3-Hydroxypropionic acid-producing recombinant yeast and process for producing 3-Hydroxypropionic acid using the same

Abstract

Recombinant yeast Saccharomyces cerevisiae comprising an active metabolic pathway for the biosynthesis of 3-HP according to [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP], wherein the yeast comprises: an exogenous gene encoding AADH, with an amino acid sequence selected from SEQ ID Nos: 145-176; an endo- or exogenous gene encoding ACC, with an amino acid sequence selected from SEQ ID Nos: 99-106; an exogenous gene encoding MCR. with an amino acid sequence selected from SEQ ID Nos: 192-207 and an exogenous gene encoding HPDH, with an amino acid sequence selected from SEQ ID Nos: 127-130, 132-133, 137, 140, 142 and 208-275.

Description

Technical FieldThe present invention relates to a 3-hydroxypropionic acid (3-HP)-producing recombinant yeast and a method for producing 3-HP using the same, and more particularly relates to a 3-HP-producing recombinant yeast comprising: an exogenous gene encoding AADH; an endo- or exogenous gene encoding ACC, an exogenous gene encoding MCR; and an exogenous gene encoding HPDH, and producing 3-HP via the biosynthetic pathway [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP] and a method using the same.General State of the Art3-HP (3-hydroxypropionic acid, C3) is an isomer of lactic acid (2-hydroxypropionic acid), and has a carboxylic acid group and a hydroxyl group at both ends thereof, and is thereby a useful material that can be converted into various chemical substances such as 1,3-propanediol, acrylic acid, acrylamide, a polymer, and the like. For the aforementioned reasons, 3-HP was even selected by the U.S. Department of Energy 2004 as one of the promising chemical substances that can be produced from biomass. In particular, acrylic acid as a predominantly used form of 3-HP could be a particularly marketable material used in a coating material, an adhesive, an additive, a diaper or the like. Theoretically, 3-HP can be produced from various biomass such as glucose by fermentation at a yield of 100%, and a fermentation process using microorganisms is suitable for satisfying the need for an environmentally friendly and renewable material.Heretofore, there is no case of commercial production of 3-HP using biomass, but research has been carried out on various processes, with the safety of an economical 3-HP producing strain having been found to be the major obstacle. Bacteria as representative microorganisms which produce organic acids and are widely used in industry such as food industry or the like are known. However, there are disadvantages in employing the production of organic acids using industrial bacteria such as E. colion an industrial scale in the chemical industry such as 3-HP production. As the amount of organic acid production is increased, the hydrogenated form of the acid increases and acidity is increased (pH decreases), thereby decreasing the activity of most of the E. coli. In the case of preparing organic acid at high concentration, the bacteria require a base such as sodium hydroxide (NaOH) and ammonium hydroxide (NH 4 OH) to maintain a neutral pH. This causes an increase in cost of the fermentation process depending on the injected base, and makes an extraction and separation process difficult or significantly increases the cost.In a recent case of employing organic acid production in the chemical industry, an organic acid is produced from glucose using yeast. Compared with bacteria, yeast has high resistance to an organic acid, so that the activity of the yeast is not significantly lowered even at high acidity (low pH), making the yeast a more suitable host for producing the organic acid. In particular, yeast has conventionally been widely used as an industrial bio-catalyst for producing sorbitol, technical alcohol or the like, and can be cultured in mass culture and cannot be contaminated with bacteriophage, so that the utility of yeast in the chemical industry is far superior to bacteria. Yeast has advantages in the production of an organic acid, but there are several disadvantages also in the use of the yeast for the production of an organic acid such as 3-HP. First, it is difficult to genetically modify yeast as compared to bacteria, and to express a specific metabolic enzyme, the subcellular locus for expression of the metabolic pathway would have to be identified together with the specific metabolic enzyme, which, unlike bacteria, is due to the form of cells divided into intracellular structural bodies such as mitochondria, peroxisomes or the like. For example, a representative metabolic intermediate, such as acetyl-CoA, is produced in yeasts mainly in the mitochondria. However, when a target product is produced in the cytosol, a method for producing acetyl-CoA in the cytosol is also required. Moreover, since there are a large number of different yeasts in the field of fungi and not all yeasts satisfy the requirements of high productivity, resistance to organic acids and mass culture, a host suitable for producing the organic acids is effectively selected.It is known that some yeasts efficiently produce ethanol from glucose which is hexose, and some yeast species also produce an organic acid. At the time of modification in producing a target product using microorganisms, it is important to maintain an overall balance of oxidation and reduction in a metabolic reaction, and suitably a metabolic reaction of fermenting ethanol from gusose is also a maintained reaction. Even in the case of genetically modifying yeast to produce an organic acid, the above-described balance should be suitably maintained, while in a case of producing lactic acid by modification of yeast, the balanced introduction of lactic acid dehydrogenase (LDH) of another reduction reaction for complementing a reduction reaction for producing ethanol from acetaldehyde is important.It is known that a small amount of 3-HP is produced in a small number of microorganisms such as Chloroflexus aurantiacus, and 3-HP is partially formed from a decomposition process of dimethyl sulfopropionate in microorganisms such as Alcaligenes faecalisor a decomposition process of uracil in yeast. By discovering the naturally present 3-HP as described above, research has been conducted on 3-HP metabolic pathways and corresponding enzymes, and based on the research, recently carried out on a technology for producing 3-HP or a PHA which is polymer of 3-HP by introducing a gene required for 3-HP biosynthesis into E. coli. Moreover, in order to maximize productivity and production yield of 3-HP, which is present only as a metabolic intermediate or produced only in small amounts in nature, technologies such as metabolic engineering technology, system biology technology, synthetic biology technology, or the like have been required to be used.As the technology of metabolic engineering has been developed, it has become possible to predict production metabolic pathways of various materials using microorganisms, and a metabolic pathway for producing 3-HP from glucose can be roughly divided into an acryloyl-CoA metabolic pathway, a β-alanine metabolic pathway, a malonyl-CoA metabolic pathway and a glycerol metabolic pathway after metabolic intermediates.The acryloyl-CoA pathway means a pathway of converting pyruvate or phosphoenolpyruvate (PEP) obtained from glycolysis of glucose into acryloyl-CoA via lactate or β-alanine, and then converting the acryloyl-CoA into 3-HP by a hydration reaction and a reduction reaction (pyruvate or PEP → lactate or β-alanine → acryloyl-CoA → 3-HP). Acryloyl-CoA is a metabolite observed during the process of decomposition of propionic acid, and since the value of Gibbs free energy is positive for the formation of the metabolite, a side reaction is an unfavourable reaction. Moreover, the substrate specificity of acryloyl-CoA thioesterases is low, so that the acryloyl-CoA pathway is not suitable as a pathway for mass production of 3-HP.The β-alanine pathway means a pathway of converting pyruvate or oxaloacetate to amino acid by means of a transamination reaction and finally converting to 3-HP via β-alanine by a transamination reaction (pyruvate or oxaloacetate→amino acid→β-alanine→3-HP; US 2012 / 0135481A1). Since the transamination reaction of β-alanine to 3-HP proceeds via malonate semialdehyde, which is highly toxic to microorganisms, a 3-HP dehydrogenase having high activity is required. Moreover, since the transamination reaction generally generates a radical form of an amino acid molecular structure in a stationary state, an enzyme of this reaction has a structure for alleviating the reactivity of the radical. Since this radical has a strong reactivity with oxygen, anaerobic conditions or a coenzyme for stabilizing radical molecules are essentially required for a smooth transamination reaction.The malonyl-CoA pathway is a pathway of converting acetyl-CoA into malonyl-CoA by carboxylation and then converting malonyl-CoA into 3-HP by a reduction reaction (acetyl-CoA → malonyl-CoA → 3-HP), wherein the glycerol pathway is a pathway of converting glucose into glycerol, converting glycerol into 3-hydroxypropionaldehyde by a reaction of dehydration, and then converting 3-hydroxypropionaldehyde into 3-HP (glucose → glycerol → 3-hydroxypropionaldehyde → 3-HP). Since the malonyl-CoA pathway and the glycerol pathway proceed via an intermediate product generally produced by microorganisms such as E. colior the like, these pathways have been predominantly studied as the pathways for 3-HP production (US 2013 / 0071893 A1). Since malonyl-CoA can be converted to 3-HP by malonate reductase and 3-HP dehydrogenase, and glycerol can be converted to 3-HP by glycerol dehydratase and aldehyde dehydrogenase, a method for converting glucose or glycerol to 3-HP using modified E. coliwas well known. A dehydration reaction of glycerol, which is a reaction accompanied by radicals similar to the transamination reaction, essentially requires coenzyme B12to conduct the reaction in the presence of oxygen.In view of industrial fermentation, the β-alanine pathway or the glycerol pathway are not suitable as a pathway for the production of 3-HP using yeast because it is difficult to use a coenzyme such as coenzyme B12as a material for a culture medium due to its cost, and bio-organisms such as yeast cannot biosynthese or absorb the corresponding material in cells. Recently, studies have been published to modify the key enzymes to overcome this problem [U.S. Pat. No. 7,655,451 B2].Malonyl-CoA is synthesized from acetyl-CoA in the cytosol and can thereby be reduced to 3-HP. In the case of bacteria such as E. coli, acetyl-CoA is generated in the cytosol of pyruvate and can thereby be used as a substrate of the TCA cycle or other metabolic reaction. However, as described above, in yeast having independent subcellular compartments, acetyl-CoA is generally synthesized in the mitochondria and used as a substrate of the TCA cycle, whereby acetyl-CoA is generated in the cytosol via acetate-generating reaction which is a side reaction of ethanol-generating reaction or citric acid cycle reaction. All reactions of generating acetyl-CoA from acetate or citric acid are reactions that consume ATP, and since yeast also consumes energy to obtain acetyl-CoA in cytosol against bacteria, yeast may be disadvantageous in terms of energy.In yeast, environments such as a state of reduction of cytosol, folding after protein synthesis, codon utilization and the like are different from those in bacteria, so that at the time of expressing an exogenous enzyme derived from bacteria, an activity thereof cannot be shown or the activity may be significantly lowered. Moreover, since the activity may be significantly changed due to the presence or absence of oxygen or other metal ions, even in the case of exogenous enzymes having the same functions, results of expression thereof in yeast may be different according to the origin of the enzymes. In the case of xylose isomerase (XI) which is an important enzyme of xylose metabolism, at the time of expressing XI derived from bacteria in yeast, an activity thereof was largely significantly low, but it has been shown that yeast was successfully modified so as to carry out xylose metabolism at relatively high activity by introducing XI derived from anaerobic fungus. Thus, in the case of introducing a metabolic pathway derived from bacteria or archaea, such as the malonyl-CoA metabolic pathway, into yeast, a gene having high activity should be secured by genes performing the same functions of different origin.In connection with a method for genetically modifying Saccharomyces cerevisiae among various yeast strains to produce 3-HP, there are numerous publications and patents, but in the case of Saccharomyces cerevisiae, since 3-HP is produced via the [pyruvic acid → acetaldehyde → acetic acid → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP] pathway, there are problems in that this pathway to produce 3-HP is complicated and the productivity of 3-HP is relatively low (US 2010 / 0248233A1; Y. Chen et al., Metabolic Engineering, 22:104-109, 2014).As a result of the attempt to solve the above problems, in order to overcome the disadvantages of yeast where ATP is consumed in a process for obtaining acetyl-CoA in cytosol, the present inventors have found a shorter metabolic pathway of [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP] that directly converts acetaldehyde to acetyl-CoA without passing through an acetate intermediate, and have confirmed, The present invention has been accomplished by the use of a recombinant yeast comprising this pathway, unlike the case of using E. coli, not only reducing the use of pH adjusting materials and thereby reducing the generation of salts, but also allowing 3-HP to be generated from biomass having a high concentration and a high yield even at a low pH, thus completing the present invention.Brief Description of the InventionIt is an object of the present invention to provide a recombinant yeast comprising an active biosynthetic 3-HP pathway of [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP] which converts acetaldehyde directly to acetyl-CoA rather than through acetate.Another object of the invention is to provide a method for producing 3-HP using the recombinant yeast.Solution of the ProblemTo achieve the above objects, the present invention provides a recombinant yeast comprising an active pathway for biosynthesis of 3-HP of [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP], wherein the yeast comprises: an exogenous gene encoding AADH; an endo- or exogenous gene encoding ACC; an exogenous gene encoding MCR; and an exogenous gene encoding HPDH.In the present invention, the yeast is acid-resistant and selected from, for example, the group consisting of the genera Saccharomyces, Kazaxantaniaand Candida. Yeast species of particular interest include, but are not limited to, Saccharomyces cerevisiae, Kazaxantania exigua, Kazaxantania bulderi, and Candida humilis.Moreover, the present invention provides a method for producing 3-HP, comprising: (a) culturing the recombinant yeast of any one of claims 1 to 10 in a medium including at least one carbon source, thereby producing 3-HP; and (b) isolating 3-HP from the culture.Brief Description of the DrawingsFig. 