A microbial platform for renewable propane synthesis based on a fermentative butanol pathway

Background Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. Results We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). Conclusions This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde

Development of yogurt with sweet potato as an ingredient

The objectives of this study were to establish critical parameters in the processing of yogurt with sweet potato as an ingredient and to determine the effect of sweet potato on sensory, physical, chemical, and nutritional attributes of the yogurt. Sweet potato level, sugar level, and incubation time had a significant effect on the production of lactic acid in the yogurt. When the percentage of sugar was increased at a given level of sweet potato and period of incubation, acidity production in the yogurt was relatively low. An increased in level of sweet potato caused a lowering of acidity for all levels of sugar at a given period of incubation. Percentage of sweet potato and sugar had an inhibitory effect on the activity of the starter culture. The predicted periods of times necessary to reach 0.85% titratable acidity in yogurt with 5% sugar and with 12, 14, 16, and 18% sweet potato were 6.25, 6.42, 6.50, and 7.25 hours, respectively. The amount of gelatin had a significant effect on firmness of the yogurt, but sweet potato had no effect. As gelatin was increased, firmness of the yogurt was increased until the gelatin content was increased to 3 g per 500 g of yogurt. The expert panel found that amount of gelatin and percentage of sweet potato had no effect on the yogurt attributes of flavor, body and texture, and appearance and color. Panelists scored the samples significantly different for each of the attributes. From the hedonic panel, gender of the panelists, the type of yogurt preferred by the panelists, the frequency of buying yogurt by the panelists and the willingness to buy the sweet potato yogurt if made commercially available had significant effects on the scores given by the panel. Panelist preferred yogurt of 16 and 18% sweet potato over the yogurt with 14% sweet potato. As level of sweet potato was increased, the percentages of moisture and fat were decreased and the percentage of carbohydrate was increased. At 14% sweet potato, yogurt had 81.3% moisture, 8.5% gat, and 66.3% carbohydrate. At 18% sweet potato, yogurt had 79.7% moisture, 4.9% fat, and 69.8% carbohydrate (DWB). Sweet potato contributed dietary fiber to the yogurt. Level of sweet ptoato did not affect the percentages of protein (mean 19.0%), ash (mean 3.9%), and total dietary fiber (mean 2.43%) (DWB). Caloric content was decreased as the level of sweet potato was increased. The calculated values indicated that yogurt with 14% sweet potato had 1,726 kjoules energy, and yogurt with 18% sweet potato had 1,651 kjoules energy (DWB). Percentage of sweet potato affected the level of vitamin C and pro-vitamin A of the yogurt. As percentage of sweet potato was increased, the vitamin contents were increased to 0.41 mg for vitamin C and to 1,252 retinol equivalents of vitamin A per 100 g sample (DWB). Level of sweet potato did affect the level of riboflavin (mean 0.41 mg) and niacin (mean 0.62 mg) per 100 g sample (DWB). However, the vitamin level of the experimental samples was higher than that for yogurt reported in the literature, except for vitamin C. As percentage of sweet potato was increased, the amounts of calcium and zinc in the yogurt were decreased. The levels of six other minerals were not affected by level of sweet potato. Percentage of sweet potato affected the levels of fructose, glucose, sucrose, and lactose of the yogurt but did not affect the level of maltose. Levels of sweet potato and sugar affected the Hunter color, L, a and b , values of the yogurt. Time of incubation had only an effect on L and b values. As level of sweet potato was increased, the yogurt developed a darker and more red and yellow color. The yogurt with the higher sugar level was darker, less red and more yellow. As time of incubation was increased, the yogurt became lighter, less red, and more yellow. During fermentation, the bacterial culture increased at approximately 0.44 log CFU/g each hour

Anion binding host systems based on calix[4]arenes and nanoparticles

A range of novel host molecules with various degrees of pre-organisation for the supramolecular complexation of anionic guest species have been synthesized. Both organic core and nanoparticle-based derivatives of the ligands have been prepared and the properties of the new host ligands studied with particular reference to their anion binding behaviour. Two types of calix[4]arene derived cationic hosts for anions with, respectively, 1,3-altemate and cone conformations have been prepared. The affinity of the tetrasubstituted calix[4]arene hosts for a variety of anions has been probed with Ή NMR spectroscopic titration. The ԼՅ-alternate system binds dicarboxylate anions in a ditopic manner while the cone compounds have the highest affinity for bromide anion and are deprotonated by carboxylates. The potentially fluorescent 1,3-altemate calix[4]arene that contains a pyridinium functionality coupled via a methylene spacer to a pyrene group undergoes selective chloride-induced conformational change which results in strong increase in both monomer and excimer emission. Gold nanoparticles protected with 5-[l ,2]dithiolan-3yl-pentanoic acid pyridin-3-ylamide remain stable as colloidal solution in methanol and the UV aborption spectra demonstrate the nanoparticles' response to exposure of a variety of anions by red shift with concomitant decrease in intensity. Titration of the colloidal solution with silver tetrafluoroborate results in an increase in absorption indicating possible interaction of silver cations with the pyridyl nitrogen atom

Engineering and biocatalytic applications of methylaspartate ammonia lyase:asymmetric synthesis of aspartic acid derivatives

