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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.

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.

Fatty acid hydratases are unique to microorganisms. Their native function is the oxidation of unsaturated C–C bonds to enable detoxification of environmental toxins. Within this enzyme family, the oleate hydratases (Ohys), which catalyze the hydroxylation of oleic acid to 10-( R )-hydroxy stearic acid (10-HSA) have recently gained particular industrial interest. 10-HSA is considered to be a replacement for 12-( R )-hydroxy stearic acid (12-HSA), which has a broad application in the chemical and pharmaceutical industry. As 12-HSA is obtained through an energy consuming synthesis process, the biotechnological route for sustainable 10-HSA production is of significant industrial interest. All Ohys identified to date have a non-redox active FAD bound in their active site. Ohys can be divided in several subfamilies, that differ in their oligomerization state and the decoration with amino acids in their active sites. The latter observation indicates a different reaction mechanism across those subfamilies. Despite intensive biotechnological, biochemical and structural investigations, surprising little is known about substrate binding and the reaction mechanism of this enzyme family. This review, summarizes our current understanding of Ohys with a focus on sustainable biotransformation.

The chemical, pharmaceutical, and cosmetic industries are currently confronted with the challenge of transitioning from traditional chemical processes to more sustainable biocatalytic methods. To support that aim, we developed various heterogeneous biocatalysts for an industrially relevant enzyme called oleate hydratase that converts oleic acid to 10-hydroxystearic acid, a fatty emollient substance useful for various technical applications. We used cheap support matrices such as silica, chitosan, cellulose, and agarose for further scale-up and economic feasibility at the industrial level alongside more sophisticated supports like metal–organic frameworks. Different physical and chemical binding approaches were employed. Particularly, by immobilizing oleate hydrates on a 3-aminopropyltriethoxysilane surface-functionalized cellulose matrix, we developed an enzyme immobilizate with almost 80% activity of the free enzyme. The long-term goal of this work was to be able to use the developed heterogeneous biocatalyst for multiple reuse cycles enabling profitable biocatalysis. Despite high initial conversion rate by the developed cellulose-based immobilizate, a depletion in enzyme activity of immobilized oleate hydratase was observed over time. Therefore, further enzyme modification is required to impart stability, the optimization of operational conditions, and the development of carrier materials that enable economical and sustainable enzymatic conversion of oleic acid to meet the commercial demand. Graphical abstract

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 this study, a novel chitinolytic Jeongeupia species “wiesaeckerbachi” was isolated from soil samples, characterized biochemically, and sequenced with the long‐read platform PacBio Sequel IIe. In silico analysis unraveled genomic differences to the closest related type strain Jeongeupia naejangsanensis TAS4‐2 in addition to an usually extensive chitinolytic machinery.

Background The growing demand for sustainable lipid sources has fostered interest in single-cell oils from oleaginous yeasts as renewable alternatives to plant-derived and fossil-based oils, with applications in food, fuel, and material production. The oleaginous yeast Cutaneotrichosporon oleaginosus is of industrial relevance due to its ability to accumulate in excess of 60% (w/w) of its dry cell weight as lipids, while metabolizing a broad range of substrates. However, economic feasibility depends on improving productivity and adapting fatty acid profiles to application requirements. Results This study investigated the influence of temperature, pH, and dissolved oxygen concentration (DO) on lipid production and fatty acid composition in C. oleaginosus ATCC 20509. A three-level, three-factor Box–Behnken design was applied to assess their effects on lipid titer, oleate lipid titer, and the proportions of saturated and unsaturated fatty acids. Response surface methodology was used to develop quadratic models, identify optimized conditions, and predict fatty acid compositions. Temperature and pH significantly affected both overall lipid titer and degree of saturation. In fed-batch cultivation with consumption-based acetic acid feeding and glucose as the initial carbon source, lipid productivity increased to 0.38 g/L/h under the optimized oleate lipid titer condition (27.6 °C, pH 5.6, 10% DO) and to 0.39 g/L/h under the optimized saturated fatty acid condition (30 °C, pH 7.0, 10% DO), corresponding to 46% and 50% increases compared to literature values (0.26 g/L/h; 28 °C, pH 6.5, 50% DO). The fatty acid profile could thus be precisely modulated by adjusting the process parameters, achieving a difference in the saturation degree of more than 10%. Temperature was identified as the main factor influencing saturation, while pH enabled adjustment of the C16/C18 ratio, resulting in a modulation of palmitic acid fraction within the total triglycerides of up to 13%. Conclusion These findings highlight the potential of optimizing cultivation parameters based on reaction surface methodology to simultaneously improve lipid productivity and functionality by tailoring the fatty acid profile to the desired application requirements, without resorting to genetic engineering. Moreover, these insights support a circular bio-based economy by enabling an efficient production of tailored microbial oils as renewable alternatives to plant-derived lipids. Graphical Abstract

