METHOD FOR REDUCING MISINCORPORATION OF NON-CANONICAL BRANCHED-CHAIN AMINO ACIDS

Abstract

The present invention relates to a method for producing a recombinant polypeptide of interest in a microbial host cell, comprising (a) introducing a polynucleotide encoding the polypeptide of interest into a microbial host cell which has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase activity (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell, and (b) expressing said polypeptide of interest in said microbial host cell. Moreover, the present invention relates to a method for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest expressed in a microbial host cell.

Claims

1. A method for producing a recombinant polypeptide of interest in a microbial host cell, comprising the steps of (d) introducing a polynucleotide encoding the polypeptide of interest into a microbial host cell which has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell, and (e) expressing said polypeptide of interest in said microbial host cell.

2. The method of claim 1, wherein the produced polypeptide of interest shows lower misincorporation of non-canonical branched-chain amino acids as compared to a polypeptide which has been produced by expression in an unmodified microbial host cell.

3. The method of any one of claims 1 and 2, wherein the method further comprises the isolation of the polypeptide from the cell, and the purification of the polypeptide.

4. The method of claim 3, wherein the purification comprises the enrichment of polypeptides which do not comprise non-canonical branched-chain amino acids.

5. The method of claims 1 and 4, wherein said enzymatic activity is increased by introducing and expressing a polynucleotide encoding a polypeptide having said enzymatic activity in said microbial host cell.

6. The method of any one of claim 5, wherein said microbial host cell does not express an endogenous polypeptide having said enzymatic activity. The method of any one of claims 1 to 6, wherein the polypeptide of interest is a therapeutic peptide or polypeptide.

8. The method of any one of claims 1 to 7, wherein the polynucleotide encoding the polypeptide of interest and/or the polynucleotide encoding the polypeptide having said enzymatic activity is operably linked to an inducible promoter.

9. The method of any one of claims 1 to 8, wherein said microbial host cell is an Escherichia coli cell.

10. A method for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest expressed in a microbial host cell, said method comprising (a) modulating an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase (EC 4.3.1.19) in the microbial host cell, (b) introducing a polynucleotide encoding the polypeptide of interest into said microbial host cell, and (c) expressing said polypeptide of interest in said microbial host cell.

11. The method of claim 10, wherein at least one non-canonical branched-chain amino acid is selected from norvaline, norleucine and beta-methylnorleucine.

12. Use of a polypeptide having an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase (EC 4.3.1.19), or of a polynucleotide encoding said polypeptide for reducing misincorporation of at least one non-canonical branched-chain amino acid into a recombinant polypeptide of interest produced in a microbial host cell.

13. Use of a microbial host cell for producing a recombinant polypeptide of interest, wherein the microbial host cell has been modified such that an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase (EC 4.3.1.19) is modulated in said microbial host cell as compared to the enzymatic activity in an unmodified microbial host cell.

14. The method according to any one of claims 1 to 11, or the use of claim 12 or 13, wherein the enzymatic activity is modulated.

15. The method according to any one of claims 1 to 11 and 14, or the use of claim 12, 13 or 14, wherein the polypeptide of interest is a proinsulin.

16. A microbial host cell comprising (a) a recombinant polynucleotide encoding a polypeptide of interest, and (b) a recombinant polynucleotide encoding a polypeptide having an enzymatic activity selected from the group consisting of ketol-acid reductoisomerase (NADP(+)) activity (EC 1.1.1.86), acetohydroxyacid synthase activity (EC 2.2.1.6), aspartate kinase activity (EC 2.7.2.4), homoserine dehydrogenase activity (EC 1.1.1.3), and L-threonine dehydratase (EC 4.3.1.19).

17. A bioreactor comprising the microbial host cell of claim 16.

18. The bioreactor of claim 17, wherein bioreactor has a volume of at least 10 L.

Description

FIGURES

[0193] FIG. 1: Plasmid map of pSW3_lacl.sup.+, expressing mini-proinsulin. Plasmid pSW3_lacl.sup.+ confers resistance to ampicillin and expresses an 11 kDa recombinant protein (mini-proinsulin) under the control of an IPTG inducible promoter. Plasmid also co-expresses the repressor Lacl, which is under the control of a lacl.sup.+ promoter variant. Generated with Snapgene®.

[0194] FIG. 2: Plasmid map of 16ABZ5NP_1934177, containing fragment 1. Generated with Snapgene®.

[0195] FIG. 3: Plasmid map of pCP20. Generated with Snapgene®.

[0196] FIG. 4: Plasmid map of pETcocol, including ori2 and its elements (repE, sopA, sopB, sopC). Generated with Snapgene®.

[0197] FIG. 5: Plasmid map of the arabinose-tunable plasmid pACG_araBAD. It includes ori2 and its elements (repE, sopA, sopB, sopC) which ensure 1 copy plasmid per cell. Plasmid confers resistance to chloramphenicol. Exogenous genes can be cloned by restriction cloning with enzymes Nhel and Notl. Cloned genes are under the control of an arabinose promoter. AraC is necessary for the activation of the arabinose promoter and is also present in the plasmid. Additional unique restriction sites (Smil, Xhol, XmaJl, Mssl) are present in order to allow exchange of the origin of replication, antibiotic resistance marker and promoter region. Generated with Snapgene®.

[0198] FIG. 6: Plasmid map of the arabinose-tunable plasmid pACG_araBAD_ilvIH. This plasmid results upon restriction cloning of gene ilvIH into the original pACG_araBAD plasmid with enzymes Nhel and Notl. This plasmid allows regulation of ilvIH gene expression by addition of arabinose into the medium. Generated with Snapgene®.

