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.

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.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.

Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus.

In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids. Graphical abstract.

Microbial lipid production by oleaginous yeasts grown on Scenedesmus obtusiusculus microalgae biomass hydrolysate.

Due to increasing oil prices and climate change concerns, biofuels have become increasingly important as potential alternative energy sources. However, the use of arable lands and valuable resources for the production of biofuel feedstock compromises food security and negatively affect the environment. Single cell oils (SCOs), accumulated by oleaginous yeasts, show great promise for efficient production of biofuels. However, the high production costs attributed to feedstocks or raw materials present a major limiting factor. The fermentative conversion of abundant, low-value biomass into microbial oil would alleviate this limitation. Here, we explore the feasibility of utilizing microalgae-based cell residues as feedstock for yeast oil production. We developed an efficient, single-step enzymatic hydrolysis to generate Scenedesmus obtusiusculus hydrolysate (SH) without thermo-chemical pretreatment. With this eco-friendly process, glucose conversion efficiencies reached 90-100%. Cutaneotrichosporon oleaginosus, Cryptococcus curvatus and Rhodosporidium toruloides were cultivated on SH as sole nutrients source. Only C. oleaginosus was able to accumulate intracellular lipids, with a 35% (g lipid/g DCW) content and a yield of 3.6 g/L. Our results demonstrate the potential valorization of algal biomass into desired end-products such as biofuels.

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.

Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate.

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10-50 °C) and pH (5-9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.

Comparative Genome-Wide Analysis of Two Caryopteris x Clandonensis Cultivars: Insights on the Biosynthesis of Volatile Terpenoids.

Caryopteris x Clandonensis, also known as bluebeard, is an ornamental plant containing a large variety of terpenes and terpene-like compounds. Four different cultivars were subjected to a principal component analysis to elucidate variations in terpenoid-biosynthesis and consequently, two representative cultivars were sequenced on a genomic level. Functional annotation of genes as well as comparative genome analysis on long read datasets enabled the identification of cultivar-specific terpene synthase and cytochrome p450 enzyme sequences. This enables new insights, especially since terpenoids in research and industry are gaining increasing interest due to their importance in areas such as food preservation, fragrances, or as active ingredients in pharmaceutical formulations. According to BUSCO assessments, the presented genomes have an average size of 355 Mb and about 96.8% completeness. An average of 52,090 genes could be annotated as putative proteins, whereas about 42 were associated with terpene synthases and about 1340 with cytochrome p450 enzymes.

Differential RNA-Seq Analysis Predicts Genes Related to Terpene Tailoring in Caryopteris × clandonensis.

Enzymatic terpene functionalization is an essential part of plant secondary metabolite diversity. Within this, multiple terpene-modifying enzymes are required to enable the chemical diversity of volatile compounds essential in plant communication and defense. This work sheds light on the differentially transcribed genes within Caryopteris × clandonensis that are capable of functionalizing cyclic terpene scaffolds, which are the product of terpene cyclase action. The available genomic reference was subjected to further improvements to provide a comprehensive basis, where the number of contigs was minimized. RNA-Seq data of six cultivars, Dark Knight, Grand Bleu, Good as Gold, Hint of Gold, Pink Perfection, and Sunny Blue, were mapped on the reference, and their distinct transcription profile investigated. Within this data resource, we detected interesting variations and additionally genes with high and low transcript abundancies in leaves of Caryopteris × clandonensis related to terpene functionalization. As previously described, different cultivars vary in their modification of monoterpenes, especially limonene, resulting in different limonene-derived molecules. This study focuses on predicting the cytochrome p450 enzymes underlying this varied transcription pattern between investigated samples. Thus, making them a reasonable explanation for terpenoid differences between these plants. Furthermore, these data provide the basis for functional assays and the verification of putative enzyme activities.

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.

Biosynthetic Potential of Hypogymnia Holobionts: Insights into Secondary Metabolite Pathways.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus). They are known to produce a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here we provide a comprehensive view of the biosynthetic gene clusters of all organisms comprising a lichen thallus: fungi, green algae, and bacteria. We present two high-quality PacBio metagenomes, in which we identified a total of 460 biosynthetic gene clusters. Lichen mycobionts yielded 73-114 clusters, other lichen associated ascomycetes 8-40, green algae of the genus Trebouxia 14-19, and lichen-associated bacteria 101-105 clusters. The mycobionts contained mainly T1PKSs, followed by NRPSs, and terpenes; Trebouxia reads harbored mainly clusters linked to terpenes, followed by NRPSs and T3PKSs. Other lichen-associated ascomycetes and bacteria contained a mix of diverse biosynthetic gene clusters. In this study, we identified for the first time the biosynthetic gene clusters of entire lichen holobionts. The yet untapped biosynthetic potential of two species of the genus Hypogymnia is made accessible for further research.

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.

