and subjected to 4 bursts of 20 s at speed control 6.
  • the lysates are centrifuged for 20 min at 4° C. and 36,000 g. Total protein determinations are performed according to the Lowry method (Lowry et al., 1951).
  • Oxaloacetate acetyl hydrolase (EC 3.7.1.1.1) activity is measured using a modification of the direct optical determination of oxaloacetate (OAA) at 255 nm as described in (Lenz et al., 1976). The disappearance of the OAA enol tautomer is checked at 255 nm at 25° C. in a Hitachi Model 100-60 spectrophotometer (Hitachi, Tokyo, Japan), using quartz cuvettes.
  • OAA oxaloacetate
  • the 1 mL reaction mixture contains 100 mM imidazole-HCl (pH 7.5), 0.9 mM MnCl 2 .2H 2 O, 1 mM OAA, 20 ⁇ L cell extract (controls with different volumes of cell extracts confirm the linear relationship between enzyme activity and the amount of cell extract).
  • the reaction is started by adding the cell extract.
  • the carbon yield Y p/s is calculated in grams of doxalate produced per gram of glucose consumed.
  • the inactivation of the pgkA gene (Locus tag An08g02260), leading to the non-functionality of the glycolysis pathway, and that of the gsdA gene (Locus tag An02g12140), inhibiting the oxidative part of the pentose phosphate pathway, are used to integrate the six genes for the functional expression of the PRK and RuBisCO enzymes, namely RbcS, RbcL, RbcX, GroES, GroEL and PRK for CO 2 fixation.
  • Plasmid pFC332 (Addgene #87845) described in Sarkari et al. (Bioresour Technol. 2017 December; 245(Pt B):1327-1333) contains a gRNA expression cassette, a cassette allowing the functional expression of the Cas9 endonuclease and an Hph cassette allowing the selection of this plasmid.
  • the plasmid also contains the fragment AMA1_2.8 which allows transient propagation of the plasmid.
  • an origin of replication for E. coli is also present.
  • the gRNA cassette between FS A and FS B can be easily exchanged.
  • This plasmid is modified by amplifying the different parts of this plasmid, in order to eliminate the antibiotic selection cassette and modify the 20 nucleotides allowing the specificity of gRNA in favor of the sequences described in Table 27 to form plasmids pEQ0610 to target pgkA and pEQ0611 to target gsdA.
  • the donor plasmid consists of an In-Fusion® HD Cloning Kit User Manual—Clontech assembly between plasmid pUC19 (GenBank: M77789.2) and the genomic targeting sequences (LA and RA) of approximately 1500 bp each, homologous to the locus chosen for integration.
  • the LA and RA sequences are adjacent at 5′ and 3′ respectively to the locus sequence targeted by the guide RNA.
  • the genomic DNA/guide RNA heterodimer is recognized by the Cas9 endonuclease for double-stranded cleavage (locus 1: pgkA; locus 2: gsdA) (Table 28).
  • the RA and LA fragments are amplified with primers for the pgkA gene and the gsdA gene (Table 29).
  • the amplicon sequences are given in the sequence listing (SEQ ID NO: 55 to SEQ ID NO: 58).
  • An extension of 18 nucleotides on all forward primers of the three fragments is added according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech, to allow a functional assembly of the plasmids (pEQ0600 or pEQ0601) and the introduction of two restriction sites for type II restriction endonucleases (restriction enzymes I-CeuI and I-Sce)I which have large asymmetric recognition sites (12 to 40 base pairs).
  • the promoters and terminators are identified on the basis of GenBank data.
  • the selected promoters are determined from the +1 transcription point and go up 1.4 kb upstream in order to cover both the “core” sequences (TATA box) and the trans-activating sequences allowing the optimal functionality of the promoter concerned.
  • the cut-off is made 500 bp after the stop codon of the gene.
  • each integration block of four expression cassettes is defined as follows: the first level consists of simple elements, namely promoters, coding sequences (CDS) and terminators.
  • the promoter (Table 30) and terminator (Table 31) elements whose sequences are provided in the sequence listing (SEQ ID NO: 59 to 62), are amplified and assembled with the engineering CDS according to Table 32.
  • the CDS whose sequences are provided in the sequence listing (SEQ ID NO: 63 to 66), are amplified according to the protocol provided with the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) to obtain the functional expression cassettes compiled in the table.
