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Gluconobacter Oxydans WSH-003 for the Direct Production of 2-Keto-L-Gulonic Acid from D-Sorbitol

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Gluconobacter Oxydans WSH-003 for the Direct Production of 2-Keto-L-Gulonic Acid from D-Sorbitol Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol Lili Gao a,b, Yudong Hu a,b, Jie Liu c, Guocheng Du a,b, Jingwen Zhou a,b,n, Jian Chen a,b,n a School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China b Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China c Jiangsu Jiangshan Pharmaceutical Co., Ltd. Jingjiang 214500, China article info abstract Article history: 2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step Received 19 November 2013 fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture Received in revised form system complicated and redundant. Thus, replacement of the conventional two-step fermentation 18 March 2014 process with a one-step process could be revolutionary in vitamin C industry. In this study, different Accepted 21 April 2014 combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) Available online 30 April 2014 from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an Keywords: industrial strain used for the conversion of D-sorbitol to L-sorbose. The optimum combination produced Vitamin C 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and L-sorbose dehydrogenase SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4 g/L L-sorbosone dehydrogenase of 2-KLG after 168 h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those One-step fermentation Linker peptides dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering fi Pyrroloquinoline quinine of G. oxydans, the nal 2-KLG production was improved to 39.2 g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C. & 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. 1. Introduction original chemical Reichstein process and became the dominant industrial production route in early 1990s (Reichstein et al., 1934). Vitamin C (L-ascorbic acid, L-AA) is an essential nutrient and The conventional two-step fermentation route is the most antioxidant for humans, and is widely used in pharmaceutical, successful method for L-AA production, and has been used on an food, beverage, cosmetics, and feed industries (Fossati et al., 2011; industrial scale for several decades. However, unlike most of the Zou et al., 2012). Currently, L-AA is produced at an industrial scale common biotechnological processes, the conventional two-step by the so-called conventional two-step fermentation process, in fermentation of L-AA involves three microorganisms and requires which L-sorbose is first formed from D-sorbitol using Gluconobacter an additional second sterilization process. This significantly oxydans, and then L-sorbose is catalyzed in Ketogulonicigenium increases the cost of both raw materials and energy requirement vulgare with Bacillus megaterium as the companion bacterium to (Zhu et al., 2011). Furthermore, the mix-culture system composed obtain 2-keto-L-gulonic acid (2-KLG), a precursor that could be of B. megaterium and K. vulgare makes both strain improvement easily converted to L-AA by a simple acidification process (Takagi et and process optimization difficult. Therefore, a one-step fermenta- al., 2010). The conversions of D-sorbitol to L-sorbose, L-sorbose to tion process is considered to be more cost-effective and revolu- L-sorbosone, and L-sorbosone to 2-KLG are catalyzed by sorbitol tionary in the L-AA industry worldwide. dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbo- Two different routes have mostly been employed for the one- sone dehydrogenase (SNDH), respectively (Gao et al., 2013). This step fermentation of L-AA. The first route is the one-step process method was developed in the 1960s, and completely replaced the based on the conventional two-step fermentation process, in which introduction of SDH and SNDH in G. oxydans leads to the direct conversion of D-sorbitol to 2-KLG because the bacterium produces SLDH and possesses the essential pyrroloquinoline n Corresponding authors at: School of Biotechnology, Ministry of Education, Jiangnan quinine (PQQ) biosynthesis and regeneration system (Gao et al., University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Fax: 86 510 85918309. þ E-mail addresses: [email protected] (J. Zhou), 2012). The second route is the one-step process based on novel [email protected] (J. Chen). two-step fermentation process, in which D-glucose is converted to Reproduced from Metabolic Engineering 24: 30-37 (2014). Jian Chen: Participant of the 21st UM, 1993-1994. 