1 shows a pathway for producing 3-HP from glucose from the recombinant yeast of the present invention (modified malonyl-CoA pathway) and important enzymes. FIG. 2 shows a relative ranking of the ACCl activity of acetyl-CoA carboxylase enzymes. Figure 3 shows results confirming relative activity (left) and expression levels (SDS-PAGE) (right) of the archaea MCR variants. Figure 4 shows yeast expression plasmids pSK-84 and pSK-85 for expressing enzymes in conjunction with the 3-HP pathway. Figure 5 shows results of testing the culture conditions that could affect the production rates of 3-HP. Figure 6 shows 3-HP production with a more established 3-HP production strain using fed-batch (tablet-splicing) conditions of culturing.DESCRIPTION OF THE INVENTIONUnless otherwise specified, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art to which the present invention pertains. In general, the nomenclature used herein is well known and is commonly used in the art.In general, ATP is consumed in yeast in the method for producing acetyl-CoA by conversion to AMP because acetyl-CoA is produced in the metabolic pathway of [acetaldehyde → acetate → acetyl-CoA], and acetate is produced in cytosol of yeast (Fig. 1; Y. Chen et al., Metabolism Engineering, 22:104-109, 2014).However, the present invention contemplates a metabolic pathway in which the disadvantages of ATP-consuming yeast have been overcome and improved by directly producing acetyl-CoA in cytosol (Figure 1) from acetaldehyde rather than acetate in the method of producing acetyl-CoA in cytosol (Figure 1). As a result, in the case of using recombinant yeast of the present invention, it has been shown that 3-HP is produced in a high concentration and a high yield from glucose even at a low pH.Therefore, in one aspect, the present invention is directed to a recombinant yeast comprising an active biosynthetic 3-HP pathway of [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP], wherein the yeast comprises: an exogenous gene encoding AADH; an endo- or exogenous gene encoding ACC; an exogenous gene encoding MCR; and an exogenous gene encoding HPDH.The 3-HP biosynthetic pathway of [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP] is a pathway for producing 3-HP from a carbon source such as glucose, etc.: (i) "pyruvate → acetaldehyde" means a pathway for producing acetaldehyde from pyruvate using pyruvate decarboxylase (PDC) without producing an intermediate; (ii) "Acetaldehyde → Acetyl-CoA" means a pathway for generating acetyl-CoA from acetaldehyde using acetylating acetaldehyde dehydrogenase (AADH) without generating an intermediate such as acetate; (iii) "Acetyl-CoA → Malonyl-CoA" means a pathway for generating malonyl-CoA from acetyl-CoA using acetyl-CoA carboxylase (ACC) without generating an intermediate; (iv) "malonyl-CoA→3-HP" or "malonyl-CoA→malonate semialdehyde→3-HP" means a pathway for generating 3-HP from malonyl-CoA using bifunctional malonyl-CoA reductase (MCR) without generating an intermediate or a pathway for generating 3-HP from malonate semialdehyde by biosynthesis of malonate semialdehyde using monofunctional malonyl-CoA reductase (FIG. 1 ).In the present invention, the PDC gene is not subjected to engineering, but engineering of the PDC gene to increase the 3-HP production rate can be carried out, for example, by amplifying the PDC gene present in yeast by applying the well-known art in the art.In an exemplary embodiment of the present invention, genes encoding metabolic pathway enzymes having a specific function are extracted by bioinformatic genome mining of candidate metabolic enzymes.In one embodiment, the gene encoding AADH is an amino acid sequence encoding AADH having at least 60%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity to an AADH amino acid sequence selected from the group consisting of amino acid sequences shown below in Tables 1 to 3, but not limited thereto, as long as they have a function of biosynthetic acetyl-CoA from acetaldehyde. [Table 1] AADH (type adhE) [Table 1] AADH (type adhE)adhE (SEQ ID NO: 145)NP_415757.116129202Escherichia coli K-12 substr. MG1655ADHEecadhE (SEQ ID NO: 146)AY282576.133578054Piromyces sp. E2ADHEpmadhE (SEQ ID NO: 147)NP_370672.115923138Staphylococcus aureus subsp. aureus Mu50ADHEsaP343_14875 (SEQ ID NO:148)EST10864.1558501608Sporolactobacillus laevolacticus DSM 442ADHEslUCRPA7_2908 (SEQ ID NO:149)EOO01596.1500258690Togninia minima UCRPA7ADHEtm(SEQ ID NO: 150)WP_020582522522071313Endozoeomonas elysiolaADHEeeRW 1 006 000 90 (SEQ ID NO: 151)GAF43117.1589262551Rhodococcus wrtislaviensisNBRC6805ADHErw[Table 2] AADH (Type eutE)[Table 2] AADH (Type eutE)euE (SEQ ID NO: 152)YP_0014592232.1157161914Escherichia coli HSEUTEec(SEQ ID NO: 153)YP_003003316.1251788595Dictya zeae Ech 1591EUTEdz(SEQ ID NO:154)NP_470466.116800198Listeria innocua Clip11262LIN1129liC79000285 (SEQ ID NO: 155)EMP53767.1468911480Morganella morganiiSC01AADHmmC66_02610 (SEQ ID NO: 156)ENO90114.1479302014Thauera linaloolescentis DSM 12138AADHtl(SEQ ID NO: 157)WP_018205006.1516997301Atrial bacterium SCGC AAA252-M02AADHabMaqu_1235 (SEQ ID NO: 158)ABM18325.1120324010Marinobacter aquaeolei VT8AADHmalCLS_23700 (SEQ ID NO:159)CBK77783.1295091676Clostridium cf. saccharolyticum K10AADHcsPlabr_4078 (SEQ ID NO: 160)ADY61655.1324970877Plantomyces brasiliensis DSM 5305AADHpbGCWU000342_00 651 (SEQ ID NO: 161)EEP29295.1229793181Shuttle Wordia satellite DSM 14600AADHssTola_1697 (SEQ ID NO: 162)ACQ93307.1237500714Tolumonas auensis DSM 9187AADHtaHMPREREF9024_01 049 (SEQ ID NO:163)EFA26759.1270280925Pediococcus acidilactici7_4AADHpaBN552_01640 (SEQ ID NO:164)CDB 76812.1524431109Bluetia sp. CAG:237AADHbsHMPREF0179_00 640 (SEQ ID NO:165)EFV45545.1316924378Bilophila wadsworthia 3_1_6AADHbMahau_0819 (SEQ ID NO: 166)AEE96017.1332699076Machella australiansis 50-1 BONAADHma2ALO_06783 (SEQ ID NO: 167)EGO64744.1337276312Acetoneema longum DSM 6540AADHalHMPREF9200_0641 (SEQ ID NO:168)EGS34769.1341591638Veillonella sp. oral taxon 780 str. F042222AADHvsoVEJY3_084440 (SEQ ID NO:169)AEX22176.1369841032Vibrio sp. EJY3AADHvsOpiT1DRAFT_04559 (SEQ ID NO:170)EIG00023.1391221602Opiateaceae bacterium TAV1AADHobHSACCH_00271 (SEQ ID NO: 171)CCU77919.1460789193Halanaerobium saccharolyticum DSM 6643AADHhsHigh 5813 (SEQ ID NO: 172)ACY18289.1262082320Haliangium ochraceum DSM 14365AADHho[Table 3] AADH[Table 3] AADHsucD (SEQ ID NO: 173)EEN82978.1229317069Porphyromonas endodontalis ATCC 35406AADHpeAZOBR_p480045 (SEQ ID NO: 174)CCD03730.1356882712Azospirillum brasilense Sp245AADHabrHMPREF1987_01259 (SEQ ID NO:175)ERI82808.1543978929Peptostreptococcaceae bacterium 113 str. W5053AADHpbaTerro_0974 (SEQ ID NO: 176)AFL87295.1390411791Terriglobus roseus DSM 18391AADHtrThe present invention encompasses a gene encoding ACC. In one embodiment, the gene encoding ACC is a nucleic acid encoding ACC having an amino acid sequence of at least 60%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity to an ACC amino acid sequence selected from the group consisting of amino acid sequences set forth in Table 4 below, but is not limited thereto, provided it has the function of biosynthetic malonyl-CoA from acetyl-CoA. [Table 4] ACC (eukaryotic multidomain type) [Table 4] ACC (eukaryotic multidomain type)ACC1 (SEQ ID NO: 177)CAA96294.11302498Saccharomyces cerevisiae S288cACC1scYALI0C11407g (SEQ ID NO:178)XP_501721.150548503Yarrowia lipolytica CLIB122ACC1y1HMPREF1544_10 598 (SEQ ID NO:179)EPB82652.1511001160Mucor circinelloidesf. circinelloides 1006PhLACC1mcACC1ke (SEQ ID NO: 102)Kazaxantania exiguaACC1keACC1ch (SEQ ID NO: 106)Candida humilisACC1chCGB F3610C (SEQ ID NO: 180)XP 003194770. 1321260100Cryptococcus gattii W276ACC1cgAGABI1DRAFT_ 70405 (SEQ ID NO:181)EKM81867.1409081508Agaricus bisporus var. bumrettii JB137-S8ACC1abBATDDEDRAFT_18 673 (SEQ ID NO:182)EGF84402.1328774365Brarachochytrium dendrobatidis JAM81ACC1bdRHTO_02004 (SEQ ID NO:183)EMS21133.1472583500Rhodosporidium toruloides NP11ACC1rtPITG-18706 (SEQ ID NO:184)EEY68805.1262110753Phytophthora infestans T30-4ACC1piTCM_034957 (SEQ ID NO:185)XP 007018852. 1590598290Theobroma cacaoACC1tcOt01g03240 (SEQ ID NO: 186)CAL50235.1116000555Ostreococcus tauriACC1otNGATSA_3002800 (SEQ ID NO:187)AFJ69228.1387219039Nannochloropsis gaditana CCMP526ACC1ngaccA (SEQ ID NO: 188)EAL63219.160465120Dictyostelium discoideumAX4ACC1ddLOC101893358 (SEQ ID NO:189)XP_005182000. 1557764587Musca domesticaACC1mdACACA (SEQ ID NO:190)ABX09993.1159895418Sus scrofaACC1ss(SEQ ID NO: 191)Uniprot: H2Y65Ciona savignyiACC1csThe present invention encompasses a gene encoding MCR. In one embodiment, an MCR may be bifunctional in that it has a function of converting malonyl-CoA to malonate semialdehyde and a function of converting malonate semialdehyde to 3-HP; or monofunctional in that it has a function of converting malonyl-CoA to malonate semialdehyde.In the present invention, the gene encoding the bifunctional MCR is a nucleic acid encoding MCR having an amino acid sequence of at least 60%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity to an MCR amino acid sequence selected from the group consisting of amino acid sequences shown in Table 5 below, but is not limited thereto, so long as it simultaneously has the function of converting malonyl-CoA to malonate semialdehyde and the function of converting malonate semialdehyde to 3-HP. [Table 5] MCR bifunctional type) [Table 5] MCR bifunctional type)Mcr (SEQ ID NO: 192)AAS20429.142561982Chloroflexus aurantiacusMCRcaCagg_1256 (SEQ ID NO: 193)ACL24164.1219542426Chloroflexus aggregans DSM 9485MCRcagOSCT_0547 (SEQ ID NO: 194)EFO81531.1308227877Oscillochloris trichoides DG-6MCRotRcas_2929 (SEQ ID NO: 195)ABU58991.1156234208Roseiflexus castellholzii DSM 13941MCRrcOMB55_00007 690 (SEQ ID NO:196)EHQ57048.1374302864gamma proteobacterium HIMB55MCRgpCatheter_B0159 (SEQ ID NO: 197)AEP13163.1347588634Chloroaciidobacterium thermophilum BMCRct(SEQ ID NO: 198)WP_022680613. 1550932202Sandarakinorhabdus limnophilaMCRsl(SEQ ID NO: 199)WP_023839102. 1564013708Blastomonas sp. CACIA14H2MCRbsIn another embodiment, the MCR gene may be monofunctional with the function of converting malonyl-CoA to malonate semialdehyde, a gene encoding an enzyme that converts malonate semialdehyde to 3-HP may be further included.In the present invention, the gene encoding monofunctional MCR is a nucleic acid encoding MCR having an amino acid sequence of at least 60%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity to an MCR amino acid sequence selected from the group consisting of amino acid sequences shown in Table 6 below, but is not limited thereto as long as it has the function of converting malonyl-CoA to malonate semialdehyde. [Table 6] MCR [Table 6] MCRMsed_0709 (SEQ ID NO: 200)ABP94884.1145701742Metallosphaera sedula DSM 5348MCRmsmcr / scr (SEQ ID NO: 201)BAB67276.115623288Sulfolobus tokodaii DSM 16993MCRst(SEQ ID NO: 202)WP 020198954. 