  • Each integration block of four genes is organized to include four different pairs (promoter/terminator) in order to limit trans interference.
  • Each integration block of six genes is organized to include six different pairs (promoter/terminator) in order to limit transcriptional interference
  • the different multiple expression cassettes (RbcS, RbcL and RbcX) or (GroES, GroEL and PRK) are amplified and assembled around an antibiotic selection cassette (Table), according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech, to form donor plasmids (pEQ0602 or pEQ0603).
  • the A. niger strain CBS 513-88 is grown at 30° C. in a 1 L Erlenmeyer flask with 250 mL of transformation medium (Kusters-van Someren et al., Curr Genet. 1991 September; 20(4):293-9). After growth for 16 h at 250 rpm, the mycelium is collected by filtration on Miracloth (Calbiochem) and washed with deionized water.
  • Protoplasts are prepared in the presence of 5 g/L lysis enzymes from Trichoderma harzianum (Sigma Saint Louis, Mo., USA), 0.075 Uml-1 chitinase from Streptomyces griseus (Sigma) and 460 Uml-1 glucuronidase from Helix pomatia (Sigma) in KMC (0.7 M KCl, 50 mM CaCl 2 , 20 mM Mes/NaOH, pH 5.8) for 2 hours at 37° C. and 120 rpm. Protoplasting is monitored every 30 minutes with a microscope. The protoplasts are filtered through a Miracloth filter and collected by centrifugation at 2000 ⁇ g and 4° C. for 10 minutes.
  • the protoplasts are washed with cold STC (1.2 M sorbitol, 10 mM Tris/HCl, 50 mM CaCl 2 , pH 7.5) and then resuspended in 100 pi of STC and used directly for the transformation.
  • cold STC 1.2 M sorbitol, 10 mM Tris/HCl, 50 mM CaCl 2 , pH 7.5
  • Plasmid pEQ0610 is co-transformed with a donor fragment to integrate part of the engineering into the genome while inactivating the pgkA gene.
  • plasmid pEQ0611 is co-transformed with a donor fragment to integrate the other part of the engineering into the genome while inactivating the gsdA gene.
  • strains are directly selected on minimal medium plates with an addition of hygromycin B or bleomycin allowing direct selection on the integration event. Due to the presence of the origin of replication AMA1_2.8, plasmid pCAS_pyrG2 is easily lost causing only transient expression of the Cas9 protein, thus reducing the risk of non-targeted adverse effects.
  • Linear cassettes (10 ⁇ g) and plasmid (5 ⁇ g) are mixed with 100 ⁇ L of STC solution containing at least 10 7 protoplasts and 330 ⁇ L of freshly prepared polyethylene glycol (PEG) solution (25% PEG 6000, 50 mM CaCl 2 , 10 mM Tris/HCl, pH 7.5) and kept on ice for 20 minutes. After mixing with an additional 2 mL PEG solution and incubating at room temperature for 10 minutes, the protoplast mixture is diluted with 4 mL of STC.
  • PEG polyethylene glycol
  • the selection of transformants is carried out on MM plates with 150 ⁇ g/mL hygromycin B added or MM plates with 50 ⁇ g/mL bleomycin added. All transformants are purified by isolating single colonies from the selection medium at least twice. The insertion of the fragments is verified by sequencing the target locus with the appropriate control primers. Genomic DNA from fungal cells is isolated with a modified protocol, using the Wizard® Genomic DNA Purification Kit (Promega, Wisconsin, USA). The mycelium is cultured overnight in CM (30° C., 150 rpm) in 290 pi of 50 mM EDTA solution and 10 pi of lyticase (10 mg/mL) to remove the cell wall.
  • the suspension is centrifuged and the supernatant is discarded.
  • the mycelium pellet is resuspended in 300 ⁇ L of nuclei lysis solution and 100 ⁇ L of protein precipitation solution.
  • the samples are incubated on ice for 5 minutes and centrifuged.
  • the DNA is precipitated with isopropanol and washed with 70% ethanol.
  • the DNA pellet is rehydrated with a DNA rehydration solution containing RNase (100 ⁇ g/mL). The successful transformation and integration of the expression cassettes was verified by PCR.
