Thermal treatment improves a process of crude glycerol valorization for the production of a heterologous enzyme by Yarrowia lipolytica.

Valorization of crude glycerol requires a potent bifunctional biocatalyst, such as Yarrowia lipolytica, capable of high-density growth on this substrate, and having i.a. high propensity for heterologous protein synthesis. Increasing evidence suggests that controlled administration of stress, i.a. thermal treatment, has a positive impact on bioprocess performance. In this study, we systematically adjusted thermal treatment conditions (20 to 42 °C) in order to maximize heterologous protein production by Y. lipolytica growing in crude glycerol-based medium. Our results showed nearly 30% enhancement in the enzyme production triggered by temporary exposure to decreased temperature. Here developed mathematical model indicated optimal treatment conditions (20 °C, 153') that were later applied to a process with biodiesel-derived glycerol and technical substrates. Techno-economic analysis of a pilot-scale-waste-free process was conducted. Quantitative description of the associated costs and economic gain due to exploitation of industrial substrates, as well as indication of current bottlenecks of the process, are also provided.

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling.

Nowadays considerable effort is being pursued towards development of consolidated microbial biocatalysts that will be able to utilize complex, non-pretreated substrates and produce valuable compounds. In such engineered microbes, synthesis of extracellular hydrolases may be fine-tuned by different approaches, like strength of promoter, type of secretory tag, and gene copy number. In this study, we investigated if organization of a multi-element expression cassette impacts the resultant Yarrowia lipolytica transformants' phenotype, presuming that different variants of the cassette are composed of the same regulatory elements and encode the same mature proteins. To this end, Y. lipolytica cells were transformed with expression cassettes bearing a pair of genes encoding exactly the same mature amylases, but fused to four different signal peptides (SP), and located interchangeably in either first or second position of a synthetic DNA construction. The resultant strains were tested for growth on raw and pretreated complex substrates of different plant origin for comprehensive examination of the strains' acquired characteristics. Optimized strain was tested in batch bioreactor cultivations for growth and lipids accumulation. Based on the conducted research, we concluded that the positional order of transcription units (TU) and the type of exploited SP affect final characteristics of the resultant consolidated biocatalyst strains, and thus could be considered as additional factors to be evaluated upon consolidated biocatalysts optimization. KEY POINTS: • Y. lipolytica growing on raw starch was constructed and tested on different substrates. • Impact of expression cassette design and SP on biocatalysts' phenotype was evidenced. • Consolidated biocatalyst process for lipids production from starch was conducted.

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology.

The oleaginous yeast Yarrowia lipolytica is an established host for the bio-based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine-tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic-resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three-gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed-batch cultures-Insight into the role of osmolarity.

Fed-batch cultivation is the preferred bioprocessing strategy applied in microbial production of proteins. Feeding strategy is crucial parameters to be optimized upon development of a fed-batch process. In this study, we investigated impact of different feeding strategies on production of recombinant enzymatic protein in Yarrowia lipolytica cultures. From amongst tested strategies, comprising intermittent and continuous feedings, also in cascade with respiratory factors, intermittent feeding executed after complete exhaustion of glycerol from the medium, with moderate amplitude of osmolarity, was the most beneficial in terms of the secretory enzyme amount, its volumetric productivity and specific activity. Because adopted feeding strategies strongly modulated osmolarity of the cultures, the effect of osmotic pressure on production of the target heterologous protein was investigated in a series of batch cultivations with addition of osmoactive compounds (NaCl, sorbitol, sucrose, and glycerol) at different concentrations. Although obvious promoting effect of the osmoactive substances on the enzyme production was clear, no straightforward correlation between the medium osmolarity and the target enzyme's specific activity could be observed. These results suggest that not only the level of osmolarity but also chemical character of the osmoactive compound have both important impact on the production of secretory proteins in Y. lipolytica cultures.

Rapid micro-assays for amylolytic activities determination: customization and validation of the tests.

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60-600 µM of reducing sugar, R2 = 0.9855; 0-2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples.

