Mastering targeted genome engineering of GC-rich oleaginous yeast for tailored plant oil alternatives for the food and chemical sector.

BACKGROUND: Sustainable production of triglycerides for various applications is a major focus of microbial factories. Oleaginous yeast species have been targeted for commercial production of microbial oils. Among all the oleaginous yeasts examined in a previous comparative study, Cutaneotrichosporon oleaginosus showed the highest lipid productivity. Moreover, a new lipid production process for C. oleaginosus with minimal waste generation and energy consumption resulted in the highest lipid productivity in the history of oleaginous yeasts. However, productivity and product diversity are restricted because of the genetic intractability of this yeast. To date, successful targeted genetic engineering of C. oleaginosus has not yet been reported. RESULTS: The targeted gene editing was successfully carried out in C. oleaginosus using CRISPR/Cas system. A tailored enzyme system isolated to degrade the C. oleaginosus cell wall enabled the isolation of viable spheroplasts that are amenable to in-cell delivery of nucleic acids and proteins. The employment of both Cas9 protein and Cas mRNA was effective in obtaining strains with URA5 knockout that did not exhibit growth in the absence of uracil. Subsequently, we successfully created several strains with enhanced lipid yield (54% increase compared to that in wild type) or modified fatty acid profiles comparable with those of cocoa butter or sunflower oil compositions. CONCLUSION: This study establishes the first targeted engineering technique for C. oleaginosus using the CRISPR/Cas system. The current study creates the foundation for flexible and targeted strain optimizations towards building a robust platform for sustainable microbial lipid production. Moreover, the genetic transformation of eukaryotic microbial cells using Cas9 mRNA was successfully achieved.

Screening of volatile organic compounds (VOCs) from liquid fungal cultures using ambient mass spectrometry.

The potential of fungi for use as biotechnological factories in the production of a range of valuable metabolites, such as enzymes, terpenes, and volatile aroma compounds, is high. Unlike other microorganisms, fungi mostly secrete secondary metabolites into the culture medium, allowing for easy extraction and analysis. To date, the most commonly used technique in the analysis of volatile organic compounds (VOCs) is gas chromatography, which is time and labour consuming. We propose an alternative ambient screening method that provides rapid chemical information for characterising the VOCs of filamentous fungi in liquid culture using a commercially available ambient dielectric barrier discharge ionisation (DBDI) source connected to a quadrupole-Orbitrap mass spectrometer. The effects of method parameters on measured peak intensities of a series of 8 selected aroma standards were optimised with the best conditions being selected for sample analysis. The developed method was then deployed to the screening of VOCs from samples of 13 fungal strains in three different types of complex growth media showing clear differences in VOC profiles across the different media, enabling determination of best culturing conditions for each compound-strain combination. Our findings underline the applicability of ambient DBDI for the direct detection and comparison of aroma compounds produced by filamentous fungi in liquid culture.

The Time-Resolved Salt Stress Response of Dunaliella tertiolecta-A Comprehensive System Biology Perspective.

Algae-driven processes, such as direct CO2 fixation into glycerol, provide new routes for sustainable chemical production in synergy with greenhouse gas mitigation. The marine microalgae Dunaliella tertiolecta is reported to accumulate high amounts of intracellular glycerol upon exposure to high salt concentrations. We have conducted a comprehensive, time-resolved systems biology study to decipher the metabolic response of D. tertiolecta up to 24 h under continuous light conditions. Initially, due to a lack of reference sequences required for MS/MS-based protein identification, a high-quality draft genome of D. tertiolecta was generated. Subsequently, a database was designed by combining the genome with transcriptome data obtained before and after salt stress. This database allowed for detection of differentially expressed proteins and identification of phosphorylated proteins, which are involved in the short- and long-term adaptation to salt stress, respectively. Specifically, in the rapid salt adaptation response, proteins linked to the Ca2+ signaling pathway and ion channel proteins were significantly increased. While phosphorylation is key in maintaining ion homeostasis during the rapid adaptation to salt stress, phosphofructokinase is required for long-term adaption. Lacking ß-carotene, synthesis under salt stress conditions might be substituted by the redox-sensitive protein CP12. Furthermore, salt stress induces upregulation of Calvin-Benson cycle-related proteins.

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol.

BACKGROUND: Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. RESULTS: In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l-1 and 395 mg l-1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. CONCLUSION: The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis.

Dietary Modulation of the Human Gut Microbiota and Metabolome with Flaxseed Preparations.