  • niger gsdA PmbfA p -RbcL-trpc; PcoxA p- RbcS-TniaD; CBS picdA p -Hph-TgpdA; PsrpB p -RbcX-glaAt 513-88 pgkA :: PmbfA p -GrES-trpc; PcoxA p- GroEL-TniaD; picdA p -Ble-TgpdA; PsrpB p -PRK-glaAt
  • Conidia (10 8 /L) from strains EQ1500 and EQ1502 are inoculated and cultured at 30° C. on a rotary shaker (180 rpm) in shaker flasks containing Vogel medium without MnSO 4 with a total glucose content of 15% and a total nitrogen content of 0.2% and 10% CO 2 .
  • the determination of glucose and organic acids was performed as described above (Blumhoff et al., 2013; Steiger et al., 2016) on an HPLC (Shimadzu, Kyoto; Japan) equipped with an Aminex HPX-87 H column (300 ⁇ 7.8 mm, Bio-Rad, Hercules, Calif.).
  • a refractive index detector (RID-10 A, Shimadzu) is used for the detection of glucose and citric acid, while a PDA detector (SPD-M20A, Shimadzu) at 300 nm is used to detect cis-aconitic and trans-aconitic acid.
  • the column is used at 60° C. at a flow rate of 0.6 mL/min and with a 0.004 M H 2 SO 4 aqueous solution as mobile phase. The culture was carried out in three biological replicates.
  • a culture sample is centrifuged at 14,000 ⁇ g for 5 min. The supernatant is filtered through a filter with a 0.45 pm pore size. The filtrate is maintained at ⁇ 20° C. until analysis.
  • the concentration of citrate and of oxalate is detected and quantified with ultraviolet light at 210 nm using an Amethyst C18-H column (250 ⁇ 4.6 mm, Sepax Technologies, Newark, Del., USA). Elution is carried out at 30° C. with 0.03% H 3 P0 4 at a flow rate of 0.8 mL/min. Reducing sugar is detected with the 3,5-dinitrosalicylic acid method.
  • Biomass determination 5 mL of sample is filtered through Miracloth (Calbiochem, San Diego, Calif., USA) to collect hyphae and washed with distilled water. The hyphae are heated to 105° C. in a “Miracloth”.
  • DCW dry cell weight
  • citric acid production yield is 18% higher in the engineered strain EQ1502 than in the wild-type strain EQ1500.
  • Inactivation of the pgkA gene (Locus tag (ATEG_00224), leading to the non-functionality of the glycolysis pathway, and that of the gsdA gene (Locus tag ATEG_01623), inhibiting the oxidative part of the phosphate pentose pathway, are used to integrate the six genes allowing the functional expression of the PRK and RuBisCO enzymes, namely rbcS, rbcL, rbcX, groES, groEL and prk allowing CO 2 fixation.
  • a sequence of 20 nucleotides punctuated by an NGG motif was determined (Table 35). In both cases, this sequence is specific to the targeted gene but also unique in the Aspergillus terreus genome.
  • These sequences are used to express a guide RNA (gRNA) which, by forming a heteroduplex with the homologous region of the Aspergillus terreus genome, directs the action of the CAS9 endonuclesae to induce a double-stranded break specifically on the selected locus.
  • gRNA guide RNA
  • the sequence identified in the second intron the first 20 nucleotides have a unique pattern in the genome, even allowing two mismatches.
  • gsdA the sequence identified in the fourth intron, the first 20 nucleotides have a unique pattern in the genome, even allowing two mismatches.
  • Plasmid pFC332 (Addgene #87845) described in Sakari et al. (Bioresour technol. 2017; 245(Pt B):1327-1333) contains a gRNA expression cassette, a cassette for the functional expression of the Cas9 endonuclease and an Hph cassette for the selection of this plasmid.
  • the plasmid also contains the fragment AMA1_2.8 which allows transient propagation of the plasmid.
  • an origin of replication for E. coli is also present.
  • this plasmid is modified by amplifying the different parts of this plasmid in order to eliminate the antibiotic selection cassette and to modify the 20 nucleotides allowing the specificity of gRNA in favor of the sequences described in Table 35 to form plasmids pEQ0615 to target pgkA and pEQ0616 to target gsdA in the Aspergillus terreus genome.