Engineering the architecture of erythritol-inducible promoters for regulated and enhanced gene expression in Yarrowia lipolytica.

The non-conventional model yeast Yarrowia lipolytica is of increasing interest as a cell factory for producing recombinant proteins or biomolecules with biotechnological or pharmaceutical applications. To further develop the yeast's efficiency and construct inducible promoters, it is crucial to better understand and engineer promoter architecture. Four conserved cis-regulatory modules (CRMs) were identified via phylogenetic footprinting within the promoter regions of EYD1 and EYK1, two genes that have recently been shown to be involved in erythritol catabolism. Using CRM mutagenesis and hybrid promoter construction, we identified four upstream activation sequences (UASs) that are involved in promoter induction by erythritol. Using RedStarII fluorescence as a reporter, the strength of the promoters and the degree of erythritol-based inducibility were determined in two genetic backgrounds: the EYK1 wild type and the eyk1Δ mutant. We successfully developed inducible promoters with variable strengths, which ranged from 0.1 SFU/h to 457.5 SFU/h. Erythritol-based induction increased 2.2 to 32.3 fold in the EYK1 + wild type and 2.9 to 896.1 fold in the eyk1Δ mutant. This set of erythritol-inducible hybrid promoters could allow the modulation and fine-tuning of gene expression levels. These promoters have direct applications in protein production, metabolic engineering and synthetic biology.

Genetic engineering of Ehrlich pathway modulates production of higher alcohols in engineered Yarrowia lipolytica.

Microbial cells can produce a vast spectrum of chemical compounds, including those most desired by the global chemical market, for example, higher alcohols, which are promising alternative fuels and chemical feedstock. In the current research, we investigated the effects of the Ehrlich pathway genetic engineering on higher alcohols production in Yarrowialipolytica, which directly follows our previous findings concerning elucidation of putative molecular identities involved in this pathway. To this end, we constructed two alternative expression cassettes composed of previously identified genes, putatively involved in the Ehrlich pathway in Y. lipolytica, and cloned them under the control of constitutive pTEF promoter, and by this released them from extensive native regulation. The effects of the pathway engineering were investigated upon provision of different Ehrlich pathway-inducing amino acids (L-Phe, L-Leu, L-Ile and L-Val). In general, amplification of the Ehrlich pathway in many cases led to increased formation of a respective higher alcohol from its precursor. We observed interesting effects of aminotransferase BAT2 deletion on synthesis of 2-phenylethanol and its acetate ester, significant relationship between L-Val and L-Phe catabolic pathways and extensive 'cross-induction' of the derivative compounds synthesis by non-direct precursors.

Pichia cactophila and Kluyveromyces lactis are Highly Efficient Microbial Cell Factories of Natural Amino Acid-Derived Aroma Compounds.

The pivotal role of non-conventional yeast (NCY) species in formation of valuable aroma compounds in various food commodities is widely acknowledged. This fact inspires endeavors aiming at exploitation of food-derived NCYs as biocatalysts in natural aromas production. In this study, we isolated, characterized and evaluated aroma-producing capacity of two NCY representatives-Pichia cactophila 7.20 and Klyuveromyces lactis 6.10 strains. The strains were isolated from food-related habitats-goat-milk regional cheese and Swiss-type ripening cheese, respectively. Aroma profiles generated by the two strains cultured in a general rich medium were analyzed through solvent extraction and GC-MS analysis of the compounds retained in the culture media. Finally, the strains were tested in bioconversion cultures with branched chain- or aromatic amino acids as the sole nitrogen source, to assess capability of the strains towards formation of amino acid-derived aromas. The results showed extraordinary capacity of both strains for production of 2-phenylethanol (at more than 3 g/L) and isoamyl alcohol (approx. 1.5 g/L). A distinctive trait of 2-phenylethyl acetate synthesis at high concentrations (0.64 g/L) was revealed for P. cactophila 7.20 strain. Highly valued disulfide dimethyl as well as methionol acetate were identified amongst the aroma compounds synthesized by the strains.