1519043079Sulfolobals archaeon Acd1MCRsa1SacRon12I_1178 0 (SEQ ID NO: 203)AGE74568.1449039143Sulfolobus acidocaldarius Ron12 / IMCRsa2SacRon12I_1070 5 (SEQ ID NO: 204)AGE74357.1449038932Sulfolobus acidocaldarius Ron12 / IMCRsa3(SEQ ID NO: 205)BAJ50751.1343485097Candidatus Caldarchaeum subterraneumMCRccMetK1_000284 80 (SEQ ID NO: 206)EHP68415.1373523495Metallosphaera yellowstonensis MK1MCRmyTREAZ_1307 (SEQ ID NO:207)AEF80380.1333734431Treponema azotonutricium ZAS-9MCRtaIn the present invention, the gene encoding an enzyme capable of converting malonate semialdehyde to 3-HP is a nucleic acid encoding an enzyme having an amino acid sequence of at least 60%, preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity to an HPDH, HIBADH, HBDH, or BDH amino acid sequence selected from the group consisting of amino acid sequences shown in Tables 7-10 below, but is not limited thereto, so long as the gene encoding the protein has the function of biosynthetic 3-HP from malonate semialdehyde.The HPDH amino acid sequences shown in Table 7 below, HIBADH amino acid sequences shown in Table 8 below, HBDH amino acid sequences shown in Table 9 below, and BDH amino acid sequences shown in Table 10 below are amino acid sequences of a protein having the function of biosynthesis of 3-HP from malonate semialdehyde. [Table 7] HPDH [Table 7] HPDHEC3431_0375 (SEQ ID NO: 208)EFV00080.1315619553Escherichia coli 3431HPDHecYMR226C (SEQ ID NO:209)DAA10125.1285814230Saccharomyces cerevisiae S288cHPDHscMsed 1993 (SEQ ID NO: 210)ABP96133.1145702991Metallosphaera sedula DSM 5348HPDHmsSTK_15070 (SEQ ID NO: 211)BAK54608.1342306519Sulfolobus tokodaii str. 7HPDHstBWG_0862 (SEQ ID NO:212)ACR64730.1238862732Escherichia coli BW2952HPDHecbATEG_09041 (SEQ ID NO:213)XP 001217663.1115436862Aspergillus terreus NIH2624HPDHat(SEQ ID NO:214)YP 902607.1118581357Pelobacter propionicus DSM 2379HPDHppSnov_0928 (SEQ ID NO: 215)YP_003692871.1298290932Starkeya novella DSM 506HPDHsn(SEQ ID NO: 216)YP_004145243.1319785768Pseudoxanthomonas suwonensis 11-1HPDHps(SEQ ID NO:217)WP_002641751.1488717875Simonsiella muelleriHPDHsm(SEQ ID NO: 218)WP_006802623.1493855747Helicobacter winghamensisHPDHh(SEQ ID NO:219)WP 007116408.1494180330Enhydrobacter aerosaccusHPDHea(SEQ ID NO: 220)WP_018365922.1517177104Streptococcus delphisHPDHsd(SEQ ID NO: 221)WP_019460509.1518290301Roseomonas sp. B5HPDHrsYDF1 (SEQ ID NO: 222)EAZ63492.1126213385Pichia stipitis CBS 6054HPDHpstKAFR0B03360 (SEQ ID NO: 223)CCF56633.1372462351Kazaxantania africana CBS 2517HPDHkaydfG (SEQ ID NO: 224)EGC72291.1325160162Haemophilus parainfluenzae ATCC 33392HPDHhpK788_004913 (SEQ ID NO: 225)ETY79751.1575860535Burkholderia earibensis MBA4HPDHbAMED_69 (SEQ ID NO:226)ADJ48621.1299798246Amycolatopsis mediterranei U32HPDHamCFU_3402 (SEQ ID NO:227)AEK63226.1340553851Collimonas fungivorans Ter331HPDHcfRahaq2_2300 (SEQ ID NO: 228)AEX52155.1371588425Rahnella aquaticis ATCC 33071HPDHraLS215_1598 (SEQ ID NO:229)ACP35603.1227456916Sulfolobus islandicus L.S.2.15HPDHsi(SEQ ID NO: 230)WP 003467297.1489562770Xanthomonas translucensHPDHxt(SEQ ID NO: 231)WP 007747336.1495021561Cronobacter dublinensisHPDHcd(SEQ ID NO:232)WP_021506918.1545151592Pantoea dispersaHPDHpd(SEQ ID NO:233)EHT00469.1376387763Klebsiella oxytoca 10-5245HPDHko(SEQ ID NO: 234)ESM32057.1555088912Enterobacter cloacae BWH 31HPDHecl[Table 8] HIBADH[Table 8] HIBADHmmsB (SEQ ID NO: 235)ADR61938.1313500572Pseudomonas putida BIRD-1HIBATHHppPA3569 (SEQ ID NO:236)AAG06957.19949723Pseudomonas aeruginosa PAO1HIBAHpaBC_4042 (SEQ ID NO:237)AAP10961.129897686Bacillus cereus ATCC 14579HIBAHbQWA_01835 (SEQ ID NO:238)EJC65559.1393165510Alcaligenes faecalis NCIB 8687HIBATHHaf(SEQ ID NO:239)JI420577.1327223309Lytechinus variegatusHIBAHLV(SEQ ID NO: 240)GAXL01007172.1596424618Chyphotes mellipesHIBAHCMPOPTR_0001s46990g (SEQ ID NO:241)XP0023090566.1224061611Populus trichocarpaHIBAHpt(SEQ ID NO: 242)WP 007234036.1494440757marine gamma proteobacterium HTCC2080HIBAHmgp(SEQ ID NO: 243)WP 009244364.1496538096Clostridiales sp.HIBATHHcs(SEQ ID NO: 244)WP 017931623.1516543998Robiginitomaculum antarcticumHIBATHHRa(SEQ ID NO:245)WP 018914915.1517744707Thiomonas sp. FB-6HIBAHts(SEQ ID NO:246)WP 022530055.1548582704Lactobacillus shenzhenensisHIBATHHlsABAZ39_230 55 (SEQ ID NO: 247)EZQ03930.1612167293Azospirillum brasilenseHIBAHab(SEQ ID NO:248)EMI09340.1460132162Anoxybacillus sp. DT3-1HIBATHHasT458 21320 (SEQ ID NO:249)EST53365.1558617142Brevibacillus panacihumi W25HIBAHbpxccb100_3039 (SEQ ID NO: 250)CAP52402.1167734194Xanthomonas campestris pv. campestrisHIBAHxcBeenme03 34 79 (SEQ ID NO:251)ACA92632.1169818050Burkholderia cenocepacia MC0-3HIBAHbmHoch 3369 (SEQ ID NO: 252)ACY15871.1262079902Haliangium ochraceum DSM 14365HIBAHhommsB (SEQ ID NO: 253)ADP96674.1311693801Marinobacter adhaers HP15HIBATHHmaivdF (SEQ ID NO: 254)AAN54737.124347484Shewanella oneidensis MR-1HIBAHSO[Table 9] HBDH[Table 9] HBDHgbd (SEQ ID NO: 255)AAC41425.1695279Cupriavidus necatorHBDHcn4hbD (SEQ ID NO:256)EDK35022.1146348486Clostridium kluyveri DSM 555HBDHckGOS_1589287 (SEQ ID NO:257)EDB80735.1142959799marine metagenomic sp.HBDHmmHMPREF0080_00276 (SEQ ID NO:258)EHM43401.1364565684Anaeroglobus geminateus F0357HBDHagBN605_01179 (SEQ ID NO:259)CCD07748.1524585315Dorea sp. CAG:317HBDHdsBN791_01127 (SEQ ID NO:260)CDE92329.1524795667Fusobacteriumsp. CAG:815HBDHfsOdosp_2059 (SEQ ID NO:261)ADY33063.1324312510Odoribacter splanchnicus DSM 220712HBDHosBpro-2526 (SEQ ID NO: 262)ABE44443.191697614Polaromonas sp. JS666HBDHpsCsalb_1756 (SEQ ID NO: 263)ABE59108.191796969Chromohalobacter salexiens DSM 3043HBDHcsBRPE64_DC DS02300 (SEQ ID NO:264)BAN27166.1506947049Burkholderia sp. RPE64HBDHbs[Table 10] BDH[Table 10] BDHbdhA (SEQ ID NO: 265)GAA17557.1346057674Pseudomonas aeruginosa NCMG1179BDHpaBresu 2563 (SEQ ID NO:266)YP 003819493.1302383670Brevundimonas subvibrioides ATCC 15264BDHbs(SEQ ID NO: 267)YP 004110707.1316935725Rhodopseudomonas palustris DX-1BDHrp(SEQ ID NO:268)WP 008960707.1496247322Bradyrhizobium sp. STM 3809BDHbss(SEQ ID NO:269)WP_009158463.1496449618Thalassobium sp. R2A62BDHts(SEQ ID NO: 270)WP 010548788.1498234632gamma proteobacterium HIMB30BDHgp(SEQ ID NO: 271)WP 018183273.1516955964Kaistia granuliBDHkgh16_A1334 (SEQ ID NO: 272)CAI92474.1113526129Ralstonia eutrophaH16BDHrebdhA (SEQ ID NO: 273)EOY63580.1509564889Klebsiella pneumoniae KP-7BDHkpAZOBR_p140023 (SEQ ID NO: 274)CCD00057.1356879155Azospirillum brasilense Sp245BDHabbdhA (SEQ ID NO: 275)ABF07432.193353343Cupriavidus metalidurans CH34BTHcmIn a specific embodiment of the present invention, the recombinant yeast comprising an active pathway for biosynthesis of 3-HP may have the genes listed in Table 11, such as an exogenous gene encoding AADH having an amino acid sequence selected from the group consisting of SEQ ID NO:74 to SEQ ID NO:98, an endogenous or exogenous gene encoding ACC having an amino acid sequence selected from the group consisting of SEQ ID NO:99 to SEQ ID NO:106, an exogenous gene encoding MCR having an amino acid sequence selected from the group consisting of SEQ ID NO:107 to SEQ ID NO:116, and an exogenous gene encoding HPDH having an amino acid sequence, which is selected from the group consisting of SEQ ID NO:117 to SEQ ID NO:144. [Table 11] [Table 11]AADHsAADHabSEQ ID NO:74AADHalSEQ ID NO:75AADHbsSEQ ID NO: 76AADHbSEQ ID NO: 77AADHcsSEQ ID NO:78AADHhoSEQ ID NO:79AADHhsSEQ ID NO:80AADHmalSEQ ID NO:81AADHma2SEQ ID NO:82AADHmmSEQ ID NO:83AADHpaSEQ ID NO:84AADHpbSEQ ID NO:85AADHpeSEQ ID NO:86AADHrSEQ ID NO:87AADHslSEQ ID NO:88AADHssSEQ ID NO:89AADHtaSEQ ID NO:90AADHtlSEQ ID NO:91AADHtmSEQ ID NO:92AADHvsSEQ ID NO:93ADHEecSEQ ID NO:94AHEpmSEQ ID NO:95EUTEdzSEQ ID NO:96EUTEecSEQ ID NO:97LIN1129liSEQ ID NO:98ACC1sACC1sc_S659ASEQ ID NO:99ACC1sc_S659A / S1157ASEQ ID NO:100ACC1sc_S1157ASEQ ID NO:101ACC1keSEQ ID NO:102ACC1mcSEQ ID NO:103ACC1scSEQ ID NO:104ACCylSEQ ID NO:105ACC1chSEQ ID NO:106bifunctional HPDH-MCRsHPDH-MCRbsSEQ ID NO:107HPDH-MCRcaSEQ ID NO:108HPDH-MCRcagSEQ ID NO:109HPDH-MCRctSEQ ID NO:110HPDH-MCRgbSEQ ID NO:111HPDH-MCRotSEQ ID NO:112HPDH-MCRrcSEQ ID NO:113HPDH-MCRslSEQ ID NO:114HPDH-MCRea_variant_3SEQ ID NO:115HPDH-MCRca_variant_6SEQ ID NO:116HPDHsBTHcmSEQ ID NO:117BDHkpSEQ ID NO:118HBDHosSEQ ID NO:119HBDHpsSEQ ID NO:120HIBATHHasSEQ ID NO:121HIBAHbSEQ ID NO:122HIBATHHmaSEQ ID NO:123HIBAHpaSEQ ID NO:124HIBAHxcSEQ ID NO:125HPDHamSEQ ID NO:126HPDHbsSEQ ID NO:127HPDHcaSEQ ID NO:128HPDHcagSEQ ID NO:129HPDHctSEQ ID NO:130HPDHecSEQ ID NO:131HPDHedSEQ ID NO:132HPDHgbSEQ ID NO:133HPDHhSEQ ID NO:134HPDHkaSEQ ID NO:135HPDHmsSEQ ID NO:136HPDHotSEQ ID NO:137HPDHpsSEQ ID NO:138HPDHraSEQ ID NO:139HPDHrcSEQ ID NO:140HPDHsiSEQ ID NO:141HPDHslSEQ ID NO:142HPDHsmSEQ ID NO:143HPDHstSEQ ID NO:144In the present invention, the yeast may be acid-resistant, and the acid-resistant yeast may be selected from the group consisting of, for example, the genera Saccharomyces, Kazaxantaniaand Candida. Yeast species of particular interest include, but are not limited to, Saccharomyces cerevisiae, Kazaxantania exigua, Kazaxantania bulderi, and Candida humilis.According to the present invention, the recombinant yeast may be acid-resistant, and in order to produce the acid-resistant recombinant yeast, a yeast host having acid resistance against an organic acid (particularly, 3-HP and / or an organic acid formed as a by-product in the production of 3-HP) is preferably used.The acid resistant yeast may be selected from the group consisting of, but is not limited to, Saccharomyces cerevisiae, Kazaxantania exigua, Kazaxantania bulderi, and Candida humilis.The term "acid-resistant yeast" used in the specification refers to yeast having acid resistance against organic acids such as 3-HP or the like, and the acid resistance can be evaluated by confirming the growth in a medium containing various concentrations of organic acid. In this case, "acid-resistant yeast" may be yeast having a high growth rate and biomass consumption rate when cultivated in a medium having high organic acid concentrations, as compared with ordinary yeast.Acid-resistant yeast according to the present invention may be yeast capable of maintaining at least 10% of the glucose consumption rate (or the like) or at least 10% of the specific growth rate in the medium containing over 1 M or more of organic acid (particularly, 3-HP) below a pH lower than the pKa of the organic acid (particularly, 3-HP) as compared with yeast grown in the medium containing no organic acid. Acid-resistant yeast according to the present invention may be yeast that can maintain at least 10% of the glucose consumption rate (or the like) or at least 10% of the specific growth rate below pH 2-4 as compared to pH 7.The genetically modified microorganism of the present invention can be produced by introducing a gene into a chromosome of a microorganism or introducing a modified vector into a microorganism.A host where the efficiency of introducing DNA is high and the efficiency of expressing introduced DNA is high is generally used as the altered microorganism, and in one embodiment of the present invention, yeast is used, but not limited to, any kind of microorganism may be used as long as the targeted DNA is sufficiently expressed.The altered microorganism can be produced by a transformation method. "transformation" means introducing DNA into a host; thereby, DNA can be duplicated, as a factor of a chromosome or by insertion into a chromosome, which is a phenomenon that artificially causes genetic alteration. In conventional transformation methods, there are electroporation, acetic acid-lithium-PEG methods and the like.In addition, in the present invention, a well-known genetic engineering method can be used as a method for introducing a gene into a chromosome of a host microorganism, and for example, there is a method using retrovirus vector, adenovirus vector, adeno-associated virus as vector, herpes simplex virus vector, poxvirus vector, lentivirus vector, nonviral vector, etc. "vector" means a DNA construct having a DNA sequence to be operably linked to an appropriate control sequence capable of expressing DNA in a host. A vector may be a plasmid, a phage particle, or just a latent genome insert. When a vector is transformed into a suitable host, it may be duplicated or functioning independently of a host genome, or in some cases, it may be integrated into a genome itself. A plasmid is the type most widely used as a vector.A typical plasmid vector that can be used for the task has a structure comprising (a) a replication initiation point that enables replication to be effectively performed to contain plasmid vectors per host cell, (b) an antibiotic resistant gene or an auxotrophic marker gene that enables a host cell transformed with a plasmid vector to be selected, and (c) a restriction site of a restriction enzyme that can be introduced with a foreign DNA fragment. Even if there is no appropriate restriction site of a restriction enzyme, a vector and foreign DNA can be easily ligated when the linker or synthetic oligonucleotide adapter is used according to a general method.A nucleic acid is "operably linked" when arranged in a functional relationship to other nucleic acid sequences. It may be a gene and control sequence(s) linked in a process that allows gene expression when a suitable molecule (for example a transcription activating protein) is linked to the control sequence(s). For example, the DNA for a presequence or secretion leader is operably linked to the DNA for a polypeptide when a preprotein involved in the secretion of a polypeptide is expressed; a promoter or enhancer is operably linked to a coding sequence when the transcription of a sequence is affected; a ribosome binding domain is operably linked to a coding sequence when the transcription of a sequence is affected; or a ribosome binding domain is operably linked to a coding sequence when it is arranged to be easily translated.Generally, "operably linked" refers to contacting a linked DNA sequence, or contacting the secretory leader and displaying it in the leader frame. However, the amplifier for making the contact is not required. The linkage of the enhancer sequence is accomplished by ligation at a suitable restriction enzyme site. When the domain is not shown, a synthetic oligonucleotide adapter or oligonucleotide linker is used according to a general method.It will be understood that all the vectors do not function equally to express the DNA sequences of the present invention. Likewise, all the host cells do not act to the same extent in the same expression system. However, one skilled in the art will appropriately select a vector, expression control sequence and host cell without departing from the scope of the present invention and without undue experimentation. For example, a host cell must be considered in selecting a vector. This is because the vector is to be replicated therein. Also, the replication number and the ability to direct the number of replications of a vector and the