3HP tolerance

Abstract

Cells and cell cultures are provided that have improved tolerance to 3-hydroxypropionic acid (3HP). Genetic modifications to provide a mutated or overexpressed SFA1 gene or other enhancement of 3HP detoxification via a glutathione-dependent dehydrogenase reaction, including medium supplementation with glutathione, may be combined with a 3HP producing metabolic pathway.

Claims

1. A cell having a metabolic pathway producing 3-hydroxypropionic acid (3HP), said cell exhibiting tolerance for 3HP and having one or more genetic modifications that provide for an enhanced activity of 3HP detoxification by a reaction pathway that includes a glutathione-dependent dehydrogenase reaction, wherein said one or more genetic modifications comprises one or more mutations in a gene encoding a glutathione-dependent formaldehyde dehydrogenase conferring tolerance for 3HP, and wherein the one or more mutations is at a position equivalent to the position aa276 Cys and/or at aa283 Met of the SFA1 gene of Saccharomyces cerevisiae.

2. A cell as claimed in claim 1, wherein said one or more mutations are in a gene encoding a protein having the sequence SEQ ID NO 1 or a protein with more than 80% homology to SEQ ID NO 1.

3. A cell as claimed in claim 1, wherein said mutation in gene SFA1 is Cys276.fwdarw.Ser, Cys276.fwdarw.Val, Cys276.fwdarw.Thr, Cys276.fwdarw.Gly, Cys276.fwdarw.Ala and/or Met283.fwdarw.Ile, Met283.fwdarw.Ala, Met283.fwdarw.Val.

4. A cell as claimed in claim 1, wherein a said genetic modification produces overexpression of a native, or heterologous, or mutated glutathione-dependent formaldehyde dehydrogenase to confer said 3HP tolerance.

5. A cell as claimed in claim 1, wherein the cell is genetically modified for increased production of glutathione.

6. A cell as claimed in claim 5, wherein the cell overexpresses genes that enhance the production of amino acid precursors for glutathione biosynthesis.

7. A cell as claimed in claim 1, wherein said metabolic pathway comprises the enzyme malonyl-CoA reductase and/or the enzyme malonyl-CoA reductase (malonate semialdehyde-forming) in combination with the enzyme 3-hydroxyisobutyrate dehydrogenase and/or the enzyme hydroxypropionate dehydrogenase, or wherein said metabolic pathway comprises a malonyl-CoA reductase gene and an acetyl-CoA carboxylase gene, or wherein said metabolic pathway comprises beta-alanine pyruvate aminotransferase and/or gamma-aminobutyrate transaminase in combination with hydroxyisobutyrate dehydrogenase and/or hydroxypropionate dehydrogenase or wherein said metabolic pathway comprises glycerol dehydratase and alcohol dehydrogenase or wherein said metabolic pathway comprises lactate dehydrogenase, propionate CoA-transferase, lactoyl-CoA dehydratase, enoyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase.

8. A cell as claimed in claim 1, wherein said cell is a yeast cell or is a bacterial cell.

9. A method of producing 3HP comprising cultivating a 3HP producing cell under 3HP producing conditions in a culture medium so as to produce 3HP, wherein toxicity of 3HP is reduced by an enhanced activity of 3HP detoxification by a reaction pathway that includes a glutathione-dependent dehydrogenase reaction, and wherein said culture medium is supplemented with glutathione.

10. A method of producing 3HP comprising cultivating a 3HP producing cell under 3HP producing conditions in a culture medium so as to produce 3HP, wherein toxicity of 3HP is reduced by an enhanced activity of 3HP detoxification by a reaction pathway that includes a glutathione-dependent dehydrogenase reaction, and wherein the cell overexpresses the glutathione biosynthetic genes gamma-glutamylcysteine synthetase and glutathione synthetase.

11. A method as claimed in any one of claim 9, wherein the cell overexpresses genes that enhance the production of amino acid precursors for glutathione biosynthesis.

Description

DESCRIPTION OF THE DRAWINGS

(1) The invention will be further described and illustrated with reference to the accompanying drawings in which:

(2) FIG. 1 illustrates pathways leading from pyruvate to 3HP; The following abbreviations are used:

(3) PYCpyruvate carboxylase,

(4) AATaspartate aminotransferase,

(5) PanDaspartate decarboxylase,

(6) BAPATbeta-alanine pyruvate aminotransferase,

(7) GabTgamma-aminobutyrate transaminase,

(8) HIBADH3-hydroxyisobutyrate dehydrogenase,

(9) HPDHhydroxypropionate dehydrogenase,

(10) HPDH3-hydroxypropionate dehydrogenase,

(11) LDHlactate dehydrogenase,

(12) PCTpropionate CoA-transferase,

(13) LCDlactoyl-CoA dehydratase,

(14) ECHenoyl-CoA hydratase,

(15) HIBCH3-hydroxyisobutyryl-CoA hydrolase.

(16) MCR1malonyl-CoA reductase (malonate semialdehyde-forming),

(17) MCR2bi-functional malonyl-CoA reductase,

(18) GDglycerol dehydratase,

(19) ALDaldehyde dehydrogenase

(20) FIG. 2 shows growth curves for S. cerevisiae on minimal medium in the presence of different concentrations of 3HP, as described in Example 1;

(21) FIG. 3 shows improved 3HP tolerance of strains of S. cerevisiae having overexpressed native or mutated SFA1 genes as found in Example 2;

(22) FIG. 4 shows improved 3HP tolerance of strains of S. cerevisiae having mutated SFA1 genes at varying levels of 3HP in the minimal medium, as found in Example 3;

(23) FIG. 5 shows 3HP titers and yields in strains having 3HP pathway genes in combination with or without having overexpressed native or mutant SFA1 genes, as found in Example 4;

(24) FIG. 6 shows the proposed 3HP detoxification pathway in S. cerevisiae;

(25) FIG. 7 shows the effect of GSH on the growth of strains of S. cerevisiae having the native or mutated SFA1 gene as well as the sfa1 strains grown on minimal medium containing 50 g/L 3HP, as described in Example 5;

(26) FIG. 8 shows the growth of SFA1-alleles replacement strains grown on minimal medium containing 50 g/L 3HP, as described in Example 8;

(27) FIG. 9 shows the effect of GSH addition on the growth of several yeast strains grown on minimal medium containing 50 g/L 3HP, as described in Example 9;

(28) FIG. 10 shows the effect of GSH addition on the growth of several E. coli strains grown on M9 containing 20 g/L 3HP, as described in Example 9.

EXAMPLES

(29) The following examples demonstrate the effectiveness of the invention. The following tables summarise materials used in the Examples and results obtained.

