Enzymes for the production of glycolic acid and other hydroxycarboxylic acids

Abstract

Alcohol dehydrogenases and aldehyde dehydrogenases having kinetic properties advantageous for the industrial production of glycolic acid or other hydroxycarboxylic acids, from ethylene glycol or other aliphatic polyhydric alcohols having a terminal hydroxyl group, are described herein. Microorganisms expressing one or both dehydrogenases, as well as their use in fermentative processes are also described.

Description

[0001] ENZYMES FOR THE PRODUCTION OF GLYCOLIC ACID AND OTHER HYDROXYCARBOXYLIC ACIDS

[0002] The present description relates to alcohol dehydrogenases and aldehyde dehydrogenases having kinetic properties advantageous for the industrial production of glycolic acid or other hydroxycarboxylic acids, from ethylene glycol or other aliphatic polyhydric alcohols having a terminal hydroxyl group, as well as their expression in a microorganism for use in fermentative processes. All documents referenced herein are incorporated by reference in their entirety.

[0003] BACKGROUND

[0004] Hydroxycarboxylic acids represent commercially valuable molecules useful as raw materials for ingredients in a variety of industrial and / or household products. Glycolic acid is an example of an ingredient of considerable industrial significance due to growing market demand, with existing applications across many sectors, including skincare, textiles, and cleaning. Traditional methods for glycolic acid synthesis involve complex chemical processes that oxidize petroleum -derived formaldehyde in the presence of metal catalysts. These conventional processes frequently have significant constraints, including harsh reaction conditions requiring large energy input, and excess byproduct generation, which necessitates energy-intensive purification steps, leaving a large environmental impact. In an attempt to address these issues, some groups have focused on developing enzymatic processes as environmentally friendly, high-specificity alternative manufacturing routes, with ethylene glycol as a potential substrate. Example microorganisms and fermentation strategies for the production of glycolic acid from ethylene glycol include those described in Boronat et al., 1983, Kataoka et al., 2001, Wada et al., 2005, Morishige et al., 2007, and Hua et al., 2018. However, a significant obstacle to achieve commercial feasibility is the need for high-performing enzymes suitable for industrial use.

[0005] SUMMARY

[0006] In a first aspect, described herein is a microorganism engineered to overexpress an alcohol dehydrogenase that catalyzes the conversion of ethylene glycol to glycolaldehyde, and / or an aldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to any one of SEQ ID NOs: 2 to 4 and 10-59; or a catalytically active fragment thereof. In some embodiments, the aldehyde dehydrogenase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to any one of SEQ ID NOs: 61 to 229; or a catalytically active fragment thereof.

[0007] In a further aspect, described herein is a composition comprising an isolated alcohol dehydrogenase and / or an isolated aldehyde dehydrogenase as described herein.

[0008] In a further aspect, described herein is a process for producing glycolaldehyde, as a product or intermediate, from ethylene glycol. The process generally comprises contacting ethylene glycol with an isolated alcohol dehydrogenase described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of ethylene glycol to glycolaldehyde.

[0009] In a further aspect, described herein is a process for producing glycolic acid, as a product or intermediate, from glycolaldehyde, the process comprising contacting glycolaldehyde with an isolated aldehyde dehydrogenase described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid.

[0010] In a further aspect, described herein is a process for producing glycolic acid, as a product or intermediate, from ethylene glycol, the process comprising contacting ethylene glycol with the isolated aldehyde dehydrogenase described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of ethylene glycol to glycolaldehyde; and contacting the glycolaldehyde with an isolated aldehyde dehydrogenase described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid. In some embodiments, the microorganism is engineered to overexpress both an aldehyde dehydrogenase and an aldehyde dehydrogenase described herein.

[0011] In a further aspect, described herein is a fermentation broth comprising ethylene glycol, glycolaldehyde, and / or glycolic acid; and a microorganism and / or a composition described herein.

[0012] General Definitions

[0013] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0014] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0015] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0017] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1 shows a schematic representation of the oxidation of ethylene glycol to glycolic acid in a two-step enzymatic process.

[0020] Fig. 2A illustrates a multiple sequence alignment between the enzymes goxADH, gmoADH, and gdiADH (SEQ ID NOs: 1-3), with bolded residues indicating positions of potential engineering. Fig. 2B illustrates a multiple sequence alignment between the enzymes goxADH, gmoADH, gdiADH, mtaADH, and mmaADH (SEQ ID NOs: 1-4 and 10).

[0021] Fig. 3 illustrates a multiple sequence alignment between the enzyme gmoADH (SEQ ID NO: 2) and orthologs thereof sharing at least 85% amino acid sequence identity (SEQ ID NOs: 11-42).

[0022] Fig. 4 shows a phylogenetic tree constructed with respect to the multiple sequence alignment of Fig. 3.

[0023] Fig. 5 illustrates a multiple sequence alignment between the enzyme gdiADH (SEQ ID NO: 3) and orthologs thereof sharing at least 85% amino acid sequence identity (SEQ ID NOs: 43-59).

[0024] Fig. 6 shows a phylogenetic tree constructed with respect to the multiple sequence alignment of Fig. 5.

[0025] Fig. 7 illustrates a multiple sequence alignment between the enzyme aviALDH (SEQ ID NO: 61) and orthologs thereof sharing at least 85% amino acid sequence identity (SEQ ID NOs: 63-161). Fig. 8 illustrates a multiple sequence alignment between the enzyme ppuALDH (SEQ ID NO: 62) and orthologs thereof sharing at least 85% amino acid sequence identity (SEQ ID NOs: 162-229).

[0026] SEQUENCE LISTING

[0027] This application contains a Sequence Listing in computer readable form created May 28, , 2025.

[0028] The computer readable form is incorporated herein by reference.

