Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in <i>E. coli</i>

Moreb, Eirik A.; Ye, Zhixia; Efromson, John P.; Hennigan, Jennifer N.; Menacho‐Melgar, Romel; Lynch, Michael

doi:10.1021/acssynbio.0c00182

Cited by 26 publications

(51 citation statements)

References 19 publications

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“…The ald* was expressed on a high copy plasmid using the robust low phosphate inducible yibD gene promoter (yibDp). 17 Additionally, in several strains the native E. coli alanine exporter ( alaE ) was also expressed. Refer to Supplemental Tables S1 and S2 for a list of plasmids and strains used in this study.…”

Section: Strain Designmentioning

confidence: 99%

“…This panel of alanine "Valve" strains were constructed and evaluated for alanine production in standardized, two-stage, 96-well plate based, minimal media micro-fermentations as reported by Moreb et al 17 Alanine titers after 24 hours of production are given in Figure 2d.…”

Section: Alanine Production In Micro-fermentationsmentioning

confidence: 99%

“…Minimal media microfermentations were performed as previously reported by Moreb et al 17 In the robustness studies variations in batch glucose and or aeration were performed as described in Supplemental Materials (Tables S6). OTR was estimated as a function of shaking speed and fill volume according to Duetz and Witholt. 51 For growth associated alanine micro-fermentations, during production FGM3 with 40 mM phosphate was used instead of No phosphate FGM3 media.…”

Section: Fermentationsmentioning

confidence: 99%

“…For example, we have recently reported that improved promoter robustness corresponds to increased scalability from high throughput studies to instrumented bioreactors. 17 In order to address the primary challenge of robustness in the more complicated production of chemicals via engineered metabolism, in this study we investigate the use of dynamic metabolic control in two-stage cultures to improve process robustness and scalability.…”

Section: Introductionmentioning

confidence: 99%

“…[18][19][20][21][22][23][24][25][26][27] Two-stage dynamic control, which decouples growth from production, offers additional potential benefits in large scale bioprocesses, enabling metabolic states to be pushed beyond the boundaries required for growth. 8,21,22,24,28 We have recently implemented two-stage dynamic control in the commonly used and well characterized microbe, E. coli , by utilizing synthetic metabolic valves, which rely on the combination of proteolysis and gene silencing to dynamically reduce levels of key enzymes in the context of a standardized two-stage phosphate depleted process (as illustrated in Figure 1) 17,21,22,29 Proteolysis is accomplished by appending C-terminal degron (DAS+4) tags to a given gene and silencing via expression of the native E. coli CRISPR CASCADE system as well as silencing gRNAs (from pCASCADE plasmids). 30,31 Importantly, in these studies where we produced the organic acid citramalate 22 and the sugar alcohol xylitol, 21 we identified that dynamic deregulation of metabolism was a fundamental mechanism to improve stationary phase metabolic fluxes.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Strain Designmentioning

confidence: 99%

Section: Alanine Production In Micro-fermentationsmentioning

confidence: 99%

Section: Fermentationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Hennigan

et al. 2020

Preprint

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Substrate‐activated expression of a biosynthetic pathway in Escherichia coli

Fox

Prather

2021

Biotechnology Journal

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BackgroundMicrobes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of D-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Methods and ResultsActivation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production D-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13±0.03 g/L D-glyceric acid within six hours of galacturonate substrate addition. ConclusionThis work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.

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Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

Menacho‐Melgar

Moreb

et al. 2020

Biotech & Bioengineering

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We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales.Expression levels using this approach have reached as high as 55% of total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of~30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/-0.7 g/L (~270 mg/gCDW).The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

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Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli

Cited by 26 publications

References 19 publications

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

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

Substrate‐activated expression of a biosynthetic pathway in Escherichia coli

Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

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