(30) TABLE-US-00001 TABLE1 Primers Primername Primersequence,5->3 SEQIDNO KO_sfa1_fw CAGAATTTGTTGGCCTATTTTCTTA SEQIDNO16 KO_sfa1_rv CAATACGTTGGTAGTTAGGAACAGG SEQIDNO17 KO_sfa1_test_fw GATGCTCATCACAGACTACT SEQIDNO18 KanMX_2/3_START_rv AGTGACGACTGAATCCGGTG SEQIDNO19 PTEF1_fw ACCTGCACUTTGTAATTAAAACTTAG SEQIDNO20 PTEF1_rv CACGCGAUGCACACACCATAGCTTC SEQIDNO21 SFA1_U1_fw AGTGCAGGUAAAACAATGTCCGCCGCTACTGTT SEQIDNO22 SFA1_U1_rv CGTGCGAUTCATTTTATTTCATCAGACTTCAAGA SEQIDNO23 SFA1_UPrevU1 AGCTGTTCUCTATTTTATTTCATCAGACTTCAAGACG SEQIDNO24 SFA1_DWfwd_U2 AGTGGCCUGAGTACTTAATTAAACTAAGTAAGCATGACTC SEQIDNO25 KO-SFA1_UPrev2 GTCTACCGTGATTTCTTCAACACTT SEQIDNO26 NB336K1LEUFwd1 TGGAAGAGGCAAGCACGTTAGC SEQIDNO27 NB335K1LEURev1 CAGAAGCATAACTACCCATTCC SEQIDNO28 NB326URA3fwdU AGAACAGCUGAAGCTTCGTACG SEQIDNO29 NB327URA3Rev2U AGGCCACUAGTGGATCTGATATCAC SEQIDNO30 PPGK1_rv CACGCGAUGCACACACCATAGCTTC SEQIDNO31 gsh1_U1_fw AGTGCAGGUAAAACAATGGGACTCTTAGCTTTGGG SEQIDNO32 gsh1_U1_rv CGTGCGAUTCAACATTTGCTTTCTATTGAAGGC SEQIDNO33 gsh2_U1_fw ATCTGTCAUAAAACAATGGCACACTATCCACCTTCC SEQIDNO34 gsh2_U1_rv CACGCGAUTCAGTAAAGAATAATACTGTCC SEQIDNO35 met14_U1_fw AGTGCAGGUAAAACAATGGCTACTAATATTACTTGGC SEQIDNO36 met14_U1_rv CGTGCGAUTCACAAATGCTTACGGATGATTTTTTC SEQIDNO37 met16_U1_fw ATCTGTCAUAAAACAATGAAGACCTATCATTTG SEQIDNO38 met16_U1_ev CACGCGAUTCAGGCATCTTGCTTTAAAAATTGC SEQIDNO39 SFA1_G614C_fw ATACCGTTGCAGTATTTGGCTCCGGGACTGTAG SEQIDNO40 SFA1_G614C_rv CTACAGTCCCGGAGCCAAATACTGCAACGGTAT SEQIDNO41 SFA1_G710C_fw GCCATTGACATTAACAATAAGAAAAAACAATATTCTTCTCA SEQIDNO42 ATTTGGTGCCAC SFA1_G710C_rv GTGGCACCAAATTGAGAAGAATATTGTTTTTTCTTATTGTT SEQIDNO43 AATGTCAATGGC SFA1_G869C_fw GAGAGATGCTTTGGAAGCCTCTCATAAAGGTTGGG SEQIDNO44 SFA1_G869C_rv CCCAACCTTTATGAGAGGCTTCCAAAGCATCTCTC SEQIDNO45 SFA1_T826G_fw GGGGTCTGGATTTTACTTTTGACGGTACTGGTAATACCAAAATTATG SEQIDNO46 SFA1_T826G_rv CATAATTTTGGTATTACCAGTACCGTCAAAAGTAAAATCCAGACCCC SEQIDNO47 SFA1_TG826GC_fw GGGGTCTGGATTTTACTTTTGACGCTACTGGTAATACCAAAATTATG SEQIDNO48 SFA1_TG826GC_rv CATAATTTTGGTATTACCAGTAGCGTCAAAAGTAAAATCCAGACCCC SEQIDNO49 SFA1_TG826GT_fw GGGGGTCTGGATTTTACTTTTGACGTTACTGGTAATACCAAAATTATGAG SEQIDNO50 SFA1_TG826GT_rv CTCATAATTTTGGTATTACCAGTAACGTCAAAAGTAAAATCCAGACCCCC SEQIDNO51 SFA1_T826A_fw GGGGTCTGGATTTTACTTTTGACACTACTGGTAATACCAAAATTATG SEQIDNO52 SFA1_T826A_rv CATAATTTTGGTATTACCAGTAGTGTCAAAAGTAAAATCCAGACCCC SEQIDNO53 SFA1_A847G_fw CTTTTGACTGTACTGGTAATACCAAAATTGTGAGAGATGCTTTGG SEQIDNO54 SFA1_A847G_rv CCAAAGCATCTCTCACAATTTTGGTATTACCAGTACAGTCAAAAG SEQIDNO55 SFA1_AT847GC_fw TTACTTTTGACTGTACTGGTAATACCAAAATTGCGAGAGATGCTTTGGAAG SEQIDNO56 SFA1_AT847GC_rv CTTCCAAAGCATCTCTCGCAATTTTGGTATTACCAGTACAGTCAAAAGTAA SEQIDNO57

(31) TABLE-US-00002 TABLE 2 Primers and templates used to generate gene fragments for gene reaplcement Fragment name Gene Fw_primer Rv_primer Template DNA SFA1_UP Upstream region of SFA1 KO_sfa1_fw KO-SFA1_UPrev2 gDNA of S. cerevisiae SFA1wt_UP Upstream region of SFA1 KO_sfa1_fw SFA1_UPrevU1 gDNA of S. cerevisiae including SFA1-wt SFA1.sup.G827C_UP SFA1 upstream region fused KO_sfa1_fw SFA1_UPrevU1 Fragment SFA1_UP_wt and by PCR to the SFA1.sup.G827C gene SFA1.sup.G827C SFA1G.sup.849A_UP SFA1 upstream region fused KO_sfa1_fw SFA1_UPrevU1 Fragment SFA1_UP_wt and by PCR to the SFA1.sup.G849A gene SFA1.sup.G849A SFA1_DOWN Downstream region of SFA1 SFA1_DWfwd_U2 SFA1_DW_test_rv gDNA of S. cerevisiae LEU2_U_2/3_START the first of loxP-KlLEU2 NB326URA3fwdU NB335KlLEURev1 Plasmid pUG73 marker LEU2_U_2/3_END the last part of loxP- NB336KlLEUFwd1 NB327URA3Rev2U Plasmid pUG73 KlLEU2 marker SFA1wt_UP_LEU2_U_2/ SFA1 upstream region KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP_wt and 3_START including SFA1-wt fused by LEU2_U_2/3_START PCR to the first of KlLEU2 marker SFA1.sup.G827C_UP_LEU2_U_2/ SFA1 upstream region KO_sfa1_fw NB335KlLEURev1 Fragment SFA1.sup.G827C_UP 3_START including SFA1.sup.G827C fused by and LEU2_U_2/3_START PCR to the first of KlLEU2 marker SFA1.sup.G849A_UP_LEU2_U_2/ SFA1 upstream region KO_sfa1_fw NB335KlLEURev1 Fragment SFA1.sup.G849A_UP 3_START including SFA1.sup.G849A fused by and LEU2_U_2/3_START PCR to the first of KlLEU2 marker LEU2_U_2/ the last part of KlLEU2 NB336KlLEUFwd1 SFA1_DW_test_rv Fragment LEU2_U_2/3_END 3_END_SFA1_DOWN marker fused by PCR to SFA1 and SFA1_DOWN downstream region SFA1.sup.G614C SFA1 gene where nt 614 was SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.G614C changed from G to C (Cys205Ser) SFA1.sup.G710C SFA1 gene where nt 710 was SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.G710C changed from G to C (Cys237Ser) SFA1.sup.G869C SFA1 gene where nt 869 was SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.G869C changed from G to C (Cys290Ser) SFA1.sup.T826G SFA1 gene where nt 826 was SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.T826G changed from T to G (Cys276Gly) SFA1.sup.TG826GC SFA1 gene where nt 826 and SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.TG826GC 827 were changed from T to G and G to C, respectively (Cys276Ala) SFA1.sup.TG826GT SFA1 gene where nt 826 and SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.TG826GT 827 were changed from T to G and G to T, respectively (Cys276Val) SFA1.sup.TG826AC SFA1 gene where nt 826 and SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.TG826AC 827 were changed from T to A and G to C, respectively (Cys276Thr) SFA1.sup.A847G SFA1 gene where nt 847 was SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.A847G changed from A to G (Met283Val) SFA1.sup.AT847GC SFA1 gene where nt 847 and SFA1_U1_fw SFA1_U1_rv pESC-LEU-SFA1.sup.AT847GC 848 were changed from A to G and T to C, respectively (Met283Ala) SFA1.sup.G614C_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.G614C and SFA1.sup.G614C gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.G710C_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.G710C and SFA1.sup.G710C gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.G869C_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.G869C and SFA1.sup.G869C gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.T826C_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.T826C and SFA1.sup.T826C gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.TG826Gc_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.TG826GC and SFA1.sup.TG826GC gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.TG826GT_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.TG826GT and SFA1.sup.TG826GT gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.TG826AC_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.TG826AC and SFA1.sup.TG826AC gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.A847C_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.A847C and SFA1.sup.A847C gene and the first LEU2_U_2/3_START of KlLEU2 marker SFA1.sup.AT847GC_UP_LEU2_U_2/ SFA1 upstream region fused KO_sfa1_fw NB335KlLEURev1 Fragment SFA1_UP, 3_START by PCR to the including SFA1.sup.AT847GC and SFA1.sup.AT847G gene and the first LEU2_U_2/3_START of KlLEU2 marker

