Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum

Abstract

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 hyaluronan in fedbatch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L −1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.

Hyaluronic Acid: The Influence of Molecular Weight on Structural, Physical, Physico-Chemical, and Degradable Properties of Biopolymer

“…Apart from the animal of origin, hyaluronic acid can be separated based on bacteria, for example, from Streptococcus genus ( uberis, equisimilis, zooepidermicus, pyogenes, equi ), Pasteurella multocida [ 6 , 7 , 8 , 9 , 10 ], and Corynebacterium glutamicum [ 11 ]; from the green algae Chlorella purposely infected by the Chlorovirus [ 6 , 7 , 12 ]; Saccharomycetes ( Cryptococcus neoformans [ 6 , 7 ]); and from molluscan shellfish, such as the bivalve mollusc Mytilus galloprovincialis [ 6 , 13 ]. At the same time, hyaluronic acid has not been disclosed in fungus, insects, or plants [ 6 , 14 ].…”

Primary recovery of hyaluronic acid produced in Streptococcus equi subsp. zooepidemicus using PEG–citrate aqueous two-phase systems

“…Microorganisms that naturally synthesise HA, such as Streptococcus equi subsp. zooepidemicus , or heterologous expression systems, such as Bacillus subtilis , Pichia pastoris , Lactococcus lactis , and Corynebacterium glutamicum have been genetically or metabolically engineered to develop strains that produce high molecular weight HA and increased product yield (Chen et al 2009 ; Cheng et al 2019 ; Jeong et al 2014 ; Jia et al 2013 ; Kaur and Jayaraman 2016 ; Wang et al 2020 ; Widner et al 2005 ).…”

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production

“…This method was reported previously to confirm the production of HA by L. lactis [ 45 ] and by the yeast K. lactis [ 11 ]. The formation of a capsule-like layer was also observed in HA-producing strains of C. glutamicum [ 46 ], although a phase-contrast microscopy analysis was applied instead of SEM. In this case, the capsule-like layer affected nutrient uptake and cell metabolism, which impaired HA production [ 46 ].…”