expression of other proteins encoded by the vector, for example antibiotic markers, should be considered.In another embodiment, the present invention is directed to a method for producing 3-HP comprising: (a) culturing the recombinant yeast of any one of claims 1 to 10 in a medium comprising at least one carbon source, thereby producing 3-HP; and (b) isolating 3-HP from the culture.In the present invention, the carbon source may be one or more selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, galactose, cellulose, glucose oligomers, and glycerol, but is not limited thereto.In the present invention, the culturing is preferably carried out under a condition that microorganisms such as E. coliare no longer active (e.g., metabolite formation, etc.). In one embodiment, the culturing is performed at a pH of 1.0 to 6.5, preferably at a pH of 1.0 to 6.0, more preferably at a pH of 2.6 to 4.0, but is not limited thereto.MODE OF THE INVENTIONHereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided as an example to easily explain the description and scope of the technical spirit of the present invention. Accordingly, the scope of the present invention is not limited thereby or changed therefrom. In addition, various modifications and changes can be made by those skilled in the art to which the present invention pertains from this description.Example 1: Selection of Host Yeast Strains by Tolerance to 3-HPAn essential feature of a 3-HP production organism is the good tolerance to high concentrations of 3-HP, which allows product accumulation during fermentation with minimal loss of strain performance. A comprehensive, diverse array of 718 wild-type yeast strains were screened to identify those strains that can tolerate high concentrations of 3-HP at low pH and that can grow and metabolize glucose under these conditions (Table 12).A series of growth assays based on agar plates and liquid medium microtiter plates were first used to screen the entire series of strains for acid tolerance. Thereafter, a subset of the strains were evaluated for their ability to tolerate high levels of 3-HP at low pH in shake flask cultures. None of these screening methods is a perfect indicator of 3-HP tolerance in an industrial environment, but a combination of many methods provides a comprehensive and stable way to determine prospective 3-HP producing yeast strains.First, the growth of 718 yeast strains was evaluated on YPD solid agar medium containing varying amounts of 3-HP: 0 g / L 3-HP (pH 6.62), 50 g / L 3-HP (pH 3.44), 75 g / L 3-HP (pH 3.28), 100 g / L 3-HP (pH 3.17) and 125 g / L 3-HP (pH 3.08). The strains were then screened in this screening procedure by their ability to tolerate the varying amounts of 3-HP.The growth of the 718 yeast strains was then evaluated in the absence (SCD medium, 0 g / L 3-HP, pH 6.0) or presence (SCD medium, 70 g / L 3-HP, pH 3.5) of 3-HP in microtiter plate liquid cultures using Bioscreen C devices which can automatically incubate, shake, and measure the turbidity of the cultures. Existing software for modeling microbial growth curves was then set up to determine the delay phase, maximum growth rate, and final cell density for each strain under all experimental conditions. For this screening procedure, each strain was scored for its maximum growth rate in the absence of 3-HP, its maximum growth rate in the presence of 3-HP, and the relative difference between these two values of maximum growth rate.Using a liquid handling robot, the growth and glucose consumption rate of the 718 yeast strains in liquid YPD medium was evaluated at 85 g / L 3-HP (pH 3.5) in microtiter plates. Automated flow was used to inoculate growth plates, dilute and centrifuge samples for OD measurement at fixed time points, and collect supernatants for HPLC analysis used to measure residual glucose levels at fixed time points. Unlike the Bioscreen C growth assays, the robotic microtiter plate growth assay allowed greater aeration and higher maximum cell densities to be achieved and also allowed glucose consumption rates to be evaluated as the two previous assays rather than just the growth rate. For this screening assay, each strain was screened by its maximum cell density obtained in the presence of 3-HP and its ability to consume glucose in the presence of 3-HP at low pH.The individual scores from the different assays for evaluation of 3-HP tolerance were averaged to obtain the final result of 3-HP tolerance for each of the strains (Table 12). These screens showed that the yeast species Candida apicola, Candida humilis, Issatchenkia orientalis, Kazaxantania bulderi, Kazaxantania exigua, Pichia membranifaciens, Saccharomyces cerevisiaeand Yarrowia lipolyticawere good general tolerance to 3-HP at low pH under varying conditions.These eight 3-HP tolerant yeast species were then further tested for tolerance to 3-HP at low pH in shake flask cultures. For these cultivations (defined SCD medium, high initial biomass, low aeration), the ability of the strain to grow, consume glucose and produce ethanol was evaluated in the presence of varying amounts of 3-HP at varying pH values: 100 g / L 3-HP (pH 4.0), 100 g / L 3-HP (pH 3.5), 100 g / L 3-HP (pH 3.0) and 80 g / L 3-HP (pH 2.6). These shake flask cultures showed that certain C. humilis, K. bulderi, K. exigua and S. cerevisiae yeast strains have a very stable tolerance to high levels of 3-HP at low pH, since they had the highest glucose consumption rates, biomass production rates and ethanol production rates of the various yeast strains under these harsh conditions. In contrast, C. apicola, I. orientalis, P. membranifaciensand Y. lipolyticastrains were unable to act under these very limiting growth conditions. These detailed follow-up analyses using different shaking flask cultures confirmed that certain C. humilis, K. bulderi, K. exigua and S. cerevisiae yeast strains have a great potential as 3-HP production hosts because they show a high natural tolerance to 3-HP under industrially relevant conditions. [Table 12] Tolerance of yeast strain to 3-HP [Table 12] Tolerance of yeast strain to 3-HPVSK-1Saccharomyces pastorianus1.54VSK-2Saccharomyces cerevisiae1.38VSK-3Saccharomyces cerevisiae1.03VSK-4Saccharomyces cerevisiae0.83VSK-5Saccharomyces cerevisiae1.71VSK-6Saccharomyces pastorianus1.57VSK-7Saccharomyces pastorianus3.79VSK-8Saccharomyces pastorianus0.63VSK-9Saccharomyces pastorianus1.58VSK-10Saccharomyces pastorianus0.08VSK-11Saccharomyces pastorianus1.67VSK-12Saccharomyces pastorianus1.65VSK-13Saccharomyces pastorianus1.24VSK-14Saccharomyces pastorianus1.54VSK-15Saccharomyces pastorianus1.04VSK-16Saccharomyces pastorianus1.46VSK-17Saccharomyces pastorianus1.17VSK-18Saccharomyces pastorianus1.17VSK-19Saccharomyces pastorianus1.28VSK-20Saccharomyces pastorianus1.53VSK-21Saccharomyces pastorianus0.67VSK-22Saccharomyces pastorianus0.58VSK-23Saccharomyces cerevisiae1.67VSK-24Saccharomyces cerevisiae2.13VSK-25Saccharomyces pastorianus1.97VSK-26Saccharomyces pastorianus3.89VSK-27Saccharomyces pastorianus1.44VSK-28Saccharomyces pastorianus0.61VSK-29Saccharomyces pastorianus1.44VSK-30Saccharomyces pastorianus1.64VSK-31Saccharomyces pastorianus1.25VSK-32Saccharomyces pastorianus1.49VSK-33Saccharomyces pastorianus1.78VSK-34Saccharomyces pastorianus1.36VSK-35Saccharomyces pastorianus1.36VSK-36Saccharomyces pastorianus1.39VSK-37Saccharomyces pastorianus1.47VSK-38Saccharomyces cerevisiae3.82VSK-39Saccharomyces pastorianus1.22VSK-40Saccharomyces pastorianus1.31VSK-41Saccharomyces pastorianus3.13VSK-42Saccharomyces pastorianus1.44VSK-43Saccharomyces pastorianus1.44VSK-44Saccharomyces pastorianus1.36VSK-45Saccharomyces pastorianus1.28VSK-46Saccharomyces pastorianus1.57VSK-47Saccharomyces pastorianus1.32VSK-48Saccharomyces pastorianus0.44VSK-49Saccharomyces pastorianus1.18VSK-50Saccharomyces cerevisiae1.61VSK-51Saccharomyces pastorianus1.86VSK-52Saccharomyces pastorianus0.94VSK-53Saccharomyces pastorianus1.11VSK-54Saccharomyces pastorianus1.53VSK-55Saccharomyces pastorianus1.36VSK-56Saccharomyces pastorianus0.63VSK-57Saccharomyces pastorianus0.81VSK-58Saccharomyces pastorianus0.25VSK-59Saccharomyces pastorianus1.44VSK-60Saccharomyces pastorianus1.31VSK-61Saccharomyces pastorianus1.40VSK-62Saccharomyces pastorianus2.43VSK-63Saccharomyces pastorianus1.44VSK-64Saccharomyces cerevisiae2.42VSK-65Saccharomyces cerevisiae2.49VSK-66Saccharomyces pastorianus3.42VSK-67Saccharomyces cerevisiae2.78VSK-68Saccharomyces pastorianus0.86VSK-69Saccharomyces pastorianus0.65VSK-70Saccharomyces pastorianus2.57VSK-71Saccharomyces pastorianus1.58VSK-72Saccharomyces pastorianus1.19VSK-73Saccharomyces pastorianus1.33VSK-74Saccharomyces cerevisiae2.71VSK-75Saccharomyces pastorianus1.17VSK-76Saccharomyces pastorianus0.83VSK-77Saccharomyces pastorianus0.79VSK-78Saccharomyces cerevisiae1.46VSK-79Saccharomyces cerevisiae1.13VSK-80Saccharomyces cerevisiae1.25VSK-81Saccharomyces cerevisiae1.97VSK-82Saccharomyces cerevisiae1.65YSK-83Saccharomyces cerevisiae2.72VSK-84Saccharomyces cerevisiae1.74VSK-85Saccharomyces cerevisiae2.47VSK-86Saccharomyces cerevisiae3.00VSK-87Saccharomyces cerevisiae3.40VSK-88Saccharomyces cerevisiae2.13VSK-89Saccharomyces pastorianus1.25VSK-90Saccharomyces pastorianus1.13VSK-91Saccharomyces pastorianus2.00VSK-92Saccharomyces pastorianus3.01VSK-93Saccharomyces pastorianus2.38VSK-94Saccharomyces cerevisiae3.11VSK-95Saccharomyces cerevisiae3.10VSK-96Saccharomyces cerevisiae3.10VSK-97Saccharomyces cerevisiae3.26VSK-98Saccharomyces cerevisiae3.51VSK-99Saccharomyces cerevisiae3.63VSK-100Saccharomyces cerevisiae2.64VSK-101Saccharomyces cerevisiae2.69VSK-102Saccharomyces cerevisiae3.06VSK-103Saccharomyces cerevisiae3.01VSK-104Saccharomyces pastorianus1.44VSK-105Saccharomyces cerevisiae3.06VSK-106Saccharomyces pastorianus1.72VSK-107Saccharomyces pastorianus1.39VSK-108Saccharomyces pastorianus1.44VSK-109Saccharomyces pastorianus1.97VSK-110Saccharomyces pastorianus1.74VSK-111Saccharomyces pastorianus2.72VSK-112Saccharomyces cerevisiae3.10VSK-113Saccharomyces cerevisiae3.39VSK-114Saccharomyces cerevisiae1.42VSK-115Saccharomyces pastorianus1.44VSK-116Saccharomyces cerevisiae3.51VSK-117Saccharomyces cerevisiae3.75VSK-118Saccharomyces bayanus3.42VSK-119Saccharomyces cerevisiae3.15VSK-120Saccharomyces cerevisiae2.32VSK-121Saccharomyces cerevisiae3.51VSK-122Saccharomyces cerevisiae3.11VSK-123Saccharomyces cerevisiae3.49VSK-124Saccharomyces cerevisiae2.96VSK-125Saccharomyces cerevisiae3.51VSK-126Saccharomyces cerevisiae0.97VSK-127Saccharomyces cerevisiae2.75VSK-128Saccharomyces cerevisiae3.68VSK-129Saccharomyces kudriavzevii2.61VSK-130Saccharomyces cerevisiae3.01VSK-131Saccharomyces cerevisiae3.35VSK-132Saccharomyces cerevisiae2.90VSK-133Saccharomyces cerevisiae3.07VSK-134Saccharomyces cerevisiae3.49VSK-135Saccharomyces cerevisiae0.75VSK-136Saccharomyces cerevisiae2.36VSK-137Saccharomyces cerevisiae2.83VSK-138Saccharomyces bayanus1.61VSK-139Saccharomyces cerevisiae3.07VSK-140Saccharomyces cerevisiae3.35VSK-141Saccharomyces cerevisiae2.75VSK-142Saccharomyces cerevisiae3.04VSK-143Saccharomyces bayanus1.43VSK-144Saccharomyces sp.3.11VSK-145Saccharomyces sp.3.28VSK-146Saccharomyces sp.1.89VSK-147Saccharomyces cerevisiae3.25VSK-148Saccharomyces cerevisiae3.01VSK-149Saccharomyces bayanus3.38VSK-150Saccharomyces cerevisiae3.22VSK-151Saccharomyces cerevisiae2.75VSK-152Saccharomyces cerevisiae3.13VSK-153Saccharomyces cerevisiae0.08VSK-154Saccharomyces cerevisiae2.46VSK-155Saccharomyces cerevisiae3.38VSK-156Saccharomyces cerevisiae3.06VSK-157Saccharomyces cerevisiae2.00VSK-158Saccharomyces pastorianus2.75VSK-159Saccharomyces cerevisiae1.10VSK-160Saccharomyces cerevisiae1.83VSK-161Saccharomyces bayanus3.15VSK-162Saccharomyces cerevisiae1.79VSK-163Saccharomyces cerevisiae2.47VSK-164Saccharomyces cerevisiae2.43VSK-165Saccharomyces bayanus1.67VSK-166Saccharomyces cerevisiae2.90VSK-167Saccharomyces cerevisiae2.86VSK-168Saccharomyces cerevisiae2.17VSK-169Saccharomyces cerevisiae2.76VSK-170Saccharomyces cerevisiae3.46VSK-171Saccharomyces cerevisiae3.65VSK-172Saccharomyces cerevisiae3.89VSK-173Saccharomyces cerevisiae3.11VSK-174Saccharomyces cerevisiae3.61VSK-175Saccharomyces cerevisiae2.67VSK-176Saccharomyces cerevisiae2.67VSK-177Saccharomyces cerevisiae1.88VSK-178Saccharomyces cerevisiae3.56VSK-179Saccharomyces cerevisiae3.82VSK-180Saccharomyces cerevisiae2.54VSK-181Saccharomyces cerevisiae3.64VSK-182Saccharomyces cerevisiae3.07VSK-183Saccharomyces cerevisiae3.32VSK-184Saccharomyces cerevisiae3.89VSK-185Saccharomyces cerevisiae1.96VSK-186Saccharomyces cerevisiae3.51VSK-187Saccharomyces cerevisiae3.39VSK-188Saccharomyces cerevisiae3.07VSK-189Saccharomyces cerevisiae3.71VSK-190Saccharomyces cerevisiae3.13VSK-191Saccharomyces pastorianus2.04VSK-192Saccharomyces cerevisiae3.14VSK-193Saccharomyces cerevisiae3.99VSK-194Saccharomyces cerevisiae3.53VSK-195Saccharomyces cerevisiae3.90VSK-196Saccharomyces cerevisiae3.46VSK-197Saccharomyces cerevisiae2.99VSK-198Saccharomyces cerevisiae4.11VSK-199Saccharomyces cerevisiae3.93VSK-200Saccharomyces cerevisiae3.40VSK-201Saccharomyces cerevisiae1.83VSK-202Saccharomyces cerevisiae2.96VSK-203Saccharomyces cerevisiae2.64VSK-204Saccharomyces cerevisiae2.28VSK-205Saccharomyces cerevisiae3.42VSK-206Saccharomyces cerevisiae3.17VSK-207Saccharomyces cerevisiae3.13VSK-208Saccharomyces cerevisiae3.69VSK-209Saccharomyces cerevisiae3.64VSK-210Issatchenkia orientalis3.07VSK-211Issatchenkia orientalis2.40VSK-212Issatchenkia orientalis2.44VSK-213Issatchenkia orientalis2.60VSK-214Issatchenkia orientalis2.85VSK-215Issatchenkia orientalis2.35VSK-216Issatchenkia orientalis3.04VSK-217Zygosaccharomyces kombulaensis0.67VSK-218Candida glabrata3.67VSK-219Candida glabrata3.38VSK-220Kazaxantania exigua3.89VSK-221Kazaxantania exigua3.89VSK-222Issatchenkia