(32) TABLE-US-00003 TABLE 3 Primers and templates used to generate gene fragments for USER cloning by PCR Fragment name Gene Fw_primer Rv_primer Template DNA PTEF1 Promoter of TEF1 gene from S. cerevisiae PTEF1_fw PTEF1_rv gDNA of S. cerevisiae SFA1-wt SFA1 gene (WT sequence) SFA1_U1_fw SFA1_U1_rv gDNA of S. cerevisiae SFA1.sup.G827C SFA1 gene where nt 827 was changed from G to C SFA1_U1_fw SFA1_U1_rv gDNA of S. cerevisiae SFA1.sup.G849A SFA1 gene where nt 849 was changed from G to A SFA1_U1_fw SFA1_U1_rv gDNA of S. cerevisiae <-PTEF1- Fused promoters of TEF1 and PGK1 genes from PTEF1_fw PPGK1_rv plasmid pSP-GM1 PPGK1-> S. cerevisiae GSH1<- Gamma glutamylcysteine synthetase (GSH1) from gsh1_U1_fw gsh2_U1_rv gDNA of S. cerevisiae S. cerevisiae GSH2-> Glutathione synthetase (GSH2) from S. cerevisiae gsh1_U1_fw gsh2_U1_rv gDNA of S. cerevisiae MET14<- Adynylylsulfate kinase (MET14) from S. cerevisiae gsh1_U1_fw met14_U1_rv gDNA of S. cerevisiae MET16-> 3-phosphoadenylsulfate reductase (MET16) from met16_U1- met16_U1-rv gDNA of S. cerevisiae fw S. cerevisiae

(33) TABLE-US-00004 TABLE 4 Plasmids Plasmid name Parent plasmid Selection marker Cloned fragment Promoter Terminator pESC-LEU LEU2 pESC-LEU-USER LEU2 pUG73 KlLEU2 pESC-LEU-SFA1-wt pESC-LEU-USER LEU2 SFA1-wt PTEF1 TADH1 pESC-LEU-SFA1.sup.G827C pESC-LEU-USER LEU2 SFA1.sup.G827C PTEF1 TADH1 pESC-LEU-SFA1.sup.G849A pESC-LEU-USER LEU2 SFA1.sup.G849A PTEF1 TADH1 pESC-URA pESC-URA-USER pESC-URA-GSH1-GSH2 pESC-URA-USER pESC-HIS pESC-HIS-USER pESC-HIS-MET14-MET16 pESC-HIS-USER pESC-LEU-SFA1.sup.G614C pESC-LEU-USER pESC-LEU-SFA1.sup.G710C pESC-LEU-USER pESC-LEU-SFA1.sup.G869C pESC-LEU-USER pESC-LEU-SFA1.sup.T826G pESC-LEU-USER pESC-LEU-SFA1.sup.TG826GC pESC-LEU-USER pESC-LEU-SFA1.sup.TG826GT pESC-LEU-USER pESC-LEU-SFA1.sup.T826A pESC-LEU-USER pESC-LEU-SFA1.sup.A847G pESC-LEU-USER pESC-LEU-SFA1.sup.AT847GC pESC-LEU-USER

(34) TABLE-US-00005 TABLE 5 Yeast strains Strain name Genotype CEN.PK 113-7D MATa URA3 HIS3 LEU2 TRP1 CEN.PK 113-32D MATa leu2 CEN.PK 102-5B MATa ura3 leu2 his3 sfa1 MATa leu2 sfa1::KanMX SCE-R1-48 MATa leu2 sfa1::KanMX + pESC-LEU SCE-R1-49 MATa leu2 sfa1::KanMX + pESC-LEU-SFA1-wt SCE-R1-50 MATa leu2 sfa1::KanMX + pESC-LEU-SFA1.sup.G827C SCE-R1-51 MATa leu2 sfa1::KanMX + pESC-LEU-SFA1.sup.G849A ST609 MATa leu2 sfa1::SFA1-wt:KlLEU2 ST610 MATa leu2 sfa1::SFA1.sup.G821C:KlLEU2 ST611 MATa leu2 sfa1::SFA1.sup.G849A:KlLEU2 ST637 MATa leu2 ACC1-CaMCR:KlURA3 ACSse-ALD6:KlLEU2 PDC1:SpHIS5 ST726 MATa ura3 leu2 his3 sfa1:: SFA1.sup.G827C:KlLEU2 ST727 MATa ura3 leu2 his3 sfa1:: SFA1.sup.G849A:KlLEU2 ST728 MATa ura3 leu2 his3 sfa1:: SFA1wt:KlLEU2

(35) TABLE-US-00006 TABLE 6 Strains and 3HP titers in strains producing 3HP via malonyl-CoA pathway Strain Parent Plasmid with 3HP titer (g/L) 3HP titer (g/L) name strain LEU2 marker in Delft media in FIT media wt CEN.PK113- 0.1 0.01 0.4 0.02 7D R2-129 ST637 pESC-LEU 1.8 0.4 5.4 0.9 R2-130 ST637 pESC-LEU- 2.1 0.4 5.9 1.0 SFA1-wt R2-131 ST637 pESC-LEU- 1.8 0.4 5.7 0.5 SFA1.sup.G827C R2-132 ST637 pESC-LEU- 2.0 0.2 5.8 0.9 SFA1.sup.G849A

Example 1. Toxicity of 3-Hydroxypropionic Acid (3HP)

