An engineered enzyme embedded into PLA to make self-biodegradable plastic.

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6-8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an 'enzymated' polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20-24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

An engineered PET depolymerase to break down and recycle plastic bottles.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.

Early lysosome defects precede neurodegeneration with amyloid-ß and tau aggregation in NHE6-null rat brain.

Loss-of-function mutations in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome in males. Christianson syndrome involves endosome dysfunction leading to early cerebellar degeneration, as well as later-onset cortical and subcortical neurodegeneration, potentially including tau deposition as reported in post-mortem studies. In addition, there is reported evidence of modulation of amyloid-ß levels in experimental models wherein NHE6 expression was targeted. We have recently shown that loss of NHE6 causes defects in endosome maturation and trafficking underlying lysosome deficiency in primary mouse neurons in vitro. For in vivo studies, rat models may have an advantage over mouse models for the study of neurodegeneration, as rat brain can demonstrate robust deposition of endogenously-expressed amyloid-ß and tau in certain pathological states. Mouse models generally do not show the accumulation of insoluble, endogenously-expressed (non-transgenic) tau or amyloid-ß. Therefore, to study neurodegeneration in Christianson syndrome and the possibility of amyloid-ß and tau pathology, we generated an NHE6-null rat model of Christianson syndrome using CRISPR-Cas9 genome-editing. Here, we present the sequence of pathogenic events in neurodegenerating NHE6-null male rat brains across the lifespan. NHE6-null rats demonstrated an early and rapid loss of Purkinje cells in the cerebellum, as well as a more protracted neurodegenerative course in the cerebrum. In both the cerebellum and cerebrum, lysosome deficiency is an early pathogenic event, preceding autophagic dysfunction. Microglial and astrocyte activation also occur early. In the hippocampus and cortex, lysosome defects precede loss of pyramidal cells. Importantly, we subsequently observed biochemical and in situ evidence of both amyloid-ß and tau aggregation in the aged NHE6-null hippocampus and cortex (but not in the cerebellum). Tau deposition is widely distributed, including cortical and subcortical distributions. Interestingly, we observed tau deposition in both neurons and glia, as has been reported in Christianson syndrome post-mortem studies previously. In summary, this experimental model is among very few examples of a genetically modified animal that exhibits neurodegeneration with deposition of endogenously-expressed amyloid-ß and tau. This NHE6-null rat will serve as a new robust model for Christianson syndrome. Furthermore, these studies provide evidence for linkages between endolysosome dysfunction and neurodegeneration involving protein aggregations, including amyloid-ß and tau. Therefore these studies may provide insight into mechanisms of more common neurodegenerative disorders, including Alzheimer's disease and related dementias.

COVID-19 vaccine boosters for young adults: a risk benefit assessment and ethical analysis of mandate policies at universities.

In 2022, students at North American universities with third-dose COVID-19 vaccine mandates risk disenrolment if unvaccinated. To assess the appropriateness of booster mandates in this age group, we combine empirical risk-benefit assessment and ethical analysis. To prevent one COVID-19 hospitalisation over a 6-month period, we estimate that 31 207-42 836 young adults aged 18-29 years must receive a third mRNA vaccine. Booster mandates in young adults are expected to cause a net harm: per COVID-19 hospitalisation prevented, we anticipate at least 18.5 serious adverse events from mRNA vaccines, including 1.5-4.6 booster-associated myopericarditis cases in males (typically requiring hospitalisation). We also anticipate 1430-4626 cases of grade ≥3 reactogenicity interfering with daily activities (although typically not requiring hospitalisation). University booster mandates are unethical because they: (1) are not based on an updated (Omicron era) stratified risk-benefit assessment for this age group; (2) may result in a net harm to healthy young adults; (3) are not proportionate: expected harms are not outweighed by public health benefits given modest and transient effectiveness of vaccines against transmission; (4) violate the reciprocity principle because serious vaccine-related harms are not reliably compensated due to gaps in vaccine injury schemes; and (5) may result in wider social harms. We consider counterarguments including efforts to increase safety on campus but find these are fraught with limitations and little scientific support. Finally, we discuss the policy relevance of our analysis for primary series COVID-19 vaccine mandates.

Measurement of the Spin Absorption Anisotropy in Lateral Spin Valves.

The spin absorption process in a ferromagnetic material depends on the spin orientation relative to the magnetization. Using a ferromagnet to absorb the pure spin current created within a lateral spin valve, we evidence and quantify a sizable orientation dependence of the spin absorption in Co, CoFe, and NiFe. These experiments allow us to determine the spin-mixing conductance, an elusive but fundamental parameter of the spin-dependent transport. We show that the obtained values cannot be understood within a model considering only the Larmor, transverse decoherence, and spin diffusion lengths, and rather suggest that the spin-mixing conductance is actually limited by the Sharvin conductance.

