Biotechnological production of value-added chemicals from cis-aconitate with the help of genetically engineered oleophilic yeasts

Hintergrund: Die Synthese von Chemikalien aus fossilen Rohstoffen wird wegen ihrer begrenzten Verfügbarkeit und ihren negativen Auswirkungen auf die Umwelt zunehmend kritisch bewertet. Eine Alternative bietet die „Weiße Biotechnologie“, insbesondere die Fermentation nachwachsender Rohstoffe mithilfe von Hefen. Die oleophilen Hefen Pseudozyma (P.) tsukubaensis und Yarrowia (Y.) lipolytica sind natürliche Säureproduzenten. Ihre Hauptprodukte sind Metabolite des Tricarbonsäurezyklus: Citrat (CA), α-Ketoglutarat und Malat. In kleineren Mengen werden auch andere Stoffe wie Isocitrat (ICA) oder Itaconat (ITA, nur von P. tsukubaensis) sekretiert. Das Interesse an den beiden Letztgenannten hat in den vergangenen Jahrzehnten stetig zugenommen. Bis heute gibt es allerdings keinen etablierten Wirtsorganismus für die ICA-Produktion. ITA hingegen wird mithilfe von Aspergillus terreus synthetisiert. Jedoch stößt die ITA-Produktivität dieses Hyphenpilzes auch mit großem wissenschaftlichem Aufwand an ihre Grenzen. Daher wird ein neuer Wirtsorganismus benötigt. Ergebnisse: In dieser Studie wurden ein vielversprechender P. tsukubaensis-Stamm für die Produktion von ITA und ein Y. lipolytica-Stamm für ICA konstruiert. Zunächst wurde das Genom von P. tsukubaensis sequenziert. Infolgedessen wurde ein Gencluster für die Synthese und den Export von ITA identifiziert, das homolog zu dem von Ustilago maydis ist. Die Überexpression von vier der fünf Clustergene erhöhte die ITA-Sekretion nicht deutlich. Das fünfte Gen kodiert den vermeintlichen Transkriptionsfaktor Ria1p, der vermutlich das Gencluster steuert. Die Überexpression des PtRIA1 Gens führte zu einer signifikant erhöhten ITA-Produktion von bis zu 31,4 g/l in Mikrotiterplatten. Durch die Optimierung der Wachstumsbedingungen wurden im Bioreaktor innerhalb von 7 d 113,6 g/l ITA ohne die Notwendigkeit eines Triggers produziert. Für die ICA-Produktion wurden zwei mutmaßliche mitochondriale Citrat-Transportproteine in Y. lipolytica identifiziert, welche von den Genen YlCTP1 sowie YlYHM2 kodiert werden. Die Funktionsweise der beiden Proteine scheint sich stark voneinander zu unterscheiden. Die Deletion von YlCTP1 führte zu einer leichten Verschiebung des ICA:CA-Verhältnisses, aber die Gesamtmenge beider Säuren nahm stark ab. Durch die Deletion von YlYHM2 stieg die ICA:CA-Produktrate von 12 % auf 95 % im Vergleich zum Wildtyp. Innerhalb von 5 d wurden bis zu 131,9 g/l ICA mit Sonnenblumenöl, bzw. 22,0 g/l ICA mit Glukose als einzige Kohlenstoffquelle in einem Bioreaktor unter kontrollierten Produktionsbedingungen erreicht. Durch die zusätzliche Hemmung des Isocitratlyase-Proteins mit ITA stieg das ICA:CA-Verhältnis bis 98 %. Fazit: Mittels Metabolic Engineering wurden im Rahmen dieser Arbeit die beiden Hefestämme P. tsukubaensis HR12 und Y. lipolytica ΔYHM2 erzeugt. Mit ihrer Hilfe ist es möglich, die hochwertigen Chemikalien ITA oder ICA in hohen Mengen (> 100 g/l) aus nachwachsenden Rohstoffen wie Glukose oder sogar Pflanzenölen herzustellen.Background: The synthesis of chemicals from fossil fuels is being evaluated increasingly critically, mainly due to its expected exhaustion and negative impact on the environment. An alternative offers ‘white biotechnology’, especially the fermentation of renewable resources with the help of yeasts. The oleophilic yeast species Pseudozyma (P.) tsukubaensis and Yarrowia (Y.) lipolytica are both natural organic acid producers. Their main products are metabolites of the tricarboxylic acid cycle, namely citrate, α-ketoglutarate and malate. In smaller amounts, other compounds like isocitrate (ICA) or itaconate (ITA, solely with P. tsukubaensis) are also secreted. The interest for the latter two has been rising steadily during the last decades. However, to this date, there is no established host organism for the ICA production. ITA, on the other hand, is being synthesised with Aspergillus terreus. Even with great scientific effort, the ITA productivity of this hyphal fungus appears to reach its limits. Therefore, a different host organism is needed. Results: In this study, a promising P. tsukubaensis strain has been constructed for the production of ITA and a Y. lipolytica strain for ICA. First, the genome of the ITA producer P. tsukubaensis has been sequenced. As a result, a gene cluster for the synthesis and export of ITA, homologous to that of Ustilago maydis, has been identified. By overexpressing four of the five cluster genes, respectively, none to low increases in ITA secretion were observed. The fifth gene is encoding the putative transcription factor Ria1p which probably controls the gene cluster. The overexpression of the gene PtRIA1 led to a significantly increased ITA production of up to 31.4 g/l in micro-wells. By optimizing the growth conditions 113.6 g/l ITA could be produced within 7 d under controlled conditions in a bioreactor without the need of a trigger like phosphate limitation. For the production of ICA, two putative mitochondrial citric acid transporter proteins were identified in Y. lipolytica. One carrier protein is encoded by the novel gene YlYHM2, the other one by YlCTP1. The mode of function for the two deduced proteins appears to be very distinct from one another. The deletion of YlCTP1 led to a minor shift in the ICA:CA ratio but the total amount of acids decreased greatly. By deleting YlYHM2, the ICA:CA product ratio could be increased from 12 % to 95 % compared to the wild type strain. Within 5 d up to 131.9 g/l ICA with sunflower oil and 22.0 g/l with glucose as the sole carbon source could be achieved under controlled production conditions in a bioreactor. Further inhibition of the isocitrate lyase protein with ITA increased the ICA:CA ratio to 98 %. Conclusion: Within this work, the two yeast strains P. tsukubaensis (HR12) and Y. lipolytica (ΔYHM2) have been created via metabolic engineering. With their help, it is possible to produce the value-added chemicals ITA or ICA on a high scale (> 100 g/l) from renewable resources like glucose or even vegetable oils