[Pulse wave velocity as an early marker of diastolic heart failure in patients with hypertension]. / Predkosc fali tetna jako wczesny marker rozkurczowej niewydolnosci serca u chorych na nadcisnienie tetnicze.

According to the WHO, hypertension is one of the major causes of death worldwide. It leads to a number of severe complications. Diastolic heart failure, that is heart failure with preserved ejection fraction (HFPEF), is especially common. New, but simple, indices for the early detection of patients who have not yet developed complications or are in their early developmental stages are still searched for. AIM: The aim of this study is to examine the correlation between pulse wave velocity (PWV) and markers of diastolic heart failure (DHF) assessed in echocardiography in patients with hypertension and no symptoms of heart failure. MATERIALS AND METHODS: The study was comprised of 65 patients with treated hypertension. Patients with symptoms of heart failure, those with diabetes and smokers were excluded. Arterial stiffness was measured with the Mobil-O-Graph NG PWA. Pulse wave velocity (PWV) was estimated. The following markers of diastolic heart failure were assessed in the echocardiographic examination: E/A ratio - the ratio of the early (E) to late (A) ventricular filling velocities, DT - decceleration time, E/E' - the ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity E' in tissue Doppler echocardiography. RESULTS: PWV was statistically significantly higher in the DHF group. In the group of patients with heart failure, the average E/A ratio was significantly lower as compared to the group with no heart failure. CONCLUSIONS: Oscillometric measurement of pulse wave velocity is non-invasive, lasts a few minutes and does not require the presence of a specialist. It allows for an early detection of patients at risk of diastolic heart failure even within the conditions of primary health care.

[Was it necessary to modify the classification of chronic kidney disease stages designed by NKF KDOQI? - "Nefrotest" study results]. / Czy istniala koniecznosc modyfikacji klasyfikacji przewleklej choroby nerek wg NKF KDOQI? - wyniki badania "Nefrotest".

INTRODUCTION: The introduction of the classification of chronic kidney disease (CKD) by NKF KDOQI guidelines in 2002, including the staging and risk assessment of this disease, was a landmark event. The division of CKD into stages 1-5 turned out to be very useful and sensitive tool in the hands of both scientists and clinical practitioners; it established common nomenclature pertaining to CKD all over the world. This stratification profoundly changed the approach to CKD, transforming it from a somewhat neglected clinical problem to the phenomenon named "the epidemic of CKD". However, after a short period if clinical experience a heated debate was initiated in the literature, indicating the shortcomings of the adopted classification. The most questionable areas included methodological issues as well as dissimilar prognoses for patients depending on the cause of kidney dysfunction, the presence of proteinuria and comorbidities. AIM: The aim of this study was to evaluate the prevalence of CKD and the risk factors based on NKF KDOQI classification of 2002 in the population of Ostróda administrative district. MATERIAL AND METHOD: In total 437 individuals (F 277, M 160) aged 52.7±18.0 were examined. The study was conducted in Ostróda among randomly selected inhabitants of Ostróda adminstrative district. Serum creatinine was determined by a modified Jaffe method and eGFR was calculated (MDRD formula) for each individual. The correlations between serum creatinine and eGFR, gender and age were studied. Additionally, 326 of the examined participants were interviewed to establish CKD risk factors: kidney disease in the family, being overweight and/or obese, arterial hypertension, diabetes, smoking, heart attack, stroke. RESULTS: 58.6% of the examined individuals demonstrated abnormal eGFR values (<90 ml/min/l.73 m2), whereas serum creatinine above the laboratory norm was found in 1.3% of patients. Significant CKD risk factors included an increased prevalence of obesity (78.3%), arterial hypertension (38.6%), and smoking (26.8%); 23.9% reported kidney disease in the family. CONCLUSIONS: Based on our study, it can be concluded that CKD prevalence evaluated according to the classification of 2002 seems to be overestimated, and the main factor contributing to a false CKD diagnosis is a physiological decline in eGFR values with aging. The modification of CKD classification carried out by NKF in 2012 requires further observation and evaluation of its usefulness in daily clinical practice.

