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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.
The production of food-grade substances and complex biocatalysts used as additives or active ingredients – mainly for food applications – can be achieved in the eukaryotic expression system of Aspergillus niger. Food proteins or food enzymes e.g., casein, ovalbumin, phytase or glucoamylase are highly complex polymers. Most of them could be used as nitrogen or energy source for animals and humans, while others are industrial relevant biomass-degrading enzymes used for biological waste processing and food production.
However, the successful production of novel recombinant proteins can be challenging, resource- and time consuming. Therefore, A. niger mutant libraries are needed to understand the “adjusting screws” to produce high yields of recombinant proteins, preferably even in a kind of generic, transferable system. In order to establish a universal and multipurpose expression platform, there is the need to overcome the lack of high throughput assays first.
To tackle this problem, we designed a modular, quantitative and feasible high-throughput screening system to express and screen recombinant proteins regarding their stability and functionality in A. niger. For this purpose a dual-luciferase reporter gene system, which is applicable in small scale will be established for A. niger. After the generation of an A. niger secretion mutant library, the system will be transferred and tested to other proteins of interest. The technology can be integrated into bio-regenerative life support systems for the autonomous production of e.g., food, food additives and food enzymes on earth as well as in deep-space.