[0029] DETAILED DESCRIPTION

[0030] Described herein are enzymes having kinetic properties advantageous for industrial processes relating to the conversion of an aliphatic polyhydric alcohol having a terminal hydroxyl group to its corresponding aldehyde, and / or its subsequent conversion to a corresponding alpha-hydroxy acid, as well as related engineered microorganisms and processes employing the enzymes. In general, the enzymes described herein comprise a first enzyme having alcohol dehydrogenase activity for catalyzing the oxidation of the aliphatic polyhydric alcohol having a terminal hydroxyl group to its corresponding aldehyde, and a second enzyme having aldehyde dehydrogenase activity for converting the corresponding aldehyde to the corresponding alpha-hydroxy acid. A schematic of the two-step enzymatic process based on the two-carbon aliphatic polyhydric alcohol, ethylene glycol, is shown in Fig. 1.

[0031] In a first aspect, described herein is a microorganism engineered to express or overexpress a first enzyme having alcohol dehydrogenase activity that catalyzes the conversion of ethylene glycol to glycolaldehyde; and / or a second enzyme having aldehyde dehydrogenase activity that catalyzes the conversion of glycolaldehyde to glycolic acid. In some embodiments, the first and the second enzymes may be expressed or overexpressed in the same or different microorganisms. As used herein, the expressions “first enzyme” and “second enzyme” are employed solely for ease of reference and do not imply that both enzymes must be present in a microorganism, composition, or enzyme cocktail described herein. As used herein in the context of enzymes, the expressions “that catalyzes,” “catalytically active,” and “having [e.g., alcohol dehydrogenase / aldehyde dehydrogenase / glycolate oxidase / pyruvate formate-lyase / ethanol dehydrogenase] activity” and refers to a minimal activity that the enzyme exhibits or possesses (as opposed to an intended use of the enzyme), without necessarily excluding that the enzyme may exhibit or possess other activities (e.g., catalytic activity on other substrates). As used herein, the expression “overexpression” or “overexpress” refers to the level of expression of a given polypeptide or mRNA transcript in a host microorganism as compared to the level of expression of that same polypeptide or mRNA transcript in a corresponding wild-type or unengineered host microorganism.

[0032] In some embodiments, the first enzyme may be an alcohol dehydrogenase that catalyzes the conversion of ethylene glycol to glycolaldehyde comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NOs: 2 to 4 and 10-59; or a catalytically active fragment thereof. As used herein, the expression “alcohol dehydrogenase” is intended to refer to the desired reaction to be catalyzed as described herein and does not exclude enzymes assigned other names in the scientific literature, as long as the enzymes otherwise meet the structural and catalytic characteristics of the enzymes described herein.

[0033] In some embodiments, the first enzyme may be an alcohol dehydrogenase from a Gluconobacter species, such as G. morbifer, G. diazotrophicus, G. aidae, G. oxydans, G. roseus, G. potus, G. thailandicus, G. frateurii, G. japonicus, G. cerinus, or G. cerinus. In some embodiments, the first enzyme may be an alcohol dehydrogenase from a Gluconacetobacter species, such as G. diazotrophicus, G. asukensis, G. tumulicola, G. aggeris, G. sacchari, G. dulcium, G. takamatsuzukensis, G. liquefaciens, G. azotocaptans , G. johannae, or G. tumuli soli. In some embodiments, the first enzyme may be an alcohol dehydrogenase from an organism listed in Table 1, 5 or 6.

[0034] In some embodiments, the first enzyme may be an alcohol dehydrogenase that is oxygeninsensitive, such as an alcohol dehydrogenase that employs an oxygen-insensitive cofactor such zinc (instead of iron). Enzymes that employ iron as cofactor may be prone to degradation via metal -catalyzed oxidation, which becomes exacerbated upon exposure to high aeration fermentation conditions.

[0035] In some embodiments, the first enzyme may be an alcohol dehydrogenase that is not Gox0313 from Gluconobacter oxydans, and / or does not comprise the amino acid sequence of SEQ ID NO: 1. While Zhang et al., 2015 reported the production of glycolaldehyde from oxidation of ethylene glycol using the zinc-dependent medium-chain alcohol dehydrogenase (Gox0313) from Gluconobacter oxydans, the authors also reported that the reverse reaction was more favored for this enzyme. Specifically, the authors at section 3.3 reported that, compared to the oxidation of ethylene glycol, Gox0313 not only had a higher affinity and catalytic rate for glycolaldehyde reduction to ethylene glycol, but also higher turnover and catalytic efficiency for glycolaldehyde as a substrate compared to ethylene glycol. Thus, in some embodiments, described herein are alcohol dehydrogenases having catalytic properties more advantageous for industrial applications. In some embodiments, the first enzyme may be an alcohol dehydrogenase that catalyzes the conversion of ethylene glycol to glycolaldehyde at a higher maximum velocity (Vmax) and / or at a higher turnover number (kcat) than the zinc -dependent medium -chain alcohol dehydrogenase Gox0313 from Gluconobacter oxydans set forth in SEQ ID NO: 1. Such enzyme characteristics may be assessed in vitro on purified enzymes under the same reaction conditions by, for example, titrating substrate concentrations and measuring product formation over time.

[0036] In some embodiments, the first enzyme may be an alcohol dehydrogenase that is a Gox0313 variant that differs or differs only from SEQ ID NO: 1 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 2 to 4 or 10. In some embodiments, these amino acid substitutions may comprise replacing the amino acid of SEQ ID NO: 1 with the corresponding amino acid of any one of SEQ ID NOs: 2 to 4 or 10, for example as shown in the multiple sequence alignment of Fig. 2A and 2B.

[0037] In some embodiments, the first enzyme may be a variant of the G. morbifer enzyme (gmoADH) of SEQ ID NO: 2, wherein the variant differs or differs only from SEQ ID NO: 2 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 11 to 42. In some embodiments, these amino acid substitutions may comprise replacing the amino acid of SEQ ID NO: 2 with the corresponding amino acid of any one of SEQ ID NOs: 11 to 42, for example as shown in the multiple sequence alignment of Fig. 3.

