Production Platform Research Articles

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

BackgroundRecent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism’s vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers.ResultsWe utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure.Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates.ConclusionsThis study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica

BackgroundIsopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method.ResultsMoorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h−1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh.ConclusionsThe successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Offshore oil and gas infrastructure electrification and offshore wind: a legal exploration

Abstract The oil and gas and renewable energy sectors are increasingly coming together as traditional hydrocarbon producers are exploring the large-scale integration of renewables in fossil-fuel projects. One option is the electrification of oil and gas production platforms in the offshore environment utilizing offshore wind energy to power the asset’s operations. This article assesses the rationale for such a shift as well as regulatory avenues and barriers to the electrification of offshore oil and gas assets through offshore wind energy technology. While the article’s explorations are of a general conceptual nature, this article studies existing and planned developments in Norway and Atlantic Canada. This article discusses key factors to determine the legal nature of different electrification project models and proposes solutions to identify the likely legislative and regulatory regime(s) which will govern. The article concludes that reforms for a forward-thinking legal and regulatory environment for the offshore space will have to re-examine the role of existing (platform) and new (wind but also oil and gas) offshore infrastructure and the associated regime that applies to these projects. Currently, in the Norwegian and Canadian contexts, these discussions are somewhat exploratory. They do, however, underline the importance of planning the co-development of offshore wind energy projects with existing and future oil and gas projects and to highlight the need for greater clarity in the evolving design of legal and regulatory frameworks to support the future of the global offshore energy sector.

High-level expression of anti FLAG tag antibody in plants

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Phaeodactylum tricornutum as a stable platform for pilot scale production and investigation of the viability of spirulina fucoxanthin as a commercial lipolysis active novel compound

The nutraceutical market has experienced a significant growth over the last years, due to changing consumer preferences towards products of natural and organic origin. One of the main areas of interest in this field is microalgae-derived carotenoids, whose antioxidant activity protects against oxidative damage in human cells. The diatom Phaeodactylum tricornutum has been widely studied due to its high fucoxanthin content (antioxidant carotenoid). However, pilot and industrial scale experiments to realize continuous and stable production of P. tricornutum and extracted fucoxanthin are limited. For this reason, this research focuses on the study of the fucoxanthin production, going through culture, harvesting, scaling procedure and encapsulation stage. Finally, the optimal culture conditions for a fucoxanthin production of 2.5 mg/day/L were verified. Furthermore, the antioxidant capacity showed a decrease during storage, but still presented a capacity of 18 months, with improved performance when encapsulated with spirulina. The study carried out on encapsulated fucoxanthin-spirulina shows an inhibitory effect on lipogenesis at all concentrations tested in the 3T3-L1 cell line and a lipolytic efficacy at concentrations of 0.2 mg/mL and 0.1 mg/mL in the 3T3-L1 cell line.

How marketers influence product platform decisions: A configurational approach

AbstractThis study identifies the political behavior marketers use to ensure the market fit of a new product platform. Drawing on two political NPD literature streams and configuration theory, the authors explain how (i) soft and hard influence tactics, (ii) reason, (iii) marketers' experience (with product and portfolio decisions), and (iv) the type of product platform (smart vs. conventional), together determine marketers' political effectiveness in gaining support for design modifications from the development team. The framework and hypotheses are tested using data from a sample of 100 influence attempts by marketers. A fuzzy set qualitative comparative analysis reveals no single, optimal solution but three “influence recipes.” First, we find a generic solution for both smart and conventional product platforms that combines marketer experience with reason and coalition building. Second, we note two additional solutions for smart product platforms: one consisting of marketer experience with coalition building and another one combining marketer experience with reason and assertiveness. Regression results reveal a positive relationship between marketers' influence behavior and platform performance in the marketplace, in support of a complementary and functional role of marketers' political behaviors in innovation management.

