Bottleneck In Production Research Articles

Acidic Water Oxidation on Quantum Dots of IrOx/Graphdiyne

AbstractEfficient acidic water oxidation utilization in the oxygen evolution reaction (OER) is still an important bottleneck for hydrogen production. From fundamental principles, a controllable graphdiyne (GDY) induced growth strategy is established; highly uniform size distribution of oxidized iridium quantum dots is prepared on the surface of graphdiyne (IrOxQD/GDY). The result shows that, the catalyst exhibits excellent activity and durability for acidic OER, with a current density of 10 mA cm−2 at a small overpotential of 236 mV versus the reversible hydrogen electrode (RHE) and a Tafel slope of 70 mV dec−1. The performance is greatly superior to previously reported electrocatalysts. Remarkably, the acidic electrolyzer using IrOxQD/GDY as both cathode and anode electrodes can reach 10 mA cm−2 only at a much low cell voltage of 1.49 V (vs RHE). The results show the superior advantages of graphdiyne in effectively increasing numbers of the catalytically active sites for improving the charge transfer behavior and protecting the metal catalysts from corrosion.

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

Bottlenecks in Production and Marketing of Apple in Mountainous and Inaccessible Areas of Chenab Valley

Background: An attempt has been made in this study to identify the limitations encountered by the apple farmers in production and marketing which stall its growth in the study area. The present study has been carried out in the Jammu region of Jammu and Kashmir union territory with special emphasis on selected districts which fall under Chenab Valley, which is mostly hilly terrain and mountainous, as these regions had the highest area and production under apple crop.Methods: The primary data has been collected by survey method by interviewing the apple farmers as well as different market functionaries directly through an especially prepared and pre-tested schedule. The Identified constraints of the farmers in the production and marketing of apple had been ranked by making use of the Garrett’s Ranking Technique.Result: The analysis of the study revealed large number of constraints in the production and marketing of the commercial varieties of apple in a selected area, out of which occurrence of apple disease such as Stem Black, Powdery Mildew, Scab, Nectaria Twig Blight, Alternative Twig Blight, Peppery Dark/Pink Disease. Similarly, some of the common pests reported are: Woolly Aphids, San Jose Scale, Stem Borer, Root Borer, Bark Borer had been the major problem faced by the Growers, high labor cost, lack of latest technical knowledge, high cost of transportation, lack of transportation facilities and costly packing material have been identified as the major problems. In order to overcome the marketing problems of the study area apple producers should be organized as federation, cooperatives and union. The study calls for all government and non-governmental organizations to work together to expand and improve apple production and establish apple marketing hub for tackling the bottlenecks in the expansion of apple crop in the study area.

Mechanisms underlying lactic acid tolerance and its influence on lactic acid production in Saccharomyces cerevisiae.

One of the major bottlenecks in lactic acid production using microbial fermentation is the detrimental influence lactic acid accumulation poses on the lactic acid producing cells. The accumulation of lactic acid results in many negative effects on the cell such as intracellular acidification, anion accumulation, membrane perturbation, disturbed amino acid trafficking, increased turgor pressure, ATP depletion, ROS accumulation, metabolic dysregulation and metal chelation. In this review, the manner in which Saccharomyces cerevisiae deals with these issues will be discussed extensively not only for lactic acid as a singular stress factor but also in combination with other stresses. In addition, different methods to improve lactic acid tolerance in S. cerevisiae using targeted and non-targeted engineering methods will be discussed.

Assessment of challenges of crop production and marketing in Bench-Sheko, Kaffa, Sheka, and West-Omo zones of southwest Ethiopia