[0038] In some embodiments, the first enzyme may be a variant of the G. diazotrophicus enzyme (gdiADH) of SEQ ID NO: 3, wherein the variant differs or differs only from SEQ ID NO: 3 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 43 to 59. In some embodiments, these amino acid substitutions may comprise replacing the amino acid of SEQ ID NO: 3 with the corresponding amino acid of any one of SEQ ID NOs: 43 to 59, for example as shown in the multiple sequence alignment of Fig. 5.

[0039] In some embodiments, the second enzyme may be an aldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NOs: 61 to 229; or a catalytically active fragment thereof. As used herein, the expression “aldehyde dehydrogenase” is intended to refer to the desired reaction to be catalyzed as described herein and does not exclude enzymes assigned other names in the scientific literature, as long as the enzymes otherwise meet the structural and catalytic characteristics of the enzymes described herein.

[0040] In some embodiments, the second enzyme may be an aldehyde dehydrogenase from an Azotobacter species, such as A. vinelandii. In some embodiments, the second enzyme may be an aldehyde dehydrogenase from a Cupriavidus species, such as C. basilensis, C. sp. BIC8F, or C. sp. WS. In some embodiments, the second enzyme may be an aldehyde dehydrogenase from a. Pseudomonas species, such as P. aeruginosa, P. asiatica, P. azerbaijanoccidentalis, P. azotoformans, P. brassicacearum, P. brenneri, P. carnis, P. cedrina, P. cyclaminis, P. fluorescens, P. fluorescens, P. fluorescens ABAC62, P. juntendi, P. kairouanensis, P. karstica, P. lurida, P. mandelii, P. marginalis, P. marginalis pv. marginalis, P. migulae, P. monteilii, P. orientalis, P. pisciculturae, P. proteolytica, P. putida, P. putida, P. reinekei, P. shahriarae, P. sivasensis, P. sp. 18.1.10, P. sp. 1912-s, P. sp. 2822-17, P. sp. 2995-1, P. sp. ACN8, P. sp. ANT H12B, P. sp. A-R-19, P. sp. B14(2022), P. sp. BIGbO381, P. sp. CFBP13528, P. sp. ES3-33, P. sp. GL-RE-29, P. sp. GM78, P. sp. GM79, P. sp. G0M6, P. sp. KBS0802, P. sp. KBW05, P. sp. M47T1, P. sp. Marseille-Q1929, P. sp. MWU16-30323, P. sp. NFACC42-2, P. sp. NFACC45, P. sp. NIBR-H-19, P. sp. PB101, P. sp. PD9R, P. sp. Ql-7, P. sp. S35, P. sp. S9, P. sp. SJZ080, P. sp. TH32, P. sp. TNT2022 ID1044, P. sp. Tril, P. sp. Xaverov 83, P. sp. YuFO20, P. sp. YuFO8, P. sp. Z18(2022), P. umsongensis, P. urethralis, P. veronii, or P. yamanorum. In some embodiments, the second enzyme may be an aldehyde dehydrogenase from a Stutzerimonas species, such as .S', azotifigens, S. azotifigens, S. balearica, S. frequens, S. nitrititolerans, S. stutzeri, or .S', zhaodongensis. In some embodiments, the second enzyme may be an aldehyde dehydrogenase from a Gammaprote obacteria bacterium, a Marinobacterium species (c.g.. 7. profundum), a Pseudomonadaceae bacterium T75, or a Rhodococcus species (e.g., R. qingshengii, or R. ruber). In some embodiments, the second enzyme may be an aldehyde dehydrogenase from an organism listed in Table 10 or 11.

[0041] E. coli aldehyde dehydrogenase aldA (ecoALDH) has been reported to catalyze the intracellular enzymatic conversion of glycolaldehyde to glycolic acid (e.g., Wada et al. 2005; Morishige et al., 2007; Pandit et al., 2019; Pandit et al., 2021). However, this enzyme is shown herein to exhibit catalytic properties (e.g., low catalytic turnover and susceptibility to substrate inhibition; as shown in Example 6 and Table 7) that may be disadvantageous or sub-optimal for industrial applications, particularly when paired with higher glycolaldehyde levels produced from the first enzymes describe herein. Thus, in some embodiments, described herein are aldehyde dehydrogenases having catalytic properties more advantageous for industrial applications than ecoALDH. In some embodiments, the aldehyde dehydrogenase catalyzes the conversion of glycolaldehyde to glycolic acid at a higher maximum velocity (Vmax) and / or at a higher turnover number (kcat) than the aldehyde dehydrogenase aldA from Escherichia coli set forth in SEQ ID NO: 60. In some embodiments, the second enzyme is not aldA from Escherichia coli and / or does not comprise the amino acid sequence of SEQ ID NO: 60.

[0042] In some embodiments, the second enzyme may be a variant of the A. vinelandii enzyme (aviALDH) of SEQ ID NO: 61, wherein the variant differs or differs only from SEQ ID NO: 61 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 63 to 161. In some embodiments, these amino acid substitutions may comprise replacing the amino acid of SEQ ID NO: 61 with the corresponding amino acid of any one of SEQ ID NOs: 63 to 161, for example as shown in the multiple sequence alignment of Fig. 7.

[0043] In some embodiments, the second enzyme may be a variant of the P. putida enzyme (ppuALDH) of SEQ ID NO: 62, wherein the variant differs or differs only from SEQ ID NO: 62 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 162 to 229. In some embodiments, these amino acid substitutions may comprise replacing the amino acid of SEQ ID NO: 61 with the corresponding amino acid of any one of SEQ ID NOs: 162 to 229, for example as shown in the multiple sequence alignment of Fig. 8.