Nieuwe milieuvriendelijke syntheseroute voor niet-natuurlijke aminozuren Het onderzoek van Hans Raj heeft een nieuwe, schone en milieuvriendelijke syntheseroute opgeleverd voor verschillende niet-natuurlijke aminozuren die met de traditionele chemische methoden moeilijk zijn te synthetiseren. Raj heeft hiertoe het enzym methylaspartaat ammonia-lyases (MAL), dat de synthese van aminozuren kan faciliteren, zo aangepast dat het bij meer reacties als katalysator kan worden ingezet. Deze nieuwe biokatalytische methodologie biedt interessante mogelijkheden voor de ‘groene’, milieuvriendelijke synthese van een groot scala aan gesubstitueerde asparaginezuren, wat waardevolle stoffen zijn voor neurobiologisch onderzoek en chirale uitgangsstoffen voor farma- en neutriceutica. Aminozuren zijn belangrijke stoffen voor biologisch onderzoek en interessante uitgangsstoffen voor de productie van lage-calorie zoetstoffen en medicijnen. Tot op heden is de synthese van optisch zuivere aminozuren lastig en tijdrovend met behulp van traditionele, organisch chemische methoden. Een belangrijk, competitief alternatief voor de synthese van optisch zuivere aminozuren wordt geboden door de toepassing van enzymen, zoals MAL. In zijn natuurlijke vorm katalyseert MAL de additie van ammonia aan 2-methylfumaarzuur, wat 3-methylasparaginezuur als product oplevert. Helaas vertoont MAL geen enkele katalytische activiteit met niet-natuurlijke substraten zoals grote alkylamines, complexe aryloxyfumaraten en monocarbonzuren zodat het ongeschikt is voor biotechnologische toepassingen. In zijn proefschrift beschrijft Raj het onderzoek naar het verhogen van de diastereoselectiviteit van MAL - en de hoeveelheid substraten die dit enzym accepteert - door middel van het aanbrengen van gerichte mutaties (structure-based engineering). Hij beschrijft voorbeelden van verhoging van de diastereoselectiviteit van wild-type MAL en ook toepassingen van de gemuteerde MALs in de stereoselectieve synthese van optisch zuivere asparaginezuurderivaten. Bovendien wist hij op deze manier de hoeveelheid substraten die door MAL wordt geaccepteerd succesvol te verhogen. De bruikbaarheid van deze gemuteerde MALs toonde hij vervolgens aan door ze toe te passen bij de synthese van diverse nieuwe asparaginezuurderivaten

Synthesis of furo[2,3-d]pyrimidines, thieno[2,3- d]pyrimidines, pyrrolo[2,3-d]pyrimidines as classical and nonclassical antifolates, receptor tyrosine kinase (RTK) inhibitors and antimitotic agents

An introduction, background and research progress in the areas of antifolates, receptor tyrosine kinase (RTK) inhbitors and anti mitotic agents has been discussed. Thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFTase) are important folate dependent enzymes that are targets for cancer chemotherapy and the treatment of infectious diseases. Classical antifolates, in most cases, are substrates for folypoly - γ - glutamate synthase (FPGS) and rely on folate transporter systems to enter cells. As a part of this study, twenty - eight compounds were designed on the basis of existing clinically active compounds and crystal structures, synthesized and evaluated as single and/or muliple targeted classical and nonclassical antifolates to decrease toxicity and improve the activity and selectivity of existing therapeutic agents. In additio n, these structures provides an extension to the structure activity relationship in the antifolate area. RTK inhibitors and antimitotic agents are important antitumor agents and are extensively used in the clinic for the treament of various types of cancer s. Pgp overexpression is one of the common reasons for drug resistance to existing antitumor agents and consequently the reason for some chemotherapeutic failures. A furo[2,3- d ]pyrimidine compound was discovered to have dual RTK inhibitory activity along with antimitotic activity that circumvent pgp over expression. Antimitotic activity via the binding at the colchicine site is one of the mechanisms of action. Molecular modelling and biological evaluation suggest the importance of conformational restriction for activity. Fifty - seven furo[2,3- d ]pyrimidines and six thieno[2,3- d ]pyrimidines were designed on iv the basis of crustal structures and synthesized as potential RTK inhibitors with antimitotic antitumor activity. Four pyrrolo[2,3- d ]pyrimidines were design ed and synthesized as antimitotic anticancer agents that also reverse pgp action

An approach to increasing awareness of IAQ

This project explores the approach to increasing the awareness of indoor air quality using a literature research to evaluate historical context, critiquing combined contaminants, reviewing fragrances, appraising awareness, resources and government policy, both UK and Worldwide. Two surveys were conducted with local authorities and Occupational Health and Safety (OHS) Practitioners in order to explore risk perception and awareness within a real world situation. The literature research and project activities raised further discussion points regarding the application of risk management, cost effective modelling, impact of body burden, the increase trend of scent marketing, understanding and influencing society risk perception and evaluation of the leadership of IAQ at local and government levels. This project highlights some key recommendations including the requirement to label products, particularly products like perfume who claim brand protection, to enable the consumer to understand the ingredients and make choices about their purchases; Funding for the development of bio-monitoring and multi-pollutant frameworks to build on existing silo contaminant research and create a harmonised and structured approach in understanding psychological and physiological impact interactions from a mixture of pollutants; And the establishment of an IAQ body to lead and engage stakeholders to deliver effective IAQ models. As a legacy of the project, the first accredited IOSH UK IAQ certificate and website was developed, implemented and appraised. This project is submitted as partial fulfilment of the requirements for the degree of Doctor of Professional Studies with an overall programme plan of ‘setting the agenda and raising awareness of IAQ within the UK’