Exploiting fatty acid metabolic pathway for production of short chain fatty acids in E. coli

Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of short chain fatty acids (SCFAs), such as such as butyric acid (C4), as they are attractive precursors to replace petroleum-based fuels and chemicals. In this study, we explored the fatty acid metabolism for production of butyric acid in E. coli with the help of three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron. We found that E. coli strain transformed with gene for TesBT and grown in presence of 8 g/L glucose produced maximum butyric acid titer at 1.46 g/L, followed by that of TesBF at 0.85 g/L and TesAT at 0.12 g/L, showing that these thioesterases were efficiently converting short chain fatty acyl-ACP into corresponding acid. The titer of butyric acid varied significantly depending upon the strain genotype and plasmid copy number. Deletion of genes involved in initiating the fatty acid degradation such as fatty acyl-CoA synthetase and acyl-CoA dehydrogenase and overexpression of FadR, which is a dual transcriptional regulator, exerts negative control over fatty acid degradation pathway, reduced up to 30% of butyric acid titer. This observation suggested that β-oxidation pathway is working synergistically with fatty acid synthesis pathway in production of butyric acid. Moreover, accelerating the fatty acid elongation cycle by overexpressing acetyl-CoA carboxyltransferase (Acc) and 3-hydroxy-acyl-ACP dehydratase (FabZ) or by deleting FabR, the transcription suppressor of elongation, did not improve the butyric acid titer, rather favored the long chain fatty acid production. Use of chemical inhibitor cerulenin, which limits the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay showed that cerulenin also inhibited the short chain specific thioesterases, though inhibitory concentration varied according to the type of thioesterase used. Further improvement in butyric acid was achieved by process optimization. Owing to the same pathway for both cell growth and butyric acid production, a balance was achieved between the two by growing the cells under nutrient and oxygen limiting condition. Keeping these factors in mind, a fed-batch cultivation strategy was devised for production of butyric acid in phosphorous and carbon limiting condition. Finally, we obtained 14.3 g/L of butyric acid and 17.5 g/L of total free fatty acid. The strategy used in this study resulted in highest reported titers of butyric acid and free fatty acids in engineered E. coli and could be used to replace the traditional chemical methods for production of butyric acid

Effect of Trading Companies Share on Investors Attitude and Financial Behavior

Pakistan is under developing country and it has an unpredictable market nature of shareholder-investors observe the company’s performance. This research could help to companies in understanding financial behavior, attitude and investors’ satisfaction in stock trade. Financial behavior is comparatively new subject in Pakistan therefore; this study has examined the financial behavior and attitude of investors. The behavioral finance that has been attempt to understand the positive experiences influences investors’ financial behavior. This study has find out that investor satisfaction is strongest in influence of positive financial behavior of investor and trader in stock trading; positive experience and investors satisfaction are strengthens the investment decision of investors and increases behavior loyalty to prefer over competitor. The findings of this study has showing that investment gains results in more positive financial behavior and experience which leads investors satisfaction and preference the company over competitor. However negative financial behavior and complain induce by loss and loss also results decrease in behavior and attitudinal loyalty which leads the disappointment and regret. Purpose – The main purpose of this study is to find factors that effects the positive experiences with stock trading on investors’ and trader’s satisfaction, attitudinal loyalty and financial behavior in Pakistan. Design /methods and approach – The research framework links with experiences in stock trade for positive (negative) experiences, attitude and financial behavior is developed. The research framework is measured with data from sample of Karachi; the data is analyzed in smart PLS which is variance based structural equation modeling using partial least square path modeling, non-parametric software. Research Limitation – This study is focused on trading experience with company’s active investors and traders in banking industry in Pakistan. The future research could be research in other sectors with inter-related issue of investors and traders in stock trade. Originality/ Value – This is the first study in this research area; this study is determined the experiences with positive (negative) financial behavior, attitude and investors satisfaction of investors and traders in stock trade. Therefore adding in this area of study, which will help in understanding the investors and traders attitude and financial behavior in financial market. Keywords – Financial Behavior, Investor satisfaction and Attitudes, Traders or Brokers and Investors Behavior, Positive and negative Experience. Paper type – Research Paper DOI: 10.7176/RJFA/11-12-09 Publication date:June 30th 202

CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium.