Flaxseeds are typically consumed either as whole flaxseed, ground flaxseed, flaxseed oil, partially defatted flaxseed meal, or as a milk alternative. They are considered a rich source of vitamins, minerals, proteins and peptides, lipids, carbohydrates, lignans, and dietary fiber, which have shown hypolipidemic, antiatherogenic, anticholesterolemic, and anti-inflammatory property activity. Here, an in vitro batch culture model was used to investigate the influence of whole milled flaxseed and partially defatted milled flaxseed press cake on the gut microbiota and the liberation of flaxseed bioactives. Microbial communities were profiled using 16S rRNA gene-based high-throughput sequencing with targeted mass spectrometry measuring lignan, cyclolinopeptide, and bile acid content and HPLC for short-chain fatty acid profiles. Flaxseed supplementation decreased gut microbiota richness with Firmicutes, Proteobacteria, and Bacteroidetes becoming the predominant phyla. Secoisolariciresinol, enterodiol, and enterolactone were rapidly produced with acetic acid, butyric acid, and propionic acid being the predominant acids after 24 h of fermentation. The flaxseed press cake and whole flaxseed were equivalent in microbiota changes and functionality. However, press cake may be superior as a functional additive in a variety of foods in terms of consumer acceptance as it would be more resistant to oxidative changes.

Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers.

BACKGROUND: Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. RESULTS: C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. CONCLUSIONS: Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology.

BACKGROUND: The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. RESULTS: In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. CONCLUSIONS: Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

Engineering Escherichia coli FAB system using synthetic plant genes for the production of long chain fatty acids.

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.

Detailed structure-function correlations of Bacillus subtilis acetolactate synthase.

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.

Optimization of Rhodococcus erythropolis JCM3201T Nutrient Media to Improve Biomass, Lipid, and Carotenoid Yield Using Response Surface Methodology.

The oleaginous bacterium Rhodococcus erythropolis JCM3201T offers various unique enzyme capabilities, and it is a potential producer of industrially relevant compounds, such as triacylglycerol and carotenoids. To develop this strain into an efficient production platform, the characterization of the strain's nutritional requirement is necessary. In this work, we investigate its substrate adaptability. Therefore, the strain was cultivated using nine nitrogen and eight carbon sources at a carbon (16 g L-1) and nitrogen (0.16 g L-1) weight ratio of 100:1. The highest biomass accumulation (3.1 ± 0.14 g L-1) was achieved using glucose and ammonium acetate. The highest lipid yield (156.7 ± 23.0 mg g-1DCW) was achieved using glucose and yeast extract after 192 h. In order to enhance the dependent variables: biomass, lipid and carotenoid accumulation after 192 h, for the first time, a central composite design was employed to determine optimal nitrogen and carbon concentrations. Nine different concentrations were tested. The center point was tested in five biological replicates, while all other concentrations were tested in duplicates. While the highest biomass (8.00 ± 0.27 g L-1) was reached at C:N of 18.87 (11 g L-1 carbon, 0.583 g L-1 nitrogen), the highest lipid yield (100.5 ± 4.3 mg g-1DCW) was determined using a medium with 11 g L-1 of carbon and only 0.017 g L-1 of nitrogen. The highest carotenoid yield (0.021 ± 0.001 Abs454nm mg-1DCW) was achieved at a C:N of 12 (6 g L-1 carbon, 0.5 g L-1 nitrogen). The presented results provide new insights into the physiology of R. erythropolis under variable nutritional states, enabling the selection of an optimized media composition for the production of valuable oleochemicals or pigments, such as rare odd-chain fatty acids and monocyclic carotenoids.

Adaptation of Proteome and Metabolism in Different Haplotypes of Rhodosporidium toruloides during Cu(I) and Cu(II) Stress.

Rhodosporidium toruloides is a carotenogenic, oleogenic yeast that is able to grow in diverse environments. In this study, the proteomic and metabolic responses to copper stress in the two haplotypes IFO0559 and IFO0880 were assessed. 0.5 mM Cu(I) extended the lag phase of both strains significantly, while only a small effect was observed for Cu(II) treatment. Other carotenogenic yeasts such as Rhodotorula mucilaginosa are known to accumulate high amounts of carotenoids as a response to oxidative stress, posed by excess copper ion activity. However, no significant increase in carotenoid accumulation for both haplotypes of R. toruloides after 144 h of 0.5 mM Cu(I) or Cu(II) stress was observed. Yet, an increase in lipid production was detected, when exposed to Cu(II), additionally, proteins related to fatty acid biosynthesis were detected in increased amounts under stress conditions. Proteomic analysis revealed that besides the activation of the enzymatic oxidative stress response, excess copper affected iron-sulfur and zinc-containing proteins and caused proteomic adaptation indicative of copper ion accumulation in the vacuole, mitochondria, and Golgi apparatus.

Value-Added Squalene in Single-Cell Oil Produced with Cutaneotrichosporon oleaginosus for Food Applications.