(36) The toxicity of 3HP on growth of S. cerevisiae CEN.PK 113-7D strain (WT) was determined by evaluating the ability of the WT strain to grow on a chemically defined minimal medium (Delft) containing varying concentrations of 3HP (0-100 g/L). The composition of Delft medium was as follows: 20.0 g/L glucose, 5.0 g/L (NH.sub.4).sub.2SO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L MgSO.sub.4.7H.sub.2O, 1.0 mL/L trace metal solution, 0.05 g/L antifoam A (10794, Sigma-Aldrich), and 1.0 mL/L vitamin solution. The trace element solution included 15 g/L EDTA, 0.45 g/L CaCl.sub.2.2H.sub.2O, 0.45 g/L ZnSO.sub.4.7H.sub.2O, 0.3 g/L FeSO.sub.4.7H.sub.2O, 100 mg/L H.sub.3BO.sub.4, 1 g/L MnCl.sub.2.2H.sub.2O, 0.3 g/L CoCl.sub.2.6H.sub.2O, 0.3 g/L CuSO.sub.4.5H.sub.2O, 0.4 g/L NaMoO.sub.4.2H.sub.2O. The pH of the trace metal solution was adjusted to 4.0 with 2 M NaOH and heat sterilized. The vitamin solution included 50 mg/L d-biotin, 200 mg/L para-amino benzoic acid, 1 g/L nicotinic acid, 1 g/L Ca.pantothenate, 1 g/L pyridoxine HCl, 1 g/L thiamine HCl, and 25 mg/L m. inositol. The pH of the vitamin solution was adjusted to 6.5 with 2 M NaOH, sterile-filtered and the solution was stored at 4 C. The Delft medium was prepared as a concentrate solution and the pH was adjusted to 6.0 and sterilized by autoclaving. Glucose was autoclaved separately. The final Delft medium (Delft buffered) was obtained by adding 79.5 mL of 0.5 M citrate solution (105 g/L C.sub.6H.sub.8O.sub.7.H.sub.2O) and 20.5 mL of 1M sodium phosphate solution (178 g/L Na.sub.2HPO.sub.4.2H.sub.2O) to control the pH of the medium at pH 3.5. For media containing 3HP, the 3HP solution (ca. 30% in water; Tokyo Chemical Industry Co.) was neutralized with solid NaOH (0.133 g NaOH/1 mL of 3HP solution) and sterile-filtered before adding to the Delft buffered media.

(37) A single colony from YPD plate (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 20 g/L agar) was inoculated in a shake flask with 20 mL YPD broth and grown at 30 C., with shaking at 200 rpm, overnight. The absorbance (OD.sub.600) of the overnight culture was measured at 600 nm wave length using NanoPhotometer UV/Vis (Implen) and the culture was diluted with YPD medium to obtain inoculum with the OD.sub.600 around 0.8. The diluted culture was used to inoculate 100 L Delft buffered medium (pH 3.5) containing various concentration of 3HP (0-100 g/L) in 96-well flat bottom plate (Greiner) with the starting inoculum of OD.sub.600=0.04. The 96-well plate was incubated at 30 C. with shaking in the Synergy MX microplate reader (BioTek) and the absorbance was measured at 600 nm wavelength every 15 min for 42 hours. Experiments were done in duplicates, and the specific growth rate (h.sup.1) was calculated at various concentrations of 3HP. The resulting of 3HP sensitivity is shown in FIG. 2.

(38) It can be seen that any concentration of 3-HP much above 10 g/L produces a substantial level of inhibition and the wild type yeast is unable to grow at all in concentrations of 75 g/L.

Example 2. Metabolic Engineering for Improving 3HP Tolerance in S. cerevisae

(39) A strain with SFA1 deletion was constructed and tested for 3HP tolerance. In addition, three versions of the SFA1 gene (native and two alleles) were overexpressed in the sfa1 and WT strains and 3HP tolerance was investigated. Deletion of SFA1 did not result in any 3HP tolerance. However, overexpression of SFA1 gene (native and two alleles) enabled growth on 50 g/L 3HP of the WT and sfa1 strains. The two alleles are referred to herein as SFA1.sup.G827C (Cys.fwdarw.Ser(aa276)) and SFA1.sup.G849A (Met.fwdarw.Ile(aa283)).

(40) The sfa1 strain was constructed by replacing the target gene in the CEN.PK113-32D strain with the KanMX cassette.

(41) The gene fragments carrying the KanMX cassette and correct overhangs for SFA1 replacement (KO-SFA1) was generated by PCR amplification using primers and template as indicated in Table 2. The PCR mix contained: 31 l water, 10 l high fidelity Phusion polymerase buffer (5), 5 l 2 mM dNTP, 1 l Phusion polymerase, 1 l forward primer at 10 M concentration, 1 l reverse primer at 10 M concentration, and 1 l DNA template. The cycling program was: 95 C. for 2 min, 30 cycles of [95 C. for 10 sec, 50 C. for 20 sec, 68 C. for 1 min], 68 C. for 5 min, pause at 10 C. The gene fragments were resolved on 1% agarose gel containing SYBR-SAFE (Invitrogen) and purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

(42) The KO-SFA1 fragment was transformed into S. cerevisiae cells using the lithium acetate transformation protocol. The cells were selected on YPD plate with G418 (200 g/mL). The transformants were streak purified on YPD+G418 plate in order to obtain single colonies. The correct transformants were confirmed by PCR analysis using primers KO_sfa1_test_fw/KanMX_2/3_START_rv.

(43) To construct the overexpression plasmid containing SFA1 gene, the SFA1-wt (SEQ ID NO 1), SFA1.sup.G827C (SEQ ID NO 2), SFA1.sup.G849A (SEQ ID NO 3) genes were subcloned into pESC-LEU-USER plasmid by USER cloning. The final plasmids (Table 4) were then transformed into different background strains (WT and sfa1) using the lithium acetate transformation protocol and selected on synthetic complete agar medium without leucine (SC-Leu). Transformants were streaked purified on SC-Leu to obtain single colonies. The resulting strains are listed in Table 5.

(44) The promoter fragment (PTEF1, SEQ ID 4) was generated by PCR followed by gene purification (Table 3). The terminators were already present on the expression plasmids.

(45) The parent plasmid pESC-LEU-USER were linearized with FastDigest AsiSI (Fermentas) for 1 hour at 37 C. and nicked with Nb.BsmI for 1 hour at 37 C. The resulting linearized nicked DNA was purified from the solution and eluted in 5 mM Tris buffer, pH 8.0.

(46) The expression plasmids were created by USER-cloning using the following protocol. One l of linearized and nicked parent plasmid was mixed with 1 L of promoter fragment, 2 L of gene fragment, 0.5 L Taq polymerase buffer, 0.5 L USER enzyme (NEB). The mix was incubated at 37 C. for 25 min, at 25 C. for 25 min and transformed into chemically competent E. coli DH5alpha. The clones with correct inserts were identified by colony PCR and the plasmids were isolated from overnight E. coli cultures and confirmed by sequencing. The expression plasmids are listed in Table 4. For testing 3HP tolerance phenotype, four single colonies from each strain line were investigated for the ability to grow on media containing 3HP. The pre-cultures were prepared by inoculation a single colony in 100 L SC-Leu media in 96-well flat bottom plate. The plate was incubated at 30 C. with 250 rpm agitation at 5 cm orbit cast overnight. Five L of the overnight cultures were used to inoculate 100 L Delft buffered (pH 3.5) containing 50 g/L 3HP. The 96-well plate was incubated at 30 C. with shaking in the Synergy MX microplate reader (BioTek) and the absorbance was measured at 600 nm wavelength every 15 min for 42 hours. Experiments were done in triplicate. The resulting improvement in 3HP tolerance in the engineered strains is shown in FIG. 3.

(47) In FIG. 3, SCE-R1-48 is the sfa1 strain carrying the empty plasmid pESC-LEU. SCE-R1-49, SCE-R1-50, and SCE-R1-51 are the strains overexpressing SFA1 in its wild type form, SFA1.sup.G827C, and SFA1.sup.G849A, respectively. As seen in FIG. 3, all three strains overexpressing a form of SFA1 have improved 3HP tolerance compared to the sfa1 strain.

Example 3. Improving 3HP Tolerance in S. cerevisiae by Replacing the Native SFA1 with Either SFA1G827C or SFA1G849A

(48) To investigate whether only the SFA1 alleles can confer 3HP tolerance without overexpression, the native SFA1 gene was replaced by either SFA1.sup.G827C or SFA1.sup.G849A and tested for 3HP tolerance. By replacing the SFA1-wt with SFA1.sup.G827C or SFA1.sup.G849A, the constructed strains (ST610 and ST611) could grow on minimum media containing 3HP above 40 g/L, whereas the strain with the SFA1-wt allele without over expression (ST609) could not grow under these conditions. The results clearly showed that only one amino acid changed in SFA1 is enough for S. cerevisiae to confer 3HP tolerance.

