Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus

Abstract

2-keto-d-gluconic acid (2-KGA) is a key precursor for synthesising vitamin C and isovitamin C. However, phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens. Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids, and high utilisation of glucose. In this study, the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-d-gluconic acid (5-KGA) and d-gluconic acid (D-GA) in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49. Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81% in shake flasks. Additionally, accumulation of 5-KGA was decreased by 63.52% with the resulting G. japonicas-Δga5dh-1-ga2dh-A strain. Using an intermittent fed-batch mode in a 3 L fermenter, 2-KGA reached 235.3 g L−1 with a 91.1% glucose conversion rate. Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L−1 h−1, 11.99% higher than in the 3 L fermenter, and D-GA and 5-KGA by-products were completely converted to 2-KGA.