[Renal artery thrombosis and renal infarction, diagnostic and therapeutic difficulties--reports of two cases]. / Zakrzepica tetnicy nerkowej i zawal nerki, trudnosci diagnostyczne i terapeutyczne--opis dwóch przypadków.

Renal artery thrombosis (RAT) is a rare condition, with a poor prognosis, which can lead to renal infarction (RI). RAT has both cardiogenic etiology (arrhythmia, endocarditis, valvular heart disease, coronary disease) and noncardiogenic etiology (renal artery injury and/or instrumentation, fibromuscular dysplasia, coagulation disorders, homocystinuria, cocaine intake). In 30% of cases RAT etiology remains undetermined and then it is labeled idiopathic RAT. Because of the absence of typical symptoms and abnormalities in laboratory tests as well as often ambiguous results of imaging scans, RAT frequently remains undiagnosed. Presently RAT treatment standards are lacking due to its being a rare disease, which renders making treatment decisions difficult. However, because of a high risk of developing its serious complication in the form of RI, it seems necessary to introduce patient-tailored treatment in each case. Two cases presented in this paper demonstrate diagnostic difficulties and the necessity to introduce personalized treatment. Case 1 concerns idiopathic RAT in which anticoagulation therapy was introduced. Case 2 describes cardiogenic RAT with renal artery stenosis treated interventionally, i.e., percutaneous transluminal angioplasty (PTA) combined with antiplatelet drugs. In both cases recanalization of the renal artery was successful and a stable renal function was achieved.

[The role of combination therapy in the management of hypertension in patients with chronic kidney disease]. / Rola terapii preparatami zlozonymi w leczeniu nadcisnienia tetniczego u pacjentów z przewlekla choroba nerek.

Despite an increasing number of hypotensive drugs available on the market, an unsatisfactory percentage of patients achieve expected therapeutic effects in the management of arterial hypertension (HTN). This issue is especially significant as regards patients with chronic kidney disease (CKD), due to a high HTN prevalence in this population of patients and because inappropriately controlled arterial blood pressure (ABP) constitutes an additional factor leading to the progression of this disease. Kidneys play a significant role in the pathophysiology of HTN. Some of the impaired mechanisms for controlling ABP in CKD can be corrected pharmacologically. However, in order to achieve therapeutic effectiveness, a simultaneous intervention at many pathophysiological levels is necessary, and this is associated with the necessity to administer more than one hypotensive drug. It is actually polytherapy that is the real challenge in managing HTN, as it disturbs the so-called compliance, i.e., cooperation and a proper adherence to the physician's orders by the patient. Combined drugs facilitate a multidirectional intervention on the impaired mechanisms for controlling ABP, while offering an uncomplicated treatment protocol. They are especially recommended in groups of patients with a high cardiovascular risk, diabetes and/or with CKD. However, despite a large body of evidence for the improved effectiveness of hypotensive treatment with the use of combination therapy in the general population, there are no multicenter studies performed on the population of CKD patients. Also there are only few reports referring to this issue in the literature. The authors of the present paper have overviewed literature concerning the distinctness of the pathogenesis of HTN in patients with CKD and have evaluated the usefulness of combination therapy in the correction mechanism of the impaired pathophysiological paths. On the basis of the conducted analysis, the combinations of drugs preferred in CKD have been presented. The usefulness of combination therapy in the early stages of CKD has been evidenced. Spe cial attention has been given to the limitations and risks associ ated with combination therapy in CKD, mainly in the advance stages and when the disease progresses rapidly. It has been pointed out that it is particularly important to monitor renal pa rameters, potassium concentrations and clinical situations that require the therapy to be changed into a standard one. It appean that despite the pros and cons of this type of HTN management in CKD, an increasing number of combined drugs available on the market and the chances for a more effective control of ABP in this group of patients with a high cardiovascular risk necessitate further studies.

Patient with purulent pericarditis: a case doomed to fail?

Exudative pericarditis, and in particular a purulent type, is a rare condition which requires emergency medical intervention. In our paper we present a case report concerning a patient with purulent pericarditis.