Biosynthetic gene cluster synteny: Orthologous polyketide synthases in Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus), which together generate a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here, we provide a comparative view of the biosynthetic gene clusters of three lichen mycobionts derived from Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata. In addition, we present a high-quality PacBio metagenome of Parmelia sulcata, from which we extracted the mycobiont bin containing 214 biosynthetic gene clusters. Most biosynthetic gene clusters in these genomes were associated with T1PKSs, followed by NRPSs and terpenes. This study focused on biosynthetic gene clusters related to polyketide synthesis. Based on ketosynthase homology, we identified nine highly syntenic clusters present in all three species. Among the four clusters belonging to nonreducing PKSs, two are putatively linked to lichen substances derived from orsellinic acid (orcinol depsides and depsidones, e.g., lecanoric acid, physodic acid, lobaric acid), one to compounds derived from methylated forms of orsellinic acid (beta orcinol depsides, e.g., atranorin), and one to melanins. Five clusters with orthologs in all three species are linked to reducing PKSs. Our study contributes to sorting and dereplicating the vast PKS diversity found in lichenized fungi. High-quality sequences of biosynthetic gene clusters of these three common species provide a foundation for further exploration into biotechnological applications and the molecular evolution of lichen substances.

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.

GFP Scaffold-Based Engineering for the Production of Unbranched Very Long Chain Fatty Acids in Escherichia coli With Oleic Acid and Cerulenin Supplementation.

Currently, very long chain fatty acids (VLCFAs) for oleochemical, pharmaceutical, cosmetic, or food applications are extracted from plant or marine organism resources, which is associated with a negative environmental impact. Therefore, there is an industrial demand to develop sustainable, microbial resources. Due to its ease of genetic modification and well-characterized metabolism, Escherichia coli has established itself as a model organism to study and tailor microbial fatty acid biosynthesis using a concerted genetic engineering approach. In this study, we systematically implemented a plant-derived (Arabidopsis thaliana) enzymatic cascade in Escherichia coli to enable unbranched VLCFA biosynthesis. The four Arabidopsis thaliana membrane-bound VLCFA enzymes were expressed using a synthetic expression cassette. To facilitate enzyme solubilization and interaction of the synthetic VLCFA synthase complex, we applied a self-assembly GFP scaffold. In order to initiate VLCFA biosynthesis, external oleic acid and cerulenin were supplemented to cultures. In this context, we detected the generation of arachidic (20:0), cis-11-eicosenoic (20:1) and cis-13-eicosenoic acid (20:1).

Multi-Factorial-Guided Media Optimization for Enhanced Biomass and Lipid Formation by the Oleaginous Yeast Cutaneotrichosporon oleaginosus.

The non-conventional, oleaginous yeast Cutaneotrichosporon oleaginosus is flagged as an industrial cell factory for generation of oleochemicals and biofuels due to its substrate flexibility and high triglyceride yields. In this study, we employed a computational Response Surface Methodology to guide and streamline the experimental media optimization matrix with 12 nitrogen and 10 carbon sources in order to provide for high biomass and lipid accumulation toward an industrially relevant fermentation process. The resulting data provide new insights into C. oleaginosus physiology under variable nutritional states. Accordingly, the lipid content % (lipid weight/yeast dry weight) is controlled by a defined interplay between carbon and nitrogen. In our experimental setup, the highest biomass (18.4 ± 2.20 g/L) and lipid yield (9 ± 0.34 g/L; 49.74 ± 5.16% g lipid weight/g yeast dry cell weight) were obtained with lactose and yeast extract as carbon and nitrogen sources at an elemental weight ratio of 120:1, respectively. Interestingly, with ammonium salts as a N-source, the intracellularly accumulated triglycerides increasingly contain saturated fatty acids, which provides a new route to generate tailored fatty acid profiles for specific oleochemicals or food applications. Our data indicate that a metabolic ceiling for lipid accumulation in C. oleaginosus is obtained with the correct carbon and nitrogen source mixture.

Potency of different red light sources in photodynamic induction of cell death in a squamous cell carcinoma cell line.

LED illumination systems were found to be more efficacious than broad spectrum lamps in recent phase III trials on photodynamic treatment of actinic keratosis. However, a detailed comparison of the light doses emitted at the appropriate spectral range and its correlation to photodynamic effects is thus far not available for the most frequently used devices. Here, we compared the spectral emissions of three different PDT lamps with their potency of inducing cell death in ALA-loaded A431 cells, including a new system equipped with more advanced LEDs matching the photosensitizer absorption peak more precisely and emitting more homogeneous light over time. Cells were exposed to two different ALA concentrations, incubated for 1 or 3h and then illuminated by one of two different LED or a broad-spectrum system at four different light doses, whereupon viability was assessed. Maximal doses were selected in accordance to clinically applied light doses in recent phase III studies and the manufacturers' recommendations. The data gathered here clearly demonstrate that the two LED systems were significantly more effective in inducing cell death than the broad spectrum system. Most efficient was the newer LED system, in agreement with emission parameters that more accurately corresponded to the photosensitizer's absorption peak.