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

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 ß-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. 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.

Identification of a novel chitinase from Aeromonas hydrophila AH-1N for the degradation of chitin within fungal mycelium.

Defined organic waste products are ideal and sustainable secondary feedstocks for production organisms in microbial biotechnology. Chitin from mycelia of fungal fermentation processes represents a homogeneous and constantly available waste product that can, however, not be utilised by typical bacterial production strains. Therefore, enzymes that degrade chitin within fungal mycelia have to be identified and expressed in production organisms. In this study, chitin-degrading bacteria were enriched and isolated from lake water with mycelia of Aspergillus tubingensis as sole organic growth substrate. This approach yielded solely strains of Aeromonas hydrophila. Comparison of the isolated strains with other A. hydrophila strains regarding their chitinolytic activities on fungal mycelia identified strain AH-1N as the best enzyme producer. From this strain, a chitinase (EC:3.2.1.14) was identified by peptide mass fingerprinting. Heterologous expression of the respective gene combined with mass spectrometry showed that the purified enzyme was capable of releasing chitobiose from fungal mycelia with a higher yield than a well-described chitinase from Serratia marcescens. Expression of the newly identified chitinase in biotechnological production strains could be the first step for making fungal mycelium accessible as a secondary feedstock. Additionally, the enrichment strategy proved to be feasible for identifying strains able to degrade fungal chitin.

Synthetic Escherichia coli-Corynebacterium glutamicum consortia for l-lysine production from starch and sucrose.

In the biorefinery concept renewable feedstocks are converted to a multitude of value-added compounds irrespective of seasonal or other variations of the complex biomass substrates. Conceptionally, this can be realized by specialized single microbial strains or by co-culturing various strain combinations. In the latter approach strains for substrate conversion and for product formation can be combined. This study addressed the construction of binary microbial consortia based on starch- and sucrose-based production of l-lysine and derived value-added compounds. A commensalism-based synthetic consortium for l-lysine production from sucrose was developed combining an l-lysine auxotrophic, naturally sucrose-negative E. coli strain with a C. glutamicum strain able to produce l-lysine that secretes fructose when grown with sucrose due to deletion of the fructose importer gene ptsF. Mutualistic synthetic consortia with an l-lysine auxotrophic, α-amylase secreting E. coli strain and naturally amylase-negative C. glutamicum strains was implemented for production of valuable fine chemicals from starch.