This study was conducted to identify the challenges of crop production and marketing in southwest Ethiopia. Primary and secondary sources of data were used. Qualitative and Quantitative data types were collected from 385 respondents through interviews, focus group discussion, key informant interviews, and observations. The collected data were analyzed by using descriptive statistics and econometric models. Crop productivity was analyzed by the Cobb Douglas model and its efficiency and determinants were identified by the stochastic frontier model. The major bottlenecks of crop production were the low attitude of farmers towards improved technology, low supply and usage of improved seed varieties (94.5%), low supply and use of fertilizers (95%), knowledge and skill gap of farmers (80.1%), poor extension service (57.3%), soil acidity (94.8%), diseases and insect pest (77.8%), conflict (84.9%) and the outbreak of human diseases (60%). Marketing challenges were poor infrastructure (87.3%), lack of market linkage (62.5%), and lack of credit services (70.6%). The Cobb Douglas model result revealed that land size, local seed, improved seed, repetition of weeding, and labor force influenced crop productivity. The mean level of crop technical efficiency was 51.3%. Education level, extension service, access to credit, cooperative membership, number of livestock owned, and soil fertility were influenced crop inefficiency negatively and distance to the farm was positively related to technical inefficiency. Improving extension services and skill of farmers through practical based training and building capacity of extension workers and systems to enhance the attitude of farmers towards technology usage and proper management practices, timely provision of farm inputs, improving road and market access, and provision of credit services to producers were some of the recommendations forwarded to alleviate crop production and marketing challenges in the study areas.

Advances in metabolic engineering for vitamins production

Vitamins are organic substances that are essential for the maintenance of life activities. Generally, vitamins need to be obtained from the diet or from some synthetic source as the body cannot synthesize vitamins, or the amounts of the synthesized vitamins are insufficient. At present, vitamins are widely used in medicine, food additives, feed additives, cosmetics and other fields, and the global demand for vitamins is constantly growing. Vitamins can be produced from chemical or microbial synthesis. Chemical synthesis usually requires harsh reaction conditions, produces serious wastes, and creates great potential safety hazard. In contrast, microbial synthesis of vitamins is greener, safer, and requires much less energy input. This review summarizes the advances in metabolic engineering for vitamins production in the past 30 years, with a focus on production of water-soluble vitamins (vitamins B1, B2, B3, B5, B6, B7, B9, B12 and vitamin C precursors) and lipid-soluble vitamins (vitamin A, precursors of vitamin D, vitamin E and vitamin K). Moreover, the bottlenecks for fermentative production of vitamins are discussed, and future perspectives for developing next generation vitamins producing strains using synthetic biotechnology are prospected.

Global Transcriptional Response of Aspergillus niger to Blocked Active Citrate Export through Deletion of the Exporter Gene.

Aspergillus niger is the major industrial citrate producer worldwide. Export as well as uptake of citric acid are believed to occur by active, proton-dependent, symport systems. Both are major bottlenecks for industrial citrate production. Therefore, we assessed the consequences of deleting the citT gene encoding the A. niger citrate exporter, effectively blocking active citrate export. We followed the consumption of glucose and citrate as carbon sources, monitored the secretion of organic acids and carried out a thorough transcriptome pathway enrichment analysis. Under controlled cultivation conditions that normally promote citrate secretion, the knock-out strain secreted negligible amounts of citrate. Blocking active citrate export in this way led to a reduced glucose uptake and a reduced expression of high-affinity glucose transporter genes, mstG and mstH. The glyoxylate shunt was strongly activated and an increased expression of the OAH gene was observed, resulting in a more than two-fold higher concentration of oxalate in the medium. Deletion of citT did not affect citrate uptake suggesting that citrate export and citrate uptake are uncoupled from the system.

The overgrowth of epiphytic Ulva prolifera during seedling cultivation of Sargassum hemiphyllum can be mitigated by regulating nitrogen availability

Sargassum hemiphyllum is a common species in many countries of the world. Its culture is both economically and ecologically important. Ulva prolifera is an annoying macro-epiphyte during seedling cultivation of S. hemiphyllum and becomes a bottleneck for the successful production of Sargassum seedlings. The present study aimed to develop an effective control method of U. prolifera. The performances of the two macroalgae cultivated under co-culture and mono-culture mode using NO3− or NH4+ as nitrogen resource were evaluated to test whether the U. prolifera could be inhibited through manipulation of nitrogen availability. Increasing NO3− and NH4+ concentrations accelerated the growth and tissue N accumulation in U. prolifera better than in S. hemiphyllum. However, the maximum growth rate of U. prolifera at infinite tissue N contents was about 70% higher than that of S. hemiphyllum, and the critical N content (NC) of U. prolifera was about twice that of S. hemiphyllum, indicating a higher risk of N limitation for U. prolifera than for S. hemiphyllum. In co-culture mode, when cultivated with 5 and 10 μM NO3− or 5 μM NH4+, the tissue N content of S. hemiphyllum was comparable to or higher than its NC, whereas the tissue N content of U. prolifera was much lower than its NC; the growth rate, chlorophyll a and soluble protein contents of U. prolifera were lower than its corresponding values in mono-culture mode. The inhibition effect of S. hemiphyllum on U. prolifera was highest in the cultivation medium with 5 μM NO3−. This suggested that the competitive advantage of epiphytic U. prolifera can to some extent be mitigated by manipulating nitrogen availability in cultivation medium. We recommend using NO3− as the N resource, and set the N concentration at approximately 5–10 μM in cultivation medium.

