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.

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.

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2.

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

Identification of sesquiterpene synthases from the Basidiomycota Coniophora puteana for the efficient and highly selective ß-copaene and cubebol production in E. coli.

BACKGROUND: Terpenes are an important and extremely versatile class of secondary metabolites that are commercially used in the pharmaceutical, food and cosmetics sectors. Genome mining of different fungal collections has revealed the genetic basis for a steadily increasing number of putative terpene synthases without any detailed knowledge about their biochemical properties. The analysis and research of this rich genetic source provides a precious basis for the advancing biotechnological production of an almost endless number of valuable natural metabolites. RESULTS: Three annotated terpene synthases from the little investigated Basidiomycota Coniophora puteana were studied in this work. For biochemical characterization, the heterologous expression in E. coli was conducted leading to the identification of two sesquiterpene synthases capable of the highly selective generation of ß-copaene and cubebol. These compounds are commercially used as food and flavor additives. The new enzymes show the highest reported product selectivity for their main compounds and therefore represent the first exclusive synthases for ß-copaene (62% product selectivity) and cubebol (75% product selectivity) generation. In combination with an optimized heterologous microbial production system, we obtained product titers of 215 mg/L ß-copaene and 497 mg/L cubebol. CONCLUSION: The reported product selectivity and our generated terpene titers exceed all published biotechnological data regarding the production of ß-copaene and cubebol. This represents a promising and economic alternative to extraction from natural plant sources and the associated complex product purification.

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems.

With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Developing a seminar curriculum for the Competence Center for General Practice in Baden-Wuerttemberg - a progress report.

Aim: The seminar program of the KWBW Verbundweiterbildungplus® is offered by the Competence Center for Postgraduate Medical Education in Baden-Württemberg (KWBW) for physicians specializing in general practice (GP trainees). Attendance is a voluntary one comprised of 48 curricular units of 45 minutes each per GP trainee. This seminar program is meant to be attended in parallel to the postgraduate medical education in clinic or practice. The intention behind this project was to develop objectives, topics and a feasible structure for a seminar curriculum while taking time and financial constraints into account. Method: The Kern cycle was applied in an open, modified nominal group consensus technique in the form of an iterative process. Participating were 17 experts from the departments of general practice at the universities in Freiburg, Heidelberg, Tuebingen and Ulm, plus a pediatrician. Results: The main objective was defined as empowering GP trainees to independently provide high-quality primary care, including in rural areas. A basic curriculum was defined based on relevant frameworks, such as the 2018 Model Regulation for Postgraduate Medical Training (Musterweiterbildungsordnung/MWBO) and the Competency-based Curriculum General Practice (KCA). Overall, the seminar curriculum has 62 basic modules with 2 curricular units each (e.g. Basic Principles of General Practice, Chest Pain, Billing) and another 58 two-unit modules on variable topics (e.g. digitalization, travel medicine) adding up to 240 (124+116) curricular units. A blueprint with a rotation schedule for all of the teaching sites in Baden-Württemberg allows regular attendance by n=400 GP trainees over a period of five years, with individual variability in terms of program length. Conclusion: The model entails a five-year, flexible program to accompany the postgraduate medical education in general practice which can also be implemented in multicenter programs and those with high enrollments. The model's focus is on acquisition of core competencies for general practice. Despite the current shift to eLearning seminars due to SARS-CoV, the program's implementation is being continued, constantly evaluated, and used to further develop the KWBW Verbundweiterbildungplus® program.

Associations of time-restricted eating with health-related quality of life and sleep in adults: a secondary analysis of two pre-post pilot studies.