[0199] FIG. 7: Genetic modifications performed to wild type E. coli in this work. Genomic DNA contains a knock out of a certain gene (gene A). Expression of that gene can then be regulated by L-arabinose thanks to the presence of a tunable expression plasmid, containing such gene (pACG_araBAD_geneA). In addition, plasmid pSW3_lacl.sup.+ expresses mini-proinsulin, which allows testing ncBCAA misincorporation.

[0200] FIG. 8: Plasmid map of pKD46. Generated with Snapgene®.

[0201] FIG. 9: Plasmid map of pKD3. Generated with Snapgene®.

[0202] FIG. 10: Plasmid map of pKD4. Generated with Snapgene®.

[0203] FIG. 11: Molar concentrations of norvaline (A), norleucine (B) and β-methylnorleucine (C) normalized to OD.sub.600nm present in the intracellular soluble protein fraction calculated over time after induction of different E. coli cultivations in a 15 L reactor under standard conditions (STD) and under conditions triggering ncBCAA accumulation, i.e. pyruvate pulsing and oxygen limitation (PYR-O2). Indicated in the legend, “WT E. coli” refers to the wild type strain E. coli K-12 BW25113 pSW3_lacl.sup.+ “ilvGM-tunable E. coli” alludes to strain E. coli K-12 BW25113 pSW3_lacl.sup.+pACG_araBAD_ilvGM and “ilvIH-tunable E. coil” corresponds with strain E. coli K-12 BW25113 ΔilvIH pSW3_lacl+ 30 pACG_araBAD_ilvIH. Arrows indicate time points where 1 g/L pyrvate pulse combined with 5 min O.sub.2 limitation was applied.

[0204] FIG. 12: Molar concentrations of norvaline (A) and norleucine (B) normalized to OD.sub.600nm present in the inclusion body fraction calculated over time after induction of different E. coli cultivations in a 15 L reactor under standard conditions (STD) and under cultivation conditions triggering ncBCAA accumulation, i.e. pyruvate pulsing and oxygen limitation (PYR-O2).

[0205] Indicated in the legend, “WT E.coli” refers to the wild type strain E. coli K-12 BW25113 pSW3_lacl.sup.+ “ilvGM-tunable E. coil” alludes to strain E. coli K-12 BW25113 pSW3_lacl.sup.+ pACG_araBAD_ilvGM and “ilvIH-tunable E. coil” corresponds with strain E. coli K-12 BW25113 ΔilvIH pSW3_lacl.sup.+ pACG_araBAD_ilvIH. Arrows indicate time points where 1 g/L pyruvate pulse combined with 5 min O.sub.2 limitation was applied.

[0206] All references referred to above are herewith incorporated by reference with respect to their entire disclosure content as well as their specific disclosure content explicitly referred to in the above description.

[0207] The following examples merely illustrate the invention. They should not be construed as limiting the scope of protection in any way.

EXAMPLES

Example 1

Transformation of K12 BW25113 ΔhrA, ΔilvA, ΔilvC, ΔilvIH and ΔilvBN Mutants with Plasmid pSW3_lacl.SUP.+

[0208] ΔthrA, ΔilvA, ΔilvC Knock-Outs

[0209] Strain E. coli K12 BW25113 as well as single knock-out mutants E. coli K12 BW25113 ΔthrA, ΔilvA and ΔilvC were acquired from the E. coli Genetic Stock Center (CGSC) of Yale University. Those mutant strains belong to the so-called KEIO collection (Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., . . . & Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular systems biology, 2(1)). These strains contain plasmid pKD46 (FIG. 8) and a kanamycin resistance marker substituting target gene. CGSC identification for each acquired strain is indicated as follows:

TABLE-US-00001 Strain CGSC identification number E. coli K12 BW25113 7636 E. coli K12 BW25113 ΔthrA JW0001-1 E. coli K12 BW25113 ΔilvA JW3745-2 E. coli K12 BW25113 ΔilvC JW3747-2

[0210] Plasmid pKD46 was curated from the acquired E. coli K12 BW25113 single knock-out mutants and the respective electrocompetent cells were transformed with pCP20 (FIG. 3), a temperature-sensitive plasmid encoding a flipase. Plasmid pCP20 was curated and removal of the antibiotic resistance marker from the mutants was tested by sequencing. The final respective electrocompetent E. coli K12 BW25113 mutants were transformed with pSW3_lacl.sup.+ (FIG. 1), a high copy plasmid encoding mini-proinsulin.

ΔilvIH and ΔilvBN Knock-Outs

[0211] The knock-out strains E. coli K12 BW25113 ΔilvIH and ΔilvBN were not acquired but manually generated. Procedure to generate E. coli knock-out mutants described at “Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences, 97(12), 6640-6645” was used as a reference. Electrocompetent E. coli K12 BW25113 cells were transformed with pKD46, a temperature-sensitive recombination helper plasmid. Knock-out mutants for the operons ilvIH and ilvBN were then generated by transformation of electrocompetent E. coli K12 BW25113 cells containing pKD46 with the respective deletion cassette, previously obtained by PCR from pKD3 (FIG. 9) or pKD4 (FIG. 10). PCR-based verification was carried out to test proper integration of the deletion cassette into the genome. Plasmid pKD46 was curated and the respective electrocompetent E. coli K12 BW25113 mutants were transformed with pCP20 (FIG. 3), a temperature-sensitive plasmid encoding a flipase. Plasmid pCP20 was curated and removal of the antibiotic resistance marker from the mutants was tested by sequencing. The final respective electrocompetent E. coli K12 BW25113 mutants were transformed with pSW3_lacl.sup.+ (FIG. 1), a high copy plasmid encoding mini-proinsulin.

