Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher Mw due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.

Background L-cysteine is an essential chemical building block in the pharmaceutical-, cosmetic-, food and agricultural sector. Conventionally, L-cysteine production relies on the conversion of keratinous biomass mediated by hydrochloric acid. Today, fermentative production based on recombinant E. coli, where L-cysteine production is streamlined and facilitated by synthetic plasmid constructs, is an alternative process at industrial scale. However, metabolic stress and the resulting production escape mechanisms in evolving populations are severely limiting factors during industrial biomanufacturing. We emulate high generation numbers typically reached in industrial fermentation processes with Escherichia coli harbouring L-cysteine production plasmid constructs. So far no genotypic and phenotypic alterations in early and late L-cysteine producing E. coli populations have been studied. Results In a comparative experimental design, the E. coli K12 production strain W3110 and the reduced genome strain MDS42, almost free of insertion sequences, were used as hosts. Data indicates that W3110 populations acquire growth fitness at the expense of L-cysteine productivity within 60 generations, while production in MDS42 populations remains stable. For the first time, the negative impact of predominantly insertion sequence family 3 and 5 transposases on L-cysteine production is reported, by combining differential transcriptome analysis with NGS based deep plasmid sequencing. Furthermore, metabolic clustering of differentially expressed genes supports the hypothesis, that metabolic stress induces rapid propagation of plasmid rearrangements, leading to reduced L-cysteine yields in evolving populations over industrial fermentation time scales. Conclusion The results of this study implicate how selective deletion of insertion sequence families could be a new route for improving industrial L-cysteine or even general amino acid production using recombinant E. coli hosts. Instead of using minimal genome strains, a selective deletion of certain IS families could offer the benefits of adaptive laboratory evolution (ALE) while maintaining enhanced L-cysteine production stability.

Chitin is the second most abundant polysaccharide worldwide as part of arthropods' exoskeletons and fungal cell walls. Low concentrations in soils and sediments indicate rapid decomposition through chitinolytic organisms in terrestrial and aquatic ecosystems. The enacting enzymes, so‐called chitinases, and their products, chitooligosaccharides, exhibit promising characteristics with applications ranging from crop protection to cosmetics, medical, textile, and wastewater industries. Exploring novel chitinolytic organisms is crucial to expand the enzymatical toolkit for biotechnological chitin utilization and to deepen our understanding of diverse catalytic mechanisms. In this study, we present two long‐read sequencing‐based genomes of highly similar Jeongeupia species, which have been screened, isolated, and biochemically characterized from chitin‐amended soil samples. Through metabolic characterization, whole‐genome alignments, and phylogenetic analysis, we could demonstrate how the investigated strains differ from the taxonomically closest strain Jeongeupia naejangsanensis BIO‐TAS4‐2T (DSM 24253). In silico analysis and sequence alignment revealed a multitude of highly conserved chitinolytic enzymes in the investigated Jeongeupia genomes. Based on these results, we suggest that the two strains represent a novel species within the genus of Jeongeupia, which may be useful for environmentally friendly N‐acetylglucosamine production from crustacean shell or fungal biomass waste or as a crop protection agent.

In nature, chitin, the most abundant marine biopolymer, does not accumulate due to the action of chitinolytic organisms, whose saccharification systems provide instructional blueprints for effective chitin conversion. Therefore, discovery and deconstruction of chitinolytic machineries and associated enzyme systems are essential for the advancement of biotechnological chitin valorization. Through combined investigation of the chitin-induced secretome with differential proteomic and transcriptomic analyses, a holistic system biology approach has been applied to unravel the chitin response mechanisms in the Gram-negative Jeongeupia wiesaeckerbachi. Hereby, the majority of the genome-encoded chitinolytic machinery, consisting of various glycoside hydrolases and a lytic polysaccharide monooxygenase, could be detected extracellularly. Intracellular proteomics revealed a distinct translation pattern with significant upregulation of glucosamine transport, metabolism, and chemotaxis-associated proteins. While the differential transcriptomic results suggested the overall recruitment of more genes during chitin metabolism compared to that of glucose, the detected protein-mRNA correlation was low. As one of the first studies of its kind, the involvement of over 350 unique enzymes and 570 unique genes in the catabolic chitin response of a Gram-negative bacterium could be identified through a three-way systems biology approach. Based on the cumulative data, a holistic model for the chitinolytic machinery of Jeongeupia spp. is proposed.

Background L-cysteine is an essential chemical building block in the pharmaceutical-, cosmetic-, food and agricultural sector. Conventionally, L-cysteine production relies on the conversion of keratinous biomass mediated by hydrochloric acid. Today, fermentative production based on recombinant E. coli, where L-cysteine production is streamlined and facilitated by synthetic plasmid constructs, is an alternative process at industrial scale. However, space-time yields and process stability are still to be optimised for improved economic viability. We simulate an industrial fermentation process with Escherichia coli harbouring various L-cysteine production plasmid constructs. Results In a comparative experimental design, theE. coli K12 production strain W3110 and the reduced genome strain MDS42, almost free of insertion sequences, were used as hosts. Data indcates that W3110 populations acquire growth fitness at the expense of L-cysteine productivity within 60 generations, while production in MDS42 populations remains stable. For the first time, the negative impact of predominantly insertion sequence family 3 and 5 transposases on L-cysteine production is reported, by combining differential transcriptome analysis with NGS based deep plasmid sequencing. Furthermore, metabolic clustering of differentially expressed genes supports the hypothesis, that metabolic stress induces rapid propagation of plasmid rearrangements, leading to reduced L-cysteine yields in evolving populations over industrial fermentation time scales. Conclusion The results of this study implicate how selective deletion of insertion sequence families could be a new route for improving industrial L-cysteine or even general amino acid production using recombinant E. coli hosts. Instead of using minimal genome strains, a selective deletion of certain IS families could offer the benefits of adaptive laboratory evolution (ALE) while maintaining enhanced L-cysteine production stability.