Engineering enzymes with improved catalytic properties in non-natural environments have been concerned with their diverse industrial and biotechnological applications. Immobilization represents a promising but straightforward route, and immobilized biocatalysts often display higher activities and stabilities compared to free enzymes. Owing to their unique physicochemical characteristics, including the high-specific surface area, exceptional chemical, electrical, and mechanical properties, efficient enzyme loading, and multivalent functionalization, nano-based materials are postulated as suitable carriers for biomolecules or enzyme immobilization. Enzymes immobilized on nanomaterial-based supports are more robust, stable, and recoverable than their pristine counterparts, and are even used for continuous catalytic processes. Furthermore, the unique intrinsic properties of nanomaterials, particularly nanoparticles, also confer the immobilized enzymes to be used for their broader applications. Herein, an effort has been made to present novel potentialities of multi-point enzyme immobilization in the current biotechnological sector. Various nano-based platforms for enzyme/biomolecule immobilization are discussed in the second part of the review. In summary, recent developments in the use of nanomaterials as new carriers to construct robust nano-biocatalytic systems are reviewed, and future trends are pointed out in this article.

Kluyveromyces marxianusis an emerging non-conventional food-grade yeast that is generally isolated from diverse habitats, like kefir grain, fermented dairy products, sugar industry sewage, plants, and sisal leaves. A unique set of beneficial traits, such as fastest growth, thermotolerance, and broad substrate spectrum (i.e., hemi-cellulose hydrolysates, xylose,l-arabinose,d-mannose, galactose, maltose, sugar syrup molasses, cellobiose, and dairy industry) makes this yeast a particularly attractive host for applications in a variety of food and biotechnology industries. In contrast toSaccharomyces cerevisiae, most of theK. marxianusstrains are apparently Crabtree-negative or having aerobic-respiring characteristics, and unlikely to endure aerobic alcoholic fermentation. This is a desirable phenotype for the large-scale biosynthesis of products associated with biomass formation because the formation of ethanol as an undesirable byproduct can be evaded under aerobic conditions. Herein, we discuss the current insight into the potential applications ofK. marxianusas a robust yeast cell factory to produce various industrially pertinent enzymes, bioethanol, cell proteins, probiotic, fructose, and fructo-oligosaccharides, and vaccines, with excellent natural features. Moreover, the biotechnological improvement and development of new biotechnological tools, particularly CRISPR–Cas9-assisted precise genome editing inK. marxianusare delineated. Lastly, the ongoing challenges, concluding remarks, and future prospects for expanding the scope ofK. marxianusutilization in modern biotechnology, food, feed, and pharmaceutical industries are also thoroughly vetted. In conclusion, it is critical to apprehend knowledge gaps around genes, metabolic pathways, key enzymes, and regulation for gaining a complete insight into the mechanism for producing relevant metabolites byK. marxianus.

Sugar alcohols and organic acids that derive from the metabolism of certain microorganisms have a panoply of applications in agro-food, chemical and pharmaceutical industries. The main challenge in their production is to reach a productivity threshold that allow the process to be profitable. This relies on the construction of efficient cell factories by metabolic engineering and on the development of low-cost production processes by using industrial wastes or cheap and widely available raw materials as feedstock. The non-conventional yeast Yarrowia lipolytica has emerged recently as a potential producer of such metabolites owing its low nutritive requirements, its ability to grow at high cell densities in a bioreactor and ease of genome edition. This review will focus on current knowledge on the synthesis of the most important sugar alcohols and organic acids in Y. lipolytica.

Background: Functional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions. Results: Herein, Y. lipolytica has been metabolically engineered to increase erythritol production titer, yield, and productivity from glucose. This consisted of the disruption of anabolic pathways for mannitol and d-arabitol together with the erythritol catabolic pathway. Genes ZWF1 and GND encoding, respectively, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also constitutively expressed in regenerating the NADPH₂ consumed during erythritol synthesis. Finally, the gene RSP5 gene from Saccharomyces cerevisiae encoding ubiquitin ligase was overexpressed to improve cell thermoresistance. The resulting strain HCY118 is impaired in mannitol or d-arabitol production and erythritol consumption. It can grow well up to 35 °C and retain an efficient erythritol production capacity at 33 °C. The yield, production, and productivity reached 0.63 g/g, 190 g/L, and 1.97 g/L·h in 2-L flasks, and increased to 0.65 g/g, 196 g/L, and 2.51 g/L·h in 30-m³ fermentor, respectively, which has economical practical importance. Conclusion: The strategy developed herein yielded an engineered Y. lipolytica strain with enhanced thermoresistance and NADPH supply, resulting in a higher ability to produce erythritol, but not mannitol or d-arabitol from glucose. This is of interest for process development since it will reduce the cost of bioreactor cooling and erythritol purification.

Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast Yarrowia lipolytica, the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene (YALI0F18590g) from erythritol-producing yeast Y. lipolytica MK1 was identified (Janek et al. in Microb Cell Fact 16:118, 2017 ). However, deletion of the gene in Y. lipolytica MK1 only resulted in some lower erythritol production but the erythritol synthesis process was still maintained, indicating that other erythrose reductase gene(s) might exist in the genome of Y. lipolytica. In this study, we have isolated genes g141.t1 (YALI0D07634g) and g3023.t1 (YALI0C13508g) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 (YALI0F18590g) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source. Our results highlight that in the Y. lipolytica genome several genes encode enzymes able to reduce erythrose into erythritol. The catalytic properties of these enzymes and their cofactor dependency are different from that of already known erythrose reductase of Y. lipolytica. Constitutive expression of the newly isolated genes and engineering of NADPH cofactor metabolism led to an increase in erythritol titer. Development of fermentation strategies will allow further improvement of this productivity in the future.