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 M_w 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.

Globally, biofuel and oleochemical production based on plant oils negatively affects biodiversity. As an alternative triglyceride source, lipid production from oleaginous yeasts faces numerous challenges in feedstock availability, lipid productivity, downstream processing, and waste treatment, prohibiting the design of a cost-competitive process with regard to plant equivalents. In this study, we present a fully integrated operation for microbial oil production, which consolidates upstream and downstream processing with side-stream recycling. Co-fermentation of sugar and acetic acid was successfully implemented in fed-batch, semi-continuous, and continuous fermentation modes. Process validation was conducted at a 25 L scale with a lipid productivity of 1.2 g L⁻¹ h⁻¹. Cutaneotrichosporon oleaginosus cell debris was used as an inducer in Trichoderma reesei fermentation for on-site generation of yeast-specific cell-wall hydrolases. In situ hydrolase application allowed for efficient C. oleaginosus cell lysis (85% w/w) and simultaneous lipid release. A subsequent centrifugation step yielded 90% (w/w) recovery of intracellular lipids without the need for any organic solvent. The nutrient-rich water phase was applied as an internal sugar source for subsequent yeast fermentation cycles. With this yeast hydrolysate, the lipid productivity was considerably increased to 2.4 g L⁻¹ h⁻¹. A techno-economic analysis of the current lipid production processes estimated costs at $1.6 per kg lipid. Moreover, life cycle assessment analysis indicated an emission of 3.56 kg CO₂ equivalents for every 1 kg produced yeast oil. Accordingly, we established an integrated operation for bioconversion of acetic acid and sugar to sustainable lipids at maximum productivity coupled with minimal waste generation and energy consumption.

The oleaginous yeast Trichosporon oleaginosus ATCC 20509 can accumulate up to 70% (w/DCW) triglycerides when cultivated on chemically diverse agricultural or food waste streams. In contrast to other lipogenic yeasts T. oleaginosus is able to efficiently convert constituents of hemicellulose and chitin hydrolysates into lipids. This study focused on establishing the genetic accessibility of T. oleaginosus aimed at manipulating lipid biosynthesis in order to generate high value lipids from waste streams. We demonstrate the first transformation protocol for T. oleaginosus based on Agrobacterium tumefaciens. Strong heterologous gene expression of a codon optimized YFP reporter protein was achieved using the constitutive promotor from the endogenous glyceraldehyde-3-phosphate dehydrogenase gene. Subsequently, we evaluated the ability of T. oleaginosus to generate non-natural fatty acid profiles by heterologous expression of several fatty acid modifying enzymes. De novo lipid generation of these recombinant strains was evaluated on diverse carbon sources. Compared to the wild type, recombinant yeast strains showed an increase of α-linolenic acid production from 2.8% to 21% with respect to the total cellular fatty acid content (TFA). Further, we designed yeast strains able to generate the non-native, polyunsaturated very long chain fatty acids eicosatrienoic (16% TFA) and eicosadienoic acid (9% TFA), respectively. Alternatively, T. oleaginosus was engineered to produce the non-native (E-10, Z-12) conjugated linoleic acid, which was generated up to 2.6% TFA. This work demonstrates, that T. oleaginosus ATCC 20509 can be used as versatile biotechnology platform to transform industrial waste streams into designed, high value fatty acids.