Customized chitooligosaccharide production—controlling their length via engineering of rhizobial chitin synthases and the choice of expression system

Chitooligosaccharides (COS) have attracted attention from industry and academia in various fields due to their diverse bioactivities. However, their conventional chemical production is environmentally unfriendly and in addition, defined and pure molecules are both scarce and expensive. A promising alternative is the in vivo synthesis of desired COS in microbial platforms with specific chitin synthases enabling a more sustainable production. Hence, we examined the whole cell factory approach with two well-established microorganisms—Escherichia coli and Corynebacterium glutamicum—to produce defined COS with the chitin synthase NodC from Rhizobium sp. GRH2. Moreover, based on an in silico model of the synthase, two amino acids potentially relevant for COS length were identified and mutated to direct the production. Experimental validation showed the influence of the expression system, the mutations, and their combination on COS length, steering the production from originally pentamers towards tetramers or hexamers, the latter virtually pure. Possible explanations are given by molecular dynamics simulations. These findings pave the way for a better understanding of chitin synthases, thus allowing a more targeted production of defined COS. This will, in turn, at first allow better research of COS’ bioactivities, and subsequently enable sustainable large-scale production of oligomers

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography

Abstract The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure–function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length. Additionally, the method allows to determine the average molecular weight of the remaining polymers and its distribution. By sampling live from an ongoing enzymatic reaction, UHPSEC-RI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously—i.e., to monitor the molecular weight reduction of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a semi-quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme’s action on a given substrate

DataSheet1_Customized chitooligosaccharide production—controlling their length via engineering of rhizobial chitin synthases and the choice of expression system.pdf

Chitooligosaccharides (COS) have attracted attention from industry and academia in various fields due to their diverse bioactivities. However, their conventional chemical production is environmentally unfriendly and in addition, defined and pure molecules are both scarce and expensive. A promising alternative is the in vivo synthesis of desired COS in microbial platforms with specific chitin synthases enabling a more sustainable production. Hence, we examined the whole cell factory approach with two well-established microorganisms—Escherichia coli and Corynebacterium glutamicum—to produce defined COS with the chitin synthase NodC from Rhizobium sp. GRH2. Moreover, based on an in silico model of the synthase, two amino acids potentially relevant for COS length were identified and mutated to direct the production. Experimental validation showed the influence of the expression system, the mutations, and their combination on COS length, steering the production from originally pentamers towards tetramers or hexamers, the latter virtually pure. Possible explanations are given by molecular dynamics simulations. These findings pave the way for a better understanding of chitin synthases, thus allowing a more targeted production of defined COS. This will, in turn, at first allow better research of COS’ bioactivities, and subsequently enable sustainable large-scale production of oligomers.</p