(49) To generate the substrates for SFA1 allele replacement, the upstream fragment including the SFA1 allele for each SFA1 mutations was generated by fusion PCR using the SFA1 upstream region (SFA1-UP), SFA1-allele fragment and the first part of KlLEU2 marker as templates and using primers KO_sfa1_fw and NB335KlLEURev1 (Table 2). The downstream fragment was generated by fusion PCR using the last part of KlLEU2 marker and the SFA1 downstream region (SFA1_DOWN) as templates and using primers NB336KlLEUFwd1 and SFA1_DW_test_rv. The cycling program was: 98 C. for 2 min, 30 cycles of [98 C. for 10 sec, 55 C. for 30 sec, 68 C. for 1 min 30 sec], 68 C. for 12 min, pause at 10 C.

(50) The strain with SFA1.sup.G827C allele replacement was constructed by replacing the KanMX cassette in the sfa1 strain using SFA1.sup.G827C_UP_LEU2_U_2/3_START and LEU2_U_2/3_END_SFA1_DOWN_wt fragments, whereas the SFA1.sup.G849A strain was constructed in the same manner using SFA1.sup.G849A_UP_LEU2_U_2/3_START and LEU2_U_2/3_END_SFA1_DOWN_wt fragments. For the control strain, the SFA1-wt allele was introduced back to the sfa1 strain by using SFA1 wt_UP_LEU2_U_2/3_START and LEU2_U_2/3_END_SFA1_DOWN_wt fragments. The strains were selected on SC-Leu media. The transformants were streaked purified on SC-Leu to obtain single colonies. The correct transformants were confirmed by PCR analysis using primers KO_sfa1_test_fw and SFA1_U1-rv.

(51) For testing 3HP tolerance phenotype, two single colonies from each strain: ST609 (SFA1-wt), ST610 (SFA1.sup.G827C) and ST611 (SFA1.sup.G849A) were investigated for the ability to grow on media containing 3HP. The pre-cultures were prepared by inoculation of a single colony in 0.5 mL Delft buffered (pH 3.5) media in 24-well plate. The plate was incubated at 30 C. with 250 rpm agitation at 5 cm orbit cast overnight. Five L of the overnight cultures were used to inoculate 100 L Delft buffered (pH 3.5) containing various concentration of 3HP (0, 10, 25, 40 and 50 g/L) in 96-well flat bottom plate. The 96-well plate was incubated at 30 C. with shaking in the Synergy MX microplate reader (BioTek) and the absorbance was measured at 600 nm wavelength every 15 min for 42 hours. Experiments were done in duplicates. The resulting of 3HP tolerance in the engineered strains is shown in FIG. 4.

Example 4. Production of 3-Hydroxypropionic Acid Via Malonyl-CoA Pathway

(52) To test the influence of 3HP tolerance gene on 3HP production, three versions of the SFA1 gene (native and two alleles) were overexpressed in the 3HP high producer (ST637) strain and characterized for 3HP production. The resulting strains and 3HP production are shown in Table 6 and FIG. 5. The 3HP titers and yields were similar to the or better than the reference strain (R2-129), showing that they were compatible and even advantageous for the production of 3HP via malonyl-CoA pathway.

(53) The S. cerevisiae-3HP high producer strain has been engineered to carry several genetic modifications. Extra copies of the native acetyl-CoA synthetase (ACSse; SEQ ID NO 6), aldehyde dehydrogenase (ALD6; SEQ ID NO 7), indolepyruvate decarboxylase (PDC1; SEQ ID NO 8) and acetyl-CoA carboxylase (ACC1; SEQ ID NO 9) were introduced into the CEN.PK102-5B strain to improve the supply of precursor and redox co-factor. These genes were integrated into the genome and were under the control of either PTEF1 (SEQ ID NO 4) or PPGK1 (SEQ ID NO 5) promoters. Furthermore, the malonyl-CoA reductase gene from Chloroflexus aurantiacus (CaMCR; SEQ ID NO 10) responsible for converting malonyl-CoA into 3HP was also introduced into this strain.

(54) The plasmids conferring 3HP tolerance were transformed into 3HP high producer strain the lithium acetate transformation protocol. The cells were selected on SC-Leu. For the control experiments, the strains were transformed with an empty plasmid pESC-LEU.

(55) Six single colonies originating from four independent transformants were inoculated in 0.5 mL SC-Ura-His-Leu in 96-deep well microtiter plate with air-penetrable lid (EnzyScreen). The plates were incubated at 30 C. with 250 rpm agitation at 5 cm orbit cast overnight. 50 L of the overnight cultures were used to inoculate 0.5 mL Delft or Feed-In-Time (FIT) Fed-batch medium (m2p-labs GmBH) in 96-deep well plate. Cultivation was carried out for 72 hours at the same conditions as above.

(56) At the end of the cultivation the OD.sub.600 was measured. The cultivation was diluted 20 times in a total volume of 200 L and absorbance was measured at 600 nm wavelength on spectrophotometer (Synergy MX Microplate reader, BioTek).

(57) The culture broth was spun down and the supernatant analyzed for 3HP concentration using enzymatic assay (Table 5). Enzymatic assay was carried out as following. 20 L of standards (3HP at concentrations from 0.03 to 2 g/L in Delft medium) and samples were added to 96-well flat bottom transparent plate (Greiner). 180 l of mix (14.8 mL water, 2 mL buffer (1 mM Tris, 25 mM MgCl.sub.2, pH 8.8), 1 mL NADP+ solution (50 mg/mL), and 0.2 mL purified YdfG enzyme in PBS buffer (1500 g/mL)) was added per well using multichannel pipet. The start absorbance at 340 nm was measured; the plate was sealed and incubated at 30 C. for 1.5 hours. After that the end absorbance at 340 nm was measured again. The difference between the end and the start values corrected for the background were in linear correlation with 3HP concentrations. The concentration of 3HP in the samples was calculated from the standard curve.

Example 5. The Proposed 3HP Detoxification Pathway

(58) The proposed function of SFA1 protein (Sfa1p) in formaldehyde detoxification pathway has been reported (Yasokawa et al., 2010). Formaldehyde is spontaneously reacted with intracellular glutathione (GSH) to form S-hydroxymethylglutathione which is then converted into S-formylglutathione by the Sfa1p and required NAD(P).sup.+ as a cofactor. We proposed that 3HP detoxification by the SFA1p might be similar to that observed in formaldehyde detoxification and GSH might also be involved in this process. The proposed 3HP detoxification pathway is shown in FIG. 6.

(59) In vivo S. cerevisiae strain can convert 3HP into 3-hydroxypropionaldehyde (3HPA) by the aldehyde dehydrogenases (ALDs). As 3HPA is much more toxic than 3HP, yeast cells must efficiently eliminate this lethal compound by converting it into other less toxic compounds. 3HPA can spontaneously bind to glutathione to form S-(3-hydroxypropanoyl)glutathione which is then oxidized into S-(3-ketopropanoyl)glutathione by the Sfa1p and used NAD(P).sup.+ as a cofactor. Finally, the intermediate compound is hydrolyzed back into 3HP and released glutathione by the S-formylglutathione hydrolase encoded by Yjl068C. The proposed glutathione-dependent cyclic mechanism of 3HP detoxification has provided new insights into molecular response to 3HP in S. cerevisiae.

