Biology subjects

Hugenholtz, J.

Publications and source records attributed to Hugenholtz, J..

7 recordsLinked to original sources

bioRxiv2025

Steering fatty acid composition of yeast microbial oil via genetic modification and bioprocess adjustment

The increasing demand for palm oil has drastic effects on the ecosystem as its production is not sustainable. To that end, developing a sustainable alternative to fatty acids and oils is urgent and of utmost interest. Oils produced by oleaginous yeasts present a promising solution, particularly because the fatty acid profile of the oil produced by these yeasts is comparable to that of plant-based oils and fats. The fatty acid composition of the oil determines its physiological properties, thereby determining its potential applications. Accordingly, the production of microbial oil with an optimal composition profile for a specific application is of great importance. In this study, we evaluated the variation that occurred in fatty acid composition due to different cultivation parameters (temperature, C/N ratio, carbon, and nitrogen sources) and applied genetic modifications to improve the lipid accumulation of Cutaneotrichosporon oleaginosus and Yarrowia lipolytica. We showed that specific fatty acid profiles associated with a particular application can be obtained by carefully selecting the microorganism and cultivation conditions.

microbiology↗

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

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↗

bioRxiv2023

Exploring the potential of Bacillus subtilis as cell factory for food ingredients and special chemicals

BackgroundBacillus subtilis has been established as model microorganism for fundamental research in the laboratory on protein production/secretion and sporulation and as model bacterium for controlling spoilage in the food industry. It has also been used for production of (commercial) enzymes and several secondary metabolites such as vitamins. However, this doesnt fully reflect the potential of B. subtilis as a cell-factory. Here, various strains of B. subtilis, including food-grade, spore-deficient strains and industrially used strains, were compared for their growth and metabolic potential. Industry-relevant parameters were analyzed for all strains under various aeration regimes, under anaerobic conditions, in various nutritious and nutrient-limited cultivation media, with and without organic nitrogen sources, and with and without sugar. ResultsPractical experiments were conducted to compare industrial relevant properties like growth rates, intracellular components and extracellular metabolite profile of different B. subtilis strains. Based on growth flexibility in different media, we found that some strains like NCIB3610 and DSM1092 are adapted to inorganic or organic nitrogen source utilization, which is highly relevant when considering a biorefinery approach using various cheap and abundant waste/sidestreams. Secondly, spore-deficient strains such as 3NA, 168S and PY79S, showed advantages in microbial protein and acetolactate pathway expression, which is associated with applications in food industry for protein supplement and diacetyl production. Lastly, WB800 and PY79S exhibited potential for fermentative production of Dipicolinic acid, 2,3-Butanediol and Lactic acid that could serve as precursors for biopolymers. ConclusionThis study demonstrates the broad potential for more extensive industrial use of Bacillus subtilis in the (bio-based) chemical industry for use of sidestreams, in the personal care industry, in the food industry for food additive production, and in the bio-sustainable industry for biofuel and bio-degradable plastic precursors production. In addition, selecting different B. subtilis strains for specific purposes makes full use of the diversity of this species and increases the potential of B. subtilis in its contribution to the bio-based economy.

microbiology↗

bioRxiv2022

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii.

One-carbon (C1) compounds are promising feedstocks for sustainable production of commodity chemicals. CO2 is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted to valuable products while reducing its atmospheric levels. Acetogens are known microorganisms that can grow on CO2/H2 and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with microbes that can further process such products can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone, butanol and ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of a solventogen and acetogen in a consortium provides a potential platform to produce valuable chemicals from CO2. In this study, metabolic modelling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO2/H2 mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in serum bottles and subsequently in pH-controlled bioreactors. Community modelling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO2/H2 producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modelling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources.

microbiology↗

bioRxiv2022

Tailoring and optimizing fatty acid production by oleaginous yeasts through the systematic exploration of their physiological fitness

BackgroundThe use of palm oil for our current needs is unsustainable. Replacing palm oil with oils produced by microbes through the conversion of sustainable feedstocks is a promising alternative. However, there are major technical challenges that must be overcome to enable this transition. Foremost among these challenges is the stark increase in lipid accumulation and production of higher content of specific fatty acids. Therefore, there is a need for more in-depth knowledge and systematic exploration of the oil productivity of the oleaginous yeasts. In this study, we cultivated Cutaneotrichosporon oleaginosus and Yarrowia lipolytica at various C/N ratios and temperatures in a defined medium with glycerol as carbon source and urea as nitrogen source. We ascertained the synergistic effect between various C/N ratios of a defined medium at different temperatures with Response Surface Methodology (RSM) and explored the variation in fatty acid composition through Principal Component Analysis. ResultsBy applying RSM, we determined a temperature of 30 {degrees}C and a C/N ratio of 175 g/g to enable maximal oil production by C. oleaginosus and a temperature of 21 {degrees}C and a C/N ratio of 140 g/g for Y. lipolytica. We increased production by 71 % and 66 % respectively for each yeast compared to the average lipid accumulation in all tested conditions. Modulating temperature enabled us to steer the fatty acid compositions. Accordingly, switching from higher temperature to lower cultivation temperature shifted the production of oils from more saturated to unsaturated by 14 % in C. oleaginosus and 31 % in Y. lipolytica. Higher cultivation temperatures resulted in production of even longer saturated fatty acids, 3 % in C. oleaginosus and 1.5 % in Y. lipolytica. ConclusionsIn this study, we provided the optimum C/N ratio and temperature for C. oleaginosus and Y. lipolytica by RSM. Additionally, we demonstrated that lipid accumulation of both oleaginous yeasts was significantly affected by the C/N ratio and temperature. Furthermore, we systematically analyzed the variation in fatty acids composition and proved that changing the C/N ratio and temperature steer the composition. We have further established these oleaginous yeasts as platforms for production of tailored fatty acids.

systems biology↗