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

Abstract

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.

Metabolic Engineering for Valorization of Agri- and Aqua-Culture Sidestreams for Production of Nitrogenous Compounds by Corynebacterium glutamicum

“…A synthetic consortium composed of a chitin degrading E. coli strain and a C. glutamicum strain producing nitrogenous compounds has been used to develop chitin-based production ( Vortmann et al, 2021 ). In this proof-of-concept study, the chitin monomer N -acetylglucosamine is hydrolyzed by a lysine-auxotrophic E. coli strain to yield acetate and glucosamine.…”

“…Acetate is the only carbon source for the engineered E. coli strain, whereas the lysine-secreting C. glutamicum producer strain was engineered to only use glucosamine. Chitin degrading enzymes (chitinase, chitin deacetylase and glucosaminidase) had to be added to the growth media, but they may be synthesized and secreted by the E. coli strain used ( Vortmann et al, 2021 ).…”

“…Online off-gas analysis and particle filtering for simultaneous process state and parameter estimation have recently been used to judge performance of a single C. glutamicum strain during SSL bioprocessing ( Sinner et al, 2021 ). Furthermore, synthetic consortia have been developed for division of labor between different strains, e.g., various C. glutamicum strains or C. glutamicum and E. coli strains production based on either starch, chitin or SSL ( Sgobba et al, 2018b ; Pérez-García et al, 2021 ; Vortmann et al, 2021 ). SSL bioprocessing for production of riboflavin was improved in fed-batch fermentations based on a inoculation strategy using strains that either utilized glucose, glucose + mannose, glucose + xylose or all three sugars ( Pérez-García et al, 2021 ).…”

Co-cultivation, Co-culture, Mixed Culture, and Microbial Consortium of Fungi: An Understudied Strategy for Biomass Conversion

“…Bacterial co-cultures, mostly referred as microbial/bacterial consortia, have been investigated to much greater detail and possess wider applications. In fact, bacterial consortia are fabricated for specific biotechnological goals (Vortmann et al, 2021 ). Synthetic microbial consortia borrow ecological concepts, such as amensalism and commensalism, to engineer high performance multi-species systems (Sgobba and Wendisch, 2020 ).…”

Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum

“…Recently, it could also be demonstrated that production of sarcosine from the pentose xylose was more efficient than that from glucose ( Mindt et al, 2019b ). Additionally, less processed substrates such as lignocellulosic residuals, rich straw, wheat bran and plant mass hydrolysates, chitin, the biorefinery sidestream pyrolysis water, and wheat sidestream concentrate (WSC) from the starch and paper industries could be harnessed by C. glutamicum as carbon sources ( Gopinath et al, 2011 ; Lange et al, 2017 ; Mindt et al, 2019a ; Sasaki et al, 2019 ; Prell et al, 2021a ; Vortmann et al, 2021 ).…”