Host shifting and host sharing in a genus of specialist flies diversifying alongside their sunflower hosts.

Congeneric parasites are unlikely to specialize on the same tissues of the same host species, likely because of strong multifarious selection against niche overlap. Exceptions where >1 congeneric species use the same tissues reveal important insights into ecological factors underlying the origins and maintenance of diversity. Larvae of sunflower maggot flies in the genus Strauzia feed on plants in the family Asteraceae. Although Strauzia tend to be host specialists, some species specialize on the same hosts. To resolve the origins of host sharing among these specialist flies, we used reduced representation genomic sequencing to infer the first multilocus phylogeny of genus Strauzia. Our results show that Helianthus tuberosus and Helianthus grosseserratus each host three different Strauzia species and that the flies co-occurring on a host are not one another's closest relatives. Though this pattern implies that host sharing is most likely the result of host shifts, these may not all be host shifts in the conventional sense of an insect moving onto an entirely new plant. Many hosts of Strauzia belong to a clade of perennial sunflowers that arose 1-2 MYA and are noted for frequent introgression and hybrid speciation events. Our divergence time estimates for all of the Helianthus-associated Strauzia are within this same time window (<1 MYA), suggesting that rapid and recent adaptive introgression and speciation in Helianthus may have instigated the diversification of Strauzia, with some flies converging upon a single plant host after their respective ancestral host plants hybridized to form a new sunflower species.

A process evaluation of the improving wisely intervention: a peer-to-peer data intervention to reduce overuse in surgery.

BACKGROUND: The Improving Wisely intervention is a peer-to-peer audit and feedback intervention to reduce overuse of Mohs Micrographic Surgery (MMS). The objective of this study was to conduct a process evaluation to evaluate Mohs surgeons' perceptions of the implementation quality and perceived impact of the Improving Wisely intervention. METHODS: Surgeons in the Improving Wisely intervention arm, comprised of members of the American College of Mohs Surgeons (ACMS) who co-led the intervention, were invited to complete surveys and key informant interviews. Participants described perceptions of implementation quality (evaluated via dose, quality of implementation, reach and participant responsiveness), perceived impact of the Improving Wisely intervention (evaluated on a 1-5 Likert and qualitatively), and barriers and facilitators to changing surgeons' clinical practice patterns to reduce Mohs overuse. RESULTS: Seven hundred thirty-seven surgeons participated in the survey. 89% were supportive of the intervention. Participants agreed that the intervention would improve patient care and reduce the annual costs of Mohs surgery. Thirty surgeons participated in key informant interviews. 93% were interested in receiving additional data reports in the future. Participants recommended the reports be disseminated annually, that the reports be expanded to include appropriateness data, and that the intervention be extended to non ACMS members. Six themes identifying factors impacting potential MMS overuse were identified. CONCLUSIONS: Participants were strongly supportive of the intervention. We present the template used to design and implement the Improving Wisely intervention and provide suggestions for specialty societies interested in leading similar quality improvement interventions among their members.

Pentagalloyl glucose (PGG) partially prevents arterial mechanical changes due to elastin degradation.

BACKGROUND: Elastic fibers are composed primarily of the protein elastin and they provide reversible elasticity to the large arteries. Degradation of elastic fibers is a common histopathology in aortic aneurysms. Pentagalloyl glucose (PGG) has been shown to bind elastin and stabilize elastic fibers in some in vitro studies and in vivo models of abdominal aortic aneurysms, however its effects on native arteries are not well described. OBJECTIVE: Perform detailed studies of the biomechanical effects of PGG on native arteries and the preventative capabilities of PGG for elastin degraded arteries. METHODS: We treated mouse carotid arteries with PGG, elastase (ELA), and PGG+ELA and compared the wall structure, solid mechanics, and fluid transport properties to untreated (UNT) arteries. RESULTS: We found that PGG alone decreased compliance compared to UNT arteries, but did not affect any other structural or biomechanical measures. Mild (30 sec) ELA treatment caused collapse and fragmentation of the elastic lamellae, plastic deformation, decreased compliance, increased modulus, and increased hydraulic conductance of the arterial wall compared to UNT. PGG+ELA treatment partially protected from all of these changes, in particular the plastic deformation. PGG mechanical protection varied considerably across PGG+ELA samples and appeared to correlate with the structural changes. CONCLUSIONS: Our results provide important considerations for the effects of PGG on native arteries and a baseline for further biomechanical studies on preventative elastic fiber stabilization.

Observation of Large Unidirectional Rashba Magnetoresistance in Ge(111).

