title: Enhancement of 5-keto-d-gluconate production by a recombinant Gluconobacter oxydans using a dissolved oxygen control strategy.
abstract: The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75±0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high μ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h).
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of the Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: jf_yuan@zju.edu.cn.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of the Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: wumb@zju.edu.cn.
authors.forename: Mianbin
authors.initials: M
authors.lastname: Wu
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of the Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: linjp@zju.edu.cn.
authors.forename: Jianping
authors.initials: J
authors.lastname: Lin
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of the Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: lryang@zju.edu.cn.
authors.forename: Lirong
authors.initials: L
authors.lastname: Yang
mesh_terms: D019149:Bioreactors; D049109:Cell Proliferation; D005285:Fermentation; D005942:Gluconates / Q000138:chemical synthesis* / Q000378:metabolism; D020600:Gluconobacter oxydans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D060847:Metabolic Engineering; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D013644:Tartrates / Q000378:metabolism; D016959:Xanthomonas campestris / Q000201:enzymology / Q000235:genetics
chemical_list: C000614856:5-keto-d-gluconate; D005942:Gluconates; D013644:Tartrates; D005947:Glucose; C030691:gluconic acid; D010100:Oxygen; C029768:tartaric acid
keywords: 5-Keto-d-gluconate; Dissolved oxygen control strategy; Gluconobacter oxydans; Pyruvate decarboxylase; Secondary alcohol dehydrogenase