Increasing transgene expression and stability in recombinant CHO cells with DNA methyltransferase Dnmt3b gene knockout via CRISPR/Cas9

Chinese hamster ovary (CHO) cells are the preferred producers because of their capacity to perform proper protein folding, assembly, and post-translational modifications, similar to human cells. When CHO cells are used to produce recombinant drug proteins, it is critical to maintain stable subculture of transgenic CHO cells and high expression levels of target genes when the recombinant expression seed cell bank is expanded to a large scale bioreactor. However, at present, the instability of recombinant protein expression during prolonged culture is one of the most common bottlenecks in mammalian protein production restricting the development of CHO cell expression system. It is believed that the instability of transgene expression in CHO cells is closely related to DNA methylation. DNA methylation was mainly catalyzed by the de novo methyltransferase Dnmt3a, Dnmt3b and DNA methylsustaining-enzyme Dnmt1. DNA methyltransferase Dnmt3a and 3b play an important role in gene silencing induced by epigenetic modifications such as DNA methylation. It has been found that epigenetic modification mediated by Dnmt3b is more closely related to gene expression regulation. Therefore, in this paper, Dnmt3b gene-deficient CHO cells were established through knock out of the Dnmt3b gene by CRISPR/Cas9 genome editing technology. Furthermore, the stability and efficiency of the expression system based on the Dnmt3b KO CHO cells were evaluated by determining the expression levels of the recombinant proteins. The results indicated that the long-term stability of the green fluorescent reporter gene EGFP in the transfected Dnmt3b KO CHO cells over 30 passages increased regardless of the presence or absence of G418, compared with the normal transfected CHO cells. We also found that the expression of recombinant human-vitronectin(VN) and monoclonal antibodies (mAbs) in the stable cell pools of the transgenic Dnmt3b KO CHO cells were at least 1.6 folds higher than those of the normal CHO cells. Under the condition of not increasing the cost of culture, it is beneficial to the screening of high yielding cells and industrial production. The findings indicated that the Dnmt3b-deficient CHO cells could significantly improve the stability of target gene expression, which provide theoretical significance and application value for developing novel effective and stable CHO cell expression system.

Factors affecting the spatio-temporal variability in the production of sandfish Holothuria scabra juveniles in floating hapa ocean nursery systems

Hatchery production of the tropical sea cucumber Holothuria scabra or sandfish has been successfully demonstrated in various countries, especially during the past decade. However, the main bottleneck in up-scaling production of larger sandfish juveniles is in the nursery phase where land-based facilities are limited in the Philippines compared to other countries. This has led to some promising pioneering research in ocean-based sandfish nurseries in the Philippines. However, site-specific and seasonal inconsistencies in production levels were observed. In this study, a comprehensive experiment comparing 17 replicated nursery culture operations of sandfish juveniles using floating hapas, was conducted across four sites with distinct geo-climatic zones in the Philippines and at different months from 2013 to 2016. We evaluated the performance of the ocean-based floating hapa nursery system by determining the effects of periphyton characteristics and broad-scale differences in meteorological variables to growth and survival of juvenile sandfish within different geo-climatic zones. Our data confirmed high spatial (across sites) and temporal (among trials within a site) variability in sandfish culture performances. Wind stress was the most significant driver of this variation, which negatively influenced growth and survival of juveniles especially during Cycle 1 (first 30 d). Sandfish growth was positively related to Chl-a and mean temperature, but was negatively impacted by Wind stress, Autotropic Index (AI), total rainfall (∑Rain), and sea-surface atmospheric pressure; while survival was positively affected by phaeophytin and Ash-Free Dry Weight (AFDW) content of the hapa biofilm. In Cycle 2 (succeeding 31–60 d), growth and survival was greatly influenced negatively by the re-stocked density (number of individuals surviving from Cycle 1), while survival was further affected negatively by ∑Rain and AFDW. Our results provide significant information on suitable ocean nursery conditions, periphyton/biofilm characteristics, and spatial-temporal considerations for optimizing production of H. scabra juveniles in floating hapas. We also highlight the negative effects of increasing biomass density when on-growing larger juveniles in this system. These insights are valuable in optimizing management and operation of ocean nurseries for sandfish, not only in various potential sites in the Philippines, but also in many developing tropical countries in the Indo-Pacific.

