An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered <i>Escherichia coli</i>

Theodosiou, Eleni; Breisch, Marina; Julsing, Mattijs K.; Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

doi:10.1002/bit.26281

Cited by 29 publications

(28 citation statements)

References 29 publications

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“…Nevertheless, the deficiency of sucC in combination with aceA gene led to the carbon flux overflow to the acetate biosynthesis in E. coli (Theodosiou et al, 2017). Nevertheless, the deficiency of sucC in combination with aceA gene led to the carbon flux overflow to the acetate biosynthesis in E. coli (Theodosiou et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…It was reported that the sucC-deficient E. coli ΔputA ΔaceA was able to grow in a glucose-minimal medium because the cells could obtain succinyl-CoA from α-ketoglutarate via α-ketoglutarate dehydrogenase and succinate from fumarate via fumarate reductase (Theodosiou et al, 2017). It was reported that the sucC-deficient E. coli ΔputA ΔaceA was able to grow in a glucose-minimal medium because the cells could obtain succinyl-CoA from α-ketoglutarate via α-ketoglutarate dehydrogenase and succinate from fumarate via fumarate reductase (Theodosiou et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…By interrupting the TCA cycle by sucCD deletion in the absence of P4H, the Hyp-0 ΔsucCD strain showed the excellent grow efficiency. It was reported that the sucC-deficient E. coli ΔputA ΔaceA was able to grow in a glucose-minimal medium because the cells could obtain succinyl-CoA from α-ketoglutarate via α-ketoglutarate dehydrogenase and succinate from fumarate via fumarate reductase (Theodosiou et al, 2017). However, the sucA-deficient E. coli ΔputA ΔaceA was unable to grow in minimal medium due to the unavailability of succinyl-CoA (Theodosiou et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, taking biomass as an optimal criterion for growth-coupled Hyp production, a zero flux through SUCOAS led to a moderate Hyp productivity ( Figure 2b); while a low flux toward AKGDH resulted in a moderate Hyp productivity (Supporting Information Figure S1), which might be attributed to the essentiality of succinyl-CoA generating from AKGDH for biomass formation and peptidoglycan synthesis in C. glutamicum (Theodosiou et al, 2017). Taking Hyp productivity as an optimal criterion, the biomass and the flux through AKGDH and SUCOAS came down to zero as Hyp productivity maximizes.…”

Section: Reconstruction Of Tca Cycle To Couple With the Hyp Synthetmentioning

confidence: 99%

“…Taking Hyp productivity as an optimal criterion, the biomass and the flux through AKGDH and SUCOAS came down to zero as Hyp productivity maximizes. In contrast, taking biomass as an optimal criterion for growth-coupled Hyp production, a zero flux through SUCOAS led to a moderate Hyp productivity ( Figure 2b); while a low flux toward AKGDH resulted in a moderate Hyp productivity (Supporting Information Figure S1), which might be attributed to the essentiality of succinyl-CoA generating from AKGDH for biomass formation and peptidoglycan synthesis in C. glutamicum (Theodosiou et al, 2017). These results were consistent with the metabolic disturbance in the Hyp producer (Figure 2a).…”

Section: Reconstruction Of Tca Cycle To Couple With the Hyp Synthetmentioning

confidence: 99%

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Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production

Zhang

Shang

Wang

et al. 2018

Biotech & Bioengineering

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trans-4-Hydroxy- l-proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l-proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ·hr in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Reconstruction Of Tca Cycle To Couple With the Hyp Synthetmentioning

confidence: 99%

Section: Reconstruction Of Tca Cycle To Couple With the Hyp Synthetmentioning

confidence: 99%

See 3 more Smart Citations

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production

Zhang

Shang

Wang

et al. 2018

Biotech & Bioengineering

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Genome-Reduced Corynebacterium glutamicum Fit for Biotechnological Applications

Wendisch

2019

Minimal Cells: Design, Construction, Biotechnological Applications

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Genome minimization ultimately leads to the smallest genome sustaining life of a given cell, however, growth of this cell may be very slow and may require multiple supplements e.g. to overcome amino acid auxotrophies. By contrast, genome reduction of industrially relevant bacteria such as Corynebacterium glutamicum does not aim at generating minimal cells. Rather chassis cells are developed that are as fit as the wild type with respect to a target function: for example growth of C. glutamicum in glucose minimal medium. Thus, a balance between reducing the burden of expressed genes while maintaining fast growth with glucose without the requirement for supplements such as amino acids is required. Here, the application of this concept to C. glutamicum is discussed. Moreover, an outlook on how the advent of genome editing by CRISPR-Cas9 or CRISPR-Cpf1 impacts genome reduction and how highly parallel genome editing must be met by highly parallel strain characterization is presented. Finally, metabolic engineering approaches for the overproduction of amino acids, organic acids, terpenoids and diamines making use of genome-reduced C. glutamicum strains are detailed.

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Challenges in the Application of Synthetic Biology Toward Synthesis of Commodity Products by Cyanobacteria via “Direct Conversion”

Burbano

Hellingwerf

et al. 2018

Synthetic Biology of Cyanobacteria

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Cyanobacterial direct conversion of CO to several commodity chemicals has been recognized as a potential contributor to support the much-needed sustainable development of human societies. However, the feasibility of this "green conversion" hinders on our ability to overcome the hurdles presented by the natural evolvability of microbes. The latter may result in the genetic instability of engineered cyanobacterial strains leading to impaired productivity. This challenge is general to any "cell factory" approach in which the cells grow for multiple generations, and based on several studies carried out in different microbial hosts, we could identify that three distinct strategies have been proposed to tackle it. These are (1) to reduce microbial evolvability by decreasing the native mutation rate, (2) to align product formation with cell growth/fitness, and, paradoxically, (3) to efficiently reallocate cellular resources to product formation by uncoupling it from growth. The implementation of either of these strategies requires an advanced synthetic biology toolkit. Here, we review the existing methods available for cyanobacteria and identify areas of focus in which specific developments are still needed. Furthermore, we discuss how potentially stabilizing strategies may be used in combination leading to further increases of productivity while ensuring the stability of the cyanobacterial-based direct conversion process.

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An artificial TCA cycle selects for efficient α‐ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli

Cited by 29 publications

References 29 publications

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production

Genome-Reduced Corynebacterium glutamicum Fit for Biotechnological Applications

Challenges in the Application of Synthetic Biology Toward Synthesis of Commodity Products by Cyanobacteria via “Direct Conversion”

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