Example 2

Design and Generation of an araC-PBAD Tunable Expression Vector (pACG_araBAD)

[0212] An arabinose-based tunable expression plasmid, allowing regulation of genes of study, previously knocked-out, was obtained by the junction of 3 different DNA segments: Fragment 1 contains the araC-PBAD promoter region (Guzman et al., 1995, Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of bacteriology, 177(14), 4121-4130), UTRs, T7 terminator, a cloning site allowing gene cloning with rare-cutting restriction enzymes Nhel and Notl and a C-terminal 6×his-tag sequence allowing expression of fusion proteins. Fragment 2 contains a chloramphenicol resistance cassette while fragment 3 includes the ori2 origin of replication and genes sopA, sopB, sopC and repE.

[0213] Fragment 1 was chemically synthesized and subsequently cloned in plasmid 16ABZ5NP_1934177 (FIG. 2). Fragment 1 was then amplified by PCR from plasmid 16ABZ5NP_1934177. Fragments 2 and 3 were directly amplified by PCR from plasmids pCP20 (FIG. 3) and pETcocol (FIG. 4), respectively. The three DNA segments were joined together according to the In-Fusion cloning strategy (Takara Bio USA; Ref.: 639649) to generate plasmid pACG_araBAD (FIG. 5).

Example 3

Cloning of the Target Genes into the Tunable Expression Vectors

[0214] Genes of study (ilvA, ilvC, ilvIH, ilvBN, and thrA) were amplified by PCR from the E. coli K12 BW25113 genomic DNA and they were subsequently cloned into the previously generated tunable expression plasmid (FIG. 5) by using restriction enzymes Nhel and Notl. ilvGM was amplified from an E. coli strain where ilvG was not mutated. As example, plasmid map of the resulting arabinose-tunable plasmid pACG_araBAD_ilvIH is shown in FIG. 6.

Example 4

Transformation of the Tunable Expression Vectors with Cloned Genes into the Respective Mutant Containing pSW3_lacl.SUP.+

[0215] The final respective electrocompetent E. coli K12 BW25113 mutants containing pSW3_lacl.sup.+ were transformed with the tunable expression plasmid expressing the corresponding gene of study.

[0216] After all genetic modifications, the generated E. coli mutant strains look like as described in FIG. 7. A total of 6 E. coli mutant strains were then generated: [0217] E. coli K12 BW25113 ΔilvC expressing pSW3_lacl.sup.+ and pACG_araBAD_ilvC [0218] E. coli K12 BW25113 ΔilvIH expressing pSW3_lacl.sup.+ and pACG_araBAD_ilvIH [0219] E. coli K12 BW25113 ΔilvBN expressing pSW3_lacl.sup.+ and pACG_araBAD_ilvBN [0220] E. coli K12 BW25113 ΔthrA expressing pSW3_lacl.sup.+ and pACG_araBAD_thrA [0221] E. coli K12 BW25113 ΔilvA expressing pSW3_lacl.sup.+ and pACG_araBAD_ilvA [0222] E. coli K12 BW25113 expressing pSW3_lacl.sup.+ and pACG_araBAD_ilvGM

Example 5

Evaluation of L-Arabinose Induction Effect on Cell Growth of Mutant E. coli Strains

[0223] The aim of this experiment was to evaluate the effect of addition of different concentrations of L-arabinose on the expression of genes under the control of the araBAD promoter and, as a consequence, the effect on cell growth of the generated mutant E. coli strains.

[0224] E. coli cells were grown in a defined mineral salt medium containing (per L): 0.67 g Na.sub.2SO.sub.4, 0.82 g (NH.sub.4).sub.2SO.sub.4, 0.17 g NH.sub.4Cl, 4.87 g K.sub.2HPO.sub.4, 1.2 g NaH.sub.2PO.sub.4×2H.sub.2O and 0.33 g (NH.sub.4).sub.2-H-citrate. The medium was supplemented with 0.67 ml/L trace elements solution and 0.67 ml/L MgSO.sub.4 solution (1.0 M). The trace element solution comprised (per L): 0.5 g CaCl.sub.2×2H.sub.2O, 0.18 g ZnSO.sub.4×7H.sub.2O, 0.1 g MnSO.sub.4×H2O, 16.7 g FeCl.sub.3×6H.sub.2O, 0.16 g CuSO.sub.4×5H.sub.2O, 0.18 g CoCl.sub.2×6H.sub.2O. Additionally, a 0.1 M Na-Phosphate buffer was used for further buffering of the medium.