Example 6. The Role of GSH in 3HP Detoxification Pathway

(60) The role of GSH in 3HP detoxification was investigated. The WT, SFA1-allles, and sfa1 strains were grown in the minimal medium with or without 50 g/L 3HP and supplemented with various concentration of external GSH (0-30 mM) and tested for 3HP tolerance. As seen in FIG. 7, GSH addition (2.5 mM) restored the growth of the WT S. cerevisiae in the presence of 50 g/L 3HP up to the level slightly higher than that of the SFA1-alleles strains grown without GSH supplement. Furthermore, GSH addition has also improved the growth rate of the strain carrying the SFA1 alleles. The growth of the sfa1 strain could not be restored by addition of GSH, further supporting the proposed role of GSH in 3HP detoxification. In addition, GSH has no significant effect on all strains grown in the minimal medium without 50 g/L 3HP.

(61) For testing 3HP tolerance phenotype, the pre-cultures were prepared by inoculation of a single colony in 1 mL Delft buffered (pH 3.5) media containing 10 g/L 3HP. The cultures were incubated at 30 C. with 250 rpm overnight. Five L of the overnight cultures were used to inoculate 100 L Delft buffered (pH 3.5) with or without 50 g/L 3HP and various concentration of GSH (0, 1, 2.5, 5, 10, and 20 mM) in 96-well flat bottom plate. The 96-well plate was incubated at 30 C. with shaking in the Synergy MX microplate reader (BioTek) and the absorbance was measured at 600 nm wavelength every 15 min for 42 hours. Experiments were done in triplicates.

Example 7. Enhanced 3HP Tolerance in S. cerevisiae by Improving Glutathione Production (Prophetic)

(62) From the previous example, addition of GSH enabled the viability the WT strain and improved the growth of the SFA1 allele's strains. However, the GSH addition is not economically suitable for industrial production of 3HP. Therefore, increasing intracellular pool of GSH by overexpression of genes involved in glutathione biosynthesis would be an alternative approach to reduce production cost.

(63) In S. cerevisiae, GSH is synthesized by two consecutive ATP-dependent reactions. The -glutamyl-cysteine (-GC) synthetase (GCS, EC 6.3.2.2) encoded by GSH1 catalyzes the conjugation of L-glutamic acid and L-cysteine into -GC. The glutathione synthetase (GS, EC 6.3.2.3) encoded by GSH2 (SEQ ID 12) catalyzes the synthesis of GSH from -GC and glycine (Grant et al., 1997).

(64) As the glutathione production requires three amino acid substrates (L-cysteine, L-glutamic acid and glycine), increasing the intracellular concentration of the substrates e.g. L-cysteine should improve the glutathione productivity. In this study, two genes in the cysteine biosynthetic pathway, MET14 and MET16, were overexpressed in combination with GSH1 and GSH2 genes and the constructed strains were tested for the effect of excess intracellular GSH concentration on 3HP tolerance.

(65) To construct the expression vector for glutathione production, the fragments carrying the GSH1 (SEQ ID NO 11) and GSH2 (SEQ ID NO 12) genes, and the PTEF1-PPGK1 double promoter fragment (SEQ ID NO 13) containing the correct overhangs for USER cloning were generated by PCR amplification using primers and templates as indicated in Table 2. The three fragments were then ligated into pESC-URA-USER by USER cloning as previously described in example 3 to generate pESC-URA-GSH1-GSH2 plasmid.

(66) To construct the expression vector for improving cysteine production, the fragments carrying the MET14 (SEQ ID NO14) and MET16 (SEQ ID NO15) genes containing the correct overhangs for USER cloning were generated by PCR amplification using primers and templates as indicated in Table 2. The final plasmid pESC-HIS-MET14-MET16 was constructed by ligation of MET14, MET16 and PTEF1-PPGK1 fragments into pESC-HIS-USER by USER cloning as previously described in Example 3.

(67) The pESC-URA-GSH1-GSH2 plasmid was co-transformed with either pESC-HIS or pESC-HIS-MET14-MET16 plasmids into different background strains (ST726, ST727, and ST728) using the lithium acetate transformation protocol. The cells were selected on SC-Ura-His-Leu media. The transformants were streak purified on SC-Ura-His-Leu plate in order to obtain single colonies.

(68) The resulting strains were tested for 3HP tolerance phenotype as described in Example 2.

Example 8. Identification of Point Mutations in the Sfa1p Protein that Confers 3HP Tolerance Phenotype

(69) The two mutations at Cys276 and Met283 residues in the Sfa1p (previously mentioned in Example 2) were investigated whether substitution of these amino acid residues with other amino acids apart from serine (Ser) and isoleucine (Ile), respectively, will also result in 3HP tolerance phenotype. Furthermore, the cysteine residues (Cys205, Cys237, and Cys290) in the Sfa1p were also selected for site-directed mutatgenesis and tested whether mutation in these residues will also improve 3HP tolerance in S. cerevisiae. Initial results have not shown improvement by mutation at these sites. Thus, substitution of amino acids Cys205, Cys237 and Cys290 in the Sfa1p by Ser205, Ser237, and Ser290, respectively, did not result in 3HP tolerance phenotype in S. cerevisiae.

(70) Substitution of the Cys276 residue with other amino acids e.g. threonine (Thr), glycine (Gly), valine (Val) and alanine (Ala) also gives 3HP tolerance phenotype in S. cerevisiae. In addition, the constructed strains where the Met283 residue in the Sfa1p was replaced with either Val or Ala also were also able to grow in the presence of 50 g/L 3HP. The growth of the resulting strains on minimal medium containing 50 g/L 3HP was shown in FIG. 8.

(71) The PCR site-directed mutagenesis was performed to replace the Cys276 residue with either Gly, Ala, Val or Thr using primers and templates as listed in Table 2. The Met283 was changed into either Val or Ala, whereas the Cys205, Cy237 and Cys290 residues were also substituted with Ser residue (Table 2). The PCR mix contained: 30 l water, 10 l high fidelity Phusion polymerase buffer (5), 2.5 l 10 mM dNTP, 1 l Phusion polymerase, 1.5 l forward primer at 10 M concentration, 1.5 l reverse primer at 10 M concentration, 1.5 l DNA template (200 ng), and 1.5 l DMSO (100%). The cycling program was: 98 C. for 2 min, 18 cycles of [98 C. for 10 sec, 55 C. for 30 sec, 68 C. for 9 min], 68 C. for 12 min, pause at 10 C. The PCR reaction was treated with 1 l DpnI enzyme and incubated at 37 C. for 1 hour to remove the plasmid template. The treated PCR samples were transformed into chemically competent E. coli DH5alpha and selected on LB containing 100 g/mL ampicillin. The plasmids were isolated from each E. coli transformants and the correct mutations in the SFA1 gene were confirmed by sequencing using primer SFA1_U1_rv.

(72) To generate the substrates for SFA1 allele replacement, the upstream fragment for each SFA1 mutations was generated by fusion PCR using the SFA1-UP fragment, SFA1-allele fragment and the first 2/3 KlLEU2 fragment as templates and using primers KO_sfa1_fw and NB335KlLEURev1 (Table 2).

(73) The strains carrying the point mutation in the SFA1 gene were constructed by replacing the KanMX cassette in the sfa1 strain with the corresponding upstream fragment (Table 2) and downstream fragment (LEU2_U_2/3_END_SFA1_DOWN fragments) of the SFA1 gene as previously mentioned in Example 3. The strains were selected on SC-Leu media. The transformants were streaked purified on SC-Leu to obtain single colonies. The correct transformants were confirmed by PCR analysis using primers KO_sfa1_test_fw and SFA1_U1-rv and the mutation in the SFA1 gene was verified by sequencing using primer SFA1_U1_rv.

(74) The constructed strains were tested for 3HP tolerance phenotype as described in Example 3.

