Sustainable biosurfactant production from secondary feedstock-recent advances, process optimization and perspectives.

Biosurfactants have garnered increased attention lately due to their superiority of their properties over fossil-derived counterparts. While the cost of production remains a significant hurdle to surpass synthetic surfactants, biosurfactants have been anticipated to gain a larger market share in the coming decades. Among these, glycolipids, a type of low-molecular-weight biosurfactant, stand out for their efficacy in reducing surface and interfacial tension, which made them highly sought-after for various surfactant-related applications. Glycolipids are composed of hydrophilic carbohydrate moieties linked to hydrophobic fatty acid chains through ester bonds that mainly include rhamnolipids, trehalose lipids, sophorolipids, and mannosylerythritol lipids. This review highlights the current landscape of glycolipids and covers specific glycolipid productivity and the diverse range of products found in the global market. Applications such as bioremediation, food processing, petroleum refining, biomedical uses, and increasing agriculture output have been discussed. Additionally, the latest advancements in production cost reduction for glycolipid and the challenges of utilizing second-generation feedstocks for sustainable production are also thoroughly examined. Overall, this review proposes a balance between environmental advantages, economic viability, and societal benefits through the optimized integration of secondary feedstocks in biosurfactant production.

From bumblebee to bioeconomy: Recent developments and perspectives for sophorolipid biosynthesis.

Sophorolipids are biobased compounds produced by the genera Starmerella and Pseudohyphozyma that gain exponential interest from academic and industrial stakeholders due to their mild and environmental friendly characteristics. Currently, industrially relevant sophorolipid volumetric productivities are reached up to 3.7 g∙L-1∙h-1 and sophorolipids are used in the personal care and cleaning industry at small scale. Moreover, applications in crop protection, food, biohydrometallurgy and medical fields are being extensively researched. The research and development of sophorolipids is at a crucial stage. Therefore, this work presents an overview of the state-of-the-art on sophorolipid research and their applications, while providing a critical assessment of scientific techniques and standardisation in reporting. In this review, the genuine sophorolipid producing organisms and the natural role of sophorolipids are discussed. Subsequently, an evaluation is made of innovations in production processes and the relevance of in-situ product recovery for process performance is discussed. Furthermore, a critical assessment of application research and its future perspectives are portrayed with a focus on the self-assembly of sophorolipid molecules. Following, genetic engineering strategies that affect the sophorolipid physiochemical properties are summarised. Finally, the impact of sophorolipids on the bioeconomy are uncovered, along with relevant future perspectives.

Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola.

Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

Unraveling and resolving inefficient glucolipid biosurfactants production through quantitative multiomics analyses of Starmerella bombicola strains.

Glucolipids (GLs) are glycolipid biosurfactants with promising properties. These GLs are composed of glucose attached to a hydroxy fatty acid through a ω and/or ω-1 glycosidic linkage. Up until today these interesting molecules could only be produced using an engineered Starmerella bombicola strain (∆ugtB1::URA3 G9) producing GLs instead of sophorolipids, albeit with a very low average productivity (0.01 g·L-1 ·h-1 ). In this study, we investigated the reason(s) for this via reverse-transcription quantitative polymerase chain reaction and Liquid chromatography-multireaction monitoring-mass spectrometry. We found that all glycolipid biosynthetic genes and enzymes were downregulated in the ∆ugtB1 G9 strain in comparison to the wild type. The underlying reason for this downregulation was further investigated by performing quantitative metabolome comparison of the ∆ugtB1 G9 strain with the wild type and two other engineered strains also tinkered in their glycolipid biosynthetic gene cluster. This analysis revealed a clear distortion of the entire metabolism of the ∆ugtB1 G9 strain compared to all the other strains. Because the parental strain of the former was a spontaneous ∆ura3 mutant potentially containing other "hidden" mutations, a new GL production strain was generated based on a rationally engineered ∆ura3 mutant (PT36). Indeed, a 50-fold GL productivity increase (0.51 g·L-1 ·h-1 ) was obtained with the new ∆ugtB1::URA3 PT36 strain compared with the G9-based strain (0.01 g·L-1 ·h-1 ) in a 10 L bioreactor experiment, yielding 118 g/L GLs instead of 8.39 g/L. Purification was investigated and basic properties of the purified GLs were determined. This study forms the base for further development and optimization of S. bombicola as a production platform strain for (new) biochemicals.

Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola.

In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.

Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1.

TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effects of glucocorti-coids are mediated by induction of this miR, resulting in reduced TNFR1 expression.

Matrix metalloproteinase 13 modulates intestinal epithelial barrier integrity in inflammatory diseases by activating TNF.

Several pathological processes, such as sepsis and inflammatory bowel disease (IBD), are associated with impairment of intestinal epithelial barrier. Here, we investigated the role of matrix metalloproteinase MMP13 in these diseases. We observed that MMP13(-/-) mice display a strong protection in LPS- and caecal ligation and puncture-induced sepsis. We could attribute this protection to reduced LPS-induced goblet cell depletion, endoplasmic reticulum stress, permeability and tight junction destabilization in the gut of MMP13(-/-) mice compared to MMP13(+/+) mice. Both in vitro and in vivo, we found that MMP13 is able to cleave pro-TNF into bioactive TNF. By LC-MS/MS, we identified three MMP13 cleavage sites, which proves that MMP13 is an alternative TNF sheddase next to the TNF converting enzyme TACE. Similarly, we found that the same mechanism was responsible for the observed protection of the MMP13(-/-) mice in a mouse model of DSS-induced colitis. We identified MMP13 as an important mediator in sepsis and IBD via the shedding of TNF. Hence, we propose MMP13 as a novel drug target for diseases in which damage to the gut is essential.