Relating magnetotransport properties to specific spin textures at surfaces or interfaces is an intense field of research nowadays. Here, we investigate the variation of the electrical resistance of Ge(111) grown epitaxially on semi-insulating Si(111) under the application of an external magnetic field. We find a magnetoresistance term that is linear in current density j and magnetic field B, hence, odd in j and B, corresponding to a unidirectional magnetoresistance. At 15 K, for I=10 µA (or j=0.33 A m^{-1}) and B=1 T, it represents 0.5% of the zero field resistance, a much higher value compared to previous reports on unidirectional magnetoresistance (UMR). We ascribe the origin of this magnetoresistance to the interplay between the externally applied magnetic field and the pseudomagnetic field generated by the current applied in the spin-splitted subsurface states of Ge(111). This unidirectional magnetoresistance is independent of the current direction with respect to the Ge crystal axes. It progressively vanishes, either using a negative gate voltage due to carrier activation into the bulk (without spin-splitted bands), or by increasing the temperature due to the Rashba energy splitting of the subsurface states lower than ∼58k_{B}. We believe that UMR could be used as a powerful probe of the spin-orbit interaction in a wide range of materials.

Disruption of amyloid precursor protein ubiquitination selectively increases amyloid ß (Aß) 40 levels via presenilin 2-mediated cleavage.

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid ß (Aß) peptide, derived from cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aß40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aß40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aß40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane.

Anatomy of a Neotropical insect radiation.

BACKGROUND: Much evolutionary theory predicts that diversity arises via both adaptive radiation (diversification driven by selection against niche-overlap within communities) and divergence of geographically isolated populations. We focus on tropical fruit flies (Blepharoneura, Tephritidae) that reveal unexpected patterns of niche-overlap within local communities. Throughout the Neotropics, multiple sympatric non-interbreeding populations often share the same highly specialized patterns of host use (e.g., flies are specialists on flowers of a single gender of a single species of host plants). Lineage through time (LTT) plots can help distinguish patterns of diversification consistent with ecologically limited adaptive radiation from those predicted by ecologically neutral theories. Here, we use a time-calibrated phylogeny of Blepharoneura to test the hypothesis that patterns of Blepharoneura diversification are consistent with an "ecologically neutral" model of diversification that predicts that diversification is primarily a function of time and space. RESULTS: The Blepharoneura phylogeny showed more cladogenic divergence associated with geography than with shifts in host-use. Shifts in host-use were associated with ~ 20% of recent splits (< 3 Ma), but > 60% of older splits (> 3 Ma). In the overall tree, gamma statistic and maximum likelihood model fitting showed no evidence of diversification rate changes though there was a weak signature of slowing diversification rate in one of the component clades. CONCLUSIONS: Overall patterns of Blepharoneura diversity are inconsistent with a traditional explanation of adaptive radiation involving decreases in diversification rates associated with niche-overlap. Sister lineages usually use the same host-species and host-parts, and multiple non-interbreeding sympatric populations regularly co-occur on the same hosts. We suggest that most lineage origins (phylogenetic splits) occur in allopatry, usually without shifts in host-use, and that subsequent dispersal results in assembly of communities composed of multiple sympatric non-interbreeding populations of flies that share the same hosts.

[Entrustable professional activities : Promising concept in postgraduate medical education]. / "Entrustable professional activities" : Zukunftsweisendes Konzept für die ärztliche Weiterbildung.

Entrustable professional activities (EPAs) are characterized as self-contained units of work in a given typical clinical context, which may be entrusted to a trainee for independent execution at a certain point of training. An example could be the intraoperative anesthesia management of an ASA 1 patient for an uncomplicated surgical intervention as an EPA in early postgraduate anesthesia training. The EPAs can be described as an evolution of a competency-based medical educational concept, applying the concept of the competencies of a person to specific workplace contexts. In this way the expected level of skills and supervision at a certain stage of training have a more practical meaning and the danger of fragmentation of individual competencies in the competence-based model is avoided. It is a more holistic view of a trainee. Experience with this new concept is so far limited, therefore, further studies are urgently needed to determine whether and how EPAs can contribute to improvements in further training.

Variations in assessment and certification in postgraduate anaesthesia training: a European survey.

BACKGROUND: Postgraduate specialty training has traditionally been based on a time- and rotation-based model, but competency-based models are emerging. Because anaesthesia training evolves differently across Europe, variations in assessment and certification processes are expected, but the extent of similarities and differences is unknown. The aim of this study was to compare anaesthesia training programmes in Europe, focusing on assessment and certification processes. METHODS: We performed an online survey among national representatives of the Union of European Medical Specialists/European Board of Anaesthesiology. RESULTS: All 36 countries participated. Duration of training had a median of 5 yr (range 2.75-7). Mean number of different assessment tools was 7.45 (range 4-13), with more tools being used in competency-based programmes [mean 9.1 (sd 2.97) vs 7.0 (sd 1.97); P=0.03]. Most countries had a nationally uniform certification process. Based on a qualitative analysis of the survey findings, a categorization of countries emerged, reflecting the approach to assessment and certification. We observed two main streams of countries with an underlying knowledge or procedural focus within a time- and rotation-based apprenticeship model. These main streams are evolving, to different extents, towards a third orientation, competency-based training. CONCLUSIONS: Assessment and certification processes in European anaesthesia training are diverse. In many countries, a time-based apprenticeship model is evolving towards a competency-based certification process. This diversity precludes comparison of competence of graduating anaesthetists across Europe.