orientalis2.01VSK-223Issatchenkia orientalis3.13VSK-224Issatchenkia orientalis2.60VSK-225Kazaxantania exigua3.26VSK-226Pichia membranifaciens3.11VSK-227Pichia membranifaciens3.18VSK-228Pichia membranifaciens2.76VSK-229Pichia membranifaciens3.26VSK-230Pichia membranifaciens3.35VSK-231Pichia membranifaciens3.13VSK-232Pichia membranifaciens3.06VSK-233Pichia membranifaciens3.18VSK-234Pichia membranifaciens2.56VSK-235Kazaxantania exigua4.13VSK-236Saccharomycodes ludwigii2.94VSK-237Zygosaccharomyces kombulaensis3.18VSK-238Zygosaccharomyces kombulaensis2.96VSK-239Candida glabrata3.49VSK-240Candida glabrata1.61VSK-241Issatchenkia orientalis2.79VSK-242Kazaxantania buldereri3.38VSK-243Kazaxantania buldereri3.60VSK-244Candida magnoliae3.08VSK-245Issatchenkia orientalis1.63VSK-246Kazaxantania buldereri3.44VSK-247Issatchenkia orientalis0.00VSK-248Issatchenkia orientalis3.08VSK-249Issatchenkia orientalis3.07VSK-250Kazaxantania exigua3.00VSK-251Candida glabrata2.49VSK-252Issatchenkia orientalis2.35VSK-253Kazaxantania exigua2.33VSK-254Saccharomycodes ludwigii2.43VSK-255Issatchenkia orientalis2.68VSK-256Kazaxantania exigua3.56VSK-257Kazaxantania exigua4.00VSK-258Issatchenkia orientalis2.24VSK-259Issatchenkia orientalis2.79VSK-260Issatchenkia orientalis2.53VSK-261Pichia burtonii0.42VSK-262Candida boidinii1.86VSK-263Pichia kluyveri1.56VSK-264Torulaspora delbrueckii3.19VSK-265Kazaxtania servazzii2.40VSK-266Zygosaccharomyces rouxii1.15VSK-267Pichia fermentans2.00VSK-268Yarrowia lipolytica2.86VSK-269Candida boidinii2.46VSK-270Candida intermedia0.65VSK-271Candida parapsilosis1.58VSK-272Yarrowia lipolytica2.10VSK-273Candida parapsilosis2.24VSK-274Debaryomyces hansenii0.25VSK-275Pichia guilliermondii2.32VSK-276Kazaxtania servazzii0.72VSK-277Rhodotorula glutinis0.72VSK-278Cryptococcus albidus0.74VSK-279Rhodosporidium toruloidess0.67VSK-280Debaryomyces occidentalis1.17VSK-281Rhodotorula mucilaginosa1.54VSK-282Candida auringiensis1.58VSK-283Candida succinyla2.08VSK-284Ambrosiozymea monospora0.71VSK-285Candida arabinofermentans0.56VSK-286Kluyveromyces marxianus2.06VSK-287Lachancea thermotolerans2.28VSK-288Cryptococcus albidus0.25VSK-289Debaryomyces occidentalis1.76VSK-290Rhodotorula mucilaginosa2.85VSK-291Rhodotorula glutinis0.00VSK-292Zygosaccharomyces lentus0.33VSK-293Rhodosporidium toruloidess0.38VSK-294Cryptococcus albidus0.00VSK-295Torulaspora globosa2.07VSK-296Candida stellata1.36VSK-297Cryptococcus laurentii0.53VSK-298Williopsis saturnus0.76VSK-299Cysoxilobasidium bisporidii1.94VSK-300Cryptococcus curvatus1.78VSK-301Sporidiobolus salmonella salmonicolor2.17VSK-302Pichia jadini0.86VSK-303Geotrichum klebegii0.67VSK-304Cryptococcus laurentii0.19VSK-305Debaryomyces hansenii0.82VSK-306Yarrowia lipolytica1.36VSK-307Candida rugosa1.97VSK-308Candida pararugosa1.68VSK-309Debaryomyces occidentalis0.68VSK-310Arxula adenine vorans1.38VSK-311Pichia stipitis2.17VSK-312Cryptococcus albidus2.19VSK-313Candida haemoulonii2.18VSK-314Debaryomyces hansenii0.38VSK-315Pichia angusta0.82VSK-316Rhodotorula minuta0.40VSK-317Pichia mandshurica1.03VSK-318Zygosaccharomyces baiii0.94VSK-319Cryptococcus albidosimilis0.44VSK-320Cryptococcus wiringae0.61VSK-321Filobasidium globebiporum0.00VSK-322Filobasidium globebiporum0.03VSK-323Bulleromyces albus0.39VSK-324Candida anglica0.56VSK-325Candida anglica0.65VSK-326Candida fermentati2.28VSK-327Candida natalensis0.76VSK-328Candida pararugosa1.08VSK-329Candida picinguabensis0.71VSK-330Candida silvae1.07VSK-331Candida solani0.63VSK-332Candida cyclindracea1.22VSK-333Cryptococcus curvatus0.47VSK-334Cryptococcus macerans0.58VSK-335Cryptococcus macerans0.33VSK-336Cryptococcus magnus1.57VSK-337Cryptococcus magnus0.57VSK-338Cryptococcus victoriae0.74VSK-339Cryptococcus victoriae0.79VSK-340Cryptococcus wiringae1.19VSK-341Cryptococcus myceliis0.33VSK-342Dioszegia hungarica2.01VSK-343Hanseniaspora sp.2.32VSK-344Hanseniaspora uvarum2.89VSK-345Pichia fabianii0.85VSK-346Rhodotorula pinicola0.36VSK-347Rhodotorula pinicola0.39VSK-348Sporobolomyces ruberrimus0.50VSK-349Sporobolomyces roseus0.36VSK-350Williopsis californica0.60VSK-351Pichia pastoris0.64VSK-352Pichia pastoris1.01VSK-353Pichia pastoris0.96VSK-354Pichia mandshurica3.00VSK-355Pichia heedii1.26VSK-356Pichia punctisspora3.32VSK-357Kazaxantania1.19VSK-358Schizosaccharomyces pombe3.49VSK-359Torulaspora delbrueckii0.36VSK-360Yarrowia lipolytica2.82VSK-361Yarrowia lipolytica2.14VSK-362Zygosaccharomyces baiii2.08VSK-363Zygosaccharomyces baiii2.39VSK-364Zygosaccharomyces bisporus0.58VSK-365Candida fluviatilis0.76VSK-366Saccharomycopsis capsularis0.68VSK-367Zygosaccharomyces rouxii2.51VSK-368Candida fluviatilis0.68VSK-369Candida humilis0.36VSK-370Candida catenulata0.47VSK-371Debaryomyces hansenii0.38VSK-372Pichia guilliermondii1.90VSK-373Candida intermedia0.74VSK-374Candida lactis condensi0.78VSK-375Pichia fermentans1.22VSK-376Candida pignaliae0.60VSK-377Candida pseudolambica0.82VSK-378Candida rugosa2.69VSK-379Candida sorboxylosa2.33VSK-380Kregervanrija fluxuum0.28VSK-381Citeromyces matritensis0.60VSK-382Debaryomyces polymorphus1.11VSK-383Debaryomycessp.1.46VSK-384Dekkera anomalousa0.75VSK-385Dekkera bruxellensis0.67VSK-386Dekkera bruxellensis0.44VSK-387Pichia burtonii0.64VSK-388Pichia burtonii0.64VSK-389Kluyveromyces yarrowii0.53VSK-390Kodamaea ohmeri2.15VSK-391Metschnikowia pulcherrima0.83VSK-392Eromothecium coryli0.71VSK-393Pichia anomala1.22VSK-394Kluyveromyces marxianus2.07VSK-395Saturnisspora mendoncae0.61YSK-396Pichia minuta0.53VSK-397Pichia nakassei0.50VSK-398Pichia silvicola0.50VSK-399Pichia stipitis0.60VSK-400Pichia tannicola2.29VSK-401Pichia toletana0.00VSK-402Schizosaccharomyces japonicus0.17VSK-403Pichia hplophila0.46VSK-404Zygosaccharomyces baiii0.83VSK-405Zygosaccharomyces bisporus1.32VSK-406Bulleromyces albus0.00VSK-407Pseudozymea antarctica0.00VSK-408Pichia stipitis0.25VSK-409Cryptococcus wiringae0.08VSK-410Sporobolomyces ruberrimus3.10VSK-411Cryptococcus diffluens0.29VSK-412Cryptococcus curvatus0.33VSK-413Lipomyces tetrasporus0.46VSK-414Candida shehatae0.42VSK-415Lipomyces lipofere0.00VSK-416Lipomyces starkyi0.00VSK-417Candida apis1.21VSK-418Candida sorbophila1.08VSK-419Candida oleophilica0.75VSK-420Sporidiobolus salmonella salmonicolor0.50VSK-421Candida apicola1.79VSK-422Zygosaccharomyces lentus0.29VSK-423Candida saitoana0.33VSK-424Pichia guilliermondii1.86VSK 425Kluyveromyces lactis0.88VSK-426Pichia jadini1.65VSK-427Metschnikowia pulcherrima1.50VSK-428Toruloidosis0.29VSK-429Schizosaccharomyces japonicus1.29VSK-430Lachancea thermotolerans0.75VSK-431Candida saitoana0.71VSK-432Dekkera anomalousa0.42VSK-433Kluvveromyces marxianus1.69VSK-434Kluyveromyces marxianus1.17VSK-435Candida maltosa2.19VSK-436Pichia fabianii1.33VSK-437Candida viswanathii0.29VSK-438Candida catenulata0.42VSK-439Schizosaccharomyces pombe1.93VSK-440Kluyveromyces lactis0.29VSK-441Kazaxantania unispora3.28VSK-442Kazaxantania unispora3.24VSK-443Pachysolen tannophilus0.75VSK-444Pachysolen tannophilus0.92VSK-445Pichia subpelliculosa1.76VSK-446Trigonopsis variabilis1.44VSK-447Candida versatilis1.99VSK-448Pichia farinosa0.54VSK-449Pichia farinosa2.04VSK-450Kodamaea ohmeri2.25VSK-451Pichia triangaria2.08VSK-452Candida diddensiae2.17VSK-453Pichia quercum1.96VSK-454Sporidiobolus johnsonii0.65VSK-455Debaryomyces coudertii0.67VRK-456Candida apicola2.33VSK-457Candida humilis4.25VSK-458Rhodotorula mucilaginosa0.29VSK-459Dekkera anomalousa0.33VSK-460Zygosaccharomyces baiii1.18VSK-461Rhodotorula glutinis0.33VSK-462Sporobolomyces roseus0.25VSK-463Pichia anomala2.21VSK-464Candida zeylanoides2.03VSK-465Zygosaccharomyces rouxii2.29VSK-466Pichia anomala2.17VSK-467Zygosaccharomyces bisporus0.42VSK-468Lachancea fermentati1.51VSK-469Zygosaccharomyces rouxii0.46VSK-470Torulaspora microellipsoides0.67VSK-471Zygotorulaspora florentinus1.61VSK-472Zygosaccharomyces mellis0.33VSK-473Lachancea cidri2.26VSK-474Zygotorulaspora mrkakii2.18VSK-475Candida sake0.42VSK-476Candida silvae1.21VSK-477Sporopahydermia lactivora0.46VSK-478Sporopahydermia lactivora0.46VSK-479Clavisspora lusitaniae0.88VSK-480Cryptococcus laurentii0.46VSK-481Clavisspora lusitaniae0.63VSK-482Naumovia dairensis0.63VSK-483Candida membranifaciens0.46VSK-484Candida tenuis0.46VSK-485Candida membranifaciens0.46VSK-486Cystoilobasidium infirmo-miniatum0.50VSK-487Candida oleophilica1.08VSK-488Rhodotorula minuta0.42VSK-489Pichia farinosa2.29VSK-490Candida solani0.61VSK-491Candida sake0.63VSK-492Hanseniaspora uvarum2.21VSK-493Pichia angusta1.79VSK-494Candida entomophila0.50VSK-495Candida methanosorbosa0.46VSK-496Candida diddensiae0.42VSK-497Candida sonoensis1.50VSK-498Saccharomyces cerevisiae0.88VSK-499Zygosaccharomyces kombulaensis1.10VSK-500Candida mesenterica0.54VSK-501Pichia punctisspora0.54VSK-502Pichia sp.1.63VSK-503Pichia sp.2.97VSK-504Saccharomyces paradoxus2.07VSK-505Pichia fermentans1.03VSK-506Kregervanrija fluxuum2.58VSK-507Zygosaccharomyces mellis1.46VSK-508Lachancea fermentati2.25VSK-509Cryptococcus liquuffaciens0.17VSK-510Filobasidium capsuligenum0.36VSK-511Wickerhamomyces abnormalus1.81VSK-512Didopatascus ingens2.00VSK-513Candida santamariae0.42VSK-514Filobasidium capsuligenum1.06VSK-515Didopatascus ingens2.50VSK-516Filobasidium capsuligenum0.58VSK-517Candida anatoniae0.64VSK-518Lindnera fabianii1.15VSK-519Pichia mexicana0.46VSK-520Sporopahydermia cereana2.10VSK-521Sporopahydermia cereana0.33VSK-522Candida sonoensis1.54VSK-523Pichia cactophila1.47VSK-524Pichia cactophila1.25VSK-525Saccharomyces pastorianus2.63VSK-526Saccharomyces cerevisiae1.72VSK-527Saccharomyces cerevisiae2.50VSK-528Saccharomyces cerevisiae2.25VSK-529Saccharomyces cerevisiae2.75VSK-530Saccharomyces cerevisiae2.71VSK-531Saccharomyces cerevisiae1.54VSK-532Saccharomyces cerevisiae2.79VSK-533Saccharomyces cerevisiae3.07VSK-534Saccharomyces cerevisiae2.38VSK-535Saccharomyces cerevisiae2.22VSK-536Saccharomyces cerevisiae2.47VSK-537Saccharomyces cerevisiae3.08VSK-538Saccharomyces cerevisiae2.35VSK-539Saccharomyces cerevisiae2.82VSK-540Saccharomyces cerevisiae2.83VSK-541Saccharomyces cerevisiae2.88VSK-542Saccharomyces cerevisiae2.50VSK-543Saccharomyces cerevisiae2.44VSK-544Saccharomyces cerevisiae2.63VSK-545Saccharomyces cerevisiae3.04VSK-546Saccharomyces cerevisiae2.83VSK-547Saccharomyces cerevisiae2.44VSK-548Saccharomyces cerevisiae2.44VSK-549Saccharomyces cerevisiae2.79VSK-550Saccharomyces cerevisiae2.93VSK-551Saccharomyces cerevisiae3.15VSK-552Saccharomyces cerevisiae2.83VSK-553Saccharomyces cerevisiae2.85VSK-554Saccharomyces cerevisiae3.19VSK-555Saccharomyces cerevisiae2.96VSK-556Saccharomyces cerevisiae2.92VSK-557Saccharomyces cerevisiae2.56VSK-558Saccharomyces cerevisiae2.21VSK-559Saccharomyces cerevisiae2.24VSK-560Saccharomyces cerevisiae2.96VSK-561Saccharomyces cerevisiae3.33VSK-562Saccharomyces cerevisiae2.97VSK-563Saccharomyces cerevisiae3.22VSK-564Saccharomyces cerevisiae3.32VSK-565Saccharomyces cerevisiae3.35VSK-566Saccharomyces cerevisiae3.36VSK-567Saccharomyces cerevisiae3.18VSK-568Saccharomyces cerevisiae3.29VSK-569Saccharomyces cerevisiae3.22VSK-570Saccharomyces cerevisiae2.81VSK-571Saccharomyces cerevisiae3.75VSK-572Saccharomyces cerevisiae3.07VSK-573Saccharomyces cerevisiae3.36VSK-574Saccharomyces cerevisiae3.22VSK-575Saccharomyces cerevisiae3.07VSK-576Saccharomyces cerevisiae2.93VSK-577Saccharomyces cerevisiae3.60VSK-578Saccharomyces cerevisiae3.60VSK-579Saccharomyces cerevisiae3.00VSK-580Saccharomyces cerevisiae2.99VSK-581Saccharomyces cerevisiae3.50VSK-582Saccharomyces cerevisiae3.58VSK-583Saccharomyces cerevisiae3.11VSK-584Saccharomyces cerevisiae3.75VSK-585Saccharomyces cerevisiae3.43VSK-586Saccharomyces cerevisiae2.93VSK-587Saccharomyces cerevisiae3.08VSK-588Saccharomyces cerevisiae3.60VSK-589Saccharomyces cerevisiae3.79VSK-590Saccharomyces cerevisiae2.96VSK-591Saccharomyces cerevisiae3.18VSK-592Saccharomyces cerevisiae3.47VSK-593Saccharomyces cerevisiae3.07VSK-594Saccharomyces cerevisiae3.33VSK-595Saccharomyces cerevisiae3.79VSK-596Saccharomyces cerevisiae3.51VSK-597Saccharomyces cerevisiae3.32VSK-598Saccharomyces cerevisiae3.53VSK-599Saccharomyces cerevisiae2.46VSK-600Saccharomyces cerevisiae3.49VSK-601Saccharomyces cerevisiae3.36VSK-602Saccharomyces cerevisiae3.51VSK-603Saccharomyces cerevisiae3.64VSK-604Saccharomyces cerevisiae3.50VSK-605Saccharomyces cerevisiae1.50VSK-606Saccharomyces cerevisiae3.92VSK-607Saccharomyces cerevisiae3.28VSK-608Saccharomyces cerevisiae3.32VSK-609Saccharomyces cerevisiae3.38VSK-610Saccharomyces cerevisiae3.39VSK-611Saccharomyces cerevisiae3.56VSK-612Saccharomyces cerevisiae3.24VSK-613Saccharomyces cerevisiae3.21VSK-614Saccharomyces cerevisiae3.32VSK-615Saccharomyces cerevisiae3.40VSK-616Saccharomyces cerevisiae3.24VSK-617Kluyveromyces marxianus1.46VSK-618Kluyveromyces marxianus2.89VSK-619Kluyveromyces marxianus1.13VSK-620Kluyveromyces marxianus1.33VSK-621Kluyveromyces marxianus1.46VSK-622Kluyveromyces marxianus1.46VSK-623Kluyveromyces marxianus0.92VSK-624Kluyveromyces marxianus0.96VSK-625Kluyveromyces marixanus1.39VSK-626Kluyveromyces marxianus0.92VSK-627Kluyveromyces marxianus1.71VSK-628marxianus2.17VSK-629Kluvveromyces marxianus2.40VSK-630Kluyveromyces marxianus1.36VSK-631Kluyveromyces marxianus1.40VSK-632Kluyveromyces marxianus1.61VSK-633Kluyveromyces marxianus0.75VSK-634Kluyveromyces marxianus0.83VSK-635Pichia fermentans1.33VSK-636Pichia fermentans1.92VSK-637Pichia fermentans1.81VSK-638Pichia fermentans2.24VSK-639Pichia fermentans0.96VSK-640Pichia fermentans1.72VSK-641Pichia fermentans0.33VSK-642Debaryomyces hansenii0.46VSK-643Debaryomyces hansenii0.54VSK-644Debaryomyces hansenii0.08VSK-645Debaryomyces hansenii2.83VSK-646Debaryomyces hansenii1.46VSK-647Debaryomyces hansenii1.74VSK-648Debaryomyces hansenii0.33VSK-649Debaryomyces hansenii0.50VSK-650Debaryomyces hansenii0.38VSK-651Debaryomyces hansenii0.71VSK-652Debaryomyces hansenii0.83VSK-653Debaryomyces