Increased Ethanol Production by Disrupting the Competitive Phosphoenolpyruvate Synthesis Pathway and Enhancing the Expression of Ethanol-producing Genes in Synechocystis Sp. PCC6803

2020 ◽  
Author(s):  
E-Bin Gao ◽  
Ye Penglin ◽  
Yunxiang Xu ◽  
Zhu Yangjie ◽  
Kwasi Kyere-Yeboah ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Large Scale ◽  
Biosynthetic Pathway ◽  
Pyruvate Decarboxylase ◽  
Clean Energy ◽  
Cell Factory ◽  
Scale Production ◽  
Microbial Cell Factory ◽  
Dry Cell Weight ◽  
Pcc 6803

Abstract BackgroundEthanol is a very important clean energy and it has many applications in medical and chemical fields. Large-scale production of ethanol has mainly been carried out through the fermentation of crops such as grain but its output and cost issues have attracted widespread attention. ResultsWith the ability to fix carbon dioxide directly, cyanobacteria have been used as a photosynthetic microbial cell factory to generate biofuels and chemicals. Here, we constructed the biosynthetic pathway of ethanol in cyanobacterium Synechocystis sp. PCC 6803 through the following approaches. (1) We used homologous substitution to introduce pyruvate decarboxylase (pdc) gene from Zymomonas mobilis and NADPH-dependent aldehyde reductase (yqhD) gene from Escherichia coli into the neutral site of Synechocystis sp. PCC 6803. (2) The native superpromoter Pcpc560, consisting of two promoters from the cpcB gene and 14 predicted transcription factor binding sites (TFBSs) from Synechocystis sp. PCC6803 genome, was used to drive the over-expression of ethanol-producing genes. (3) To further increase ethanol production, we used molecular biotechnology to inhibit the metabolic pathway that direct the carbon flux of intermediate pyruvate metabolism to phosphoenolpyruvate (PEP) through disrupting the cyanobacterial endogenous PEP synthase. These approaches led to the production of 2.79g/g dry cell weight ethanol directly from light and greenhouse gas CO2 in Synechocystis after cultivating for 9 days. ConclusionOur study provides insights into the biosynthetic pathway for ethanol production in Synechocystis indicating that knocking out the competitive pathway of the initial precursor and enhancing the expression of exogenous genes can effectively increase the amount of the targeted chemicals.

scholarly journals Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses

Metabolites ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 45
Author(s):  
Qi Yang ◽  
Wenli Lin ◽  
Jiawei Xu ◽  
Nan Guo ◽  
Jiachen Zhao ◽  
...  
Keyword(s):  
Steady State ◽  
Dissolved Oxygen ◽  
Penicillium Chrysogenum ◽  
Large Scale ◽  
Scale Up ◽  
Redox Status ◽  
Oxygen Availability ◽  
Industrial Scale ◽  
Cell Factory ◽  
Microbial Cell Factory

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

A new Procedure for Large Scale Production and Freezing of Lymphokine Activated Killer (LAK) Cells to be used in Adoptive Immunotherapy of Cancer

1988 ◽  
Vol 74 (5) ◽  
pp. 523-530 ◽  
Author(s):  
Carlo Gambacorti-Passerini ◽  
Marina Radrizzani ◽  
Licia Rivoltini ◽  
Edoardo Marchesi ◽  
Fernando Ravagnani ◽  
...  
Keyword(s):  
Adoptive Immunotherapy ◽  
Large Scale ◽  
Interleukin 2 ◽  
Peripheral Blood Lymphocytes ◽  
Cell Factory ◽  
Scale Production ◽  
Lymphokine Activated Killer Cells ◽  
Large Scale Production ◽  
Recombinant Interleukin 2 ◽  
Immunotherapy Of Cancer

A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell Factory™, CF), gasified and incubated at 37 °C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.

scholarly journals Zymobacter palmae pyruvate decarboxylase is less efficient than that of Zymomonas mobilis for ethanol production in metabolically engineered Synechocystis sp PCC6803

2019 ◽  
Author(s):  
Lorraine Quinn ◽  
Patricia Armshaw ◽  
Tewfik Soulimane ◽  
Con Sheehan ◽  
Michael P Ryan ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Inducible Promoter ◽  
Control Strain ◽  
Zymobacter Palmae ◽  
Synechocystis Sp ◽  
Pcc 6803

