Expanding the Genetic Toolbox to Improve Metabolic Engineering in the Industrial Oleaginous Yeast, \u3cem\u3eYarrowia lipolytica\u3c/em\u3e

The oleaginous yeast, Yarrowia lipolytica, is becoming a popular host for industrial biotechnology because of its ability to grow on non-conventional feedstocks and naturally accumulate significant amounts of lipids. With new genome editing technologies, engineering novel pathways to produce lipid-derived oleochemicals has become easier. The goal, however, is to expand the genetic toolbox to improve the efficiency of metabolic engineering such that production capacities could expand from proof-of-concept shake flasks to an industrial scale. Building efficient metabolic circuits require controlling strength and timing of several enzymes in a metabolic pathway. One method to do this is through transcription – using suitable promoters to control expression of genes that code for enzymes. Native promoters have limited application because of complex regulation and non-tunable expression. Engineering hybrid promoters alleviates these issues to obtain predictable and tunable gene expression. In Y. lipolytica, how to design these promoters is not fully understood, resulting in only a handful of engineered promoters to date. In this work, we aim to develop tools for gene expression by investigating promoter architecture and designing tunable systems. In addition to Upstream Activating Sequences (UAS), tuning promoter strength can be achieved by varying sequence in the core promoter, TATA motif, and adjacent proximal sequences. UASs can modulate transcription strength and inducibility, enabling controlled timing of expression. A promoter of the acyl-CoA oxidase 2 (POX2) from the β-oxidation pathway was truncated heuristically to identify oleic acid (OA) UAS sequences. By fusing tandem repeats of the OA UAS elements, tunable yet inducible fatty acid hybrid promoters were engineered. The current approaches to identify novel UAS elements in Y. lipolytica are laborious. Therefore, we investigated DNA accessibility through nucleosome positioning to determine if a relationship between POX2 UASs and DNA accessibility can be inferred. The goal is to eventually apply this approach develop newer hybrid promoters efficiently. Finally, the hybrid fatty acid inducible promoter we developed was used to rationally engineering a Y. lipolytica strain capable of producing high amounts of free fatty acids. By localizing the fatty acyl / fatty aldehyde reductase in the peroxisome, we compartmentalized fatty alcohol production. This strategy led to upwards of 500 mg/L of fatty alcohols produced. It is a promising route to eventually make short to medium chain fatty alcohols in Y. lipolytica by utilizing the native β-oxidation machinery

Combined Effect of Nicotine and Caffeine on Orthodontic Tooth Movement in Rats

Background: The individual effects of nicotine and caffeine have been reported in previous studies but their combined effect on tooth movement needs to be elucidated. The objective of this study was to evaluate the combined effect of nicotine and caffeine on the magnitude of orthodontic tooth movement (OTM) in rats. Material and Methods: This experimental study was conducted on Sprague-Dawley rats (Animal House and Pathology Laboratory; Post Graduate Medical Institute, Lahore) in the department of Orthodontics, de’Montmorency College of Dentistry, Lahore from 8th July 2014 to 8th January 2015. Forty male Sprague-Dawley rats were divided into four equal groups: Control group (CR), nicotine group (NT), caffeine group (CF) and combined nicotine and caffeine group (CNC). Closed coil nickel titanium (NiTi) spring was placed between incisor and maxillary molar. Nicotine group (NT) was treated by intraperitoneal injections of nicotine. Caffeine was given to caffeine group and Combined nicotine and caffeine group (CNC) was treated in the same way as individual nicotine and caffeine groups daily for 14 days. All the rats were sacrificed on 15th day. Magnitude of the orthodontic tooth movement was measured using digital Vernier caliper. Means and standard deviation were calculated for orthodontic tooth movement. One-way ANOVA was used to determine the mean difference in OTM. Post hoc Tukey test was used for multiple comparisons among the groups. Results: The mean orthodontic tooth movement (OTM) was 0.32 mm ± 0.05 in control group, 0.56 mm ± 0.04 in nicotine group, 0.52 mm ± 0.034 in caffeine group and 0.8 mm ± 0.06 in combined NC group, respectively. The difference between mean OTM among the groups was statistically significant (P-value <0.001). The mean OTM in CNC group was significantly higher as compared to other groups (CR, NT, CF, NT) (P-value <0.001). Conclusions: In rats, the combined use of nicotine and caffeine results in greater orthodontic tooth movement as compared to their individual use. Key words: Bone remodeling, Caffeine, Nicotine, Orthodontic tooth movemen

(How) is formulaic language universal? Insights from Korean, German and English

The existence of common expressions, also referred to as formulaic language or phraseological units, has been evidenced in a very large number of languages. However, the extent to which languages feature such formulaic material, how formulaicity may be understood across typologically different languages and whether indeed there is a concept of formulaic language that applies across languages, are questions that have been less commonly discussed. Using a novel data set consisting of topically matched corpora in three typologically different languages (Korean, German and English), this study proposes an empirically founded universal concept for formulaic language and discusses what the shape of this concept suggests for the theoretical understanding of formulaic language going forward. In particular, it is argued that the nexus of the concept of formulaic language cannot be fixed at any particular structural level (such as the phrase or the level of polylexicality) and incorporates elements specified at varying levels of abstraction (or schematicity). This means that a cross-linguistic concept of formulaic language fits in well with a constructionist view of linguistic structure

3,3′-Diethylthiatricarbocyanine Iodide: A Highly Sensitive Chiroptical Reporter of DNA Helicity and Sequence

Using UV-vis absorption and circular dichroism (CD) spectroscopies, we explored the binding interactions of 3,3′-diethylthiatricarbocyanine iodide (Cy7) with polynucleotides of different sequences and helicity. CD showed to be a very diagnostic tool giving different spectroscopic chiroptical signatures for all explored DNA sequences upon Cy7 binding. Cy7 was able to spectroscopically discriminate between the right handed B-DNA of poly(dG-dC)2 and its left handed Z-DNA counterpart induced by spermine or Co(III)hexamine via nearly opposite induced circular dichroic signal

Alternative Substrate Metabolism in Yarrowia lipolytica

Recent advances in genetic engineering capabilities have enabled the development of oleochemical producing strains of Yarrowia lipolytica. Much of the metabolic engineering effort has focused on pathway engineering of the product using glucose as the feedstock; however, alternative substrates, including various other hexose and pentose sugars, glycerol, lipids, acetate, and less-refined carbon feedstocks, have not received the same attention. In this review, we discuss recent work leading to better utilization of alternative substrates. This review aims to provide a comprehensive understanding of the current state of knowledge for alternative substrate utilization, suggest potential pathways identified through homology in the absence of prior characterization, discuss recent work that either identifies, endogenous or cryptic metabolism, and describe metabolic engineering to improve alternative substrate utilization. Finally, we describe the critical questions and challenges that remain for engineering Y. lipolytica for better alternative substrate utilization

Recent advances in bioengineering of the oleaginous yeast <em>Yarrowia lipolytica</em>

The oleaginous yeast, Yarrowia lipolytica, is becoming increasing popular for metabolic engineering applications. Advances in synthetic biology and metabolic engineering have allowed microorganisms such as Y. lipolytica to be tailored for specific chemical production. Significant progress has been made to understand the genetics of Y. lipolytica and towards developing novel genetic engineering tools, leading to accelerated metabolic engineering efforts for a variety of different products. In this review, we discuss recent advances in genetic engineering tools and metabolic engineering achievements specific to Y. lipolytica. Topics covered in this review include genetic manipulation and expression systems, lipid-based products, peroxisome-based products and alternative sugar utilization

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems