Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

Abstract Background Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. Results Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. Discussion The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Download Full-text

Pollution, Toxicity and Carcinogenicity of Organic Dyes and their Catalytic Bio-Remediation

Current Pharmaceutical Design ◽

10.2174/1381612825666191021142026 ◽

2019 ◽

Vol 25 (34) ◽

pp. 3645-3663 ◽

Cited By ~ 21

Author(s):

Muhammad Ismail ◽

Kalsoom Akhtar ◽

M.I. Khan ◽

Tahseen Kamal ◽

Murad A. Khan ◽

...

Keyword(s):

Water Pollution ◽

Textile Industry ◽

Pathogenic Bacteria ◽

Diarrheal Disease ◽

Organic Dyes ◽

Chemical Reduction ◽

Cost Effective ◽

Agricultural Runoff ◽

E Coli ◽

Sanitation Services

: Water pollution due to waste effluents of the textile industry is seriously causing various health problems in humans. Water pollution with pathogenic bacteria, especially Escherichia coli (E. coli) and other microbes is due to the mixing of fecal material with drinking water, industrial and domestic sewage, pasture and agricultural runoff. Among the chemical pollutants, organic dyes due to toxic nature, are one of the major contaminants of industrial wastewater. Adequate sanitation services and drinking quality water would eliminate 200 million cases of diarrhea, which results in 2.1 million less deaths caused by diarrheal disease due to E. coli each year. Nanotechnology is an excellent platform as compared to conventional treatment methods of water treatment and remediation from microorganisms and organic dyes. In the current study, toxicity and carcinogenicity of the organic dyes have been studied as well as the remediation/inactivation of dyes and microorganism has been discussed. Remediation by biological, physical and chemical methods has been reviewed critically. A physical process like adsorption is cost-effective, but can’t degrade dyes. Biological methods were considered to be ecofriendly and cost-effective. Microbiological degradation of dyes is cost-effective, eco-friendly and alternative to the chemical reduction. Besides, certain enzymes especially horseradish peroxidase are used as versatile catalysts in a number of industrial processes. Moreover, this document has been prepared by gathering recent research works related to the dyes and microbial pollution elimination from water sources by using heterogeneous photocatalysts, metal nanoparticles catalysts, metal oxides and enzymes.

Download Full-text

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

International Journal of Bioassays ◽

10.21746/ijbio.2017.04.004 ◽

2017 ◽

Vol 6 (04) ◽

pp. 5347 ◽

Cited By ~ 3

Author(s):

Omar B. Ahmed* ◽

Anas S. Dablool

Keyword(s):

Dna Extraction ◽

Control Method ◽

Extraction Procedure ◽

Cost Effective ◽

High Yield ◽

Gram Negative Bacteria ◽

Bacterial Dna ◽

Gram Negative ◽

E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

Download Full-text

Heat Exchanger Network Retrofit of an Oleochemical Plant through a Cost and Energy Efficiency Approach

ChemEngineering ◽

10.3390/chemengineering5020017 ◽

2021 ◽

Vol 5 (2) ◽

pp. 17

Author(s):

Valli Trisha ◽

Kai Seng Koh ◽

Lik Yin Ng ◽

Vui Soon Chok

Keyword(s):

Energy Consumption ◽

Heat Exchanger ◽

Cost Effective ◽

Capital Cost ◽

Global Market ◽

Annual Saving ◽

Heat Exchanger Network ◽

First Case ◽

Payback Time ◽

Better Than

Limited research of heat integration has been conducted in the oleochemical field. This paper attempts to evaluate the performance of an existing heat exchanger network (HEN) of an oleochemical plant at 600 tonnes per day (TPD) in Malaysia, in which the emphases are placed on the annual saving and reduction in energy consumption. Using commercial HEN numerical software, ASPEN Energy Analyzer v10.0, it was found that the performance of the current HEN in place is excellent, saving over 80% in annual costs and reducing energy consumption by 1,882,711 gigajoule per year (GJ/year). Further analysis of the performance of the HEN was performed to identify the potential optimisation of untapped heating/cooling process streams. Two cases, which are the most cost-effective and energy efficient, were proposed with positive results. However, the second case performed better than the first case, at a lower payback time (0.83 year) and higher annual savings (0.20 million USD/year) with the addition of one heat exchanger at a capital cost of USD 134,620. The first case had a higher payback time (4.64 years), a lower annual saving (0.05 million USD/year) and three additional heaters at a capital cost of USD 193,480. This research has provided a new insight into the oleochemical industry in which retrofitting the HEN can further reduce energy consumption, which in return will reduce the overall production cost of oleochemical commodities. This is particularly crucial in making the product more competitive in its pricing in the global market.

Download Full-text

Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽

10.3390/genes12020283 ◽

2021 ◽

Vol 12 (2) ◽

pp. 283

Author(s):

Eyal Seroussi

Keyword(s):

Copy Number ◽

Sanger Sequencing ◽

Cytosine Methylation ◽

Direct Sequencing ◽

Information Source ◽

Gene Copy Number ◽

Cost Effective ◽

Gene Copy ◽

Base Editing ◽

Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

Download Full-text

Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab083 ◽

2021 ◽

Author(s):

Eric S Tvedte ◽

Mark Gasser ◽

Benjamin C Sparklin ◽

Jane Michalski ◽

Carl E Hjelmen ◽

...