[0044] In some embodiments, at least one of the alcohol dehydrogenase and the aldehyde dehydrogenase enzymes described herein may be heterologous or exogenous with respect to the microorganism from which they are expressed. In some embodiments, the alcohol dehydrogenase and the aldehyde dehydrogenase enzymes described herein may be encoded by polynucleotides comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the microorganism in one or more copies). In some embodiments, the alcohol dehydrogenase and the aldehyde dehydrogenase enzymes described herein may be encoded by polynucleotides comprised in an expression vector (e.g., plasmid). In some embodiments, the alcohol dehydrogenase and / or the aldehyde dehydrogenase enzymes described herein may be encoded by polynucleotide(s) operably linked to a heterologous promoter (i.e., the promoter being heterologous with respect to the enzyme under control thereof).

[0045] In some embodiments, the microorganism described herein may be engineered or further engineered to comprise genetic modifications to increase product titres. In some embodiments, the microorganism described herein may be engineered or further engineered to comprise genetic modifications to inactivate or disrupt genes (e.g., endogenous genes) that encode for glycolate oxidase, lactate dehydrogenase, pyruvate formate lyase, and / or ethanol dehydrogenase. In some embodiments, the microorganism may be one in which an endogenous glycolate oxidase, endogenous pyruvate formatelyase, or ethanol dehydrogenase activity is / are inactivated or decreased. In some embodiments, the microorganism’s endogenous glycolate oxidase (or a subunit thereof required for catalytic activity) may deleted, truncated, or mutated to impair or abrogate glycolate oxidase activity. In some embodiments, expression of the microorganism’s endogenous glycolate oxidase, lactate dehydrogenase, pyruvate formate lyase, and / or ethanol dehydrogenase (or any subunit thereof required for catalytic activity), may be inhibited, silenced, knocked-down, or repressed. In some embodiments, the microorganism described herein comprises an inactivated or impaired glcD gene encoding a glycolate oxidase subunit GlcD.

[0046] In some embodiments, the microorganism described herein may be a bacteria (e.g., Escherichia coll). yeast, or fungus. In some embodiments, the microorganism may be from a Pseudomonas species, Clostridium species, Chlorella species or other algae, Gluconohacter oxydans, Pichia naganishii, Corynehacterium species, or Corynehacterium glutamicum. In some embodiments, the microorganism may be from the species Candida hoidinii, Candida etchellsii, Candida geochares, Candida lamhica, Candida sorhophila, Candida sorhosivorans, Candida sorhoxylosa, Candida vanderwaltii, Candida zemplinina, Deharyomyces castellii, Issatchenkia orientalis (also known as Pichia kudriavzevii, Candida krusei, or Saccharomyces krusei), Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia jadinii, Pichia jadinii, Pichia memhranifaciens, Saccharomyces hayanus, Saccharomyces hulderi, Saccharomycopsis crataegensis, Zygosaccharomyces hisporus, Zygosaccharomyces komhuchaensis, or Zygosaccharomyces lentus. In some embodiments, the microorganism may be from Haloferax mediterranei, Halohactreium salinarum, Nicotiana tahacum, or Thermus thermophilus.

[0047] In some aspects, described herein are compositions comprising one or more enzymes that catalyze the conversion of an aliphatic polyhydric alcohol having a terminal hydroxyl group to its corresponding aldehyde, and / or that catalyze its subsequent conversion to a corresponding alpha-hydroxy acid. In some embodiments, described herein are compositions comprising one or more enzymes that catalyze the conversion of ethylene glycol to glycolaldehyde, and / or the conversion of glycolaldehyde to glycolic acid. In some embodiments, the compositions may comprise an isolated alcohol dehydrogenase as described herein. In some embodiments, the compositions may comprise an isolated aldehyde dehydrogenase as described herein. In some embodiments, the compositions may comprise an alkaline buffer and / or may have an alkaline pH (e.g., greater than pH 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4).

[0048] In some embodiments, the microorganisms and / or compositions described herein are for use in (i) the conversion of an aliphatic polyhydric alcohol having a terminal hydroxyl group to its corresponding aldehyde, as a product or intermediate; (ii) the conversion of the corresponding aldehyde to a corresponding alpha-hydroxy acid, as a product or intermediate; or (iii) the conversion of an aliphatic polyhydric alcohol having a terminal hydroxyl group to a corresponding alpha-hydroxy acid, as a product or intermediate. In some embodiments, the aliphatic polyhydric alcohol having a terminal hydroxyl group described herein is a substrate of an alcohol dehydrogenase described herein, and the corresponding aldehyde is a substate of an aldehyde dehydrogenase described herein.

[0049] In some aspects, described here is a polynucleotide encoding an alcohol dehydrogenase as described herein. In some aspects, described here is a polynucleotide encoding an aldehyde dehydrogenase as described herein. In some aspects, described here is a polynucleotide encoding both an alcohol dehydrogenase and aldehyde dehydrogenase as described herein. In some aspects, described herein is an expression cassette comprising a polynucleotide described herein operably linked to a heterologous promoter. In some aspects, described herein is a vector comprising a polynucleotide or expression cassette described herein. In some embodiments, the polynucleotides described herein (including the full complements thereof) may be useful as tools for screening and / or hybridization (e.g., to identify novel polypeptides potentially encoding alcohol dehydrogenases or aldehyde dehydrogenases of interest), independent the activity of the polypeptides them encode.

[0050] In some embodiments, described herein is a process for producing glycolaldehyde, as a product or intermediate, from ethylene glycol, the process comprising contacting ethylene glycol with the isolated alcohol dehydrogenase as described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of ethylene glycol to glycolaldehyde. In some embodiments, described herein is a process for producing glycolic acid, as a product or intermediate, from glycolaldehyde, the process comprising contacting glycolaldehyde with the isolated aldehyde dehydrogenase as described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid. In some embodiments, described herein is a process for producing glycolic acid, as a product or intermediate, from ethylene glycol, the process comprising contacting ethylene glycol with the isolated aldehyde dehydrogenase as described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of ethylene glycol to glycolaldehyde; and contacting the glycolaldehyde with the isolated aldehyde dehydrogenase as described herein, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid.