Abstract Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1–xylose medium

Awake craniotomy for resection of intracranial meningioma: First case series from a low- and middle-income country

Introduction: Awake craniotomy (AC) has emerged as a better modality for resection of intra-axial brain tumors. The advantages are not just related to the preservation of neurological function, but also include early recovery, short hospital stay and possibly lower costs. However, data on AC for meningioma resection is deficient, likely because of concerns related to intra-operative pain and blood loss.Methods: All patients who underwent AC, using awake through-out technique for resection of meningioma, during the last five years, were included in the study. Non-probability consecutive sampling technique was employed. Variables for demographics, and details of diagnosis and surgical procedure were recorded. The outcomes measured were length of hospital stay, worsening of neurological function during surgery and significant intra-operative or post-operative pain.Results: Seventeen patients underwent AC for resection of meningioma during the study period. Eleven of these were grade I meningioma, and six were grade II meningioma. The mean age was 45.8 ± 10.5 years. Presenting complaints were variable, with seizures being the most common (n = 7; 41.2%). The mean duration of surgery was 180.8 ± 36.2 minutes and median estimated blood loss was 450 ml (IQR: 225 ml - 737.5 ml). The mean length of stay in the hospital was 3.1 ± 1.3 days. Only one patient had a prolonged hospital stay of seven days, because of post-operative seizures. Eleven patients (58.3%) had convexity meningioma, 4 (33.3%) had parasagittal meningioma and 1 each had a parafalcine and anterior skull-base meningioma. Simpson grade I resection was performed in 6 (41.7%) patients, grade II resection in 10 (50%) patients, and grade III resection in 1 (5.9%) patient. None of our patients had deterioration in their neurological deficits after surgery and no one required emergency intubation, conversion of surgery to general anesthesia, or redo exploration.Conclusion: AC may be considered a safe modality for surgical resection of convexity and parasagittal meningioma, with no significant risk of intra-operative or post-operative pain, although it requires more evidence. It can be offered to patients who are at higher risk, or are not willing to undergo general anesthesia. Ultimately, it might also be beneficial in terms of reducing overall costs

Establishing Mixotrophic Growth of Cupriavidus necator H16 on CO2 and Volatile Fatty Acids

The facultative chemolithoautotroph Cupriavidus necator H16 is able to grow aerobically either with organic substrates or H2 and CO2 s and it can accumulate large amounts of (up to 90%) poly (3-hydroxybutyrate), a polyhydroxyalkanoate (PHA) biopolymer. The ability of this organism to co-utilize volatile fatty acids (VFAs) and CO2 as sources of carbon under mixotrophic growth conditions was investigated and PHA production was monitored. PHA accumulation was assessed under aerobic conditions, with either individual VFAs or in mixtures, under three different conditions—with CO2 as additional carbon source, without CO2 and with CO2 and H2 as additional sources of carbon and energy. VFAs utilisation rates were slower in the presence of CO2. PHA production was significantly higher when cultures were grown mixotrophically and with H2 as an additional energy source compared to heterotrophic or mixotrophic growth conditions, without H2. Furthermore, a two-step VFA feeding regime was found to be the most effective method for PHA accumulation. It was used for PHA production mixotrophically using CO2, H2 and VFA mixture derived from an anaerobic digestor (AD). The data obtained demonstrated that process parameters need to be carefully monitored to avoid VFA toxicity and low product accumulation

A complete genome sequence of Cupriavidus necator H16 (DSM 428)

The hydrogen-utilizing strain Cupriavidus necator H16 (DSM 428) was sequenced using a combination of PacBio and Illumina sequencing. Annotation of this strain reveals 6,543 protein-coding genes, 263 pseudogenes, 64 tRNA genes, and 15 rRNA genes

Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum

Abstract Background Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain. Results We began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum. Conclusions Our study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion

A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production.

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol

Expression of β-galactosidase via its native and heterologous promoter in genome integration based system.

<p>Cells were grown (A) aerobically and (B) anaerobically, harvested and used to monitor β-galactosidase activity. The data are presented as the average and standard deviation of two independent experiments.</p