Sustainable supply chains – Managing environmental impact data on product platforms

This paper addresses the importance of sustainable supply chains driven by environmental concerns in various industries. Life cycle assessment (LCA) presents a crucial tool for evaluating environmental impacts, but its case-by-case approach is costly and time-consuming. We present a product platform level solution to capture key supply chain elements and calculate emissions for each product variant using a product configuration system. The study presents a proof-of-concept implementation of a specialized software solution for generating combined greenhouse gas certificates and certificates of origin for product families. A case study on a ship engine demonstrates the solution capabilities, utilizing empirical data from a collaborative effort from an industrial company. The proposed solution is a demonstration of impact quantification for the product platform of a ship engine. In terms of the applicability of the software, a set of scenarios is presented to demonstrate decision-making parameters related to energy sources in operations and transportation modes related to supplier locations. The developed software shows the possibilities of the proposed approach to maintain authenticated environmental impact within the supply chain on a product platform level. The approach integrates the supplier and promotes transparency.

Safety analysis of fire evacuation from Drilling and Production Platforms (DPP)

Offshore platforms are vulnerable to fire accidents and it is essential to conduct safety analysis in case of an evacuation and take measures to improve worker evacuation efficiency and reduce casualties. Based on the proposed Credible and Representative Fire Scenarios (CRFS) methodology, nine fire scenarios were selected and simulated. The safety analysis of fire evacuation was performed based on the coupled injury model and the modified social force model, while the effects of wind direction, wind speed, fire location and pre-evacuation time on the probability of death (POD) were investigated. The results demonstrated that the wind from the stern led to a higher coverage rate of smoke (CRS) on the decks and higher POD values. The wind from the bow, larboard and starboard sides was shown to be more conducive to the dissipation of smoke. As a result, the overall POD values were relatively low. The magnitude of wind speed affects the accumulation behavior of smoke between different decks, and the lower the wind speed, the higher the CRS on the decks and POD of the workers.The pool fire located on the lower deck was more likely to cause injuries than the fire on the working deck. In addition, it was found that the trend between POD and pre-evacuation time shows a non-linear relationship, thus the scenario with the most severe fire threat was used as an example for regression analysis. An approach was proposed to determine the fire-evacuation safety time windows in the event of a fire accident and the time windows in different areas of the nine selected scenarios were predicted. Finally, the time windows obtained from the regression curves and the proposed method were compared.

Identifying and charactering a 4-aminobutyryl-CoA ligase for the production of butyrolactam

Butyrolactam, a crucial four-carbon molecule, serves as building block in synthesis of polyamides. While biosynthesis of butyrolactam from renewable carbon sources offers a more sustainable approach, it has faced challenges in achieving high product titer and yield. Here, an efficient microbial platform for butyrolactam production was constructed by elimination of rate-limiting step and systematic pathway optimization. Initially, a superior 4-aminobutyryl-CoA ligase was discovered and characterized among six acyl-CoA ligases from different sources, which greatly improved the pathway efficiency. Subsequent optimizations were implemented to further enhance butyrolactam production, including promoter engineering, the elimination of competing pathways, transporter engineering and improving the availability of precursors. There efforts resulted in achieving approximately 2 g/L butyrolactam in shake flask experiments. Finally, the biosynthesis of butyrolactam was scaled up in a 3-L bioreactor in 84 hours, resulting in a significantly increased production of 45.2 g/L, with a carbon yield of 0.34 g/g glucose. This study highlights the construction of a microbial platform with the capability to achieve elevated levels of butyrolactam production and unlocks its potential in sustainable manufacturing processes.

The power of plants as vaccine production platforms

The power of plants as vaccine production platforms

Harnessing Pentameric Scaffold of Cholera Toxin B (CTB) for Design of Subvirion Recombinant Dengue Virus Vaccine.