TOTAL PRODUCTIVE MAINTENANCE AS A REQUIREMENT OF WORLD CLASS MANUFACTURING

Total Productive Maintenance (TPM) is one of the approaches to World Class Manufacturing which is the application of TQM philosophy and tools in the fields of production, quality assurance and maintenance. TPM aims at eliminating the wastes due to machine downtime and product quality - By maximizing equipment effectiveness and productivity and eliminating machine losses; - By creating team ownership and involvement: - By promoting continuous improvement through problem solving activities involving people from production, quality assurance and maintenance. After outlining a possible approach to Total Productive Maintenance, the author examines how the contradiction of productive management strategies aiming at maximum availability with minimum costs can be resolved

What about sustainability? Investigating engineering students’ sustainability awareness and attitude

Engineers have a growing contribution towards attaining the Sustainable Development Goals (SDGs). Thus, graduate engineering students’ awareness and attitude will be crucial for dealing with these complex societal challenges. The purpose of this study is to investigate the sustainability awareness (SA) of engineering students from a developing country in comparison to that of those published from developed European countries. It also aims to explore engineering students’ attitudes and willingness to consider sustainability challenges as an important part of their future professional role. We have conducted a quantitative online survey (n=253) with the participation of engineering students from different majors at Bachelors and Masters levels. The data concerning students’ awareness and attitude underwent quantitative statistical analysis and was compared to that in the literature. The data was analyzed using SPSS to investigate differences and similarities between majors and validate its quality. The findings show good levels of SA and lower levels of knowledge in SDGs among engineering students compared to that presented by European studies. Thus, a gap in SDGs’ awareness exists between students from developed and developing countries. However, students’ positive attitudes and willingness to be involved in SDGs’ practices were remarkably high in understanding the leading role of engineers toward achieving the SDGs. Our results confirm engineering students’ motivation and strong positive attitude for resolving sustainability issues in developing countries despite challenging lifestyles. Our findings could be further used by engineering faculties in developing countries to minimize the gap and enhance future engineers’ contribution towards a more sustainable society

Reliability based Customer Satisfaction Evaluation

The purpose of this article is to introduce the so-called Reliability based Customer Satisfaction Evaluation Method that is founded upon the evaluator functions using the approach of logistic-type evaluation. The scorecard based customer satisfaction measurement methods, which are widely used at Electronics Manufacturing Services (EMS) provider companies, were studied to draw conclusions on the goodness, reliability and usefulness of these traditional practices. The approach followed here was to understand the sources of uncertainties of these methods, and construct mathematical models to improve their reliability. The nature of customers´ perceptions about the services they receive as functions of the scores they give through pre-agreed scorecards was mathematically modeled in evaluator functions. A method for parameter setting of evaluator functions was developed to make the functions adequately reflecting the customers´ satisfaction perceptions

Supporting efforts to measure intellectual capital through the EFQM Model with the example of Hungarian National Quality Award winners

The bottom line of managing intellectual capital is the ability to measure its elements. Year by year intellectual capital literature and company practice line up new measuring attempts, but only some of them prove to have strong empirical evidence. Notwithstanding, several companies complete their traditional financial accountings with the evaluation of assets which are invisible in the books. First, our study introduces the main issues regarding the measurement of intellectual capital elements. Then it seeks the answer for the question how the EFQM-based self-assessment method can support an organization in its efforts to evaluate some of its intellectual capital elements

Report on the Future Role of Engineers in Society and the Skills and Competences Engineering will Require

This literature review outlines the current state of the art knowledge in relation to the influence of diversity, attractiveness of the engineering profession and the skills and competencies required of engineers to achieve the SDGs. We aim to answer the overall research question: How do engineering educators develop new learning and teaching approaches (methods) that will attract a diverse group of students (and mature learners) whilst achieving the SDGs in 2030? The main objective of the A-STEP 2030 project is to create an attractive and fascinating learning environment to encourage young people with diverse backgrounds to engage in engineering studies and the profession as a whole