[0051] In some embodiments, the process described herein may comprise a growth phase during which the microorganism is cultured under conditions promoting biomass accumulation, and a production phase during which the microorganism is cultured under conditions promoting the production of the product or intermediate. In some embodiments, transition from the growth phase to the production phase is not triggered by a modification in aeration conditions from the growth phase to the production phase (e.g., a decrease in oxygen uptake rate from the growth phase to the production phase).

[0052] In some aspects, described herein is a fermentation broth comprising: (i) ethylene glycol, glycolaldehyde, and / or glycolic acid; and (ii) a microorganism as described herein, and / or a composition as described herein.

[0053] EXAMPLES

[0054] Example 1: Materials and Methods

[0055] Enzyme expression and purification

[0056] The enzymes of interest were genetically engineered in the E. coli BL21 (DE3) strain on a high copy plasmid under control of the T7 promoter. The enzymes were tagged with a hexahistidine (His-tag) sequence to facilitate purification. Post-expression, the His-tagged enzymes were selectively bound to a nickel-charged immobilized metal affinity chromatography (IMAC) resin, exploiting the high affinity between histidine residues and nickel ions. Upon washing away non-specifically bound proteins, the His- tagged enzymes were eluted with an imidazole -containing buffer and subsequently used for enzymatic assays.

[0057] Enzyme assays

[0058] Standard enzyme characterization protocols were performed to determine the activities of the enzymes. Dehydrogenase activity against the substrates was determined using a continuous assay following the increase in absorbance at 340 nm [a340 (NADH) = 6.22 mM1• and a340 (NADPH) = 6.2 mM1• cm ' |. Substrate screens were performed at 30 °C in a reaction mixture (200 pL) containing 50 mM of substrate in 100 mM Tris-HCl (pH 8.5), 1 mM NAD+, 1 mM substrate, and 10 pg of purified protein. Initial velocity patterns of the enzymatic reaction were obtained by varying the concentrations of ethylene glycol at fixed concentrations of enzyme concentration in the presence of the saturating NAD concentrations. Example 2: Expression and purification of candidate alcohol dehydrogenases for converting ethylene glycol to glycolaldehyde

[0059] Glycolaldehyde is useful as a precursor or intermediate in a number of organic synthesis reactions. Although various chemical methods have been reported for its synthesis, these methods often suffer from drawbacks such as sub-optimal yields, formation of by-products, reliance on heavy metal catalysts, and high reaction temperatures. Bio-based production of glycolaldehyde is an attractive alternative to chemical synthesis, however higher performing enzymes are needed for commercialization. While Zhang et al., 2015 reported the production of glycolaldehyde from oxidation of ethylene glycol using the zinc-dependent medium-chain alcohol dehydrogenase (Gox0313) from Gluconobacter oxydans, the authors reported enzyme characteristics that may not be favorable for industrial applications. Thus, we sought to benchmark the catalytic activity of Gox0313 for ethylene glycol against that of other zincdependent enzymes with potential alcohol dehydrogenase activity.

[0060] After identifying a plurality of potential enzymes of interest through bioinformatic searches, several candidates were selected from different branches of a phylogenetic tree for cloning, expression and characterization. Candidate alcohol dehydrogenase enzymes, along with Gox0313 (herein referred to as “goxADH”) as a control, were cloned and expressed in E. coll as described in Example 1, and the yields of several candidates following purification are shown in Table 1. Two of the candidate enzymes, rjoADH and scoADH, were found to be insoluble following purification, precluding any activity measurements.

[0061] Table 1: Candidate alcohol dehydrogenase enzymes expressed and purified Example 3: Characterization of alcohol dehydrogenase activity

[0062] The candidate alcohol dehydrogenase enzymes were next characterized for their enzymatic activity. Initial activity screening was performed in a reaction mixture consisting of 50 mM of ethylene glycol (EG) as substrate in 100 mM Tris-HCl buffer at pH 8.5. Reactions were initiated by the addition of the purified enzyme and enzymatic activity was measured by monitoring the formation of the product (glycolaldehyde) overtime as described in Example 1. Robust EG oxidation activity was observed for goxADH, gmoADH, mtaADH, gdiADH, and mmaADH. While a detectable level of EG oxidation activity was observed for ecoADHP, no EG oxidation activity was detected for gstADH and cpsADH (data not shown).

[0063] Kinetic parameters for goxADH, gmoADH, mtaADH, gdiADH, and mmaADH were then determined by conducting enzyme activity assays at varying concentrations of EG (100, 50, 25, 15, 10, 5, 2, and 1 mM). The resultant activity data were fitted to the Michaelis-Menten equation to derive kinetic constants (Kmand Vmax) for each enzyme. These constants provide insights into the enzyme’s substrate affinity and maximum catalytic rate, contributing to a better understanding of the enzyme’s operational efficiency. As seen in Table 2, mmaADH exhibited similar substrate affinity and maximum catalytic rate to goxADH. Interestingly, enzymes mtaADH, gmoADH, and gdiADH exhibited comparable substrate affinities to one another, albeit with maximum catalytic rates (Vmax) significantly higher than that of goxADH and mmaADH. The maximum catalytic rates of gmoADH and gdiADH were particularly striking, being 2.5 and 3.8 fold higher than that of goxADH, respectively.

[0064] Table 2: Michaelis-Menten kinetic constants of candidate alcohol dehydrogenases

[0065] Example 4: Structural comparisons between goxADH, mtaADH, gmoADH, and gdiADH

[0066] Pairwise sequence alignment analyses between the full-length amino acid sequences of goxADH, mtaADH, gmoADH, and gdiADH were performed using the EMBOSS Needle online tool under default settings and the results are shown in Tables 3 and 4 for identity and similarity, respectively. Interestingly, both gmoADH and gdiADH share relatively high amino acid sequence identity with goxADH yet exhibited 2.5- and 3.8-fold higher EG turnover rate, respectively. This observation suggests one or more specific amino acid residue positions that may be engineered for increasing EG turnover rate, as illustrated in the bolded residues in the multiple sequence alignment shown in Fig. 2A.

