Biology subjects

Julsing, M. K.

Publications and source records attributed to Julsing, M. K..

4 recordsLinked to original sources

bioRxiv2024

Combining metabolic engineering and fermentation optimization to achieve cost-effective oil production by Cutaneotrichosporon oleaginosus

Microbial oils, produced by oleaginous microorganisms, offer a sustainable alternative to plant-derived oils. Among these microorganisms, Cutaneotrichosporon oleaginosus attracts attention as a promising microbial cell factory for sustainable oil production due to its capacity to accumulate lipids with a similar composition to palm oil. Although C. oleaginosus can reach higher lipid contents than other oleaginous yeasts, suboptimal lipid yields and productivity limit economic feasibility. Enhanced productivity is necessary to have a feasible microbial oil production process via C. oleaginosus. In this study, we followed a combinatorial approach for strain design and bioprocess development to improve the lipid content and lipid yield. Initially, we deployed a full factorial design with genetic factors (ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS)) and carbon-to-nitrogen ratio (C/N) in the medium. The C/N ratio appeared to have the most impact on oil accumulation. Combined with genetic modifications, lipid content and lipid yield increased by 1.6-fold. In a two-stage fermentation approach at a 2L scale, the triple transformant overexpressing ACL, ACC, and TS outperformed the wild-type by achieving a lipid content of 75.4% (w/w) with lipid productivity of 0.40 g L-1 h-1 and around 0.30 g lipids/g glycerol. In all, we established a cultivation strategy and strain that reached almost the theoretical maximum yield, and highest lipid content reported for a medium containing glycerol as a carbon source. These results strengthen the basis of using C. oleaginous as a platform for microbial oil production, thereby facilitating the development of processes substituting palm oil with a sustainable alternative. HighlightsO_LIFull factorial design allowed selection of the best performer transformant. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher lipid yield. C_LIO_LITwo-stage fermentation enhanced the lipid content of wild-type and transformant. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/624102v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1371a7aorg.highwire.dtl.DTLVardef@ab5ae1org.highwire.dtl.DTLVardef@1db84caorg.highwire.dtl.DTLVardef@eb19e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Model-driven engineering of Yarrowia lipolytica for improved microbial oil production

Extensive usage of plant-based oils, especially palm oil, has led to environmental and social issues, such as deforestation and loss of biodiversity, thus sustainable alternatives are required. Microbial oils, especially from Yarrowia lipolytica, offer a promising solution due to their similar composition to palm oil, low carbon footprint, and ability to utilize low-cost substrates. In this study, we employed the Design-Build-Test-Learn (DBTL) approach to enhance lipid production in Y. lipolytica. We systematically evaluated predictions from the genome-scale metabolic model to identify and overcome bottlenecks in lipid biosynthesis. We tested the effect of predicted medium supplements and genetic intervention targets, including the overexpression of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS), diacylglycerol acyltransferase(DGA1), the deletion of citrate exporter gene (CEX1) and disruption of {beta}-oxidation pathway (MFE1). Combining TS and DGA1 overexpression in the{Delta} mfe_{Delta}cex background achieved a remarkable 200% increase in lipid content (56 % w/w) and a 230% increase in lipid yield on glycerol. These findings underscore the potential of Y. lipolytica as an efficient microbial cell factory for fatty acid production. Our study advances the understanding of lipid metabolism in Y. lipolytica and demonstrates a viable approach for developing sustainable and economically feasible alternatives to palm oil. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/606002v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1394cf6org.highwire.dtl.DTLVardef@ebdd5eorg.highwire.dtl.DTLVardef@1126ab2org.highwire.dtl.DTLVardef@1ae028_HPS_FORMAT_FIGEXP M_FIG C_FIG We followed the Design-Build-Test-Learn approach to identify and overcome bottlenecks in lipid biosynthesis in Y. lipolytica. DBTL intertwined the predictions from the metabolic model with addressed bottlenecks, investigated the effect of genetic interventions and medium supplements on lipid content, and ultimately defined an efficient strain design strategy.

molecular biology↗

bioRxiv2024

Improved genome sequence and annotation of Cutaneotrichosporon oleaginosus ATCC 20509

Cutaneotrichosporon oleaginosus is an oleaginous yeast with a high content of fatty acids and can accumulate more than 40% of its weight in lipids. It can grow on a wide range of carbon sources and side streams such as crude glycerol. The genome sequence of C. oleaginosus ATCC 20509 is reported here to contribute to its development as a biotechnological platform for producing microbial oils.

microbiology↗

bioRxiv2024

Model-driven engineering of Cutaneotrichosporon oleaginosus ATCC 20509 for improved microbial oil production

Consumption of plant-based oils, especially palm oil, is increasing at an alarming rate. This boosted demand for palm oil has drastic effects on the ecosystem as its production is not sustainable. C. oleaginosus is an oleaginous yeast with great potential as a source for microbial-based oil production which is a sustainable alternative to palm oil. However, microbial processes are not yet economically feasible to replace palm oil, unto a large extent due to limited lipid accumulation in the microbe, which limits titers and productivity. Therefore, obtaining enhanced lipid accumulation is essential to render this process commercially viable. Herein we deployed a systematic, iterative Design-Build-Test-Learn (DBTL) approach to establish C. oleaginosus as an efficient fatty acid production platform. In the design step, we identified genes and medium supplements that improved lipid content. To this end, we compared its transcriptional landscape in conditions with high and low amounts of lipid production. A metabolic map was reconstructed and integrated with the expression data. Finally, the genome-scale metabolic model of C. oleaginosus was used to explore metabolism under maximal growth and maximal production conditions. The combination of these four analyses led to the selection of four overexpression targets (ATP-citrate lyase (ACL1), acetyl-CoA carboxylase (ACC), threonine synthase (TS), and hydroxymethylglutaryl-CoA synthase (HMGS)) and five media supplements (biotin, thiamine, threonine, serine, and aspartate). We established an electroporation-based co-transformation method to implement selected genetic interventions. These findings were experimentally validated in the build and test steps of the DBTL approach by adding supplements into the medium and overexpressing the identified genes. Characterization of ACL, ACC, and TS at various C/N ratios, and the addition of medium supplements provided up to 56% (w/w) lipid content, and a 2.5-fold increase in total lipid in the glycerol and urea-based defined medium. In the learn step, quadratic models identified the optimum C/N ratios shifted towards around C/N240. These results firmly confirm C. oleaginous as a sustainable alternative to replace palm as an oil source. HighlightsO_LITranscriptional profile and metabolic model analyzed, predicting genetic targets and medium supplements. C_LIO_LIGenetic targets and medium supplements for improved oil production. C_LIO_LIThe genetic toolbox for C. oleaginosus was expanded (co-transformation method, promoters, genes, and terminators). C_LIO_LIExperimental validations showed that biotin, and threonine increased lipid content. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher oil content. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/585731v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@e3b6org.highwire.dtl.DTLVardef@65d016org.highwire.dtl.DTLVardef@40952eorg.highwire.dtl.DTLVardef@22724_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