Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli

Zhou, Li; Deng, Can; Cui, Wenjing; Liu, Zhongmei; Zhou, Zhemin

doi:10.1007/s12010-015-1874-x

Cited by 36 publications

(26 citation statements)

References 21 publications

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“…Production of both enzymes as either wet (EfPheDC) or dry (AvPAL) whole cell biocatalysts allowed simple investigation of their pH optima. The enzymes were shown to operate under very different conditions in this respect, as consistent with previous studies of PAL enzymes and the potential involvement of AADCs with bacterial acid stress responses . As such, initial experiments focussed on the development of a sequential reaction whereby cells, water and ammonium carbamate (required for PAL‐mediated amination) were removed via centrifugation and evaporation, leaving a crude isolate for conversion by EfPheDC under separate conditions.…”

Section: Methodssupporting

confidence: 88%

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

et al. 2020

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The conversion of carboxylic acids, such as acrylic acids, to amines is a transformation that remains challenging in synthetic organic chemistry. Despite the ubiquity of similar moieties in natural metabolic pathways, biocatalytic routes seem to have been overlooked for this purpose. Herein we present the conception and optimisation of a two‐enzyme system, allowing the synthesis of β‐phenylethylamine derivatives from readily‐available ring‐substituted cinnamic acids. After characterisation of both parts of the reaction in a two‐step approach, a set of conditions allowing the one‐pot biotransformation was optimised. This combination of a reversible deaminating and irreversible decarboxylating enzyme, both specific for the amino acid intermediate in tandem, represents a general method by which new strategies for the conversion of carboxylic acids to amines could be designed.

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Section: Methodssupporting

confidence: 88%

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

et al. 2020

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“…The logic of our intended dynamic switch is shown in Figure 2. To first demonstrate that we can control cell growth by dynamic regulation of ICD by this temperature‐dependent switch we proceeded as Zhou, Deng, Cui, Liu, and Zhou (2015) and replaced the promoter including the untranslated region upstream of the icd gene by the Lambda promoters p R and p L and a synthetic ribosome binding site for translation of icd . The repressor CI857 , controlling the expression from p R and p L , is expressed from the promoter for repressor maintenance ( p RM ).…”

Section: Resultsmentioning

confidence: 99%

“…The logic of our intended dynamic switch is shown in Figure 2. To first demonstrate that we can control cell growth by dynamic regulation of ICD by this temperature-dependent switch we proceeded as Zhou, Deng, Cui, Liu, and Zhou (2015) and replaced the promoter including the…”

Section: Control Of Growth Rate By Dynamic Regulation Of Isocitratementioning

confidence: 99%

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Harder

Bettenbrock

Klamt

2017

Biotech & Bioengineering

105

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Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr−1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p R/p L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.

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“…Several groups have produced alanine to titers from 80-110g/L in several microbes, even in the context of two-stage production, however in these studies significant process optimization was needed to achieve these results. [35][36][37] Similarly previous studies producing citramalate in E. coli have required significant process optimization, including the optimization of media, complex feeding strategies and oxygen control. 47,48 However this is the process optimization cycle that improved strain robustness can minimize.…”

Section: Discussionmentioning

confidence: 99%

“…coli. [35][36][37] Previous aerobic production of L-alanine has required significant process optimization, even in two-stage approaches, making this amino acid an excellent test case to evaluate the impact of two-stage dynamic control on robustness and scalability. [35][36][37]…”

Section: Introductionmentioning

confidence: 99%

Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in EngineeredE. coli

Hennigan

et al. 2020

Preprint

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We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

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Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli

Cited by 36 publications

References 21 publications

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in EngineeredE. coli

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