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFß.

The amyloid ß-peptide (Aß) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid ß protein-precursor (APP CTFß) by the γ-secretase complex. This processing produces Aß ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aß peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aß48 or Aß49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aß: the primary Aß49-46-43-40 line and a minor Aß48-45-42-38 line. The major secreted Aß species are Aß40 and Aß42, and an increased proportion of the longer, aggregation-prone Aß42 compared to Aß40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFß by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aß49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFß. We also show that instability of the CTFß TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFß dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Spin to Charge Conversion at Room Temperature by Spin Pumping into a New Type of Topological Insulator: α-Sn Films.

We present results on spin to charge current conversion in experiments of resonant spin pumping into the Dirac cone with helical spin polarization of the elemental topological insulator (TI) α-Sn. By angle-resolved photoelectron spectroscopy (ARPES), we first check that the Dirac cone (DC) at the α-Sn (0 0 1) surface subsists after covering Sn with Ag. Then we show that resonant spin pumping at room temperature from Fe through Ag into α-Sn layers induces a lateral charge current that can be ascribed to the inverse Edelstein effect by the DC states. Our observation of an inverse Edelstein effect length much longer than those generally found for Rashba interfaces demonstrates the potential of TIs for the conversion between spin and charge in spintronic devices. By comparing our results with data on the relaxation time of TI free surface states from time-resolved ARPES, we can anticipate the ultimate potential of the TI for spin to charge conversion and the conditions to reach it.

Comparison of the use of NiFe and CoFe as electrodes for metallic lateral spin valves.

Spin injection and detection in Co60Fe40-based all-metallic lateral spin valves have been studied at both room and low temperatures. The obtained spin signals amplitudes have been compared to those of identical Ni80Fe20-based devices. The replacement of Ni80Fe20 by CoFe allows increasing the spin signal amplitude by up to one order of magnitude, thus reaching 50 mΩ at room temperature. The spin signal dependence with the distance between the ferromagnetic electrodes has been analyzed using both a 1D spin-transport model and finite element method simulations. The enhancement of the spin signal amplitude when using CoFe electrodes can be explained by a higher effective polarization.

Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid ß-peptides (Aß) by γ-secretase to increase 42-to-40-residue Aß.

The presenilin-containing γ-secretase complex produces the amyloid ß-peptide (Aß) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aß critical to pathogenesis, but other Aßs of 38-49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aß. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aß: Aß49 → Aß46 → Aß43 → Aß40 and Aß48 → Aß45 → Aß42 → Aß38. Here we show that the addition of synthetic long Aß peptides (Aß45-49) directly into purified preparations of γ-secretase leads to the formation of Aß40 and Aß42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aß42 to Aß40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aß peptides to Aß40 and Aß42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aß42/Aß40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aß42/Aß40.

Switchable spin-current source controlled by magnetic domain walls.

Using nonlocal spin injection, spin-orbit coupling, or spincaloritronic effects, the manipulation of pure spin currents in nanostructures underlies the development of new spintronic devices. Here, we demonstrate the possibility to create switchable pure spin current sources, controlled by magnetic domain walls. When the domain wall is located at a given point of the magnetic circuit, a pure spin current is injected into a nonmagnetic wire. Using the reciprocal measurement configuration, we demonstrate that the proposed device can also be used as a pure spin current detector. Thanks to its simple geometry, this device can be easily implemented in spintronics applications; in particular, a single current source can be used both to induce the domain wall motion and to generate the spin signal.

Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems.

Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by X-ray crystallography. These features comprise a mobile domain called the lid that controls access to the catalytic site. Conformational rearrangements of the lid have been suggested to regulate lipase enzymatic activities. We used nuclear magnetic resonance to investigate the dynamics of Lip2 by exploring four expression systems, Escherichia coli, cell-free, Pichia pastoris and Y. lipolytica to produce uniformly labelled enzyme. The expression of Lip2 was assessed by determining its specific activity and measuring (15)N-(1)H HSQC spectra. Y. lipolytica turned out to be the most efficient expression system. Here, we report the first use of Y. lipolytica as an expression host for the production of uniform stable isotopic labelled protein for further structural and dynamics studies using NMR.

Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica.

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform ß-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ∆12 desaturase (Fad2p). We also expressed the Ricinus communis ∆12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7% of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29% of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35% of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14% of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43% of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.