226 227 2,5-diketo-gluonic acid (2,5-DKG) by Erwinia herbicola, and then experiment. The pre-culture was cultivated in 5 mL of 5% sorbitol 2,5-DKG is converted to 2-KLG by a strain of Corynebacterium sp. medium at 30 1C and 200 rpm, and transferred to 50 mL of (Anderson et al., 1985). Through overexpression of 2,5-diketo- medium (5% or 15% D-sorbitol and 1% yeast extract) in a 500-mL gluconic acid reductase from Corynebacterium ATCC 31090 in flask with 50 μg/mL of ampicillin. Furthermore, to verify whether E. herbicola ATCC 21988, a single recombinant organism that could 2-KLG was degraded by G. oxydans or not, 50 g/L of 2-KLG was directly produce 1 g/L of 2-KLG from saturated D-glucose solution added to the sorbitol medium. Batch fermentation was performed has been developed (Anderson et al., 1985). Besides, Lin et al. in a 3-L bioreactor (BioFlo 115, New Brunswick Scientific Co., (1999) fused E. herbicola and a Corynebacterium strain by proto- Edison, NJ), and the temperature of the reactor was maintained at Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 plast fusion, and observed that the resultant strain could produce 30 1C, the agitation speed was controlled at 400 rpm, and the fi for the direct production of 2-keto-L-gulonic acid from D-sorbitol 2.07 g/L of 2-KLG. Nevertheless, further signi cant increase in aeration rate was 1.5 vvm. 2-KLG production by this route has been rarely reported. fi Lili Gao a,b, Yudong Hu a,b, Jie Liu c, Guocheng Du a,b, Jingwen Zhou a,b,n, Jian Chen a,b,n Rapid development of systems biology methods has signi - 2.3. Construction of the shuttle vector cantly expanded our understanding of the conventional two-step a School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China L-AA fermentation process (Zou et al., 2012). Through genomics The genomic DNA and plasmids of the strains were extracted b Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China and proteomics analyses, the essential genetic elements related to using the FastPure DNA kit and SanPrep Column Plasmid c Jiangsu Jiangshan Pharmaceutical Co., Ltd. Jingjiang 214500, China 2-KLG metabolism have been systematically identified. In our Mini-Preps Kit (Sangon, Shanghai, China). The par-rep gene (see previous studies, all the potential dehydrogenases and gene Supplementary material) of cryptic plasmid pGOX3 from G. clusters associated with PQQ biosynthesis in G. oxydans and K. oxydans 621H was amplified with primers pr-F and pr-R article info abstract vulgare were identified based on genome sequencing (Gao et al., (Table 1). The PCR product encoding the par-rep gene was digested 2012; Liu et al., 2011). In addition, the enzymatic characteristics and inserted into the EcoRI/SacI site of the pUC18, resulting in Article history: 2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step were further demonstrated through detailed biochemical analysis pGUC (L. Zhang et al., 2010). Received 19 November 2013 fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture (Gao et al., 2013). These advances have significantly facilitated the Received in revised form system complicated and redundant. Thus, replacement of the conventional two-step fermentation fi 18 March 2014 process with a one-step process could be revolutionary in vitamin C industry. In this study, different development of the ef cient one-step process based on the 2.4. Overexpression of SDH and SNDH in G. oxydans WSH-003 Accepted 21 April 2014 combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) conventional two-step fermentation process. Available online 30 April 2014 from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an In the present study, different combinations of five SDHs and two In our previous study, five SDHs (KVU_pmdA_0245, KVU_2142, Keywords: industrial strain used for the conversion of D-sorbitol to L-sorbose. The optimum combination produced SNDHs from K. vulgare WSH-001 were introduced into G. oxydans KVU_2159, KVU_1366, and KVU_0203; GenBank Accession No. Vitamin C 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and WSH-003, and the optimum combination resulted in the production 12376006, 12375064, 12375082, 12374244 and 12373039) and L-sorbose dehydrogenase SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4 g/L of 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were L-sorbosone dehydrogenase two SNDHs (KVU_0095 and KVU_pmdB_0115; GenBank Accession of 2-KLG after 168 h.
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