hansenii1.04VSK-654Debaryomyces hansenii0.33VSK-655Debaryomyces hansenii0.42VSK-656Saccharomyces pastorianus1.25VSK-657Saccharomyces pastorianus1.04VSK-658Saccharomyces pastorianus2.51VSK-659Saccharomyces pastorianus2.54VSK-660Trichosporon pullulans2.60VSK-661Candida sake0.63VSK-662Candida sake1.17VSK-663Cryptococcus tephrensis0.63VSK-664Cryptococcus tephrensis0.50VSK-665Trichosporon brassicae0.38VSK-666Candida sp.2.46VSK-667Yarrowia lipolytica2.74VSK-668Candida zeylanoides2.08VSK-669Torulopsis sp.1.07VSK-670Meyerozymea guilliermondii2.17VSK-671Wickerhamomyces abnormalus1.38VSK-672Yarrowia lipolytica2.94VSK-673Candida boidinii1.17VSK-674Pichia membranifaciens1.63VSK-675Pichia membranifaciens2.88VSK-676Candida sp.2.36VSK-677Magnusiomyces ingens0.75VSK-678Trichosporon dulcitum0.83VSK-679Schefferomyces stipitis0.71VSK-680Yarrowia lipolytica2.99VSK-681Hanseniaspora uvarum1.83VSK-682Hanseniaspora sp.2.63VSK-683Priceomyces carsonii0.67VSK-684Trichomonascus ciferrii1.29VSK-685Trichosporon veenhuisii0.42VSK-686Sugiyamaella smithiae0.38VSK-687Trichosporon aquaticile0.71VSK-688Schwanniomyces polymorphus2.82VSK-689Priceomyces hplophilus1.92VSK-690Debaryomyces robertsiae2.38VSK-691Candida rhagii0.71VSK-692Metschnikowia reukufii0.88VSK-693Metschnikowia agaves0.88VSK-694Komagataella pastoris0.33VSK-695Lodderomyces elongisporus1.04VSK-696Saccharomyces eubayanus1.63VSK-697Candida parapsilosis2.04VSK-698Torulaspora delbrueckii3.14VSK-699Torulaspora delbrueckii1.29VSK-700Hanseniaspora uvarum2.00VSK-701Hanseniaspora osmophila2.54VSK-702Zygosaccharomyces rouxii0.92VSK-703Cryptococcus flavescens1.13VSK-704Torulaspora delbrueckii2.36VSK-705Wickerhamomyces abnormalus2.22VSK-706Rhodosporidium toruloidess0.75VSK-707Candida sp.0.71VSK-708Pichia jadini1.13VSK-709Pichia jadini1.17VSK-710Candida humilis3.60VSK-711Pichia membranifaciens3.04VSK-712Rhodosporidium toruloidess1.79VSK-713Candida zeylanoides2.64VSK-714Candida diddensiae0.46VSK-715Pichia membranifaciens1.67VSK-716Saccharomyces cerevisiae1.92VSK-717Candida mesenterica2.13VSK-718Saccharomyces cerevisiae2.79Example 2: Bioinformatic genome mining in candidate metabolic enzymesHomology-based database searches were performed to identify candidate enzymes for a particular functionality. The databases were each searched with a series of query sequences. In addition, the members of the relevant protein family were retrieved from UniProt / SwissProt based on the InterPro domain entries. Homology based searches were performed against UniProt protein databases (SwissProt and TrEMBL) and GenBank protein databases (nr, pat and env_nr) by blastp, and against GenBank nucleotide databases (tsa_nt, env_nt and pat) by tblastn. Sequences having an E value less than 1e-30 were extracted; however, in some cases additional analysis was performed on sequences having an E value less than 1e-80. Nucleotide hits were translated into protein sequences with GeneWise using the query sequence as the leader sequence in translation. The task of translation of long ACC sequences was too difficult for GeneWise, so instead the protein sequence (part matching the query sequence) was extracted from the data output of blast xml. In order to remove unnecessary sequences, the retrieved sequences were clustered, by means of BLASTCLUST or CD-HIT, into clusters with sequences that are more than 80% identical to each other. Only one representative sequence was retained from each cluster. The unnecessary set of sequences was aligned, either with the PFAM domain of the protein family or by MAFFT. World-wide alignment by MAFT was established in cases where the family of proteins was unrelated to one PFAM domain or where the sequence of the protein was divided into several PFAM domains. A phylogenetic tree was generated by multiple sequence matching using PHYLIP or FASTTREE. The tree was annotated with EC numbers, organism name, blast E values and visualized using the Genious software.2-1: Acetylating Acetaldehyde DehydrogenaseThe reduction of acetaldehyde to acetyl-CoA can be achieved by acetylating acetaldehyde dehydrogenase (AADH, E.C. 1.2.1.10).AADHs can be divided into three groups of functional homologues (Wei et al., Nat. Commun. 4:2580, 2013), among them, 1) bifunctional proteins having AADH and alcohol dehydrogenase activities (adhE type E. coligenes, GenBank No.: NP_415757 query sequence), 2) proteins involved in ethanolamine catabolism (euE type E. coligenes, GenBank No.: AAG57564 query sequence), and 3) bifunctional proteins which are part of an aldolase dehydrogenase complex involved in 4-hydroxy-2-ketovalerate catabolism (mphF type E. coligenes, GenBank No.: NP_414885). Of particular interest are the enzymes of Group 1 (adhE type) and Group 2 (eutE type).The N-terminal domain of the AdhE protein is highly homologous to aldehyde:NAD+ oxidoreductases, whereas the C-terminal region is homologous to a family of Fe2+-dependent ethanol:NAD+ oxidoreductases (Membrillo-Hernandez et al., J. Biol. Chem. 275:33869-33875, 2000). Acetylating acetaldehyde dehydrogenase activity can also be introduced into the cell by truncation of the bifunctional AdhE protein to possess only the N-terminal aldehyde reductase domain by removal of the alcohol dehydrogenase domain. Other genes with this bifunctional AADH activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 1.Many enterobacteria can use ethanolamine as a carbon and nitrogen source (Stojiljkovic et al., J. Bacteriol. 177:1357-1366, 1995). This catabole complement pathway includes a step in which acetaldehyde is converted to acetyl-CoA by acetylating acetaldehyde dehydrogenase, EutE. Novel genes with this AADH activity have been inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 2.Moreover, based on bioinformatic analyses and sequence homology to the adhE and eutE type genes, there is a group of genes noted as aldehyde dehydrogenases which can be concluded to have AADH activity. The various genes are summarized in Table 3.2-2: Eukaryotic Acetyl-CoA CarboxylaseAcetyl-CoA carboxylase (ACC, EC 6.4.1.2) is a multifunctional biotin-dependent carboxylase which is a central enzyme of fatty acid biosynthesis. It uses cofactors ATP and biotin to catalyze the conversion of acetyl-CoA to malonyl-CoA. The reaction proceeds in two steps. First, biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin with bicarbonate. Second, the carboxyl transferase transfers the carboxyl group from biotin to acetyl-CoA to form malonyl-CoA. Eukaryotic enzymes are large multidomain enzymes, whereas corresponding prokaryotic enzymes consist of several subunits encoded by different genes.The activity of ACC is regulated at the transcriptional level and also at the posttranscriptional level (e.g., by phosphorylation and aggregation) to obtain the acetyl-CoA homeostasis. ACC manipulation in various yeast species resulted in increased ACC activity and increased production of malonyl-CoA-derived products. Genes encoding enzymes having ACC activity have been detected or postulated in Saccharomyces cerevisiae(GenBank No.: CAA96294.1, query sequence), Yarrowia lipolytica(GenBank No.: XP_501721.1, query sequence) and Mucor circinelloides(GenBank No.: EPB82652.1 query sequence). Candidate acetyl-CoA carboxylase genes were identified in the newly sequenced genomes of Kazaxantania exigua(SEQ ID NO:1) and Candida humilis(SEQ ID NO:2) and cloned into our yeast expression plasmid. Other genes with ACC activity have been inferred based on bioinformatic analysis and sequence homology. The various multidomain eukaryotic ACC genes are summarized in Table 4.2-3: Bifunctional malonyl-CoA reductaseThe reduction of malonyl-CoA to 3-HP (via a malonate semialdehyde intermediate) can be achieved by a large bifunctional malonyl-CoA reductase having both functionalities of the C-terminal aldehyde dehydrogenase domain and N-terminal alcohol dehydrogenase domain. A highly substrate specific and NADPH dependent enzyme having this activity has been described in the nonsulfur phototrophic green bacterium Chloroflexus aurantiacus(GenBank No.: AAS20429.1; query sequence) involved in an autotrophic CO 2- fixation pathway called the 3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410, 2002). Other genes with this bifunctional malonyl-CoA reductase activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 5.2-4: Malonyl-CoA reductaseIn contrast to the bifunctional malonyl-CoA reductases discussed above, malonyl-CoA can also be catalyzed by two separate enzymes to 3-HP. By this route, malonyl-CoA is first reduced by malonyl-CoA reductase to malonate semialdehyde (MCR; EC 1.2.1.75) or a CoA acylating malonate semialdehyde dehydrogenase and then reduced by a 3-hydroxypropionate dehydrogenase to 3-HP (3-HPDH; EC 1.1.1.59 or EC 1.1.1.298). MCR is a NADPH-dependent enzyme used by some thermoacidophilic archaea to fix carbon autotrophically in organic material via a 3-hydroxypropionate / 4-hydroxybutyrate cycle (Berg et al., Science, 318:1782-1786, 2007). Genes encoding enzymes having this MCR activity are described in Metallosphaera sedula(GenBank No.: ABP94884.1, query sequence) and Sulfolobus tokodaii(GenBank No.: BAB67276.1 query sequence). Although these MCRs share similar aldehyde dehydrogenase activity with the bifunctional malonyl-CoA reductase enzymes of Chloroflexus aurantiacus, they do not share significant sequence similarity, suggesting that the autotrophic complement pathways have developed convergently in chloroflexus and sulfolobaceae and that various genes have been recruited to perform similar metabolic processes in these taxonomic groups (Alber et al., J. Bacteriol. 188:8551-8559, 2006). In particular, the archaea MCRs show a high sequence similarity to aspartate semialdehyde dehydrogenases. Other genes with this MCR activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 6.2-5: 3-Hydroxypropionate dehydrogenaseMalonate semialdehyde can be reduced by a reversible 3-hydroxypropionate dehydrogenase to 3-HP (HPDH; EC 1.1.1.59, NADH-dependent) or a malonate semialdehyde reductase (EC 1.1.1.298, NADPH-dependent). These enzymes are naturally involved in beta-alanine metabolism, propanoate metabolism or uracil degradation in bacteria and plants. Moreover, these enzymes are required for some thermoacidophilic archaea for fixing carbon via the 3-hydroxypropionate / 4-hydroxybutyrate cycle (Kockelkörn and Fuchs, J. Bacteriol. 191:6352-6362, 2009). Genes encoding enzymes having 3-hydroxypropionate dehydrogenase or malonate semialdehyde reductase activity were detected or postulated in Escherichia coli(GenBank No.: EFV00080.1, query sequence), Saccharomyces cerevisiae(GenBank No.: DAA10125.1, query sequence), Metallosphaera sedula(GenBank No.: ABP96133.1, query sequence), Sulfolobus tokodaii(GenBank No.: BAK54608.1, query sequence) and Escherichia coli(GenBank No.: ACR64730.1 query sequence). Other genes with 3-hydroxypropionate dehydrogenase or malonate semialdehyde reductase activity were inferred on the basis of bioinformatic analyses and sequence homology. The various genes are summarized in Table 7.2-6: 3-Hydroxyisobutyrate dehydrogenase3-Hydroxyisobutyrate dehydrogenase (HIBADH; EC 1.1.1.31) is a central enzyme involved in the metabolism of valine and the other branched chain amino acids. HIBATHH catalyzes the NADH or NADPH dependent reversible conversion of 3-hydroxyisobutyrate to methylmalonate semialdehyde. However, as a result of its broad substrate specificity, HIBATHH has also been shown to have 3-hydroxypropionate dehydrogenase activity (i.e., EC 1.1.1.59) by conversion of malonate semialdehyde to 3-HP (Yao et al., Appl. Biochem. Biotechnol. 160:694-703, 2010). Enzymes having HIBADH activity were identified in Pseudomonas putida(GenBank No.: ADR61938.1, query sequence), Pseudomonas aeruginosa(GenBank No.: AAG06957.1, query sequence), Bacillus cereus(GenBank No.: AAP10961.1, query sequence) and Alcaligenes faecalis(GenBank No.: EJC65559.1, query sequence). Other genes with this HIBADH activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 8.2-7: 4-Hydroxybutyrate dehydrogenase4-Hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.61) is an enzyme naturally involved in butanoate metabolism. HBDH catalyzes the reversible NAD+-dependent conversion of 4-hydroxybutanoate to succinate semialdehyde. However, HBDH can also convert malonate semialdehyde to 3-HP because the enzymatic reaction is similar. Enzymes with HBDH activity were identified in Cupriavidus necator (GenBank No.: AAC41425.1, query sequence) and Clostridium kluyveri (GenBank No.: EDK35022.1 query sequence). Other genes with this HBDH activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 9.2-8: 3-Hydroxybutyrate Dehydrogenase3-Hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) is an enzyme naturally involved in butanoate metabolism. BDH catalyzes the reversible NAD+-dependent conversion of 3-hydroxybutyrate to acetoacetate, but it may also oxidize other 3-hydroxy monocarboxylic acids. For example, BDH can convert malonate semialdehyde to 3-HP because the enzymatic reaction is similar. An enzyme having BDH activity was identified in Pseudomonas aeruginosa(GenBank No.: GAA17557.1 query sequence). Other genes with this BDH activity were inferred based on bioinformatic analysis and sequence homology. The various genes are summarized in Table 10.Example 3: Measurement of enzyme activitiesACC Spectrophotometric Enzyme AssaysSpectrophotometric ACC assays are coupled assays in which a product produced by the ACC reaction is further consumed in a reaction requiring cofactor NAD(P)H whose oxidation can be monitored with a spectrophotometer.Krieger et al. (2011, Anal. Biochem. 