Example 9. Improving 3HP Tolerance in Other Cells

(75) Based on the above examples, 3HP tolerance in S. cerevisiae is improved by increasing the availability of intracellular GSH either by addition of external GSH or improving GSH production. This example demonstrates that the GSH addition also helps to improve 3HP tolerance on other microorganisms e.g. E. coli and several yeast strains (Schizosaccharomyces pombe, Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Cyberlindnera jadinii, Torulaspora delbrueckii, Rhodotorula minuta).

(76) As seen in FIG. 9, addition of 5 mM GSH restored the viability of several yeast strains i.e. S. cerevisiae, S. kluyveri and K. lactis grown in the presence of 50 g/L 3HP. For some yeast strains e.g. K. marxianus, Y. lipolytica, C. jadinii, S. pombe, T. delbrueckii and Rhodotorula minuta, these strains could tolerate more than 50 g/L 3HP with decreasing in the specific growth rate compared to the cells grown without the presence of 50 g/L 3HP. However, the specific growth rate of these strains grown in the presence of 50 g/L 3HP was improved by the addition of 5 mM GSH in the cultivation medium.

(77) In FIG. 10, addition of 5 mM GSH also restored the viability of four E. coli strains i.e. W3110, CROOKS, W and BL21 grown in M9 minimal medium supplemented with 20 g/L 3HP. For MG1655, the strain could grow in M9 medium containing 20 g/L 3HP with 2.2-fold decrease in the specific growth rate compared to the cells grown in M9 medium. However, the specific growth rate of MG1655 strain was improved by the addition of 5 mM GSH in the cultivation medium containing 20 g/L 3HP.

(78) The results strongly supported that the proposed role of GSH in 3HP detoxification mechanism are similar in several microorganisms.

(79) The yeast strains were grown in 3 mL Delft medium (pH 6.0) at 250 rpm, 25 C., overnight. Three L of the overnight cultures were used to inoculate 100 L Delft medium (pH 6.0) with or without 50 g/L 3HP in 96-well flat bottom plate. The 96-well plate was incubated with shaking in the Synergy MX microplate reader (BioTek) at 25 C. and the absorbance was measured at 600 nm wavelength every 15 min for 72 hours. Experiments were done in triplicates, and the specific growth rate (h.sup.1) was calculated. The effect of GSH addition was performed by adding 5 mM GSH into the cultivation medium containing 50 g/L 3HP. The growth of each strain was determined as mentioned above.

(80) For E. coli, the effect of GSH addition on the growth of E. coli in the presence of 20 g/L 3HP was investigated. Five platform E. coli wild-type strains e.g. W3110, CROOKS, W, BL21 (DE3) and MG1655 were selected for 3HP tolerance experiment. Single colony from each strain was inoculated into 3 mL M9 medium and incubated at 250 rpm, 37 C., overnight. Two L of the overnight cultures were used to inoculate 150 L M9 medium, M9 with 20 g/L 3HP, and M9 with 20 g/L 3HP and 5 mM GSH in 96-well flat bottom plate. The pH of all E. coli tested media was at pH 7.0. The 96-well plate was incubated with shaking in the ELx808 Absorbance microplate reader (BioTek) at 37 C. and the absorbance was measured at 630 nm wavelength every 5 min for 22 hours. Experiments were done in triplicates, and the specific growth rate (h.sup.1) was calculated.

(81) The composition of M9 medium was as follows: 2.0 g/L glucose, 6.8 g/L Na HPO.sub.4, 3.0 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 1.0 g/L NH.sub.4Cl, 0.24 g/L MgSO.sub.4, 0.011 g/L CaCl.sub.2, 0.5 mL/L trace elements solution, and 1.0 mL/L Wolfe's Vitamin solution. The trace elements solution included 1.0 g/L FeCl.sub.3.6H.sub.2O, 0.18 g/L ZnSo.sub.4.7H.sub.2O, 0.12 g/L CuCl.sub.2.2H.sub.2O, 0.12 g/L MnSO.sub.4.H.sub.2O, and 0.18 g/L CoCl.sub.2.6H.sub.2O. The Wolfe's vitamin solution included 10 mg/L pyridoxine hydrochloride, 5.0 mg/L thiamine HCl, 5.0 mg/L riboflavin, 5.0 mg/L nicotinic acid, 5.0 mg/L calcium-(+) phantothenate, 5.0 mg/L para-amino benzoic acid, 5.0 mg/L thiotic acid, 2.0 mg/L d-biotin, 2.0 mg/L folic acid, and 0.1 mg/L vitamin B12. The trace elements and vitamin solutions were sterile-filtered and the solution was stored at 4 C. The M9 salt solution was prepared as a 10 concentrate solution and sterilized by autoclaving. 20% Glucose solution, 1M MgSO.sub.4, and 1M CaCl.sub.2 stock solutions were autoclaved separately.

REFERENCES

(82) Erdeniz, N., Mortensen, U. H., and Rothstein, R. Cloning-free PCR-based allele replacement methods. (1997) Genome Res 7: 1174-1183. Fernndez, M. R., Biosca, J. A., Torres, D., Crosas, B., and Pars, X. A double residue substitution in the coenzyme-binding site accounts for the different kinetic properties between yeast and human formaldehyde dehydrogenases. (1999) J Biol Chem 274(53): 37869-37875. Gietz, R. D. and Woods, R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. (2002) Methods Enzymol 350:87-96 Grant, C. M., MacIver, F. H., and Dawes, I. W. Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cervisiae due to an accumulation of the dipeptide gamma-glutamylcysteine. (1997) Mol Biol Cell 8:1699-1707. Hgler, M., Meenedez, C, Schgger, H., and Fuchs, G. Malonyl-Coenzyme A Reductase from Chloroflexus Aurantiacus, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO2 Fixation. (2002) J Bacteriol 184(9):2404-2410. Kwak S., Park, Y. C, and Seo, J. H. Biosynthesis of 3-hydroxypropionic Acid from Glycerol in Recombinant Escherichia coli Expressing Lactobacillus brevis dhaB and dhaR Gene Clusters and E. coli K-12 aldH. (2013) Bioresour Technol 135: 432-439. Luo, L. H., Kim, C. H., Heo, S. Y., Oh, B. R., Hong, W. K., Kin, S., Kim, D. H. and Seo, J. W. Production of 3-hydroxypropionic Acid Through Propionaldehyde Dehydrogenase PduP Mediated Biosynthetic Pathway in Klebsiella pneumoniae. (2012) Bioresour Technol 103(1): 1-6. Luo, L. H., Seo, J. W., Oh, B. R., Seo, P. S., Heo, S. Y., Hong, W. K., Kim, D. H. and Kim, C. H. Stimulation of Reductive Glycerol Metabolism by Overexpression of an Aldehyde Dehydrogenase in a Recombinant Klebsiella pneumoniae Strain Defective in the Oxidative Pathway. (2011). J Ind Microbiol Biotechnol 38(8): 991-999. Molin and Blomberg, Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation. (2006) Mol Microbiol 60(4), 925-938. Rathnasingh, C., Raj, S. M., Jo, J. E., and Park, S. Development and Evaluation of Efficient Recombinant Escherichia coli Strains for the Production of 3-hydroxypropionic Acid from Glycerol. (2009) Biotechnol Bioeng 104(4): 729-739. Wen, S., Zhang, T. and Tan, T. Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae. (2004) Enzyme Microbial Tech 35: 501-507.