AbstractPyruvate decarboxylase (PDC) from Zymobacter palmae (ZpPDC) has been reported to have a lower Km the Zymomonas mobilis PDC (ZmPDC). ZpPDC was combined with native slr1192 alcohol dehydrogenase (adh) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with Zmpdc. Native (Zppdc) and codon optimised (ZpOpdc) versions of the ZpPDC were cloned into a construct where the pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis PCC 6803. These constructs were transformed into wildtype Synechocystis PCC 6803. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL004) expressing the pdc from Zymomonas mobilis. The utilisation of a PDC with a lower Km from Zymobacter palmae did not result in enhanced ethanol production in Synechocystis PCC 6803.

scholarly journals Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

2021 ◽  
Author(s):  
Eline Postma ◽  
Else-Jasmijn Hassing ◽  
Venda Mangkusaputra ◽  
Jordi Geelhoed ◽  
Pilar de la Torre ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Metabolic Networks ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

scholarly journals Elevated β-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica

2021 ◽  
Vol 12 ◽  
Author(s):  
Liang Liu ◽  
Yu Ling Qu ◽  
Gui Ru Dong ◽  
Jing Wang ◽  
Ching Yuan Hu ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Large Scale ◽  
Blakeslea Trispora ◽  
Scale Production ◽  
Total Production ◽  
Metabolic Balance ◽  
Carotene Content ◽  
Large Scale Production ◽  
Dry Cell Weight ◽  
Β Carotene

β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

scholarly journals Zymobacter palmae Pyruvate Decarboxylase is Less Effective Than That of Zymomonas mobilis for Ethanol Production in Metabolically Engineered Synechocystis sp. PCC6803

2019 ◽  
Vol 7 (11) ◽  
pp. 494 ◽  
Author(s):  
Lorraine Quinn ◽  
Patricia Armshaw ◽  
Tewfik Soulimane ◽  
Con Sheehan ◽  
Michael P. Ryan ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Production ◽  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Biomass Productivity ◽  
Gene Cassette ◽  
Major Effect ◽  
Zymobacter Palmae ◽  
Synechocystis Sp ◽  
Pcc 6803

To produce bioethanol from model cyanobacteria such as Synechocystis, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.

scholarly journals Replacement of a Metabolic Pathway for Large-Scale Production of Lactic Acid from Engineered Yeasts

1999 ◽  
Vol 65 (9) ◽  
pp. 4211-4215 ◽  
Author(s):  
Danilo Porro ◽  
Michele M. Bianchi ◽  
Luca Brambilla ◽  
Rossella Menghini ◽  
Davide Bolzani ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Large Scale ◽  
Kluyveromyces Lactis ◽  
Lactic Acid Production ◽  
Pyruvate Decarboxylase ◽  
Polymer Materials ◽  
Efficient Production ◽  
Scale Production ◽  
High Productivity ◽  
Large Scale Production

ABSTRACT Interest in the production of l-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovinel-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into aK. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter−1, 0.91 g liter−1 h−1, and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those usual for bacterial processes.

Composition Engineering of Organic–Inorganic Perovskites Prepared in Air for Highly Efficient Energy Conversion

2020 ◽  
Vol 15 (5) ◽  
pp. 592-598
Author(s):  
Yang Li ◽  
Ruiquan Liao
Keyword(s):  
Solar Energy ◽  
Energy Conversion ◽  
Conversion Efficiency ◽  
Large Scale ◽  
Carrier Transport ◽  
Perovskite Solar Cells ◽  
Clean Energy ◽  
Energy Conversion Efficiency ◽  
Fabrication Process ◽  
Scale Production

The global energy crisis significantly raises the research on renewable energy materials and devices both in academic and industrial community. Besides the electrochemical energy such as batteries, the solar energy is another choice to develop renewable clean energy. During the last ten years, the perovskite solar cells (PSCs) has been a hot research topic and developed fast. However, large-scale production of PSCs is still hindered by the high cost of their fabrication process, because the perovskite films are known to be sensitive to oxygen and water. Therefore, developing a composition engineering in air for PSCs with high solar energy conversion efficiency is urgently required in the field. Herein, it is found that the crystallization and morphology of CH3NH3PbI3 (MAPbI3) perovskite films prepared in air are dependent on the processing methods. The perovskite grain size becomes larger when the concentration of CH3NH3I (MAI) solution was increased from 20 mg/mL to 70 mg/mL, which is beneficial for charge carrier transport and device performance. Thanks to the optimal perovskite fabrication process, the champion PSC has been fabricated in open air and it shows a power conversion efficiency (PCE) of 14.9%. More importantly, the PSC fabricated with our method shows good stability. This work provides an effective composition engineering to fabricate PSCs in air with both high PCE and stability.