Dengue virus is an enveloped virus with an icosahedral assembly of envelope proteins (E). The E proteins are arranged as a head-to-tail homodimer, and domain III (EDIII) is placed at the edge of the dimer, converging to a pentamer interface. For a structure-based approach, cholera toxin B (CTB) was harnessed as a structural scaffold for the five-fold symmetry of EDIII. Pivoted by an RNA-mediated chaperone for the protein folding and assembly, CTB-EDIII of dengue serotype 1 (DV1) was successfully produced as soluble pentamers in an E. coli host with a high yield of about 28 mg/L. Immunization of mice with CTB-DV1EDIII elicited increased levels of neutralizing antibodies against infectious viruses compared to the control group immunized with DV1EDIII without CTB fusion. IgG isotype switching into a balanced Th1/Th2 response was also observed, probably triggered by the intrinsic adjuvant activity of CTB. Confirming the immune-enhancing potential of CTB in stabilizing the pentamer assembly of EDIII, this study introduces a low-cost bacterial production platform designed to augment the soluble production of subunit vaccine candidates, particularly those targeting flaviviruses.

Effect of COVID-19 pandemic on vegetable growers of Maharashtra

The world is struggling to fight the pandemic, the COVID-19 and the following lockdown has affected all walks of lives. Importantly, the farming community is the major victim of this. The present study was conducted purposively in the Rahuri and Ahmednagar tahasils of Ahmednagar district of Maharashtra State with the objectives to study the profile of the vegetable growers, to assess the effect of the COVID -19 pandemic on Marketing of vegetables, to document the constraints faced and suggestions offered by vegetable growers for future strategies. The five villages from each tahsils and nine respondents from each village thus total 90 respondents i.e. 45 farmers from Rahuri tahsil and 45 farmers from Ahmednagar tahsil were selected for the study purposively. The findings concluded that, to face the pandemic situation in future like COVID-19 and to motivate vegetable growers Government should focus on vegetable growers for adoption of sustainable and natural resources like zero energy cool chambers (79%), organizing massive public awareness and trainings campaign to popularizing the Farmer Producer Organizations and e-agriculture platforms for vegetable production and marketing (93%), utilize the services of Agro-Input dealers to enhancing more participation of the farmers and to create the Producers to Consumer marketing system (91%) and providing inputs and financial support by the Govt. (79%) is highly needed . The research study recommended that the State Department of Agriculture, Krishi Vigyan Kendra, Agricultural Universities and Agricultural Inputs Dealers jointly conduct a comprehensive public awareness and training campaign on zero energy cool chamber, Farmer Producer Organizations, e-agriculture platforms and Producers to Consumer marketing system to strengthen the agricultural extension system through Public-Private Partnership (PPP).

Interface‐Driven Spontaneous Differentiation‐Repulsion Behavior in Isochemical Droplet Populations

AbstractAn important exercise for understanding the emergent behavior in biology is to study it using minimal synthetic systems. Current biomimetic systems involving directed motion and collective migration require at least three components: two immiscible solvents to form an emulsion, and external agent(s), usually surfactants, to give rise to asymmetric interactions and induce transient non‐equilibrium conditions. Here, the most minimal system thinkable, consisting of only two components, is presented, in the form of micron‐sized oil (1‐decanol) droplets dispersed in a finite water reservoir. Spontaneous emergent dynamics within chemically identical droplet populations is reported, in the form of physical differentiation of droplets in a stochastic manner leading to an immediate repulsive behavior amongst their neighbors. Using a microfluidic production platform, fluorescence microscopy experiments, and modelling, it is showed that this cyclic phenomenon of differentiation‐repulsion results from oil droplets entering the air‐water interface leading to Marangoni flows and their coupling with evaporative flux of decanol. The potential of this platform is illustrated by demonstrating control over the event frequency, its use as Marangoni tweezers to trap droplet clusters, and proof‐of‐principle reorganization of multi‐component assemblies. The presented collective behavior with exceptional chemical simplicity makes it amenable for potentially developing self‐assembled biomimetic and bioengineered structures.

Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.