[0067] Table 3: Sequence identity comparisons between goxADH, mtaADH, gmoADH, and gdiADH

[0068] Table 4: Sequence similarity comparisons between goxADH, mtaADH, gmoADH, and gdiADH

[0069] Example 5: Identification of gmoADH orthologs

[0070] Orthologs of gmoADH were identified using sequence identity searches based on a cut-off of at least 85% overall sequence identity with respect to its full-length sequence (SEQ ID NO: 2). The hits are shown in Table 5, with corresponding multiple sequence alignments and phylogenetic trees shown in Fig. 3 and Fig. 4, respectively.

[0071] Table 5: Alcohol dehydrogenases at least 85% identical to gmoADH

[0072] Example 6: Identification of gdiADH orthologs

[0073] Orthologs of gdiADH were identified using sequence identity searches based on a cut-off of at least 85% overall sequence identity with respect to its full-length sequence (SEQ ID NO: 3). The hits are shown in Table 6, with corresponding multiple sequence alignments and phylogenetic trees shown in Fig. 5 and Fig. 6, respectively.

[0074] Table 6: Alcohol dehydrogenases at least 85% identical to gdiADH

[0075] Example 7: Expression and purification of candidate aldehyde dehydrogenases for converting glycolaldehyde to glycolic acid

[0076] E. coll aldehyde dehydrogenase aldA (ecoALDH) has been reported to catalyze the intracellular enzymatic conversion of glycolaldehyde (GA) to glycolic acid (e.g., Wada et al. 2005; Morishige et al., 2007; Pandit et al., 2019; Pandit et al., 2021). In order to identify potentially higher-performing enzymes for industrial use capable of converting glycolaldehyde to glycolic acid, we evaluated a plurality of candidates based on either close, medium, or distant phylogeny to ecoALDH (SEQ ID NO: 60). A subset of candidate enzymes were cloned, expressed, and purified, along with ecoALDH as a control, as described in Example 1, and their in vitro enzymatic activities were measured in a reaction mixture consisting of 50 mM glycolaldehyde in 100 mM Tris-HCl buffer (pH 8.5). The reaction was initiated by the addition of the purified enzyme, and activity was measured by monitoring the formation of glycolic acid over time. The resultant activity data were then used to calculate enzyme performance metrics for each enzyme, such as Km(substrate affinity) and kcat (enzyme turnover), as well as the inhibitor constant Ki, to assess whether the enzymes suffer from substrate inhibition (Kokkonen et al., 2021) with respect to glycolaldehyde.

[0077] The activity results for ecoALDH and two candidate enzymes of interest, aviALDH (from Azotobacter vinelandii,' SEQ ID NO: 61) and ppuALDH (Pseudomonas putida,' SEQ ID NO: 62), are shown in Table 7. Interestingly, the catalytic turnover rates of aviALDH and ppuALDH were 5.3- and 5.8-fold higher than that of ecoALDH, suggesting that these enzymes may be advantageous for industrial use (where saturating or near-saturating concentrations of glycolaldehyde are ideally employed). Furthermore, while both aviALDH and ppuALDH exhibited greater resistance to glycolaldehyde substrate inhibition than ecoALDH, the resistance to substrate inhibition of aviALDH was particularly striking at 5.6-fold higher than that of ecoALDH (Table 7).

[0078] Table 7: Michaelis-Menten kinetic constants of ecoALDH

[0079] Example 8: Structural comparisons between ecoALDH, aviALDH, and ppuALDH

[0080] Pairwise sequence alignment analyses between the full-length amino acid sequences of ecoALDH (SEQ ID NO: 60), aviALDH (SEQ ID NO: 61), and ppuALDH (SEQ ID NO: 62) were performed using the EMBOSS Needle online tool under default settings and the results are shown in Tables 8 and 9 for identity and similarity, respectively. Table 8: Sequence identity comparisons between ecoALDH, aviALDH, and ppuALDH

[0081] Table 9: Sequence similarity comparisons between ecoALDH, aviALDH, and ppuALDH

[0082] Example 9: Identification of aviALDH orthologs

[0083] Orthologs of aviALDH were identified using sequence identity searches based on a cut-off of at least 80% overall sequence identity with respect to its full-length sequence (SEQ ID NO: 61). The hits are shown in Table 10, with corresponding multiple sequence alignments shown in Fig. 7.

[0084] Table 10: Aldehyde dehydrogenases at least 80% identical to aviALDH

[0085] Example 10: Identification of ppuALDH orthologs

[0086] Orthologs of ppuALDH were identified using sequence identity searches based on a cut-off of at least 80% overall sequence identity with respect to its full-length sequence (SEQ ID NO: 62). The hits are shown in Table 11, with corresponding multiple sequence alignments shown in Fig. 8.

[0087] Table 11: Aldehyde dehydrogenases at least 80% identical to ppuALDH

[0088] REFERENCES

[0089] Boronat et al., (1983). Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli. Journal of Bacteriology, 153(1): 134-139. Hua et al., (2018). Improving techno-economics of bioproduct glycolic acid by successive recycled-cell catalysis of ethylene glycol with Gluconobacter oxydans. Bioprocess and biosystems engineering, vol. 41, 10: 1555-1559.

[0090] Kataoka et al., (2001). Glycolic Acid Production Using Ethylene Glycol-Oxidizing Microorganisms. Biosci. Biotechnol. Biochem., 65(10), 2265-2270.

[0091] Kokkonen et al., (2021). Substrate inhibition by the blockage of product release and its control by tunnel engineering. RSC chemical biology vol. 2,2 645-655.

[0092] Morishige et al., (2007). Process For Producing Hydroxycarboxylic Acid. European Patent No. 2025760 Bl.

[0093] Pandit et al., (2019). Production of Glycolate from Ethylene Glycol and Related Microbial Engineering. International Application Publication No. WO / 2019 / 046946.