[0225] In order to generate the cultivation, 50 μL of a cryostock containing the corresponding E. coli strain were used to inoculate 5 mL of supplemented defined mineral salt medium containing 5 g/L glucose, 100 μg/mL ampicillin, 25 μg/mL chloramphenicol (only for mutant E. coli strains) and a given concentration of L-arabinose. The cultivation was incubated at 37° C. and 220 rpm in an orbital shaker for 15 h. At the end of the process, OD.sub.600nm, was measured for each cultivation. Following table summarizes results obtained:

TABLE-US-00002 Tested strain L-arabinose concentration (%) OD.sub.600 nm E. coli K12 BW25113 ΔilvC 0 0.21 pSW3_lacI.sup.+ 0.05 0.32 pACG_araBAD_ilvC 0.1 0.64 0.4 2.34 1.6 2.42 E. coli K12 BW25113 ΔthrA 0 0.16 pSW3_lacI.sup.+ 0.05 0.17 pACG_araBAD_thrA 0.2 0.37 0.4 2.88 0.8 2.79 1.6 2.69 E. coli K12 BW25113 ΔilvA 0 0.14 pSW3_lacI.sup.+ 0.025 0.78 pACG_araBAD_ilvA 0.05 2.95 0.2 2.83 0.8 2.62 1.6 2.61 E. coli K12 BW25113 ΔilvIH 0 2.84 pSW3_lacI.sup.+ 0.025 3.03 pACG_araBAD_ilvIH 0.05 3.08 0.2 3.03 0.8 2.43 1.6 2.77 E. coli K12 BW25113 ΔilvBN 0 2.66 pSW3_lacI.sup.+ 0.025 2.66 pACG_araBAD_ilvBN 0.05 2.65 0.2 2.96 0.8 2.80 1.6 2.66 E. coli K12 BW25113 0 3.10 pSW3_lacI.sup.+ 0.025 3.05 pACG_araBAD_ilvGM 0.05 3.17 0.2 3.01 0.8 2.65 1.6 2.93 E. coli K12 BW25113 0 3.10 pSW3_lacI.sup.+

Example 6

Cultivation Conditions for Evaluation of L-Arabinose Induction Effect on ncBCAA Production in Mutant E. coli Strains at Mini-Bioreactor Level

Cultivation Medium

[0226] E. coli cells were grown in a defined mineral salt medium containing (per L): 0.67 g Na.sub.2SO.sub.4, 0.82 g (NH.sub.4).sub.2SO.sub.4, 0.17 g NH.sub.4Cl, 4.87 g K.sub.2HPO.sub.4, 1.2 g NaH.sub.2PO.sub.4×2H.sub.2O and 0.33 g (NH.sub.4).sub.2-H-citrate. The medium was supplemented with 0.67 ml/L trace elements solution and 0.67 ml/L MgSO.sub.4 solution (1.0 M). The trace element solution comprised (per L): 0.5 g CaCl.sub.2×2H.sub.2O, 0.18 g ZnSO.sub.4×7H.sub.2O, 0.1 g MnSO.sub.4×H2O, 16.7g FeCl.sub.3×6H.sub.2O, 0.16 g CuSO.sub.4×5H.sub.2O, 0.18 g CoCl.sub.2×6H.sub.2O. Additionally, a 0.1 M Na-Phosphate buffer was used for further buffering of the medium.

Pre-Cultivation

[0227] 30 μL of a cryostock containing the corresponding E. coli strain were used to inoculate 30 mL of supplemented defined mineral salt medium containing 5 g/L glucose, 100 μg/mL ampicillin and 25 μg/mL chloramphenicol (only for mutant E. coli strains) in order to generate the pre-cultivation. For each mutant E. coli strain, medium also contained the minimum L-arabinose concentration necessary to recover the cell growth of the non-engineered strain, which was previously tested in Example 5. The pre-cultivation was incubated at 37° C. and 220 rpm in an orbital shaker, overnight.

Main Cultivation

[0228] OD.sub.600nm at the end of the pre-cultivation was measured and a given volume was used to inoculate a 5 mL starting volume Pall Micro24 mini-bioreactor (Microreactor Technologies Inc.) so that initial OD.sub.600nm was 0.4. The mini-reactor medium consisted of supplemented defined mineral salt medium containing 4 g/L glucose, 100 μg/mL ampicillin, 25 μg/mL chloramphenicol (only for mutant E. coli strains) and 1 μL/mL Desmophen antifoam. Medium was also supplemented with different concentrations of L-arabinose. Cultivation was carried out at 37° C. and the pH was maintained at 7 by automatic control with NH.sub.4OH and CO.sub.2. Stirrer speed was set to 800 rpm and DO set-point to 25%, maintaining the last by automatically increasing the oxygen flow into the mini-reactor. Batch phase lasted around 4 h. After batch phase was finished, 1 mL 400 g/L EnPump 200 solution and 50 μL 3000 U/L amylase solution were manually added into the mini-reactor, hence starting the fed-batch phase. EnPump 200 is a glucose polymer and when amylase is present, it constantly hydrolyses the polymer, thus delivering free glucose molecules over time, ensuring then a glucose-limited fermentation. 30 min after beginning of the fed batch phase, recombinant protein expression was induced by manual addition of an IPTG pulse to a final concentration of 0.5 mM. Fed-batch phase was active for 3.5 h.

Example 7

Amino Acid Analysis

[0229] Intracellular soluble protein fraction and inclusion body fraction were isolated from cell extracts according to protocol provided in “BugBuster Protein Extraction Reagent” kit (Merck, Cat. Nr.: 70584-4). 250 μL of the isolated intracellular soluble protein fraction were mixed with 750 μL 5M HCl. Isolated inclusion body pellets were resuspended with 200 μL dH.sub.2O and 100 μL of the resulting inclusion body suspension were mixed with 900 μL 5M HCl. Resulting solutions were introduced in crystal vials with screw caps and vials were incubated closed for 24 h at 80° C. for acid hydrolysis. Afterwards, vials were left opened in a heating block for 16-24 h at 65° C. while rotating until all liquid was evaporated. Amino acid isolation from dried hydrolyzed samples was performed according to protocol provided in “EZ:faast™ for free (physiological) amino acid analysis by GC-FID” kit (Phenomenex, Cat. Nr.: KGO-7165). After isolation process, around 120 μL of the resulting upper layer were introduced into GC vials and 2 μL were then injected into the GC analyzer. The GC was run according to following oven conditions: equilibration time of 0.5 min, 110° C. for 1 min, 30° C./min heating up to 320° C. and then 320° C. for 1 min. Nitrogen was used as a carrier gas with a constant flow rate of 1.5 mL/min. Injection was carried out with a 1:15 split ratio at 250° C.