BACKGROUND: Therapeutic fasting may improve health-related quality of life (HRQoL) and sleep but is not applicable for everyone. Time-restricted eating (TRE) offers a low threshold alternative but research on associations with HRQoL and sleep is rare. METHODS: We conducted a secondary analysis of two pilot studies in a pre-post design, which examined TRE in healthy employees at the Ulm University and in abdominal obese patients in a general practitioners office. Participants reported their HRQoL (EQ-5D visual analogue scale) before and after 3 months of restricting their daily eating to 8-9 h. They kept a diary to protocol timing of first and last meal, sleep quality (analogue scale) and duration. Pearson's correlation coefficient was applied to test bivariate correlations between continuous variables and linear regression analyses were conducted to identify associated factors with the pre-post differences in HRQoL and the differences in sleep quality. RESULTS: Ninety-nine participants (aged aged 48.9 ± 1.1, 83.8% female) reached the fasting target of 15-16 h on average on 77.2 ± 18.7% of all recorded days. HRQoL increased by 7.8 ± 12.6 and sleep quality by 9.6 ± 13.9 points, but sleep duration was not extended. Regression analysis revealed mean fasting duration and baseline sleep quality as significant factors associated with changes in HRQoL. Improvements in sleep quality correlated with baseline sleep quality and HRQoL at follow-up but not with fasting. Changes in anthropometry did not correlate with the HRQoL or sleep quality. CONCLUSIONS: TRE correlates with increased HRQoL and sleep quality independent from weight loss. TRE is easily applicable with or without medical supervision. The potential effects of TRE on health and sleep should be further investigated in larger randomized trials. TRIAL REGISTRATION: German Register for Clinical Trials (DRKS), DRKS-ID: DRKS00015057 . Registered 4 July 2018.

Opportunities and challenges of e-learning in vocational training in General Practice - a project report about implementing digital formats in the KWBW-Verbundweiterbildungplus.

Background: The task of the Competence Centers for vocational training (KW) is to increase the attractiveness and quality of vocational (=post-graduate) training in general practice. For this purpose, they offer, among other things, a structured seminar program for post-graduate trainees in general practice (GP-trainees). During the Covid-19 pandemic the seminar program of the KWBW-Verbundweiterbildungplus® in Baden-Württemberg was converted to digital formats. The goal of the paper is to evaluate the acceptance by the GP-trainees and lecturers, to describe experiences with the conversion to e-learning and to derive recommendations with regard to the future orientation of seminar programs in post-graduate as well as continuing medical education. The implementation was based on a modified Kern-cycle and aimed at offering eight teaching units of 45 minutes each to a large number of GP-trainees. It tried to maintain the high quality of content and education as well as the interactive character of the previous seminars. For this purpose, the events were designed as synchronous webinars (six units) with asynchronous preparation and post-processing (two units) according to the flipped classroom method. The evaluation by the participating GP-trainees and lecturers was performed online using a multi-center developed and pre-piloted questionnaire. Results and discussion: N=101 GP-trainees participated in the evaluation of five individual seminar days in the second quarter of 2020 (response rate 97%). 58% (N=59) of the trainees were satisfied or very satisfied with the implementation. 82% (n=83) rated pre-tasks as helpful. 99% (n=100) would participate in an online seminar again. For 52% (n=53) of the trainees, the attitude towards e-learning had changed positively. The main advantages mentioned were no travel, save in time and costs as well as increased flexibility. The main disadvantages mentioned were less personal interaction and technical obstacles. The high acceptance of the new digital format showed the fundamental potential of e-learning in continuing medical education. The experiences can be a source of inspiration for other departments and KW. However, it also shows that important goals of KW, such as the personal interaction of the peer group, could not be achieved. In the future, it is important to develop a suitable mix of presence and digital formats with the aim to improve the attractiveness as well as sustainability of continuing medical education.

Microalgae a Superior Source of Folates: Quantification of Folates in Halophile Microalgae by Stable Isotope Dilution Assay.

A multitude of human nutritional supplements based on Chlorella vulgaris biomass has recently been introduced to the specialty food market. In this study, an analysis of total folate contents in Chlorella sp. and a series of marine microalgae was conducted to evaluate folate content in alternative algae-based food production strains. For the first time, total folate content and vitamer distribution in microalgae were analyzed by stable isotope dilution assay (SIDA) using LC-MS/MS, which has demonstrated its superiority with respect to folate quantification. Consistently, high folate contents were detected in all examined microalgae samples. High folate concentrations of 3,460 ± 134 µg/100 g dry biomass were detected in freshly cultivated Chlorella vulgaris, notably also in other well-researched microalgae strains. To that end, the highest folate content currently documented for any algae sample was measured in the marine microalgae Picochlorum sp. isolate with values of 6,470 ± 167 µg/100 g dry biomass. This calls for alternative products based on other algae biomass. Our data indicate that freshwater and marine microalgae provide extremely high concentrations of folates, which warrant further studies on the regulation of pteroylpolyglutamates in algae as well as on bioaccessibility, absorption, and retention in humans.

Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches.

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme's active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modeling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.

Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis.

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C-C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins' structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2's catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis.

The genome of the kinetoplastid parasite, Leishmania major.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.