Assessing the economic impact of tsunami and nuclear power plant disasters in Shizuoka, Japan: a dynamic inter-regional input–output (IRIO) approach

Natural disasters cause damage to regional economies. This study investigated how production activities in 35 municipalities of Shizuoka Prefecture recover when such activities stop due to a nuclear power plant disaster and tsunami that may be caused by a Nankai Megathrust Earthquake (NME). We developed a dynamic inter-regional input–output model (IRIO) featuring a production bottleneck due to a shortage of inputs and other goods. Using a simulation model, the economic damage caused by single and combined disasters was evaluated based on several recovery scenarios. The effect of depopulation in Japan on post-disaster recovery processes was also examined. This analysis provides useful disaster preparedness information for local governments and companies.

Prospect of marine natural gas hydrate stimulation theory and technology system

The key to realize the commercial production of natural gas hydrate (NGH) is to increase theNGH productivity significantly in the scale of magnitude. Whether NGH production can be commercialized depends on two aspects. The first is whether the in-situ recoverable reserves are large enough to support the basic production period for commercial production. The second is whether the average productivity can reach the standard for commercial production. In this paper, we will analyze mainly about the potential stimulation technologies for NGH development, and discuss about the basic principles, the evaluation methods, and the technical bottlenecks for NGH production and stimulation. The results indicate that the main mechanisms for increasing theNGH productivity are in three respects, namely enlarging the drainage area, increasing the NGH dissociation efficiency, and improving the seepage conditions. With complex-structure wells and multiple-well patterns, combined with novel production methods and/or reservoir stimulation technologies, the NGH productivity can be increased greatly. Particularly, the complex-structure wells and well patterns are very important for increasing NGH productivity. With complex-structure wells and well patterns, combined with heat injection and/or reservoir stimulation, NGH productivity can be increased on a magnitude scale. Currently, in fundamental researches, there are some technical bottlenecks for the studies of NGH production, mainly in sample preparation, simulated reservoir monitoring, and mechanical coupling technologies. Therefore, it is suggested that the study focuses should be on the above technical bottlenecks during the basic research on how to increase the NGH productivity. It is concluded that the combined application of complex-structure wells (horizontal wells and multi-lateral wells), well-pattern production models (with multi-cluster/group well production), the novel production methods (mainly thermal stimulation, together with depressurization), and reservoir stimulation technologies (hydraulic fracturing) are the keys to increase NGH productivity in the scale of magnitude.

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

BackgroundPhytases 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.ResultsIn 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.ConclusionsOverall, 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.

Vertically Integrated Supply Chain of Batteries, Electric Vehicles, and Charging Infrastructure: A Review of Three Milestone Projects from Theory of Constraints Perspective

This research utilizes case study methodology based on longitudinal interviews over a decade coupled with secondary data sources to juxtapose Tesla with two high-profile past mega-projects in the electric transportation industry, EV-1 and Better Place. The theory of constraints serves as a lens to identify production and market bottlenecks for the dissemination of electric vehicles. The valuable lessons learned from EV1 failure and Better Place bankruptcy paved the way for Tesla’s operations strategy to build gigafactories which bears a resemblance to Ford T mass production last century. Specifically, EV1 relied on external suppliers to develop batteries, while Better Place was dependent on a single manufacturer to build cars uniquely compatible with its charging infrastructure, whereas Tesla established a closed-loop, green, vertically integrated supply chain consisting of batteries, electric cars and charging infrastructure to meet its customers evolving needs. The analysis unveils several limitations of the Tesla business model which can impede its worldwide expansion, such as utility grid overload and a shortage of raw material, which Tesla strives to address by innovating advanced batteries and further extending its vertically integrated supply chain to the mining industry. The study concludes by sketching fruitful possible avenues for future research.