411:100-105) have described a coupled assay in which malonyl-CoA produced by ACC1 is further converted to malonate semialdehyde by purified malonyl-CoA reductase (MCR) in a reaction that requires NADPH as a cofactor. ACC activity was measured by following NADPH oxidation.Diakovich et al. (2002, J. Biol. Chem. 277 :31228-31236) combined the conversion of ADP, a hydrolysis product of ATP used as cofactor in the ACC reaction, with an ADP-requiring pyruvate kinase reaction which was further coupled to the formation of pyruvate using lactate dehydrogenase. The latter enzyme requires NADH as cofactor, the oxidation of which has been followed.ACC Radioactive Enzyme AssaysThe most commonly used in vitro ACC assay is based on the use of radioactive C 14- carbonate. The incorporation of radioactive carbonate in an acid and a nonvolatile (i.e., malonyl-CoA) is followed. The 14 C-labeled sodium bicarbonate that has not been converted to malonyl-CoA is removed by an acid and heat treatment that converts the remaining NaH 14 CO 3 and the possible byproducts of the reaction to 14 C-labeled CO 2.This is described by Diakovich et al. (2002, J. Biol. Chem. 277:31228-31236) was used with minor changes to detect ACC activity in yeast lysates (Shi et al. 2014, mBIO 5:3 e01130-14). Cell lysates were prepared from yeast cells recovered during the late exponential or stationary phase. Cells were washed and then resuspended in lysis buffer containing 100 mM potassium phosphate pH 7.5, 2 mM MgCl 2, 1 mM dithiothreitol and 1x EDTA-free complete protease inhibitor (Roche). The cells were broken by glass beads and the supernatant was collected after centrifugation at 4°C.The ACC enzyme assay reaction mixture had 100 mM potassium phosphate (pH 8.0), 300 μg BSA, 3 mM ATP, 5 mM MgCl 2, 10 mM NaH 14 CO 3[ specific activity 200 μCi mmol -1(7400 kBq mmol)] and 0.5 mM acetyl-CoA. The total volume of the reaction was 100 μl comprising 20 μl of cell extract.The reaction was incubated at 30°C for 15 min and stopped by adding 50 μl of 5M HCl. The contents of the tubes were evaporated to dryness at 95°C and the residue was resuspended in 100 μl of water and mixed with 3 ml of scintillation cocktail (Ultima Gold AB, PerkinEl). The 14 C content of the samples was determined using a liquid scintillation counter (PerkinEl Tri-Carb 2810TR).AADH Enzyme AssayAADH activity was determined as described by Kozak et al. (2014, Metab. Eng. 21:46-59), by monitoring the reduction of NAD + at 340 nm at 30° C. Yeast cells for the cell lysates were collected, washed with water and then resuspended in lysis buffer containing 100 mM Tris-HCl buffer (pH 7.5) and 1X EDTA free protease inhibitor cocktail (Roche). The cells were lysed with glass beads in a Precellys 24 homogenizer at 5500 U / min for 3x40 seconds and maintained on ice between rounds. Lysates were centrifuged at 16,000 g for 20 min at 4°C and supernatants were collected. Total protein concentration was determined using the Bradford method.The enzyme assay reaction mixture contained 0.1 mM coenzyme A, 50 mM CHES buffer (pH 9.5), 0.8 mM NAD +, 0,2 mM DTT, and 10 μl of cell extract in a total reaction volume of 200 μl. The reaction was started by adding 10 mM freshly prepared acetaldehyde solution and the reduction of NAD + was monitored with a Thermo Konelab 20XT analyzer.MCR Enzyme Assay from Yeast Cell LysatesMCR enzyme activity was assayed according to the method described by Chen et al. (2014, Metab. Eng. 22:104-109) with minor changes. The method is based on monitoring the oxidation of NAD(P)H at 340 nm.Cells were collected and washed with cold wash buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM NaN 3 and then resuspended in 1 ml of degradation buffer containing 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM DTT, 1 mM EDTA, 0.5% Triton X-100, and 1X EDTA-free protease inhibitor cocktail (Roche). The cells were lysed with glass beads in a Precellys 24 homogenizer at 5500 U / min for 3x40 seconds and maintained on ice between rounds. Lysates were centrifuged at 16,000 g for 20 min at 4°C and supernatants were collected. Total protein concentration was determined using the Bradford method.The MCR assay mixture contained 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl 2, 0,3 mM NADPH or NADH. After pulling 20 μl of cell lysate into a total reaction volume of 200 μl, the reaction was pre-incubated for five minutes at 30°C, after which the reaction was started by addition of 0.15 mM malonyl-CoA. The assay was monitored at 340 nm with a Thermo Konelab 20XT analyzer.Preparation of yeast expression vectorsA series of yeast expression plasmids were prepared to evaluate the candidate genes for their expression and activity capabilities in yeast. First, a new pBlueScript-based polylinker (MCS) was designed so that all possible restriction enzyme (RE) site combinations could be used. This altered MCS was then inserted into the pRS-based series of centromeric and multicopy yeast plasmids. Subsequently, using a set of 10 unique RE sites, nine different sets of promoters and terminators were cloned into these pRS-based yeast expression vectors. Thus, this plasmid system could be utilized to constituently express up to 9 genes simultaneously, with either a low or high copy number, in a suitable yeast strain to evaluate a variety of complement pathway enzyme combinations for the production of 3-HP.Cloning of ACC genesACC1 genes transformed into the industrial S. cerevisiaeVSK-128 strain with and without SNF1 deletion are shown in Table 13. ACC genes were expressed from a multicopy plasmid where they were under the control of the PDC1 promoter. Mutant ACC1sc genes described in a publication by Shi et al. (2014) were constructed with a QuikChange II site mutagenesis kit (Agilent Technologies). Table 13. acetyl-CoA carboxylase genes converted to S. cerevisiaeVSK-128. Table 13. acetyl-CoA carboxylase genes converted to S. cerevisiaeVSK-128.ACC1scS. cerevisiae wild type ACC1ACC1sc S659AS. cerevisiae S659A mutant ACC1ACC1sc S1157AS. cerevisiae S1157A mutant ACC1ACC1sc S659A / S1157AS. cerevisiae S659A / S1157A mutant ACC1ACC1chC. humilis wild type ACCACC1keK. exigua wild type ACCACC1mcM. Circularloides wild type ACCACC1ylY. lipolytica wild type ACCACC Enzyme AssaysSpectrophotometric assays have been used in the literature to detect ACC enzyme activity with purified or partially purified ACC enzymes. Spectrophotometric assays were tested with yeast cell lysates in which ACC was overexpressed but no absorbance change was detected compared to the controls.The radioactive ACC enzyme assay is very sensitive, although pmoles / mg / min. activities can be detected. The procedure was tested for the first time with purified human ACC enzyme which is commercially available. When clear activity was found, the method was further optimized to detect ACC activity from yeast cell lysates. The strains listed in Table 13 were tested for their ACC1 activity and on the basis of the results; a list describing the relative ranking of the ACC1s (Fig. 2) was prepared. The most prospective candidate ACCyl was further examined and the results of the wild type S. cerevisiae S-128 strain and the same strain in which ACC1 was overexpressed are presented in Table 14. Table 14. Overexpression of the ACCyl gene resulted in a 2.9 fold increase in ACC1 enzyme activity compared to the endogenous ACC1 activity of the wild-type S. cerevisiaeVSK-128 strain. Table 14. Overexpression of the ACCyl gene resulted in a 2.9 fold increase in ACC1 enzyme activity compared to the endogenous ACC1 activity of the wild-type S. cerevisiaeVSK-128 strain.S. cerevisiae VSK-128 wild type25,550,742S. cerevisiaeVSK-128 (ACClyl)73,7917,234AADH In Vitro Enzyme Activity AssaysFive AADH genes were originally selected (i.e., ADHEpm, ADHEec, EUTec, EUTdz, and LIN1129li) and were converted to the CEN.PK laboratory strain. These AADHs were expressed from a multicopy plasmid where they were under control of the TEF1 promoter. All five AADH genes exhibited AADH activity, but the three eutE type AADH genes (i.e., EUTec, EUTdz, and LIN1129li) gave much higher AADH activity in yeast compared to adhE type AADHs (i.e., ADHEpm and ADHEec).Twenty additional novel AADH genes were selected from genomics to be evaluated for expression and enzyme activity in yeast. These twenty novel AADH genes were tested for in vitroenzyme activity and four of them (i.e., AADHmm, AADHab, AADHw and AADHvs) were shown to have AADH activity.AADH In Vivo Growth AssaysAll copies of the ACS2 genes were removed from the industrial S. cerevisiaeVSK-128 strain to produce a strain with defective PDH bypass that was unable to grow on glucose-based medium. Expression vectors carrying 25 different AADH variants were then converted to this ACS2 deletion strain to assess their ability to restore the growth of this strain to glucose.Twelve different AADH variants (i.e., EUTEec, EUTEdz, LIN 1129li, AADHmm, AADHtl, AADHab, AADHta, AADHbs, AADHw, AADHvs, AADHhs and ADHEec) were able to restore the growth deficit of the ACS2 deletion strain based on their ability to grow on glucose-based agar plates. There was a good correlation between the in vitroAADH enzyme activity assays and the in vivo growth recovery analyses.The growth rate of the first nine AADH variant strains found to be capable of restoring the growth of the ACS2 deletion strain were then evaluated in shake flask cultures and compared to the wild type strain containing the intact PDH bypass. All nine of these AADH variants were capable of obtaining >50% of the aerobic growth rate of the S. cerevisiaeVSK-128 strain when they grew on glucose, and six of these AADH variants were capable of obtaining ≥80% of the aerobic growth rate of the S. cerevisiaeVSK-128 strain.MCR Enzyme Assays in YeastsThe eight full length chloroflexus MCR homologs were truncated into their two functional domains and the MCR-specific domains were tested for enzyme activity in yeast along with the six archaeal MCRs. These MCRs were expressed from a multicopy plasmid where they were under control of the TEF1 promoter and were converted to the CEN.PK laboratory strain.MCRca (Chloroflexus aurantiacus) and its homologue MCRrc (Roseiflexus castellholzii) were the only two Chloroflexus MCR homologues which, using NADPH as cofactor, gave MCR enzyme activities higher than the wild type strain. No enzyme activity was observed in these eight chloroflexus MCR homologues using NADH as cofactor.Three of the archaea MCRs [Sulfolobales archaeon, Sulfolobalus acidocaldarius(x2)] gave MCR enzyme activities that were higher than the wild type strain when using NADPH as cofactor, and all six of the archaea MCRs gave MCR enzyme activities that were higher than the wild type strain when using NADH as cofactor.Heterologous Expression and Characterization of Archaea MCRsExpression constructs based on pBAT T7 promoters were prepared for four different MCRs from Archaea, Metallosphaera sedula (MCRms), Sulfolobus tokodai (MCRst), Candidatus caldarchaeum (MCRcc) and Sulfolobales archaeon (MCRsa1). These constructs contain no purification tag and were codon optimized for E. coliand were expressed under the conditions described previously in this report (Table 15). Their activity was analyzed using the following assay conditions: 0.4 mM NAD(P)H; 0.15 mM malonyl-CoA; Tris-HCl pH 7; 2 mM MgCl 2. Assays with 20 fold diluted lysates were performed in microtiter plate (MTP) format at RT. The oxidation of NADPH or NADH on A 365 was followed over time. MCR2 and MCRst showed the highest MCR activity of the four archaea genes tested. However, the MCR activities were less than those measured for the tagged MCRca. No activity could be measured on NADPH or NADH at the Candidatus caldiarachaeumMCR (MCRcc) (in the E. coli cell lysate).When these constructs were analyzed using SDS-PAGE gels, MCRsal showed the highest levels of expression in the E. coli lysate, while MCRcc could not be expressed in soluble form; see the gel (sa1>ca>st>ms>cc) (Figure 3). Constructs MCR2 and MCRst appear to have a dual cofactor preference and exhibited about 40-50% relative NADH activity compared to that measured on NADPH. MCRst may be the most active enzyme of the four archaeal MCRs tested in terms of specific activity, as it showed a relatively high level of activity at a relatively low level of expression.Example 4: Production of 3-HP by culturing recombinant yeast Culturing S. cerevisiae in shake flaskA small loop of cells was taken from strains freshly grown on selective agar-based plates and used to inoculate 20 mL of selective SC-based medium (20 g / L glucose) in a 250 mL flask and grown for 2 days (30°C, 250 rpm) until all the glucose and ethanol were consumed. The final cell density was measured and the cultures were centrifuged at 4000 rpm for 5 min. The supernatants were then analyzed by HPLC or GC / MS to determine the accumulation of 3-HP and other major metabolites in the culture supernatants. However, depending on the particular strain and target (e.g., starting amount of glucose, amount of aeration, type of medium and addition of additional substances to the medium, etc.), other culture conditions were also tested.Example 5: Culturing S. cerevisiae in the bioreactorCultures were performed in Multifors bioreactor (maximum working volume 500 mL, 2 4-blade Rusthon turbine impellers, Infors HT, Switzerland) containing 250-500 mL of medium. The cultures were maintained at 30°C, 300 or 900-950 rpm with initial 1.2, 2.4 or 3.6 volumes of gas (volume culture) -1 min -1( VVM). The pH of the culture was maintained constant to pH 5.5 ± 0.2 by addition of sterile 2 M NaOH or 2 M H 3 PO 4. Clerol FBA 3107 antifoam (Cognis France, Ponthierry Paris, France; 0.03% v / v) was added to control foam production. The gas concentration (CO 2, O 2, N 2 and Ar) was analyzed continuously in a Prima Pro Process mass spectrometer (Thermo Scientific, UK) calibrated with 3% CO 2 in Ar, 5% CO 2 with 0.99% Ar and 15% O 2 in N 2, 20 % O 2 plus 20% Ar in N 2 and 0.04% ethanol in N 2.The strains were precultured overnight in shake flasks in SCD-based selective medium and used to inoculate the bioreactor.The batch phase of the cultures (20 g / L initial glucose) was allowed to continue for 14-20 hours and glucose feed was started only after the glucose had been consumed but either after or before ethanol had been consumed (depending on the objective of the culture). The glucose feed rate was maintained at 0.38-0.65 g L -1 h -1 (depending on the objective of culturing). Supernatant samples were then analyzed by HPLC to determine the accumulation of 3-HP and other major metabolites in the cultures.Example 6: 3-HP Analysis from Cell Culture Supernatants by HPLCCulture supernatant samples were analyzed using the Waters Alliance e2695 HPLC system (Waters, Milford, USA) with the injection volume being 10 μl. As the stationary phase, Aminex HPX-87H Organic Acid Column (organic acid column) (300 mm×7.8 mm) (Bio-Rad, U.S.A.) coupled to a Fast Acid Analysis Column (fast acid analysis column) (100 mm×7.8 mm) (Bio-Rad, U.S.A.) was used in HPLC. The columns were maintained at +55°C and the eluent used was 5.0 mM H 2 SO 4( Merck KgaA, Germany) at the flow rate of 0.3 or 0.5 ml min -1. To detect 3-hydroxypropionic acid, glucose, acetate, succinate, pyruvate, glycerol, and ethanol, a Waters 2489 dual wavelength UV (210 nm) detector (Waters, Milford, USA) and a 2414 differential refractometer (Waters, Milford, USA) were used.Example 7: 3-HP analysis from cell culture supernatants by GC / MSThe test samples and standard curve were prepared in the following manner: Supernatant (0.5 ml) was acidified with 50 μl of HCl (6N) and 3-HPA (TCI) standard (in ethyl acetate) was added. 