[0094] Pandit et al., (2021). Engineering Escherichia coli for the utilization of ethylene glycol. Microbial Cell Factories, 20(1): 22. doi: 10.1186 / sl2934-021-01509-2.

[0095] Wada et al., (2005). Process For Producing Hydroxycarboxylic Acid. European Patent No. 1748076 Bl.

[0096] Zhang et al., (2015). Oxidation of ethylene glycol to glycolaldehyde using a highly selective alcohol dehydrogenase from Gluconobacter oxydans. Journal of Molecular Catalysis B: Enzymatic, 112: 69-75.

Claims

CLAIMS1. A microorganism engineered to overexpress:(a) an alcohol dehydrogenase that catalyzes the conversion of ethylene glycol to glycolaldehyde comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NOs: 2 to 4 and 10-59; or a catalytically active fragment thereof;(b) an aldehyde dehydrogenase that catalyzes the conversion of glycolaldchydc to glycolic acid comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NOs: 61 to 229; or a catalytically active fragment thereof; or(c) both (a) and (b).

2. The microorganism of claim 1, wherein:(a) said alcohol dehydrogenase catalyzes the conversion of ethylene glycol to glycolaldehyde at a higher maximum velocity (Vmax) and / or at a higher turnover number (kcat) than the zincdependent medium-chain alcohol dehydrogenase Gox0313 from Gluconobacter oxy dans set forth in SEQ ID NO: 1;(b) said aldehyde dehydrogenase catalyzes the conversion of glycolaldehyde to glycolic acid at a higher maximum velocity (Vmax) and / or at a higher turnover number (kcat) than the aldehyde dehydrogenase aldA from Escherichia coll set forth in SEQ ID NO: 60; or(c) both (a) and (b).

3. The microorganism of claim 1 or 2, wherein:(a) said alcohol dehydrogenase is from:(1) a Gluconobacter species (e.g., G. morblfer, G. diazotrophlcus, G. aldae, G. oxydans, G. roseus, G. potus, G. thailandicus, G. fiateurii, G. Japonicus, G. cerinus, or G. cerinus Y, or(2) a Gluconacetobacter species (e.g., G. diazotrophlcus, G. asukensis, G. tumulicola, G. aggeris, G. sacchari, G. dulcium, G. takamatsuzukensis, G. liquefaciens, G. azotocaptans, G.johannae, or G. tumulisoliy,(b) said aldehyde dehydrogenase is from:(1) an Azotobacter species (e.g., A. vinelandiiy, a Cupriavidus species (e.g., C. basilensis,C. sp. BIC8F, or C. sp. WS)22RECTI FIED SHEET (RULE 91.1 )(2) a. Pseudomonas species (e.g., / '. aeruginosa, P. asialica, P. azerbaijanoccidenlalis, P. azotoformans, P. brassicacearum, P. brenneri, P. carnis, P. cedrina, P. cyclaminis, P. fluorescens, P.fluorescens, P. fluor escens AB AC62, P.juntendi, P. kairouanensis, P. karstica, P. lurida, P. mandelii, P. marginalis, P. marginalis pv. marginalis, P. migulae, P. monteilii, P. orientalis, P. pisciculturae, P. proteolytica, P. putida, P. putida, P. reinekei, P. shahriarae, P. sivasensis, P. sp. 18.1.10, P. sp. 1912-s, P. sp. 2822-17, P. sp. 2995-1, P. sp. ACN8, P. sp. ANT H12B, P. sp. A-R-19, P. sp. B14(2022), P. sp. BlGbO381, P. sp. CFBP 13528, P. sp. ES3-33, P. sp. GL-RE-29, P. sp. GM78, P. sp. GM79, P. sp. GOM6, P. sp. KBS0802, P. sp. KBW05, P. sp. M47T1, P. sp. Marseille-Q1929, P. sp. MWU 16-30323, P. sp. NFACC42-2, P. sp. NFACC45, P. sp. NIBR-H-19, P. sp. PB101, P. sp. PD9R, P. sp. Ql-7, P. sp. S35, P. sp. S9, P. sp. SJZ080, P. sp. TH32, P. sp. TNT2022 ID 1044, P. sp. Tril, P. sp. Xaverov 83, P. sp. YuFO20, P. sp. YuFO8, P. sp. Z18(2022), P. umsongensis, P. urethralis, P. veronii, or P. yamanorumf(3) a Stutzerimonas species (e.g., S. azotifigens, S. azotifigens, S. balearica, S.frequens, S. nitrititolerans, S. stutzeri, or S. zhaodongensis),-(4) a Gammaproteobacteria bacterium;(5) a Marinobacterium species (e.g., M. profimdutriy,(6) aPseudomonadaceae bacterium T75; or(7) aRhodococcus species (e.g., R. qingshengii, or R ruber); or(c) both (a) and (b).

4. The microorganism of any one of claims 1 to 3, wherein said alcohol dehydrogenase is a zincdependent alcohol dehydrogenase.

5. The microorganism of any one of claims 1 to 4, wherein:(a) said alcohol dehydrogenase is not Gox0313 from Gluconobacter oxydans, and / or docs not comprise the amino acid sequence of SEQ ID NO: 1;(b) said aldehyde dehydrogenase is not aldA from Escherichia coll and / or does not comprise the amino acid sequence of SEQ ID NO: 60; or(c) both (a) and (b).