Example 8

Evaluation of L-Arabinose Induction Effect on ncBCAA Production in Mutant E. coli Strains at Mini-Bioreactor Level

[0230] Following tables summarize experimental results for each tested protein fraction, ncBCAA and mutant strain under different concentrations of L-arabinose. Bold data presented in tables correspond to the concentration of a given ncBCAA in the E. coli BW25113 pSW3_lacl.sup.+ control strain. Data in percentage format shown in tables correspond to the variation percentage of the ncBCAA concentration obtained in the mutant strain under a given concentration of L-arabinose with respect to ncBCAA concentration obtained for the E. coli BW25113 pSW3_lacl.sup.+ control strain.

Inclusion Body Fraction

A) Norvaline

[0231]

TABLE-US-00003 E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.026 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara 6.8% 0.8% L-ara −1.2% 1.6% L-ara −21.5% E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔthrA pSW3_lacI.sup.+ 0.036 (nmol aa/OD.sub.600 nm) pACG_araBAD_thrA 0.4% L-ara −36.4% 0.8% L-ara −44.9% 1.6% L-ara −40.6% E. coli BW25113 E. coli BW25113 E. coli BW25113 E. coli BW25113 pSW3_lacI.sup.+ ΔilvIH ΔilvA ΔilvBN E. coli BW25113 0.023 (nmol aa/ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ OD.sub.600 nm) pACG_araBAD_ilvIH pACG_araBAD_ilvA pACG_araBAD_ilvBN pACG_araBAD_ilvGM 0.05% L-ara −12.3% −7.6% 2.7% −61.8% 0.2% L-ara — 4.5% 48.3% −56.0% 0.8% L-ara −40.7% −17.1% 62.9% −58.4%

[0232] For strains E. coli BW25113 ΔilvC pSW3_lacl.sup.+ pACG_araBAD_ilvC and E. coli BW25113 ΔilvIH pSW3_lacl.sup.+ pACG_araBAD_ilvIH, norvaline concentration significantly decreases when adding increasing concentrations of L-arabinose into the medium. The opposite behavior is observed for strain E. coli BW25113 ΔilvBN pSW3_lacl.sup.+ pACG_araBAD_ilvBN. For strains E. coli BW25113 ΔthrA pSW3_lacl.sup.+ pACG_araBAD_thrA and E. coli BW25113 pSW3lacl.sup.+ pACG_araBAD_ilvGM, norvaline concentration shows a significant reduction but effect of increasing L-arabinose concentrations does not show a clear trend on variation of norvaline concentration. For strain E. coli BW25113 ΔilvA pSW3_lacl.sup.+ pACG_araBAD_ilvA no significant reduction of norvaline concentration was reported and effect of increasing L-arabinose concentrations does not seem to show a clear trend on variation of norvaline concentration.

B) Norleucine

[0233]

TABLE-US-00004 E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.091 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara 71.9% 0.8% L-ara 8.7% 1.6% L-ara −24.3% E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔthrA pSW3_lacI.sup.+ 0.237 (nmol aa/OD.sub.600 nm) pACG_araBAD_thrA 0.4% L-ara −57.2% 0.8% L-ara −58.4% 1.6% L-ara −60.3% E. coli BW25113 E. coli BW25113 E. coli BW25113 E. coli BW25113 pSW3_lacI.sup.+ ΔilvIH ΔilvA ΔilvBN E. coli BW25113 0.060 (nmol aa/ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ OD.sub.600 nm) pACG_araBAD_ilvIH pACG_araBAD_ilvA pACG_araBAD_ilvBN pACG_araBAD_ilvGM 0.05% L-ara −4.2% −22.3% 67.4% −100.0% 0.2% L-ara — 16.9% 132.2% −88.5% 0.8% L-ara −70.8%  −10.5% 245.9% −100.0%

[0234] For strains E. coli BW25113 ΔilvC pSW3_lacl.sup.+ pACG_araBAD_ilvC and E. coli BW25113 ΔilvIH pSW3_lacl.sup.+ pACG_araBAD_ilvIH, norleucine concentration significantly decreases when adding increasing concentrations of L-arabinose into the medium. The opposite behavior is observed for strain E. coli BW25113 ΔilvBN pSW3_lacl.sup.+ pACG_araBAD_ilvBN. For strains E. coli BW25113 ΔthrA pSW3_lacl.sup.+ pACG_araBAD_thrA and E. coli BW25113 pSW3_lacl.sup.+ pACG_araBAD_ilvGM, norleucine concentration shows a significant reduction but effect of increasing L-arabinose concentrations does not seem to show a clear trend on variation of norleucine concentration. For strain E. coli BW25113 ΔilvA pSW3_lacl.sup.+ pACG_araBAD_ilvA no significant reduction of norleucine concentration was reported and effect of increasing L-arabinose concentrations does not seem to have a clear effect on variation of norleucine concentration.