(83) In this specification, unless expressly otherwise indicated, the word or is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator exclusive or which requires that only one of the conditions is met. The word comprising is used in the sense of including rather than in to mean consisting of. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

(84) The invention may be summarised according to the following clauses: 1. A cell having a metabolic pathway producing 3-hydroxypropionic acid (3HP), said cell exhibiting tolerance for 3HP and having one or more genetic modifications that provide for an enhanced activity of 3HP detoxification by a reaction pathway that includes a glutathione-dependent dehydrogenase reaction. 2. A cell as defined in clause 1, wherein a said genetic modification is one or more mutations in a gene encoding a glutathione-dependent formaldehyde dehydrogenase conferring said tolerance. 3. A cell as defined in clause 2, wherein said one or more mutations are in a gene equivalent to SFA1 of Saccharomyces cerevisiae. 4. A cell as defined in clause 3, wherein said one or more mutations are in a gene encoding a protein having the sequence SEQ ID NO 1 or a protein with more than 80% homology to SEQ ID NO 1. 5. A cell as defined in any one of clauses 2 to 4, wherein the mutation is at a position equivalent to the position aa276 Cys and/or at aa283 Met of the SFA1 gene of Saccharomyces cerevisiae. 6. A cell as defined in clause 5, wherein said mutation in gene SFA1 is Cys276.fwdarw.Ser, Cys276.fwdarw.Val, Cys276.fwdarw.Thr, Cys276.fwdarw.Gly, Cys276.fwdarw.Ala and/or Met283.fwdarw.Ile, Met283.fwdarw.Ala, Met283.fwdarw.Val. 7. A cell as defined in any preceding clause, wherein a said genetic modification produces overexpression of a native, or heterologous, or mutated glutathione-dependent formaldehyde dehydrogenase to confer said 3HP tolerance. 8. A cell as defined in clause 7, wherein said genetic modification produces overexpression of a native or heterologous glutathione-dependent formaldehyde dehydrogenase which has the sequence SEQ ID NO 1 or is a protein with more than 80% homology to SEQ ID NO 1. 9. A cell as defined in any preceding clause, wherein the cell is genetically modified for increased production of glutathione. 10. A cell as defined in clause 9, wherein the cell overexpresses the glutathione biosynthetic genes gamma-glutamylcysteine synthetase and glutathione synthetase. 11. A cell as defined in clause 9 or clause 10, wherein the cell overexpresses genes that enhance the production of amino acid precursors for glutathione biosynthesis. 12. A cell as defined in any preceding clause, wherein said metabolic pathway comprises the enzyme malonyl-CoA reductase and/or the enzyme malonyl-CoA reductase (malonate semialdehyde-forming) in combination with the enzyme 3-hydroxyisobutyrate dehydrogenase and/or the enzyme hydroxypropionate dehydrogenase. 13. A cell as defined in any one of clauses 1 to 11, wherein said metabolic pathway comprises a malonyl-CoA reductase gene and an acetyl-CoA carboxylase gene. 14. A cell as defined in clause 13, wherein said metabolic pathway comprises the malonyl-CoA reductase gene from Chloroflexus aurantiacus (CaMCR). 15. A cell as defined in clause 14, wherein said metabolic pathway comprises the malonyl-CoA reductase gene from Chloroflexus aurantiacus (CaMCR) and the acetyl-CoA carboxylase gene (ACC1) of S. cerevisiae. 16. A cell as defined in any one of clauses 1 to 11, wherein said metabolic pathway comprises beta-alanine pyruvate aminotransferase and/or gamma-aminobutyrate transaminase in combination with hydroxyisobutyrate dehydrogenase and/or hydroxypropionate dehydrogenase. 17. A cell as defined in clause 16, wherein said metabolic pathway comprises the beta-alanine pyruvate aminotransferase from Bacillus cereus together with one of the following: 3-hydroxypropanoate dehydrogenase from Metallosphaera sedula, 3-hydroxypropanoate dehydrogenase from Sulfolobus tokadaii, 3-hydroxypropanoate dehydrogenase from E. coli (YdfGp), 3-hydroxypropanoate dehydrogenase from E. coli (RutEp), 3-hydroxyisobutyrate dehydrogenase from Pseudomonas aeruginosa, 3-hydroxyisobutyrate dehydrogenase from P. putida, 3-hydroxyisobutyrate dehydrogenase from Bacillus cereus, or 3-hydroxyisobutyrate dehydrogenase from Candida albicans. 18. A cell as defined in any one of clauses 1 to 11, wherein said metabolic pathway comprises glycerol dehydratase and alcohol dehydrogenase. 19. A cell as defined in any one of clauses 1 to 11, wherein said metabolic pathway comprises lactate dehydrogenase, propionate CoA-transferase, lactoyl-CoA dehydratase, enoyl-CoA hydratase and 3-hydroxyisobutyryl-CoA hydrolase. 20. A cell as defined in any preceding clause, wherein said cell is a yeast cell. 21. A cell as defined in clause 18, wherein the yeast cell is of the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Candida, Yarrowia, Brettanomyces, Hansenula, Lipomyces, and Issatchenkia. 22. A cell as defined in clause 18, wherein the yeast cell is a Saccharomyces cerevisiae cell. 23. A cell as defined in clause 22, wherein said genetic modification comprises one or more mutations in gene SFA1 at aa276 Cys and/or at aa283 Met. 24. A cell as defined in any one of clauses 1 to 19, wherein said cell is a bacterial cell. 25. A cell as defined in any one of clauses 1 to 19, wherein said bacterial cell is of the genus Eschericia, Lactobacillus, Lactococcus, Corynebacterium, Clostridium, or Bacillus. 26. A cell as defined in any one of clauses 20 to 25, wherein the cell is S. cerevisiae, S. pombe, S. kluyveri, K. lactis, K. marxianus, Y. lipolytica, T. delbreueckii, R. minuta, I. orientalis, P. stipites, L. starkeyi, C. guilliermondii, or E. coli. 27. A yeast exhibiting tolerance for 3HP, said yeast having a mutation in gene SFA1 conferring said tolerance. 28. A yeast as defined in clause 27, wherein the yeast is Saccharomyces cerevisiae and said mutation in gene SFA1 is at aa276 Cys and/or at aa283 Met. 29. A yeast as defined in clause 28, wherein said mutation in gene SFA1 is Cys276.fwdarw.Ser, Cys276.fwdarw.Val, Cys276.fwdarw.Thr, Cys276.fwdarw.Gly, Cys276.fwdarw.Ala and/or Met276.fwdarw.Ile, Met276.fwdarw.Ala, Met276.fwdarw.Val 30. A yeast as defined in clause 29, wherein said mutation in gene SFA1 is Cys.fwdarw.Ser(aa276) and/or Met.fwdarw.Ile(aa283). 31. A method of producing 3HP comprising cultivating a 3HP producing cell under 3HP producing conditions in a culture medium so as to produce 3HP, wherein toxicity of 3HP is reduced by an enhanced activity of 3HP detoxification by a reaction pathway that includes a glutathione-dependent dehydrogenase reaction. 32. A method as defined in clause 31, wherein said culture medium is supplemented with glutathione. 33. A method as defined in clause 32, wherein glutathione is added to the culture medium to produce a concentration therein of >2.5 mM. 34. A method as defined in clause 31 or clause 32, wherein said cell has a genetic modification conferring an enhanced glutathione production ability thereon. 35. A method as defined in clause 34, wherein the cell overexpresses the glutathione biosynthetic genes gamma-glutamylcysteine synthetase and glutathione synthetase. 36. A method as defined in clause 34 or clause 35, wherein the cell overexpresses genes that enhance the production of amino acid precursors for glutathione biosynthesis. 37. A method as defined in any one of clauses 31 to 36, wherein a concentration of 3HP in excess of 1 g/l is produced in said culture medium. 38. The use of an enhanced glutathione-dependent dehydrogenase reaction in a cell to enhance tolerance of said cell to 3HP. 39. A use as defined in clause 38, wherein said reaction is one converting S-(3-hydroxypropanoyl)glutathione to S-(3-ketopropanoyl)glutathione.