5 μl of the lactic acid solution as internal standard (Sigma Aldrich (ISOTEC), sodium L-lactate-3,3,3-d3 98 atomic %; 5.5 g / l) and about 0.2 g of NaCl were added. Since labeled 3-HP is not commercially available, this stable isotope lactic acid product was used as an internal standard because it was the available compound that was most structurally / chemically similar to 3-HP that is not present in the sample matrix. The mixture was shaken in a vortex mixer for about 3 minutes. The sample was then extracted twice with 0.5 ml ethyl acetate by mixing in a vortex mixer for about 3 minutes. The phases were separated by centrifugation at 10000 rpm for 5 minutes. The upper phases were collected in a GC vial and evaporated. The dried residues were derivatized with MSTFA (50 μl) containing 1% TMCS by incubating for 1 hour at 60°C. The standards for the calibration curve were extracted in the same manner as the samples to minimize errors.Samples were measured using an Agilent 6890 gas chromatograph (GC) combined with an Agilent 5973 mass selective detector (MSD). The injector (injection volume 1 μl 20:1 split ratio) and MSD interface temperatures were 280°C and the furnace temperature program was 50°C to 280°C at a rate of 20°C / min. The analyses were carried out on an Agilent HP-5MS capillary column (30 m, ID 200 μm, film thickness 0.25 μm; Agilent 19091S-433). The identifications of the compounds were based on a spectral search from the NIST library. 3-HP was detected by monitoring m / z 147 and m / z 219 and 3-HP dimer was detected by monitoring m / z 177. Five point calibration curves (c=1-400 mg / l) were constructed using the 3-HP reactions and an internal standard was used for normalization. Quantification was found to be linear in this concentration range. Blank samples were analyzed together with the samples.Example 8: Assessment of S. cerevisiae Plasmid Expression StrainsThe 3-HP plasmid expression strains simultaneously expressed one, two or three enzymes of the 3-HP pathway (i.e., AADH, ACC1, MCR and HPDH) from two different expression plasmids (i.e., pSK-084 and / or pSK-085) (Figure 4). These strains were used to assess the effects of the different enzymes of the 3-HP pathway (and combinations of these enzymes) on the production of 3-HP in the VSK-128 acid tolerant S. cerevisiaestrain. The strains were cultured in 20 mL of selective SC-based medium (20 g / L glucose) in 250 mL flasks and grown for 2 days (30°C, 250 rpm) until all the glucose and ethanol had been consumed.Example 9: Summary of in vivo activity analyses of metabolic pathway enzymesTwenty five AADHs, 8 ACC1s, 10 bifunctional HPDH MCRs, 6 archaeal MCRs and 28 HPDHs in Table 16 were analyzed for their ability to produce 3-HP in various S. cerevisiaestrains that also expressed additional enzymes of the 3-HP pathway as needed. Many novel enzymes of the 3-HP pathway (obtained from genome mining analyses) have been shown to be active in yeast and have many of them excellent properties (i.e., higher activities, better cofactor preference) compared to previously published enzymes of the 3-HP pathway. [Table 16] [Table 16]Genes coding for AADHsAADHabSEQ ID NO:3coding code coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding coding codingAADHalSEQ ID NO:4AADHbsSEQ ID NO:5AADHbSEQ ID NO:6AADHcsSEQ ID NO:7AADHhoSEQ ID NO:8AADHhsSEQ ID NO:9AADHma1SEQ ID NO:10AADHma2SEQ ID NO:11AADHmmSEQ ID NO:12AADHpaSEQ ID NO:13AADHpbSEQ ID NO:14AADHpeSEQ ID NO:15AADHrSEQ ID NO:16AADHslSEQ ID NO:17AADHssSEQ ID NO:18AADHtaSEQ ID NO:19AADHtlSEQ ID NO:20AADHtmSEQ ID NO:21AADHvsSEQ ID NO:22ADHEecSEQ ID NO:23AHEpmSEQ ID NO:24EUTEdzSEQ ID NO:25EUTEecSEQ ID NO:26LIN1129liSEQ ID NO:27Genes Encoding ACC1sACC1sc_S659ASEQ ID NO:28ACC1sc_S659A / S1157ASEQ ID NO:29ACC1sc_S1157ASEQ ID NO:30ACC1keSEQ ID NO:31ACC1mcSEQ ID NO:32ACC1scSEQ ID NO:33ACCylSEQ ID NO:34ACC1chSEQ ID NO:35Genes Encoding Bifunctional HPDH MCRsHPDH-MCRbsSEQ ID NO:36HPDH-MCRcaSEQ ID NO:37HPDH-MCRcagSEQ ID NO:38HPDH-MCRctSEQ ID NO:39HPDH-MCRgbSEQ ID NO:40HPDH-MCRotSEQ ID NO:41HPDH-MCRrcSEQ ID NO:42HPDH-MCRslSEQ ID NO:43HPDH-MCRca_variant_3SEQ ID NO:44HPDH-MCRca_variant_6SEQ ID NO:45Genes Encoding HPDHsBTHcmSEQ ID NO:46BDHkpSEQ ID NO:47HBDHosSEQ ID NO:48HBDHpsSEQ ID NO:49HIBATHHasSEQ ID NO:50HIBAHbSEQ ID NO:51HIBATHHmaSEQ ID NO:52HIBAHpaSEQ ID NO:53HIBAHxcSEQ ID NO:54HPDHamSEQ ID NO:55HPDHbsSEQ ID NO:56HPDHcaSEQ ID NO:57HPDHcagSEQ ID NO:58HPDHctSEQ ID NO:59HPDHecSEQ ID NO:60HPDHedSEQ ID NO:61HPDHgbSEQ ID NO:62HPDHhSEQ ID NO:63HPDHkaSEQ ID NO:64HPDHmsSEQ ID NO:65HPDHotSEQ ID NO:66HPDHpsSEQ ID NO:67HPDHraSEQ ID NO:68HPDHrcSEQ ID NO:69HPDHsiSEQ ID NO:70HPDHslSEQ ID NO:71HPDHsmSEQ ID NO:72HPDHstSEQ ID NO:73Example 10: Shake flask culture experiments with S. cerevisiae to produce 3-HPCulture conditionsVarious different culture conditions were evaluated on some early test strains to see how the different culture conditions affected the ability of the strain to produce 3-HP. S. cerevisiaestrain VKK-128 (Δura3, Δhis3) expressed two plasmids, one of the plasmids containing either the HIBADHpa or the HPDHecgene, and the second plasmid containing the MCRsa2 gene. Both strains behaved similarly during the cultures and had very similar metabolite profiles.Six different culture conditions were tested in shake flasks: 1st Aerobic, batch process, high initial glucose (120 g / L). On day 3, an additional 100 g / L glucose was added. 2. anaerobic, batch process, high initial glucose (100 g / L). The flasks were sealed and shaking was slower at 100 rpm. 3. aerobic, batch process, repeated addition of initial glucose was 20 g / L, then 40 g / L was added on each follow-up day. 4. aerobic, simulated batch fed process, many initial glucose tablets. Initially, 5 tablets were added, an additional 5 tablets were added on day 3. Various amounts of enzyme A solution (50-150 μl per day) were added daily. 5. aerobic, simulated batch fed process, repeated admixture with glucose tablets. 1 tablet was added initially, 2 tablets were added on days 1 and 2, 3 tablets were added on days 3 and 4. Various amounts of enzyme A solution (50-150 μl per day) were added daily. 6. aerobic, batch process, repeated galactose addition. Initial galactose was 20 g / L, after which 40 g / L were added on each subsequent day.It is believed in the simulated batch fed cultures that each tablet releases 0.5 g of glucose (corresponding to 20 g / L glucose in our 25 ml culture volumes). The tablets consist essentially of glucose (i.e. starch) and an enzyme A solution (i.e. amylase), whereby during the cultivations in a shaking flask the controlled slow release of glucose into the medium is possible. It was hypothesized that glucose-limited fed batch conditions may promote conversion to growth and subsequently to 3-HP production.Cell growthAll of the regular glucose-based cultures grew similarly, and the galactose-fed culture grew more slowly. On the other hand, the fed batch conditions promoted more cell growth as compared to the other culture conditions. The conditions of the fed batch (adding tablets) promoted, in particular, a strong growth early in the cultivation.3-HP ProductionMost of the growth in these cultures had generally occurred to the end of day 2, and the major portion of 3-HP had also been produced to day 2, indicating that growth and 3-HP production are linked together. The fed batch culturing conditions (tableted) produced most of the 3-HP (~0.85 g / L) and the galactose fed cultures produced the least amount of 3-HP (~0.12 g / L) and the other culturing conditions all produced about 0.3 to 0.4 g / L 3-HP. These results show that culture conditions can have a great effect on the 3-HP production levels (Figure 5).Glucose ConsumptionGlucose was rapidly consumed in all of the culturing experiments up to days 2-3, after which the glucose consumption rates decreased significantly (during days 4-5) along with growth and 3-HP production. During the cultivations of the fed batch, glucose appeared to be consumed as quickly as it was released from the tablets, suggesting that these cultivations were performed under glucose-limited conditions.Accumulation of glycerol, acetate and ethanolThe accumulation of glycerol was quite high for glucose fed cultures and much lower for galactose fed cultures. Acetate accumulation was quite similar under the different culture conditions, but fed batch conditions (many tablets) produced more acetate.In most of these shake flask cultures, large amounts of ethanol accumulate, especially in the batch glucose cultures and glucose-added cultures. Batch fed cultures aimed at representing glucose-limited conditions to reduce overflow metabolism, and this approach seems to have been successful because ethanol accumulation in batch fed cultures was significantly lower.Culturing an Establishing 3-HP Production StrainAn additional 3-HP production strain [(EutEe, HPDH-MCRca, ACC1sc_S1157A ) and (HIBADHpa, MCRsa2)] was cultured according to the most promising fed batch culture conditions (tableted) to test its performance.Again, most of the growth had occurred until day 3 and most of the 3-HP production had occurred until day 3. Under these culture conditions, the 3-HP production in this strain exceeded 1.2 g / L (Figure 6).Although specific embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that the specific description relates only to desired exemplary embodiments and should not be considered as limiting the scope of the present invention. The essential scope of the present invention is therefore defined by the appended claims and their equivalent.Industrial applicabilityThe use of the recombinant yeast and the process for producing 3-HP of the present invention enables the production of 3-HP in a high concentration and a high yield at a low pH from a usable sugar such as glucose, thereby contributing substantially to the economical production of 3-HP from biomass and its applied products.A sequence protocol as an electronic document follows. This can be called in both the DEPATISnet and the DPMA register.

Claims

A recombinant yeast Saccharomyces cerevisiae comprising an active pathway for biosynthesis of 3-HP according to [pyruvate → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate semialdehyde → 3-HP], wherein the yeast comprises: an exogenous gene encoding AADH having an amino acid sequence selected from SEQ ID Nos: 145-176; an endo- or exogenous gene encoding ACC having an amino acid sequence selected from SEQ ID Nos: 99-106; an exogenous gene encoding MCR having an amino acid sequence selected from SEQ ID NOS: 192-207 and an exogenous gene encoding HPDH having an amino acid sequence selected from SEQ ID NOS: 127-130, 132-133, 137, 140, 142 and 208-275.The recombinant yeast of claim 1, wherein the MCR is bifunctional in that the MCR simultaneously has a function of converting malonyl-CoA into malonate semialdehyde and a function of converting malonate semialdehyde into 3-HP.The recombinant yeast of claim 1, wherein the MCR is monofunctional in that it has a function of converting malonyl-CoA into malonate semialdehyde; and further comprising a gene encoding an enzyme capable of converting malonate semialdehyde into 3-HP.The recombinant yeast of claim 1, wherein the yeast is acid resistant.A method for producing 3-HP, comprising: (a) culturing the recombinant yeast of any one of claims 1 to 4 in a medium including at least one carbon source, thereby producing 3-HP; and (b) isolating 3-HP from the culture.The method for producing 3-HP according to claim 5, wherein the carbon source is one or more selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, galactose, cellulose, glucose oligomers, and glycerol.The method for producing 3-HP according to claim 5, wherein the culturing is performed at a pH in the range of 2.6 to 4.0.

Images