scholarly journals Scalable Synthesis of Mesoporous TiO2 for Environmental Photocatalytic Applications

Materials ◽  
2019 ◽  
Vol 12 (11) ◽  
pp. 1853 ◽  
Author(s):  
Francesca Petronella ◽  
Alessandra Truppi ◽  
Massimo Dell’Edera ◽  
Angela Agostiano ◽  
M. Lucia Curri ◽  
...  
Keyword(s):  
Large Scale ◽  
Clean Energy ◽  
Mesoporous Tio2 ◽  
Water Remediation ◽  
Radiation Absorption ◽  
Reaction Yield ◽  
Hydroxyl Groups ◽  
Scale Production ◽  
High Reaction ◽  
Large Scale Production

Increasing environmental concern, related to pollution and clean energy demand, have urged the development of new smart solutions profiting from nanotechnology, including the renowned nanomaterial-assisted photocatalytic degradation of pollutants. In this framework, increasing efforts are devoted to the development of TiO2-based nanomaterials with improved photocatalytic activity. A plethora of synthesis routes to obtain high quality TiO2-based nanomaterials is currently available. Nonetheless, large-scale production and the application of nanosized TiO2 is still hampered by technological issues and the high cost related to the capability to obtain TiO2 nanoparticles with high reaction yield and adequate morphological and structural control. The present review aims at providing a selection of synthetic approaches suitable for large-scale production of mesoporous TiO2-based photocatalysts due to its unique features including high specific surface area, improved ultraviolet (UV) radiation absorption, high density of surface hydroxyl groups, and significant ability for further surface functionalization The overviewed synthetic strategies have been selected and classified according to the following criteria (i) high reaction yield, (ii) reliable synthesis scale-up and (iii) adequate control over morphological, structural and textural features. Potential environmental applications of such nanostructures including water remediation and air purification are also discussed.

scholarly journals Production of Raw Starch-Digesting Amylolytic Preparation in Yarrowia lipolytica and Its Application in Biotechnological Synthesis of Lactic Acid and Ethanol

2020 ◽  
Vol 8 (5) ◽  
pp. 717
Author(s):  
Aleksandra Gęsicka ◽  
Monika Borkowska ◽  
Wojciech Białas ◽  
Paulina Kaczmarek ◽  
Ewelina Celińska
Keyword(s):  
Lactic Acid ◽  
Ethanol Production ◽  
Yarrowia Lipolytica ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Thermal Conditions ◽  
Cell Factory ◽  
The Novel ◽  
Microbial Cell Factory ◽  
Raw Starch

Sustainable economy drives increasing demand for raw biomass-decomposing enzymes. Microbial expression platforms exploited as cellular factories of such biocatalysts meet requirements of large-volume production. Previously, we developed Yarrowia lipolytica recombinant strains able to grow on raw starch of different plant origin. In the present study, we used the most efficient amylolytic strain as a microbial cell factory of raw-starch-digesting (RSD) amylolytic preparation composed of two enzymes. The RSD-preparation was produced in fed-batch bioreactor cultures. Concentrated and partly purified preparation was then tested in simultaneous saccharification and fermentation (SSF) processes with thermotolerant Kluyveromyces marxianus for ethanol production and Lactobacillus plantarum for production of lactic acid. These processes were conducted as a proof-of-concept that application of the novel RSD-preparation supports sufficient starch hydrolysis enabling microbial growth and production of targeted molecules, as the selected strains were confirmed to lack amylolytic activity. Doses of the preparation and thermal conditions were individually adjusted for the two processes. Additionally, ethanol production was tested under different aeration strategies; and lactic acid production process was tested in thermally pre-treated substrate, as well. Conducted studies demonstrated that the novel RSD-preparation provides satisfactory starch hydrolyzing activity for ethanol and lactic acid production from starch by non-amylolytic microorganisms.

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