African Leafy Vegetables for Improved Human Nutrition and Food System Resilience in Southern Africa: A Scoping Review

The economic potential of African leafy vegetables (ALVs) remains obscured by a poorly developed value chain. This scoping review assembled and examined scattered knowledge generated on ALVs across southern Africa, focusing on production, processing, marketing, and consumption. Two electronic databases (Scopus and Web of Science) were screened, and a total of 71 relevant studies were included and evaluated. The review provides a state of the art on knowledge related to utilisation of ALVs across the entire value chain. The findings show that functional properties are of prime importance in the production and consumption of ALVs. However, the lack of improved germplasm and a non-existent seed supply system are significant production bottlenecks. Pests and diseases affecting the productivity of ALVs remain mostly unexplored. Sun-drying and boiling were the most reported post-harvest processing methods, suggesting that traditional processing methods are still prominent. Many studies also confirmed the predominance of informal markets in the trading of ALVs as they fail to penetrate formal markets because of poor product positioning and exclusion from produce demand and supply forecasts. The inception of cultivar development, mechanised processing methods, and market linkages will enhance the profitability of ALVs in the region. This review enhances the gaining of insight into the state of different value chain components will assist in upscaling production, value addition of products, and enhance marketing efficiency. There is a great opportunity for basic and applied research into ALVs.

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli.

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

Evaluation of electrocoagulation, flocculation, and sedimentation harvesting methods on microalgae consortium grown in anaerobically digested abattoir effluent

Microalgae dewatering is a major bottleneck for biomass production in a large-scale microalgal production system which accounts for 20–60% of production cost. In this study, three dewatering systems of electrocoagulation, flocculation, and pH-induced flocculation were evaluated for microalgal consortium grown in anaerobically digested abattoir effluent at pH 6.5 and 9.5. At the shortest time (15 min) and the highest current density (0.08 A cm−2), the highest microalgae recoveries of 78 and 84% were obtained with the corresponding power consumptions of 1.25 and 1.07 kWh kg−1 for cultures at pH 6.5 and 9.5. For microalgae suspension at pH 6.5, the highest biomass recovery of 77% was obtained when 100 mg L−1 of FeCl3·6H2O (after 15 min) or 100 mg L−1 of Al2(SO4)3·18H2O (after 30 min) was added. However, microalgal recoveries significantly increased when FeCl3·6H2O or Al2(SO4)3·18H2O was used with the culture at pH 9.5. pH-Induced experiments showed that cultures adjusted at pH 10.5 had 36% higher biomass recovery compared to that in cultures at pH 8.5 after 2 h. The results of this study showed that cultures at higher pH (9.5) had a better microalgae recovery in all dewatering systems than cultures at lower pH (6.5).

Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD+ also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD+-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD+-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD+ by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD+ synthesis were overexpressed and led to a 221% increase in the NAD+ concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD+ content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD+ by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD+ was constructed, with the intracellular NAD+ concentration reaching 26.9 μmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD+-producing strain through global regulation metabolic engineering.

Application of value stream mapping using simulation tool in manufacturing assemble line: A case study

Value stream mapping is one of the most convenient graphic tools in the manufacturing industry to eliminate wastes. This study has been carried out for the heat sink part assembly line of a manufacturing unit. The basic objective was to employ computational tools for carrying out value stream mapping and to make improvements. In the first stage, value stream mapping was done for the existing assemble line with details such as cycle time, setup time, number of employees, etc., to identify bottlenecks. Future value stream mapping was carried out using a software tool. Simulation is employed for identifying the real-time production bottlenecks; this in turn leads to improvement in productivity. This proposed method can be implemented for any manufacturing industry to eliminate non-value-added activities (NVA). The study demonstrates that the use of appropriate computational tools ensure standard methodology for a complex problem as well as produces quick results.

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV-1 virus-like particles.

HIV-1 Gag virus-like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9-fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking-down the Gag-binding protein CNP improved 2.5-fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.