(191at) Blocking Lactic Acid Pathway for Enhanced HA Production in C. Glutamicum

Hyaluronic acid (HA), containing D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) as a disaccharide unit, plays multiple roles in human body and has been widely applied in cosmetics, surgery, medical, food industry and so on. According to the estimation, the market value of HA would exceed 10 billion US dollars until 2020. Presently, the main approach to produce HA is fermentation. With the development of bioengineering, Streptococcusor genetically engineered Bacillus could obtain HA with titer by 6-10 g/L.

Corynebacterium glutamicum (C. glutamicum) is a non-pathogenic, non-sporulating bacterium that generally recognized as safe (GRAS). It is an ideal alternative recombinant host for HA production. In our previous study, co-expressing HA synthase (HasA) from S. equisimilis and native UDP-glucose dehydrogenase gene (HasB) in engineered C. glutamicum successfully achieved 5-6 g/L HA titer in flask culture and 8-9 g/L HA titer in batch culture of 5L fermentor. In this work, a novel idea is further proposed to engineer the recombinant C. glutamicum for enhanced HA synthesis.

HA biosynthesis is an ATP consuming pathway. Every 2 mol glucose needs 5 mol ATP equivalent to form a disaccharide unit. Lactate dehydrogenase (LDH) participates transforming 1 mol pyruvic acid to lactic acid, in which 2 mol ATP equivalent was consumed. Blocking lactic acid pathway would enable more pyruvic acid flowing into TCA cycle and creating ATP. Therefore, a LDH-inactive C. glutamicum mutant (∆ldh) was constructed by single cross over homologous recombination. An engineered HA producer, C. glutamicum ∆ldh-AB with HA synthesis operon (hasA-hasB) was further obtained through introducing C. glutamicum- E. coli shuttle plasmid pXMJ19-ssehasA-hasB (AB). In 5 L fermentor, the biomass and HA titer reached 14.7 g/L and 10.6 g/L, respectively, with 40g/L glucose as carbon source. To enhance the HA accumulation, glucose feeding strategy was further applied to keep residual glucose concentration higher than 8 g/L after 20 h. In the fed-batch culture, the maximal titer of HA achieved 21.6 g/L, which is the highest result reported so far. The M_w of the HA product consisted two part, 1.28 MDa (mass percentage 14.2%) and 290 KDa (mass percentage 85.8%), respectively.