Intracellular Soluble Protein Fraction

A) Norvaline

[0235]

TABLE-US-00005 E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.300 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara −22.0% 0.8% L-ara −36.0% 1.6% L-ara −39.5% E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔthrA pSW3_lacI.sup.+ 0.253 (nmol aa/OD.sub.600 nm) pACG_araBAD_thrA 0.4% L-ara −41.3% 0.8% L-ara −44.3% 1.6% L-ara −41.6% E. coli BW25113 E. coli BW25113 E. coli BW25113 E. coli BW25113 pSW3_lacI.sup.+ ΔilvIH ΔilvA ΔilvBN E. coli BW25113 0.529 (nmol aa/ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ OD.sub.600 nm) pACG_araBAD_ilvIH pACG_araBAD_ilvA pACG_araBAD_ilvBN pACG_araBAD_ilvGM 0.05% L-ara −53.1% −19.5% −2.2% −71.9% 0.2% L-ara — −23.9% 23.5% −73.0% 0.8% L-ara −63.6% −20.8% 211.4% −75.9%

[0236] For strains E. coli BW25113 ΔilvC pSW3_lacl.sup.+ pACG_araBAD_ilvC and E. coli BW25113 ΔilvIH pSW3_lacl.sup.+ pACG_araBAD_ilvIH, norvaline concentration decreases when adding increasing concentrations of L-arabinose into the medium. The opposite behavior is observed for strain E. coli BW25113 ΔilvBN pSW3_lacl.sup.+ pACG_araBAD_ilvBN. For strains E. coli BW25113 ΔthrA pSW3_lacl.sup.+ pACG_araBAD_thrA, E. coli BW25113 ΔilvA pSW3_lacl.sup.+ pACG_araBAD_ilvA and E. coli BW25113 pSW3_lacl.sup.+ pACG_araBAD_ilvGM, norvaline concentration shows a significant reduction but effect of increasing L-arabinose concentrations does not seem to show a clear trend on variation of norvaline concentration.

B) Norleucine

[0237]

TABLE-US-00006 E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.180 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara 154.8% 0.8% L-ara 42.5% 1.6% L-ara 2.8% E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔthrA pSW3_lacI.sup.+ 0.304 (nmol aa/OD.sub.600 nm) pACG_araBAD_thrA 0.4% L-ara −30.1% 0.8% L-ara −26.6% 1.6% L-ara −36.4% E. coli BW25113 E. coli BW25113 E. coli BW25113 E. coli BW25113 pSW3_lacI.sup.+ ΔilvIH ΔilvA ΔilvBN E. coli BW25113 0.241 (nmol aa/ pSW3_lacI.sup.+ pSW3_lacl.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ OD.sub.600 nm) pACG_araBAD_ilvIH pACG_araBAD_ilvA pACG_araBAD_ilvBN pACG_araBAD_ilvGM 0.05% L-ara −17.8% −58.5% 2.8% −100.0% 0.2% L-ara — −56.1% 36.6% −81.3% 0.8% L-ara −69.9% −54.7% 127.5% −100.0%

[0238] For strains E. coli BW25113 ΔilvC pSW3_laci.sup.+ pACG_araBAD_ilvC and E. coli BW25113 ΔilvIH pSW3_lacl.sup.+ pACG_araBAD_ilvIH, norleucine concentration decreases when adding increasing concentrations of L-arabinose into the medium. The opposite behavior is observed for strain E. coli BW25113 ΔilvBN pSW3_lacl.sup.+ pACG_araBAD_ilvBN. For strains E. coli BW25113 ΔthrA pSW3_lacl.sup.+ pACG_araBAD_thrA, E. coli BW25113 ΔilvA pSW3_lacl.sup.+ pACG_araBAD_ilvA and E. coli BW25113 pSW3_lacl.sup.+ pACG_araBAD_ilvGM, norleucine concentration shows a significant reduction but L-arabinose concentration does not seem to have a clear effect on norleucine concentration.

C) β-Methylnorleucine

[0239]

TABLE-US-00007 E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.459 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara −5.5% 0.8% L-ara −25.2% 1.6% L-ara 8.5% E. coli BW25113 pSW3_lacI.sup.+ E. coli BW25113 ΔilvC pSW3_lacI.sup.+ 0.459 (nmol aa/OD.sub.600 nm) pACG_araBAD_ilvC 0.4% L-ara −5.5% 0.8% L-ara −25.2% 1.6% L-ara 8.5% E. coli BW25113 E. coli BW25113 E. coli BW25113 E. coli BW25113 pSW3_lacI.sup.+ ΔilvIH ΔilvA ΔilvBN E. coli BW25113 0.432 (nmol aa/ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ pSW3_lacI.sup.+ OD.sub.600 nm) pACG_araBAD_ilvIH pACG_araBAD_ilvA pACG_araBAD_ilvBN pACG_araBAD_ilvGM 0.05% L-ara −26.4% −10.3% −42.0% −21.3% 0.2% L-ara — −14.1% −23.2% −12.8% 0.8% L-ara −12.8% −2.2% −14.0% −18.5%

[0240] For almost all tested strains and L-arabinose concentrations a slight reduction of β-methylnorleucine concentration is reported in the intracellular soluble protein fraction. However, this reduction is not really significant if compared with norvaline and norleucine, with exception of strain E. coli BW25113 ΔilvBN pSW3_lacl.sup.+ pACG_araBAD_ilvBN induced with 0.05% L-ara, where reduction reached about 42%. In addition, effect of increasing L-arabinose concentrations does not show a clear trend on variation of β-methylnorleucine concentration.

