bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Identification of NAD-dependent xylitol dehydrogenase from Gluconobacter 2 oxydans WSH-003 3 4 Li Liu1,2,4, Weizhu Zeng1, Guocheng Du1,3, Jian Chen1,2,4, Jingwen Zhou1,2,4* 5 6 1 School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry 7 of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. 8 2 National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan 9 University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. 10 3 The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of 11 Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. 12 4 Jiangsu Provisional Research Center for Bioactive Product Processing Technology, 13 Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. 14 15 * Corresponding author: Jingwen Zhou 16 Mailing address: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 17 Jiangsu 214122, China 18 Phone: +86-510-85914317, Fax: +86-510-85914317 19 E-mail: [email protected]. 20 21 bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 22 Abstract 23 Gluconobacter oxydans plays important role in conversion of D-sorbitol to L-sorbose, 24 which is an essential intermediate for industrial-scale production of vitamin C. In the 25 fermentation process, some D-sorbitol could be converted to D-fructose and other 26 byproducts by uncertain dehydrogenases. Genome sequencing has revealed the 27 presence of diverse genes encoding dehydrogenases in G. oxydans. However, the 28 characteristics of most of these dehydrogenases remain unclear. Therefore, analyses of 29 these unknown dehydrogenases could be useful for identifying those related to the 30 production of D-fructose and other byproducts. Accordingly, dehydrogenases in G. 31 oxydans WSH-003, an industrial strain used for vitamin C production, were examined. 32 An NAD-dependent dehydrogenase, which was annotated as xylitol dehydrogenase 2, 33 was identified, codon-optimized, and expressed in Escherichia coli BL21 (DE3) cells. + 34 The enzyme exhibited high preference for NAD as the cofactor, while no activity 35 with NADP+, FAD, or PQQ was noted. Although this enzyme presented high 36 similarity with NAD-dependent xylitol dehydrogenase, it showed high activity to 37 catalyze D-sorbitol to D-fructose. Unlike the optimum temperature and pH for most of 38 the known NAD-dependent xylitol dehydrogenases (30°C–40°C and about 6–8, 39 respectively), those for the identified enzyme were 57°C and 12, respectively. The Km 40 and Vmax of the identified dehydrogenase towards L-sorbitol were 4.92 μM and 196.08 41 μM/min, respectively. Thus, xylitol dehydrogenase 2 can be useful for cofactor 42 NADH regeneration under alkaline conditions or its knockout can improve the 43 conversion ratio of D-sorbitol to L-sorbose. 44 45 Keywords: D-Fructose; D-sorbitol; cofactor regeneration; metabolic pathway. 46 bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 47 Importance 48 Production of L-sorbose from D-sorbitol by Gluconobacter oxydans is the first step 49 for industrial scale production of L-ascorbic acid. G. oxydans contains a lot of 50 different dehydrogenases, among which only several are responsible for the 51 conversion of D-sorbitol to L-sorbose, while others may responsible for the 52 accumulation of byproducts, thus decreased the yield of L-sorbose on D-sorbitol. 53 Therefore, a new xylitol dehydrogenase has been identified from 44 dehydrogenases 54 of G. oxydans. Optimum temperature and pH of the xylitol dehydrogenase are 55 different to most of the known ones. Knock-out of the dehydrogenase may improve 56 the conversion ratio of D-sorbitol to L-sorbose. Besides, the enzyme exhibits high 57 preference for NAD+ and have potential to be used for cofactor regeneration. 