Industrial Biotechnology:<i>Escherichia coli</i>as a Host

Theisen, Matthew K.; Liao, James C.

doi:10.1002/9783527807796.ch5

Cited by 15 publications

(11 citation statements)

References 199 publications

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“…(4) kcat's optimization with no relationship between kcat_11 (X 26 ) and kcat_16 (X 32 ) assuming are both independent. (5) kcat's optimization were the K cat IspA basal ratio kcat_11/ kcat_16 = 2.22 (X 26 /X 32 ) were imposed for all the experiments (6) enzymatic levels optimization allowing modifications in the Km's, K 0,5 's, and Ki's. The IspA ratio is set to one i.e., X 11 /X 16 = 1.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of the enzymes optimization (X 11, …,.X 16 , X 20 , X 21 , X 23 , X 24 , X 68 ), the boundaries allowed were between 1 and 10 fold their basal value (Tables S3. 5, S3.2, and S3.4a). The reason for the lower range of one (their basal value) and not lower is that a practical implementation of results with values lower than the basal enzyme concentration will be difficult to achieve.…”

Section: Optimization Strategymentioning

confidence: 90%

“…The main advantages of E. coli as host are, fundamentally, its rapid growth, the simplicity of its genome, the great variety of techniques developed for its genetic manipulation, the diversity of culture media and the great knowledge about its metabolism [5]. The last one may be the most important, since it allows the optimization of production processes in order to obtain the best yields.…”

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confidence: 99%

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MODEL BASED OPTIMIZACION OF THE TERPENOIDS BIOSYNTHESIS INE. coli

Alvarez‐Vasquez

González-Alcón

Gallego‐Jara

et al. 2021

Preprint

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Terpenoids are a family of compounds with high industrial interest and the development of biotechnological production methods is essential to achieve more sustainable alternatives to traditional extraction and synthesis methods. The modification and engineering of the catalytic activity (kcat) have been shown to be a feasible strategy in the biotechnological realm. Accordingly, we introduce a novel optimization strategy based in the modification of the kcat of the enzymes and applied it to the maximization of the terpenoids synthesis in E. coli. This approach is fairly general and can be applied alone or in conjunction with classic optimization strategies such as the modification of enzymatic specific activities. For this purpose we first build up a reliable dynamic mathematical model of the alternative mevalonate pathway synthesis leading terpenoids biosynthesis in E. coli through the methyl-D-erythritol 4-phosphate (MEP) pathway. This model includes the 2-C-methyl-D-erythritol 2, 4-diphosphate (MEC) pumps that mediate MEC extracellular extrusion. Although the physiological significance of the MEC extrusion is still discussed and their biological function is not clear, we find that this process is a must to guarantee bacteria homeostasis and cell viability. We have identified the enzyme IspA as a bottleneck of the terpenoids biosynthesis, which is dual substrate for the two bisubstrate final reactions. Here are presented different ways to overcome this enzymatic flux restriction by modification of the IspA kcat or by introduction of new enzymes with parallel function. Our results show that the MEP pathway optimized solutions for kcat can yield a maximum of 17.68 fold increment in the terpenoids biosynthetic flux when the kcat are modified. This maximal solution involves the modification of 8 kcat and the corresponding Km's and Kd's. Remarkably, this increase doesn't imply a change in the total enzyme concentration of the cell. This is favorable output since enzyme overproduction can compromise cell functionality. We also apply the overexpression of the enzyme activity approach and then we have compared and combined both strategies including scenarios as the deletion or import of the genes expressing enzymes not naturally present in E. coli. A combined strategy of enzymes concentrations and kcat modifications with changes up ti six enzyme levels allows a flux increment of 21.22 fold the basal value. It involves the incorporation of two IspA with similar GPP and IPP bisubstrate functions than the native one but low affinity for the DMAPP as substrate.

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Section: Discussionmentioning

confidence: 99%

Section: Optimization Strategymentioning

confidence: 90%

mentioning

confidence: 99%

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MODEL BASED OPTIMIZACION OF THE TERPENOIDS BIOSYNTHESIS INE. coli

Alvarez‐Vasquez

González-Alcón

Gallego‐Jara

et al. 2021

Preprint

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“…E. coli is a well-studied standard organism and provides beneficial properties for industrial processing like the ease of cultivation, low overall operating costs and high growth and product yields. A variety of efficient genetic tools are available [ 16 ], which make E. coli a preferred host for the production of recombinant proteins. Former obstacles of E. coli , for example, its lack of post-translational modifications, have been tackled by transferring the N-glycosylation systems of Campylobacter jejuni [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Keil

Dittrich

Lattermann³

et al. 2020

J Biol Eng

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Background Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24–0.90 mg/h per well. Conclusions The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

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“…Many genetic tools have been developed and optimized for use in E. coli and comprehensive omics data is available. However, E. coli is not always an ideal host for industrial processes, due to its low stress tolerance and risk of phage infection [14]. Therefore, it would be beneficial to extend automation platforms to include the engineering of other microorganisms that are less genetically tractable.…”

Section: Introductionmentioning

confidence: 99%

Automated Rational Strain Construction Based on High-Throughput Conjugation

Tenhaef

Stella

Frunzke

et al. 2020

Preprint

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Molecular cloning is the core of Synthetic Biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA part creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (i.e., the DNA sequence design is fixed, and not based on randomized libraries) and the target host is less genetically tractable (e.g., not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism Corynebacterium glutamicum is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of C. glutamicum Lrp.

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Industrial Biotechnology:Escherichia colias a Host

Cited by 15 publications

References 199 publications

MODEL BASED OPTIMIZACION OF THE TERPENOIDS BIOSYNTHESIS INE. coli

MODEL BASED OPTIMIZACION OF THE TERPENOIDS BIOSYNTHESIS INE. coli

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Automated Rational Strain Construction Based on High-Throughput Conjugation

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