Example 9

Screening of Potential ilvGM- and ilvIH-Tunable E. coil Strains in a 15 L Reactor Under Conditions Triggering ncBCAA Formation

[0241] During fermentation in large scale reactors, gradient zones of substrate, dissolved oxygen, pH and other parameters are formed due to inefficient mixing and E. coli cells respond to these environmental changes by modulating their metabolism (Schweder (1999). Monitoring of genes that respond to process-related stress in large-scale bioprocesses. Biotechnology and bioengineering, 65(2), 151-159). For instance, E. coli responds to glucose excess and oxygen limitation by shifting metabolism from oxidative respiration to mixed-acid fermentation, resulting in overflow metabolism (Enfors et al. (2001). Physiological responses to mixing in large scale bioreactors. Journal of biotechnology, 85(2), 175-185). Under these conditions, not only the mixed-acid fermentation products accumulate, but also pyruvate (Soini, J. et al. (2008). Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110. Microb. Cell Fact., 7: 1-14). Pyruvate excess present intracellularly increases the metabolic flux going to ncBCAA biosynthesis through the sequential keto acid chain elongation from pyruvate to α-ketocaproate over α-ketobutyrate and α-ketovalerate by the actuation of the leu operon-encoded enzymes (Apostol, I. et al. (1997). Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli. Journal of Biological Chemistry, 272(46), 28980-28988). This hypothesis is supported by the observations reported by Soini et al. (2011, Accumulation of amino acids deriving from pyruvate in Escherichia coli W3110 during fed-batch cultivation in a two-compartment scale-down bioreactor. Advances in Bioscience and Biotechnology, 2(05), 336): the combination of oxygen limitation with a constant glucose supply in a two-compartment STR-PFR scale-down bioreactor reported a significant impact on enhancing norvaline biosynthesis due to pyruvate accumulation in a recombinant E. coli cultivation. Furthermore, Soini et al. (2008) originally reported accumulation of pyruvate-based amino acids such the ncBCAAs norleucine and norvaline as well as alanine and valine in a standard STR fed-batch E. coli cultivation under glucose excess and induced oxygen limitation upon a stirrer downshift.

[0242] Concentration gradients happening in large industrial-scale bioreactors due to deficient mixing can be also simulated in small bioreactors in the laboratory. In this investigation scale-up effects are reproduced in a 15 L reactor by combining pyruvate pulsing and O.sub.2 limitation. This novel cultivation strategy might represent more accurately the physiological behavior of bacterial cultivations taking place in large scale bioreactors.

[0243] According to the mini-bioreactor screening (Example 8), strains E. coli K-12 BW25113 pSW3_lacl.sup.+ pACG_araBAD_ilvGM (ilvGM-tunable E. coil) and E. coli K-12 BW25113 ΔlvlH pSW3_lacl.sup.+ pACG_araBAD_ilvIH (ilvIH-tunable E. coil) induced with 0.8% L-arabinose showed the best performance among all screened mutants in a 10 mL mini-bioreactor, since they reported the most significant reduction of ncBCAA mis-incorporation into recombinant mini-proinsulin in comparison with the control non-engineered E. coli strain. The aim of this experiment was to verify the performance of the aforementioned potential tunable E. coli strains in a 15 L reactor under cultivation conditions triggering formation of ncBCAA, i.e. pyruvate pulses and oxygen limitation, in order to confirm its advantage as strain ensuring product quality. For comparison, the control non-engineered E. coli host (E. coli K-12 BW25113 pSW3_lacl.sup.+) was also cultivated.

Cultivation of E. coil K-12 BW25113 pSW3_lacl.sup.+ (Control Strain) Under Standard Conditions

[0244] 100 μL of a cryostock containing E. coli K-12 BW25113 pSW3_lacl.sup.+ were used to inoculate 500 mL of supplemented mineral salt medium containing 5 g/L glucose and 100 μg/mL ampicillin in order to generate the pre-culture. Composition of the mineral salt medium was as follows: 2 g/L Na.sub.2SO.sub.4, 2.468 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L NH.sub.4Cl, 14.6 g/L K.sub.2HPO.sub.4, 3.6 g/L NaH.sub.2PO4.2H.sub.2O and 1 g/L (NH.sub.4).sub.2-H-citrate. The mineral salt medium was then supplemented with 2 mL/L MgSO.sub.4 solution (1.0 M) and 2 mL/L trace elements solution. The trace element solution comprised (per L): 0.5 g CaCl.sub.2×2H.sub.2O, 0.18 g ZnSO.sub.4×7H.sub.2O, 0.1 g MnSO.sub.4×H.sub.2O, 16.7 g FeCl.sub.3×6H.sub.2O, 0.16 g CuSO.sub.4×5H.sub.2O, 0.18 g CoCl.sub.2×6H.sub.2O. The pre-culture was incubated at 37° C. and 220 rpm in an orbital shaker for 12 h, using an initial cold-start technique. OD.sub.600nm at the end of the pre-culture was measured and a given volume was used to inoculate a 7 L starting volume reactor so that initial OD.sub.600nm was 0.4. The reactor medium consisted of supplemented mineral salt medium containing 5 g/L glucose, 2 mL antifoam (Antifoam 2014, Sigma) and 100 μg/mL ampicillin. Cultivation was carried out at 37° C. and the pH was maintained at 7 by automatic control with 25% NH.sub.4OH. Airflow was set to 7 vvm and DO set-point to 20%, maintaining the last by using a cascade control altering stirrer speed (initial stirrer speed was set to 800 rpm). Batch phase lasted effectively 4 h, with an intermediate 13 h cold phase at 15° C. At the end of the batch phase, exponential feeding was started, according to following equation:

[00001]$F\left(t\right)=\frac{q_s}{S}.Math.\left(X.Math.V\right).Math.e^{{\mu }_{set}.Math.t}$

[0245] where F (t) represents the feed flow rate over time (L h.sup.−1), q.sub.s the set-point of the specific substrate uptake rate (0.514 gS gX.sup.−1 h.sup.−1), S the concentration of glucose in the feed solution (442 g/L), X the biomass concentration over time (g/L), V the volume of the reactor over time (L), μ.sub.set the set-point of the specific cell growth rate (0.3 h.sup.−1) and t the time during the fed-batch phase. The feed solution consisted of TUB mineral salt medium supplemented with 4 mL/L trace elements solution, 2 mL/L MgSO.sub.4 solution (1.0 M), 100 μg/mL ampicillin and 442 g/L glucose.