6. The microorganism of any one of claims 1 to 5, wherein:(a) said alcohol dehydrogenase is:23RECTI FIED SHEET (RULE 91.1 )(1) a Gox0313 variant that differs or differs only from SEQ ID NO: 1 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 2 to 4 or 10, optionally wherein said amino acid substitutions comprise replacing the amino acid of SEQ ID NO: 1 with the corresponding amino acid of any one of SEQ ID NOs: 2 to 4 or 10;(2) a gmoADH variant that differs or differs only from SEQ ID NO: 2 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 11 to 42, optionally wherein said amino acid substitutions comprise replacing the amino acid of SEQ ID NO: 2 with the corresponding amino acid of any one of SEQ ID NOs: 11 to 42; or(3) a gdiADH variant that differs or differs only from SEQ ID NO: 3 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 43 to 59, optionally wherein said amino acid substitutions comprise replacing the amino acid of SEQ ID NO: 3 with the corresponding amino acid of any one of SEQ ID NOs: 43 to 59;(b) said aldehyde dehydrogenase is:(1) an aviALDH variant that differs or differs only from SEQ ID NO: 61 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 63 to 161, optionally wherein said amino acid substitutions comprise replacing the amino acid of SEQ ID NO: 61 with the corresponding amino acid of any one of SEQ ID NOs: 63 to 161; or(2) a ppuALDH variant that differs or differs only from SEQ ID NO: 62 by having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions at corresponding non-conserved amino acid positions with respect to any one of SEQ ID NOs: 162 to 229, optionally wherein said amino24RECTI FIED SHEET (RULE 91.1 )acid substitutions comprise replacing the amino acid of SEQ ID NO: 62 with the corresponding amino acid of any one of SEQ ID NOs: 162 to 229.

7. The microorganism of any one of claims 1 to 6, wherein:(i) at least one of the alcohol dehydrogenase and the aldehyde dehydrogenase is heterologous or exogenous with respect to the microorganism;(ii) the alcohol dehydrogenase and the aldehyde dehydrogenase are encoded by polynucleotides comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the microorganism in one or more copies);(iii) the alcohol dehydrogenase and the aldehyde dehydrogenase are encoded by polynucleotides comprised in an expression vector (e.g., plasmid);(iv) the alcohol dehydrogenase and / or the aldehyde dehydrogenase are encoded by polynucleotide(s) operably linked to a heterologous promoter;(v) any combination of (i) to (iv).

8. The microorganism of any one of claims 1 to 7, wherein the microorganism is one in which an endogenous glycolate oxidase, endogenous pyruvate formate -lyase, or ethanol dehydrogenase activity is inactivated or decreased.

9. The microorganism of any one of claims 1 to 8, wherein the microorganism is bacteria (e.g., Escherichia coll), yeast, or fungus.

10. The microorganism of claim 9, wherein:(i) the microorganism is from Pseudomonas species, Clostridium species, Chlorella species or other algae, Gluconobacter oxydans, Pichia naganishii, Corynebacterium species, or Corynebacterium glutamicum;(ii) the microorganism is from the species Candida boidinii, Candida etchellsii, Candida geochares, Candida lambica, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida vanderwaltii, Candida zemplinina, Debaryomyces castellii, Issatchenkia orientalis (also known as Pichia kudriavzevii, Candida krusei, or Saccharomyces krusei), Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia jadinii, Pichia jadinii, Pichia membranifaciens, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomycopsis crataegensis, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, or Zygosaccharomyces lentus,' or25RECTI FIED SHEET (RULE 91.1 )(iii) the microorganism is from Haloferax medilerranei, Halobaclreium salinarum, Nicoliana tabacum, or Thermus thermophilus.

11. A composition comprising: (a) an isolated alcohol dehydrogenase as defined in any one of claims 1 to 7; (b) an isolated aldehyde dehydrogenase as defined in any one of claims 1 to 7; or (c) both (a) and (b).

12. The microorganism as defined in any one of claims 1 to 10, or the composition as defined in claim 11, for use in:(i) the production of glycolaldehyde, as a product or intermediate, from ethylene glycol;(ii) the production of glycolic acid, as a product or intermediate, from glycolaldehyde; or(iii) the production of glycolic acid, as a product or intermediate, from ethylene glycol.

13. A polynucleotide encoding the alcohol dehydrogenase and / or the aldehyde dehydrogenase as defined in any one of claims 1 to 6.

14. An expression cassette comprising the polynucleotide of claim 13 operably linked to a heterologous promoter.

15. A vector comprising the polynucleotide of claim 13 or expression cassette of claim 14.

16. A process for producing glycolaldehyde, as a product or intermediate, from ethylene glycol, the process comprising contacting ethylene glycol with the isolated alcohol dehydrogenase as defined in any one of claims 1 to 7, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of ethylene glycol to glycolaldehyde.

17. A process for producing glycolic acid, as a product or intermediate, from glycolaldchydc, the process comprising contacting glycolaldehyde with the isolated aldehyde dehydrogenase as defined in any one of claims 1 to 7, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid.

18. A process for producing glycolic acid, as a product or intermediate, from ethylene glycol, the process comprising contacting ethylene glycol with the isolated aldehyde dehydrogenase as defined in any one of claims 1 to 7, or with a microorganism overexpressing same, under reaction or fermentation26RECTI FIED SHEET (RULE 91.1 )conditions enabling the conversion of ethylene glycol to glycolaldehyde; and contacting the glycolaldehyde with the isolated aldehyde dehydrogenase as defined in any one of claims 1 to 7, or with a microorganism overexpressing same, under reaction or fermentation conditions enabling the conversion of glycolaldehyde to glycolic acid.

19. The process of any one of claims 16 to 18, wherein the microorganism is as defined in any one of claims 8 to 10.

20. The process of any one of claims 16 to 19, wherein the microorganism is a microorganism that overexpresses both the aldehyde dehydrogenase and the aldehyde dehydrogenase.

21. The process of any one of claims 16 to 20, which comprises a growth phase during which the microorganism is cultured under conditions promoting biomass accumulation, and a production phase during which the microorganism is cultured under conditions promoting the production of the product or intermediate, wherein the transition from the growth phase to the production phase is not triggered by a modification in aeration conditions from the growth phase to the production phase (e.g., a decrease in oxygen uptake rate from the growth phase to the production phase).

22. A fermentation broth comprising: (i) ethylene glycol, glycolaldehyde, and / or glycolic acid; and (ii) the microorganism as defined in any one of claims 1 to 10, and / or the composition as defined in claim 11.27RECTI FIED SHEET (RULE 91.1 )

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