58 59 60 bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 61 Introduction 62 The genus Gluconobacter is a part of the group of acetic acid bacteria, which are 63 characterized by their ability to incompletely oxidize a wide range of carbohydrates 64 and alcohols (1). Gluconobacter strains have been successfully used for the industrial 65 production of food-related products, pharmaceuticals, and cosmetics, such as vitamin 66 C (2), miglitol (3), dihydroxyacetone (DHA) (4), and ketogluconates (5). In particular, 67 Gluconobacter oxydans has applications in the production of food additives and 68 sweeteners owing to its ability to synthesize flavoring ingredients from aromatic 69 alcohols, aliphatic alcohols, and 5-ketofructose (6, 7). Besides, G. oxydans enzymes, 70 cell membranes, and whole cells are also used as sensor systems for the detection of 71 polyols, sugars, and alcohols (8-10). In recently years, some G. oxydans strains have 72 been employed for the production of enantiomeric pharmaceuticals and platform 73 compounds; for example, G. oxydans DSM2343 has been employed for the reduction 74 of various ketones used in pharmaceutical, agrochemical, and natural products (11), 75 Gluconobacter sp. JX-05 has been utilized for D-xylulose and xylitol production (12), 76 and G. oxydans DSM 2003 has been used for 3-hydroxypropionic acid production (13). 77 As all of these products are related to the dehydrogenases of G. oxydans, identification 78 of these enzymes in G. oxydans can expand the application of this bacterium. 79 Gluconobacter strains possess numerous dehydrogenases, some of which have 80 been identified, such as alcohol dehydrogenase that could convert ethanol to 81 acetaldehyde (14, 15), NADP-dependent acetaldehyde dehydrogenase that could 82 convert acetaldehyde to acetate (16), PQQ-dependent glucose dehydrogenase that 83 could convert D-glucose to D-gluconate (14), gluconate dehydrogenase that could 84 convert D-gluconate to 2- or 5-ketogluconate (17), 2-ketogluconate dehydrogenase 85 that could convert 2-ketogluconate to 2,5-diketogluconate (14), D-sorbitol bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 86 dehydrogenase that could convert D-sorbitol to L-sorbose or fructose (14, 18-21), 87 sorbose/sorbosone dehydrogenase that could convert L-sorbose to L-sorbosone or 88 2-KLG (22, 23), mannitol dehydrogenase that could convert mannitol to fructose (24, 89 25), quinate dehydrogenase that could convert quinic acid to shikimic acid (26-28), 90 glycerol dehydrogenase that could convert glycerol to DHA (28), etc. In 2005, the 91 complete genome of G. oxydans 621H was sequenced (29), which revealed 75 open 92 reading frames (ORFs) that encode putative dehydrogenases/oxidoreductases of 93 unknown functions. Identification of the functions of these unknown 94 dehydrogenases/oxidoreductases is important to expand the application of G. oxydans. 95 For instance, carbonyl reductase (GoKR) from G. oxydans DSM2343 has been 96 employed for the reduction of various ketones (11), and membrane-bound alcohol 97 dehydrogenase (mADH) and membrane-bound aldehyde dehydrogenase from G. 98 oxydans DSM 2003 have been employed for 3-hydroxypropionic acid production 99 (13). 100 In G. oxydans, the central metabolic pathway, such as citrate cycle and 101 Embden-Meyerhof-Parnas pathway (EMP), is incomplete because of the absence of 102 some genes encoding succinate dehydrogenase, phosphofructokinase, 103 phosphotransacetylase, acetate kinase, succinyl-CoA synthetase, succinate 104 dehydrogenase, isocitratelyase, and malate synthase (30, 31), which may be the reason 105 for the low biomass of G. oxydans when cultured in rich medium. In a previous study, 106 sdhCDABE genes encoding succinate dehydrogenase and flavinylation factor SdhE, 107 ndh gene encoding a type II NADH dehydrogenase, and sucCD from 108 Gluconacetobacter diazotrophicus encoding succinyl-CoA synthetase were expressed 109 in G. oxyda ns to increase its biomass yield (31). However, G. oxydans biomass only 110 increased by 60%, suggesting the presence of some unknown bottleneck. Except for bioRxiv preprint doi: https://doi.org/10.1101/634238; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 111 the TCA cycle, all the genes were identified to encode enzymes involved in oxidative 112 pentose phosphate and Entner-Doudoroff (ED) pathways (29). The pentose phosphate 113 pathway is believed to be the most important route for phosphorylative breakdown of 114 sugars and polyols to CO2 and provide carbon skeleton. It has been speculated that G. 115 oxydans has the capability to uptake and channelize several polyols, sugars, and sugar 116 derivatives into the oxidative pentose phosphate pathway; however, the gene involved 117 in this process is still unknown.