The salt-tolerant marine microalgae Dunaliella tertiolecta is reported to generate significant amounts of intracellular glycerol as an osmoprotectant under high salt conditions. This study highlights the phylogenetic distribution and comparative glycerol biosynthesis of seven new Dunaliella isolates compared to a D. tertiolecta reference strain. Phylogenetic analysis indicates that all Dunaliella isolates are newly discovered and do not relate to the D. tertiolecta reference. Several studies have identified light color and intensity and salt concentration alone as the most inducing factors impacting glycerol productivity. This study aims to optimize glycerol production by investigating these described factors singularly and in combination to improve the glycerol product titer. Glycerol production data indicate that cultivation with white light of an intensity between 500 and 2000 μmol m−2 s−1 as opposed to 100 μmol m−2 s−1 achieves higher biomass and thereby higher glycerol titers for all our tested Dunaliella strains. Moreover, applying higher light intensity in a cultivation of 1.5 M NaCl and an increase to 3 M NaCl resulted in hyperosmotic stress conditions, providing the highest glycerol titer. Under these optimal light intensity and salt conditions, the glycerol titer of D. tertiolecta could be doubled to 0.79 mg mL−1 in comparison to 100 μmol m−2 s−1 and salt stress to 2 M NaCl, and was higher compared to singularly optimized conditions. Furthermore, under the same conditions, glycerol extracts from new Dunaliella isolates did provide up to 0.94 mg mL−1. This highly pure algae-glycerol obtained under optimal production conditions can find widespread applications, e.g., in the pharmaceutical industry or the production of sustainable carbon fibers.

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

In context of the global climate change, microalgae processes are gaining momentum as a biotechnological tool for direct fixation and valorization of greenhouse gases. Algae have the metabolic capacity to photosynthetically convert CO 2 into high value products, such as food additives, under economic boundary conditions. High cost, commercial flat panel gas-lift bioreactors for microalgae cultivation at laboratory scale provide either small volumes or no sterile operation, which limits academic research. This brief report presents initial data for a new type of sterile operating flat panel gas-lift bioreactor with a unique asymmetrical U-shape. It utilizes automatable process control technologies that adhere to industrial standards to enhance data reproducibility and aid industrial scale up. The practicability was demonstrated using a Chlorella sorokiniana cultivation, which showed the typical growth behavior. Due to the sophisticated implemented control engineering technology, pivotal parameters as pH and temperature can be determined within a range of ±0.1 units, which was confirmed experimentally. The new flat panel gas-lift photobioreactor presented in this brief report fills the technology gap at laboratory scale with an autoclavable volume of 7.2 L. Moreover, it is easy to rebuild by means of the hereby provided blueprint, while exhibiting a six-fold cost reduction compared to commercially available flat panel photobioreactors.

In analogy to higher plants, eukaryotic microalgae are thought to be incapable of utilizing green light for growth, due to the “green gap” in the absorbance profiles of their photosynthetic pigments. This study demonstrates, that the marine chlorophyte Picochlorum sp . is able to grow efficiently under green light emitting diode (LED) illumination. Picochlorum sp. growth and pigment profiles under blue, red, green and white LED illumination (light intensity: 50–200 μmol m −2 s −1 ) in bottom-lightened shake flask cultures were evaluated. Green light-treated cultures showed a prolonged initial growth lag phase of one to 2 days, which was subsequently compensated to obtain comparable biomass yields to red and white light controls (approx. 0.8 g DW L −1 ). Interestingly, growth and final biomass yields of the green light-treated sample were higher than under blue light with equivalent illumination energies. Further, pigment analysis indicated, that during green light illumination, Picochlorum sp . formed unknown pigments (X1-X4). Pigment concentrations increased with illumination intensity and were most abundant during the exponential growth phase. Mass spectrometry and nuclear magnetic resonance data indicated, that pigments X1-X2 and X3-X4 are derivatives of chlorophyll b and a , which harbor C=C bonds in the phytol side chain similar to geranylgeranylated chlorophylls. Thus, for the first time, the natural accumulation of large pools (approx. 12 mg g DW −1 ) of chlorophyll intermediates with incomplete hydrogenation of their phytyl chains is demonstrated for algae under monochromatic green light (Peak λ 510 nm, full width at half maximum 91 nm). The ability to utilize green light offers competitive advantages for enhancing biomass production, particularly under conditions of dense cultures, long light pathways and high light intensity. Green light acclimation for an eukaryotic microalgae in conjunction with the formation of new aberrant geranylgeranylated chlorophylls and high efficiency of growth rates are novel for eukaryotic microalgae. Illumination with green light could enhance productivity in industrial processes and trigger the formation of new metabolites–thus, underlying mechanisms require further investigation.