Macroalgae: Marine players in vaccinology

Although seaweeds have been used for decades as food, medicinal plants, and nutritional supplements, their use as a vaccine production and delivery platform has been barely explored, despite the availability of several genetic tools for seaweed transformation. The present review introduces seaweeds and their valuable immunomodulatory compounds in perspective as vaccine production and mucosal delivery platform. Genetic engineering tools are reviewed for macroalgal transformation, and the recombinant proteins produced in this platform are summarized. Finally, the hurdles and opportunities on seaweed potential as a vaccine production platform and delivery vehicle are proposed. As expected, seaweeds can soon be exploited to produce vaccine candidates due to their biological characteristics, including accelerated growth, photosynthetic capacity, and bioactive compound production, as well as the genetic engineering tools available. With all these properties -plus the commercial seaweed industry- this new envisioned technology can be adopted to improve human and animal health.

Influence of acetate concentration on acetone production by a modified Acetobacterium woodii

Global warming is the driving force for developing production processes of chemical compounds based on CO2 reduction technologies. Bacteria can act as biological catalysts that reduce this gaseous substrate in added-value compounds. Acetobacterium woodii is one of the best-performing strains on H2–CO2 blends and naturally produces acetate. Acetone is a raw material deeply used in the chemical industry, and its global demand is increasing. Acetone-butanol-ethanol (ABE) fermentation is the oldest microbial production platform for acetone synthesis from organic substrates, and Clostridium acetobutylicum is the model strain for its production. In various wild-type acetogens and ABE-producing Clostridium species, acetate positively influences the synthesis of reduced products. In this work, a modified A. woodii strain expressing the enzymes of the acetone pathway from C. acetobutylicum was used to convert H2–CO2 streams into acetone. This study aims to assess the impact of acetate on acetone production catalyzed by such a modified A. woodii. Tests were carried out in serum bottles and a continuous stirred tank reactor up to a pressure of 10 bar, in gas-batch or in continuous gassing, providing different gas mixes. Outcomes indicated that acetone synthesis was stimulated when acetate concentration in the medium exceeded the threshold of 100–120 mM. Thus, acetic acid can affect acetone productivity in the modified A. woodii strain. This outcome should be considered in the design of fermentation processes, especially in setting up fermentations with the liquid continuous operative mode.

Development of a tag-free plant-made interferon gamma production system with improved therapeutic efficacy against viruses.

Plants offer a promising platform for cost-effective production of biologically active therapeutic glycoproteins. In previous studies, we have developed a plant expression system based on Bamboo mosaic virus (BaMV) by incorporating secretory signals and an affinity tag, which resulted in notably enhanced yields of soluble and secreted fusion glycoproteins (FGs) in Nicotiana benthamiana. However, the presence of fusion tags on recombinant glycoproteins is undesirable for biomedical applications. This study aimed to develop a refined expression system that can efficiently produce tag-free glycoproteins in plants, with enhanced efficacy of mature interferon gamma (mIFNγ) against viruses. To accommodate the specific requirement of different target proteins, three enzymatically or chemically cleavable linkers were provided in this renovated BaMV-based expression system. We demonstrated that Tobacco etch virus (TEV) protease could process the specific cleavage site (LTEV) of the fusion protein, designated as SSExtHis(SP)10LTEV-mIFNγ, with optimal efficiency under biocompatible conditions to generate tag-free mIFNγ glycoproteins. The TEV protease and secretory-affinity tag could be effectively removed from the target mIFNγ glycoproteins through Ni2+-NTA chromatography. In addition, the result of an antiviral assay showed that the tag-free mIFNγ glycoproteins exhibited enhanced biological properties against Sindbis virus, with comparable antiviral activity of the commercialized HEK293-expressed hIFNγ. Thus, the improved BaMV-based expression system developed in this study may provide an alternative strategy for producing tag-free therapeutic glycoproteins intended for biomedical applications.

Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 μg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC–MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 μM) and UO-31 (2.2 μM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett–Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 μg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus” to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.