Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batch cultivations were run under the desired conditions, generating sufficient information to define the fastest-growing strain in an environment with oscillating glucose concentrations similar to industrial-scale bioreactors.

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Automated Conditional Screening of Escherichia Coli Knockout Mutants in Parallel Adaptive Fed-Batch Cultivations

10.20944/preprints202009.0381.v1 ◽

2020 ◽

Author(s):

Sebastian Hans ◽

Benjamin Haby ◽

Niels Krausch ◽

Barz Tilman ◽

Peter Neubauer ◽

...

Keyword(s):

Escherichia Coli ◽

Developmental Stages ◽

Added Value ◽

Major Influence ◽

Cell Factory ◽

Sufficient Information ◽

Limited Information ◽

Fed Batch ◽

Input Design ◽

Scale Down

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance of the selected cell factory in larger reactors, has a major influence on the performance of the final process. To overcome this, scaledown approaches are essential to run screenings that show the real cell factory performance at industrial like conditions. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batches ran under the desired conditions generating sufficient information to define the fastest growing strain in an environment with varying glucose concentrations similar to industrial scale bioreactors.

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Accumulation of amino acids deriving from pyruvate in <i>Escherichia coli</i> W3110 during fed-batch cultivation in a two-compartment scale-down bioreactor

Advances in Bioscience and Biotechnology ◽

10.4236/abb.2011.25049 ◽

2011 ◽

Vol 02 (05) ◽

pp. 336-339 ◽

Cited By ~ 12

Author(s):

Jaakko Soini ◽

Kaisa Ukkonen ◽

Peter Neubauer

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Batch Cultivation ◽

Fed Batch ◽

Scale Down ◽

Fed Batch Cultivation

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Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids

Nature Communications ◽

10.1038/s41467-021-25243-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lixia Fang ◽

Jie Fan ◽

Shulei Luo ◽

Yaru Chen ◽

Congya Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Free Fatty Acids ◽

Stress Responses ◽

Metabolic Networks ◽

Target Identification ◽

High Titer ◽

Cell Factory ◽

Microbial Cell Factory ◽

Ffa Metabolism

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

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A feedforward–feedback substrate controller based on a Kalman filter for a fed-batch cultivation of Escherichia coli producing phytase

Computers & Chemical Engineering ◽

10.1016/j.compchemeng.2004.11.011 ◽

2005 ◽

Vol 29 (5) ◽

pp. 1113-1120 ◽

Cited By ~ 25

Author(s):

M. Arndt ◽

S. Kleist ◽

G. Miksch ◽

K. Friehs ◽

E. Flaschel ◽

...

Keyword(s):

Escherichia Coli ◽

Kalman Filter ◽

Batch Cultivation ◽

Fed Batch ◽

Fed Batch Cultivation

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Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Microbial Cell Factories ◽

10.1186/s12934-021-01615-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Zhenning Liu ◽

Xue Zhang ◽

Dengwei Lei ◽

Bin Qiao ◽

Guang-Rong Zhao

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Phosphopantetheinyl Transferase ◽

Chromosome Engineering ◽

Microbial Cell Factory ◽

E Coli ◽

Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

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The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion

Biotechnology Journal ◽

10.1002/biot.201600633 ◽

2017 ◽

Vol 12 (7) ◽

pp. 1600633 ◽

Cited By ~ 21

Author(s):

Matthias Brunner ◽

Philipp Braun ◽

Philipp Doppler ◽

Christoph Posch ◽

Dirk Behrens ◽

...

Keyword(s):

Intracellular Ph ◽

Batch Process ◽

Cell Physiology ◽

Process Performance ◽

Fed Batch ◽

Cho Cell ◽

Scale Down ◽

The Impact

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Expression Characteristics of the Transfer-RelatedkilB Gene Product of Streptomyces Plasmid pIJ101: Implications for the Plasmid Spread Function

Journal of Bacteriology ◽

10.1128/jb.183.4.1339-1345.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1339-1345 ◽

Cited By ~ 7

Author(s):

Gregg S. Pettis ◽

Naomi Ward ◽

Kevin L. Schully

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Secondary Metabolism ◽

Gene Product ◽

Gene Transcription ◽

Cellular Differentiation ◽

Developmental Stages ◽

Agar Medium ◽

Spread Function ◽

Aerial Hyphae

ABSTRACT Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the kilB gene of pIJ101 into the S. lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S. lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well.

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