Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and their synergism

Bissaro

Journal of Biological Chemistry

et al. 2018

138

229

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials. It has recently been shown that LPMOs can use HO, instead of O, as a cosubstrate. This peroxygenase-like reaction by a monocopper enzyme is unprecedented in nature and opens new avenues in chemistry and enzymology. Here, we provide the first detailed kinetic characterization of chitin degradation by the bacterial LPMO chitin-binding protein CBP21 using HO as cosubstrate. The use of C-labeled chitin provided convenient and sensitive detection of the released soluble products, which enabled detailed kinetic measurements. The for chitin oxidation found here (5.6 s) is more than an order of magnitude higher than previously reported (apparent) rate constants for reactions containing O but no added HO The / for HO-driven degradation of chitin was on the order of 10 m s, indicating that LPMOs have catalytic efficiencies similar to those of peroxygenases. Of note, HO also inactivated CBP21, but the second-order rate constant for inactivation was about 3 orders of magnitude lower than that for catalysis. In light of the observed CBP21 inactivation at higher HO levels, we conclude that controlled generation of HO seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides.

Section: Kinetics Of the H 2 O 2 -Driven Degradation Of Chitin By Cbp21mentioning

confidence: 99%

Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Bissaro

Journal of Biological Chemistry

et al. 2018

138

229

“…The newly formed chitin chain ends formed by LPMO activity, represent new points of attachment for the chitinases, thereby increasing substrate accessibility. Indeed, several studies have demonstrated this phenomenon (16, 68–70), including a study on the virulence-related LPMO from Listeria monocytogenes (71).…”

Section: Discussionmentioning

confidence: 97%

The fish pathogenAliivibrio salmonicidaLFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

Skåne

Minniti

Loose

et al. 2021

Preprint

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc)2), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both a- and b-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on β-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on β-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability.

Appl Microbiol Biotechnol

“…As the co-culture required a freshwater salinity, we investigated the chitinolytic enzymes of A. hydrophila . In fact, an E. coli strain secreting a chitinase from A. hydrophila as well as an LPMO from S. marcescens proved that simultaneous production and secretion of these two types of enzymes by E. coli is possible (Yang et al 2017 ). We have identified the A. hydrophila strain AH-1 N based on an enrichment approach with chitin as substrate (Stumpf et al 2019 ), and characterized its chitinase AH-1NChi as being rather efficient on crystalline chitin, and as acting synergistically with the LPMO AhLPMO10A from the same strain (Vortmann et al unpublished).…”

Section: Discussionmentioning

confidence: 99%

A bottom-up approach towards a bacterial consortium for the biotechnological conversion of chitin to l-lysine

Vortmann

Stumpf²,

Sgobba

et al. 2021

Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc2, chitin deacetylase to convert GlcNAc2 into GlcN2 and acetate, and glucosaminidase to cleave GlcN2 into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc2 or GlcN2 to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin. Graphical abstract Key Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.