Enhanced Biosynthesis of Hyaluronic Acid Using Engineered Corynebacterium glutamicum Via Metabolic Pathway Regulation

• Published in: Biotechnology journal Vol. 12; no. 10
• Main Authors: Cheng, Fangyu, Luozhong, Sijin, Guo, Zhigang, Yu, Huimin, Stephanopoulos, Gregory
• Format: Journal Article
• Language: English
• Published: Germany 01-10-2017
• Subjects: Adenosine Triphosphate; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Batch Cell Culture Techniques; Bioreactors; Biosynthetic Pathways - genetics; Corynebacterium glutamicum - enzymology; Corynebacterium glutamicum - genetics; Corynebacterium glutamicum - growth & development; Corynebacterium glutamicum - metabolism; DNA, Bacterial; engineered Corynebacterium glutamicum; fed‐batch culture; Fermentation; Gene Knockout Techniques; Genes, Bacterial - genetics; Glucose - metabolism; hyaluronic acid; Hyaluronic Acid - biosynthesis; L-Lactate Dehydrogenase - genetics; LDH gene knockout; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Operon; operon organization; engineered Corynebacterium glutamicum; operon organization; LDH gene knockout; fed-batch culture; hyaluronic acid
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201700191

Hyaluronic acid (HA) is a polysaccharide used in many industries such as medicine, surgery, cosmetics, and food. To avoid potential pathogenicity caused by its native producer, Streptococcus, efforts have been made to create a recombinant host for HA production. In this work, a GRAS (generally recognized as safe) strain, Corynebacterium glutamicum, is engineered for enhanced biosynthesis of HA via metabolic pathway regulation. Five enzymes (HasA‐HasE) involved in the HA biosynthetic pathway are highlighted, and eight diverse operon combinations, including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE, are compared. HasAB and HasABC are found to be optimal for HA biosynthesis in C. glutamicum. To meet the energy demand for HA synthesis, the metabolic pathway that produces lactate is blocked by knocking out the lactate dehydrogenase (LDH) gene using single crossover homologous recombination. Engineered C. glutamicum/Δldh‐AB is superior and had a significantly higher HA titer than C. glutamicum/Δldh‐ABC. Batch and fed‐batch cultures of C. glutamicum/Δldh‐AB are performed in a 5‐L fermenter. Using glucose feeding, the maximum HA titer reached 21.6 g L−1, more than threefolds of that of the wild‐type Streptococcus. This work provides an efficient, safe, and novel recombinant HA producer, C. glutamicum/Δldh‐AB, via metabolic pathway regulation.
Enhanced biosynthesis of hyaluronic acid with engineered Corynebacterium glutamicum is achieved via metabolic regulation: Eight diverse operon combinations including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE are compared and HasAB along with HasABC performed the best. LDH gene knockout is conducted by single crossover homologous recombination and the recombinant strain C. glutamicum/△ldh‐AB is constructed, meanwhile ATP supply is enhanced in the early stage of batch culture. Through glucose feeding, the maximum HA titer of C. glutamicum/△ldh‐AB in a 5 L fermenter highly reached 21.6 g L−1, more than threefolds of that of the wild Streptococcus.