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.

scholarly journals Intelligent microbial cell factory with genetic pH shooting (GPS) for cell self-responsive base/acid regulation

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Chenyi Li ◽  
Xiaopeng Gao ◽  
Xiao Peng ◽  
Jinlin Li ◽  
Wenxin Bai ◽  
...  
Keyword(s):  
Ph Regulation ◽  
Self Regulation ◽  
Scale Up ◽  
Microbial Cell ◽  
Production Costs ◽  
Cell Factory ◽  
Ph Sensing ◽  
Genetic Circuits ◽  
Microbial Cell Factory ◽  
Cell Factories

Abstract Background In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called “Genetic pH Shooting (GPS)” to realize microbial self-regulation of pH. Results In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. Conclusions The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

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 Heterotrophy as a tool to overcome the long and costly autotrophic scale-up process for large scale production of microalgae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
A. Barros ◽  
H. Pereira ◽  
J. Campos ◽  
A. Marques ◽  
J. Varela ◽  
...  
Keyword(s):  
Large Scale ◽  
Scale Up ◽  
Biomass Concentration ◽  
Growth Conditions ◽  
Pilot Scale ◽  
Industrial Scale ◽  
Scale Production ◽  
Two Stage ◽  
Chlorophyll Contents ◽  
Large Scale Production

Abstract Industrial scale-up of microalgal cultures is often a protracted step prone to culture collapse and the occurrence of unwanted contaminants. To solve this problem, a two-stage scale-up process was developed – heterotrophically Chlorella vulgaris cells grown in fermenters (1st stage) were used to directly inoculate an outdoor industrial autotrophic microalgal production unit (2nd stage). A preliminary pilot-scale trial revealed that C. vulgaris cells grown heterotrophically adapted readily to outdoor autotrophic growth conditions (1-m3 photobioreactors) without any measurable difference as compared to conventional autotrophic inocula. Biomass concentration of 174.5 g L−1, the highest value ever reported for this microalga, was achieved in a 5-L fermenter during scale-up using the heterotrophic route. Inocula grown in 0.2- and 5-m3 industrial fermenters with mean productivity of 27.54 ± 5.07 and 31.86 ± 2.87 g L−1 d−1, respectively, were later used to seed several outdoor 100-m3 tubular photobioreactors. Overall, all photobioreactor cultures seeded from the heterotrophic route reached standard protein and chlorophyll contents of 52.18 ± 1.30% of DW and 23.98 ± 1.57 mg g−1 DW, respectively. In addition to providing reproducible, high-quality inocula, this two-stage approach led to a 5-fold and 12-fold decrease in scale-up time and occupancy area used for industrial scale-up, respectively.

scholarly journals Analytical, Preparative, and Industrial-Scale Separation of Substances by Methods of Countercurrent Liquid-Liquid Chromatography

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6020
Author(s):  
Artak A. Kostanyan ◽  
Andrey A. Voshkin ◽  
Vera V. Belova
Keyword(s):  
Steady State ◽  
Closed Loop ◽  
Load Capacity ◽  
Scale Up ◽  
Industrial Scale ◽  
Separation Processes ◽  
Immiscible Liquids ◽  
Pure Compounds ◽  
High Load Capacity ◽  
Liquid Chromatographic

Countercurrent liquid-liquid chromatographic techniques (CCC), similar to solvent extraction, are based on the different distribution of compounds between two immiscible liquids and have been most widely used in natural product separations. Due to its high load capacity, low solvent consumption, the diversity of separation methods, and easy scale-up, CCC provides an attractive tool to obtain pure compounds in the analytical, preparative, and industrial-scale separations. This review focuses on the steady-state and non-steady-state CCC separations ranging from conventional CCC to more novel methods such as different modifications of dual mode, closed-loop recycling, and closed-loop recycling dual modes. The design and modeling of various embodiments of CCC separation processes have been described.

scholarly journals Data Driven Modelling of a Chemically Defined Growth Medium for Cupriavidus necator H16

2019 ◽  
Author(s):  
Christopher C Azubuike ◽  
Martin G Edwards ◽  
Angharad M. R Gatehouse ◽  
Thomas Paul Howard
Keyword(s):  
Growth Medium ◽  
Scale Up ◽  
Culture Cell ◽  
Cupriavidus Necator ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Cupriavidus Necator H16 ◽  
Fuels And Chemicals ◽  
System Optimisation ◽  
The Impact

Cupriavidus necator is a Gram-negative soil bacterium of major biotechnological interest. It is a producer of the bioplastic 3-polyhydroxybutyrate, has been exploited in bioremediation processes, and it's lithoautotrophic capabilities suggest it may function as a microbial cell factory upgrading renewable resources to fuels and chemicals. It remains necessary however to develop appropriate experimental resources to permit controlled bioengineering and system optimisation of this microbial chassis. A key resource for physiological, biochemical and metabolic studies of any microorganism is a chemically defined growth medium. Here we use 1 mL micro-well cell cultures, automated liquid handling and a statistical engineering approach to develop a model that describes the effect of key media components and their interactions on C. necator culture cell density. The model is predictive and was experimentally validated against novel media compositions. Moreover, the model was further validated against larger culture volumes at 100 mL and 1 L volumes and found to correlate well. This approach provides valuable and quantifiable insights into the impact of media components on cell growth as well as providing predictions to guide culture scale-up.

scholarly journals The future of self-selecting and stable fermentations

2020 ◽  
Vol 47 (11) ◽  
pp. 993-1004 ◽  
Author(s):  
Peter Rugbjerg ◽  
Lisbeth Olsson
Keyword(s):  
Growth Rate ◽  
Large Scale ◽  
Scale Up ◽  
Serial Passage ◽  
Cell Factory ◽  
Sequencing Data ◽  
High Performing ◽  
Cell Factories ◽  
Strain Design ◽  
High Production

AbstractUnfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production.

scholarly journals Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Theresa Zwiener ◽  
Marina Dziuba ◽  
Frank Mickoleit ◽  
Christian Rückert ◽  
Tobias Busche ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Large Scale ◽  
Gene Clusters ◽  
Genome Reduction ◽  
Cell Factory ◽  
Wild Type ◽  
Biosynthetic Gene Clusters ◽  
Microbial Cell Factory ◽  
Magnetospirillum Gryphiswaldense ◽  
Large Gene

Abstract Background Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. Results We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. Conclusion We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

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.

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