Keyword(s):

Bacterial Genome ◽

Hybrid Approach ◽

Cost Effective ◽

Fruit Fly ◽

Drosophila Ananassae ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

E Coli ◽

Hybrid Approaches ◽

Long Read

Abstract The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Download Full-text

Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Biotechnology for Biofuels ◽

10.1186/s13068-021-01936-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Laura Navone ◽

Thomas Vogl ◽

Pawarisa Luangthongkam ◽

Jo-Anne Blinco ◽

Carlos H. Luna-Flores ◽

...

Keyword(s):

Pichia Pastoris ◽

Phytic Acid ◽

Disulfide Bond ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Biochemical Characterisation ◽

Disulfide Bond Isomerase ◽

Microbial Systems ◽

Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Download Full-text

A Reference Framework to Combine Model-Based Design and AR to Improve Social Sustainability

Sustainability ◽

10.3390/su13042031 ◽

2021 ◽

Vol 13 (4) ◽

pp. 2031

Author(s):

Fabio Grandi ◽

Riccardo Karim Khamaisi ◽

Margherita Peruzzini ◽

Roberto Raffaeli ◽

Marcello Pellicciari

Keyword(s):

Positive Impact ◽

Cost Effective ◽

Digital Models ◽

Model Based ◽

Digital Contents ◽

Recent Developments ◽

Virtual Simulations ◽

Product And Process ◽

The Impact ◽

Time Information

Product and process digitalization is pervading numerous areas in the industry to improve quality and reduce costs. In particular, digital models enable virtual simulations to predict product and process performances, as well as to generate digital contents to improve the general workflow. Digital models can also contain additional contents (e.g., model-based design (MBD)) to provide online and on-time information about process operations and management, as well as to support operator activities. The recent developments in augmented reality (AR) offer new specific interfaces to promote the great diffusion of digital contents into industrial processes, thanks to flexible and robust applications, as well as cost-effective devices. However, the impact of AR applications on sustainability is still poorly explored in research. In this direction, this paper proposed an innovative approach to exploit MBD and introduce AR interfaces in the industry to support human intensive processes. Indeed, in those processes, the human contribution is still crucial to guaranteeing the expected product quality (e.g., quality inspection). The paper also analyzed how this new concept can benefit sustainability and define a set of metrics to assess the positive impact on sustainability, focusing on social aspects.

Download Full-text

Electrochemical Biosensors in Food Safety: Challenges and Perspectives

Molecules ◽

10.3390/molecules26102940 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2940

Author(s):

Antonella Curulli

Keyword(s):

Food Industry ◽

Electrochemical Sensors ◽

Cost Effective ◽

Electrochemical Biosensors ◽

Food Chains ◽

Sensing Systems ◽

Highly Sensitive ◽

Skilled Personnel ◽

Recent Developments ◽

Key Issues

Safety and quality are key issues for the food industry. Consequently, there is growing demand to preserve the food chain and products against substances toxic, harmful to human health, such as contaminants, allergens, toxins, or pathogens. For this reason, it is mandatory to develop highly sensitive, reliable, rapid, and cost-effective sensing systems/devices, such as electrochemical sensors/biosensors. Generally, conventional techniques are limited by long analyses, expensive and complex procedures, and skilled personnel. Therefore, developing performant electrochemical biosensors can significantly support the screening of food chains and products. Here, we report some of the recent developments in this area and analyze the contributions produced by electrochemical biosensors in food screening and their challenges.

Download Full-text

Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

Nature Communications ◽

10.1038/s41467-021-24924-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Weiwei Wang ◽

Yan Gao ◽

Yanting Tang ◽

Xiaoting Zhou ◽

Yuezheng Lai ◽

...

Keyword(s):

Rational Design ◽

Pathogenic Bacteria ◽

Structural Topology ◽

Antimicrobial Drugs ◽

E Coli ◽

Cytochrome Bd ◽

Cryo Electron Microscopy ◽

Functional Mechanisms ◽

The Relationship ◽

Potential Oxygen

AbstractCytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

Download Full-text

Training Development for Complex Cognitive Tasks

Proceedings of the Human Factors Society Annual Meeting ◽

10.1177/154193128703101119 ◽

1987 ◽

Vol 31 (11) ◽

pp. 1261-1265 ◽

Cited By ~ 5

Author(s):

Joan M. Ryder ◽

Richard E. Redding ◽

Peter F. Beckschi

Keyword(s):

Cognitive Skills ◽

Task Analysis ◽

Cognitive Task ◽

Cost Effective ◽

Cognitive Task Analysis ◽

Instructional Systems ◽

Training Development ◽

Recent Developments ◽

Training Methodologies ◽

Complex Cognitive Skills

This study evaluated current training methodologies, particularly Instructional Systems Development (ISD), and recent developments in cognitive science to determine how training procedures should be modified to support training for tasks which require complex cognitive skills. We contend that ISD is still viable if procedures are developed for the training of cognitive skills. An important component of ISD which needs to be modified to support training of cognitive skills is the task analysis. We discuss the need for integrating efficient and cost-effective cognitive task analysis methodologies with traditional analysis methods.

Download Full-text