  • the donor plasmid consists of an In-Fusion® HD Cloning Kit User Manual—Clontech assembly between plasmid pUC19 (GenBank: M77789.2) and genomic targeting sequences (LA and RA) of approximately 1500 bp each homologous to the locus chosen for integration.
  • the LA and RA sequences are adjacent at 5′ and 3′ respectively to the locus sequence targeted by the guide RNA.
  • the genomic DNA/guide RNA heterodimer is recognized by the Cas9 endonuclease for double-stranded cleavage (locus 1: pgkA; locus 2: gsdA) (Table 35).
  • the RA and LA fragments are amplified with the primers described in Table 36, for the pgkA gene, and Table 37, for the gsdA gene.
  • the amplicon sequences are in the sequence listing (SEQ ID NO: 67 to 70).
  • Promoters and terminators are identified on the basis of GenBank data.
  • the selected promoters are determined from the +1 transcription point and go up 1.4 kb upstream in order to cover both the “core” sequences (TATA box) and the trans-activating sequences allowing the optimal functionality of the promoter concerned.
  • the cut-off is made 500 bp after the stop codon of the gene.
  • each integration block of four expression cassettes is defined as follows: the first level consists of simple elements, namely promoters, coding sequences (CDS) and terminators.
  • the promoter (Table 30) and terminator (Table 31) elements are amplified and assembled with the engineering CDS according to Table 32.
  • the CDS are amplified according to the protocol provided with the NEBuilder® HiFi DNA Assembly Master Mix Kit (E2321) in order to obtain the functional expression cassettes compiled in the table.
  • Each integration block of four genes is organized to include four different terminator promoter pairs in order to limit trans interference
  • Each integration block of six genes is organized to include six different terminator promoter pairs in order to limit transcriptional interference.
  • the different multiple expression cassettes (RbcS, RbcL and RbcX or GroES, GroEL and PRK are amplified and assembled around an antibiotic selection cassette (Table 38), according to the protocol of the In-Fusion® HD Cloning Kit User Manual—Clontech, to form donor plasmids (pEQ0606 or pEQ0607).
  • the optimized media composition described by Hevekerl et al. (Appl Microbiol Biotechnol. 2014; 98:6983-6989) is used. It contains 0.8 g KH 2 P0 4 , 3 g NH 4 N0 3 , 1 g MgSO 4 .7H20, 5 g CaCl 2 .2 H 2 0, 1.67 mg FeCl 3 .6H 2 O, 8 mg ZnSO 4 .7H 2 O and 15 mg CuSO 4 .7H2O per liter.
  • the concentration of the cell mass is determined from the dry cell weight.
  • the cell mass present in the fermentation broth is collected by centrifugation at 10,000 g for 10 minutes and carefully rinsed three times with deionized water. The rinsed cell mass was completely dried at 80° C. until a constant weight was obtained.
  • the fermentation broth after centrifugation (10,000 g, 10 min) is stored at ⁇ 20° C. before analysis of glucose, itaconic acid and by-products (succinic acid, ⁇ -ketoglutaric acid, malic acid, cis-aconitic acid, and trans-aconitic acid) using high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • Aminex HPX-87P column 300 ⁇ 7.8 mm with ash removal cartridge and Carbo-P protection cartridge
  • Aminex HPX 87H column 300 ⁇ 7.8 mm with Microguard Cation H cartridge (Bio-Rad)
  • the Aminex HPX 87P column is maintained at 85° C. and glucose is eluted with Milli-Q acidified deionized water (Millipore, Bedford, Mass.) at a flow rate of 0.6 mL/min.
  • the Aminex HPX 87H column is maintained at 65° C. and sugars and organic acids are eluted with 5 mM H 2 SO 4 prepared using Milli-Q deionized filtered water at a rate of 0.5 mL/min. Detection is carried out using a refractive index detector for sugars and a 210 nm UV detector for organic acids. Propionic acid (1%, weight/volume) is used as internal standard to estimate the liquid lost during aerobic fermentation for 7-10 days at 33° C. under 10% CO 2 . All HPLC standards, including organic acids, are purchased from Sigma.
  • the manganese concentration (ppb level) is determined using an Optima 7000DV (Perkin-Elmer, Waltham, Mass.) inductively coupled plasma optical emission spectrometer (ICP-OES) by the procedure described by Bakota et al. (Eur J Lipid Sci Technol. 2015; 117:1452-1462.

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