Single-cell oil (SCO) produced by oleaginous microorganisms is potentially a more land-efficient and sustainable alternative to vegetable oil. The cost of SCO production can be reduced by value-added co-products like squalene, a highly relevant compound for the food, cosmetic, and pharmaceutical industry. For the first time, squalene in the oleaginous yeast Cutaneotrichosporon oleaginosus was analyzed, reaching 172.95 ± 61.31 mg/100 g oil in a lab-scale bioreactor. Using the squalene monooxygenase inhibitor terbinafine, cellular squalene was significantly increased to 2169 ± 262 mg/100 g SCO, while the yeast remained highly oleaginous. Further, SCO from a 1000 L scale production was chemically refined. The squalene content in the deodorizer distillate (DD) was found to be higher than that in DD from typical vegetable oils. Overall, this study demonstrates squalene as a value-added compound in SCO from C. oleaginosus for application in food and cosmetics without the use of genetic modifications.

Effects of Light on Growth and Metabolism of Rhodococcus erythropolis.

Rhodococcus erythropolis is resilient to various stressors. However, the response of R. erythropolis towards light has not been evaluated. In this study, R. erythropolis was exposed to different wavelengths of light. Compared to non-illuminated controls, carotenoid levels were significantly increased in white (standard warm white), green (510 nm) and blue light (470 nm) illuminated cultures. Notably, blue light (455, 425 nm) exhibited anti-microbial effects. Interestingly, cellular lipid composition shifted under light stress, increasing odd chain fatty acids (C15:0, C17:1) cultured under white (standard warm white) and green (510 nm) light. When exposed to blue light (470, 455, 425 nm), fatty acid profiles shifted to more saturated fatty acids (C16:1 to C16:0). Time-resolved proteomics analysis revealed several oxidative stress-related proteins to be upregulated under light illumination.

Efficient Green Light Acclimation of the Green Algae Picochlorum sp. Triggering Geranylgeranylated Chlorophylls.

In analogy to higher plants, eukaryotic microalgae are thought to be incapable of utilizing green light for growth, due to the "green gap" in the absorbance profiles of their photosynthetic pigments. This study demonstrates, that the marine chlorophyte Picochlorum sp. is able to grow efficiently under green light emitting diode (LED) illumination. Picochlorum sp. growth and pigment profiles under blue, red, green and white LED illumination (light intensity: 50-200 µmol m-2 s-1) in bottom-lightened shake flask cultures were evaluated. Green light-treated cultures showed a prolonged initial growth lag phase of one to 2 days, which was subsequently compensated to obtain comparable biomass yields to red and white light controls (approx. 0.8 gDW L-1). Interestingly, growth and final biomass yields of the green light-treated sample were higher than under blue light with equivalent illumination energies. Further, pigment analysis indicated, that during green light illumination, Picochlorum sp. formed unknown pigments (X1-X4). Pigment concentrations increased with illumination intensity and were most abundant during the exponential growth phase. Mass spectrometry and nuclear magnetic resonance data indicated, that pigments X1-X2 and X3-X4 are derivatives of chlorophyll b and a, which harbor C=C bonds in the phytol side chain similar to geranylgeranylated chlorophylls. Thus, for the first time, the natural accumulation of large pools (approx. 12 mg gDW -1) of chlorophyll intermediates with incomplete hydrogenation of their phytyl chains is demonstrated for algae under monochromatic green light (Peak λ 510 nm, full width at half maximum 91 nm). The ability to utilize green light offers competitive advantages for enhancing biomass production, particularly under conditions of dense cultures, long light pathways and high light intensity. Green light acclimation for an eukaryotic microalgae in conjunction with the formation of new aberrant geranylgeranylated chlorophylls and high efficiency of growth rates are novel for eukaryotic microalgae. Illumination with green light could enhance productivity in industrial processes and trigger the formation of new metabolites-thus, underlying mechanisms require further investigation.

Additive Analytics: Easy Transformation of Low-Cost Fused Deposition Modeling Three-Dimensional Printers for HPTLC Sample Application.

Additive manufacturing, known as three-dimensional (3D) printing technologies, has revolutionized production in all domains of science and technology. Although 3D printing has a high impact on research and development, its capacity to implement low-cost, flexible, and robust sample handling automation has not been exploited in full. To this end, we have created a low-cost, robust, and easy-to-utilize kit to transform an off-the-shelf fused deposition modeling 3D printer to a thin layer chromatography (TLC) sample application device. Our technology solution improves TLC convenience when higher throughput of the established method is required. The developed dual-needle sprayer allows simple and exceptionally robust automatic sample application. The device is especially well-suited for high-performance TLC-assisted method selection in counter-current chromatography. A step-by-step guide and list of required parts, including 3D printable files with instruction, can be obtained from the Supporting Information for research usage and open development.

In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization.

Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine-aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L-1 (2 mM), which compares well with other cell-free n-butanol production processes.

Cell-free metabolic engineering: production of chemicals by minimized reaction cascades.

The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.