[0246] Exponential fed-batch phase was carried out for 3 hours and afterwards expression of recombinant mini-proinsulin was induced by automatic addition of IPTG to a final concentration of 0.5 mM. Induction time was 30 minutes. During the induction phase no feed was added into the reactor. After induction, a constant feeding phase was started, so that the constant flow rate was equal to the last flow rate achieved in the exponential feeding phase. Constant feed fed-batch phase was active for 5-6 h.

Cultivation of E. coil K-12 BW25113 pSW3_lacl.sup.+ (Control Strain) Under Conditions Triggering ncBCAA Formation

[0247] Cultivation was performed as described for the standard cultivation in previous section. However, after the exponential fed-batch phase, 1 g/L pyruvate pulse was automatically added into the reactor. Pyruvate solution was constantly pumped for 5 minutes. During that time period no feed was added, airflow rate was temporary set to 0 and DO cascade control was disconnected. After the first pyruvate pulse, expression of recombinant mini-proinsulin was induced by automatic addition of IPTG to a final concentration of 0.5 mM. Induction time was 30 minutes. During the induction phase no feed was added into the reactor and airflow and DO cascade control were re-established. After induction, sequential 1 g/L pyruvate pulses were performed each 30 min as described above for a total of 4 pulses. Between pulses, constant feeding phase was activated, so that the constant flow rate was equal to the last flow rate achieved in the exponential feeding phase, and airflow and DO cascade control were re-established. Constant feed fed-batch phase was active for 5-6 h.

Cultivation of ilvGM-tunable E. coil Under Conditions Triggering ncBCAA Formation

[0248] Cultivation operation was as described in previous section and only minor changes were done in order to adapt the cultivation process to ilvGM-tunable E. coli strain. Both pre-culture and reactor medium contained additionally 25 μg/mL chloramphenicol. The reactor medium additionally contained 0.8% L-arabinose, necessary to induce expression of gene ilvGM hosted in plasmid pACG_araBAD_ilvGM. The feeding solution was also additionally supplemented with 25 μg/mL chloramphenicol and 0.8% L-arabinose.

Cultivation of ilvIH-tunable E. coil Under Conditions Triggering ncBCAA Formation

[0249] Cultivation operation was as described in previous section.

Example 10

Analysis of ncBCAA

[0250] Concentrations of ncBCAA present in the intracellular soluble protein fraction and in the inclusion body fraction over cultivation time for each tested strain in Example 9 are shown in FIG. 11 and FIG. 12, respectively.

[0251] The cultivation of the control E. coli strain subjected to pyruvate pulses combined with O.sub.2 limitation (“WT E. coli, PYR-O2”) reported a progressive accumulation of norleucine and β-methylnorleucine in the intracellular soluble protein fraction over time after induction, being that more significant for norleucine. Furthermore, norvaline concentration also increased progressively under aforementioned cultivation conditions, but only until 3 h after induction. From that time point on, norvaline concentration progressively dropped until reaching initial values at 5 h after induction. This might suggest that, after 2 h from last pyruvate pulse combined with O.sub.2 limitation, its associated effect triggering norvaline accumulation is not active anymore (FIG. 11). As expected, the aforementioned strain reported a higher level of norvaline and norleucine than the control E. coli strain cultivated under standard conditions (“WT E. coli, STD”). Such concentration difference could not be observed for β-methylnorleucine.

[0252] Both tested potential mutants in cultivations “ilvGM-tunable E. coli, PYR-O2” and “ilvIH-tunable E. coli, PYR-O2” reported a dramatic reduction of norvaline and norleucine concentrations in the intracellular soluble protein fraction, being such decrease higher for norleucine in “ilvGM-tunable E. coli, PYR-O2”. However, β-methylnorleucine concentrations did not significantly vary with respect to the control cultivation. It is noteworthy to highglight that, for most samples, norvaline could not be properly detected since concentrations were under the limit of detection of the GC-FID equipment (FIG. 11).

[0253] The cultivation of the control E. coli strain subjected to pyruvate pulses combined with O.sub.2 limitation (“WT E. coli, PYR-O2”) reported a progressive accumulation of norvaline and norleucine in the inclusion body fraction over time after induction. As expected, the aforementioned strain reported a higher level of norvaline and norleucine than the control E. coli strain cultivated under standard conditions (“WT E. coli, STD”). Again, and similar to reported in the intracellular soluble fraction, both tested potential mutants in cultivations “ilvGM-tunable E. coli, PYR-O2” and “ilvIH-tunable E. coli, PYR-O2” reported a dramatic reduction of norvaline and norleucine concentrations in the inclusion body fraction, being this decrease even higher for norleucine in “ilvGM-tunable E. coli, PYR-O2”. Norvaline could not be detected in any case for both tested mutants. β-methylnorleucine could not be detected in any tested samples (FIG. 12).