Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum

Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

doi:10.1016/j.ymben.2015.09.017

Cited by 142 publications

(102 citation statements)

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“…orynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1)(2)(3)(4)(5), but it has also been engineered, for instance, for the production of lower alcohols (6)(7)(8)(9), organic acids (10-13), diamines (14,15), and carotenoids (16)(17)(18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogeneity.…”

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confidence: 99%

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Binder

Frohwitter

Mahr

et al. 2016

Appl Environ Microbiol

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Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-␤-D-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum. In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (؉)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCEOptogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum. By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications. Corynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1-5), but it has also been engineered, for instance, for the production of lower alcohols (6-9), organic acids (10-13), diamines (14, 15), and carotenoids (16-18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogene...

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confidence: 99%

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Binder

Frohwitter

Mahr

et al. 2016

Appl Environ Microbiol

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“…This challenges strain development for the production of L-phenylalanine. Thus, the design of suitable biosensors would provide a convenient tool for strain analysis and screening, and might be especially useful for the identification of non-intuitive targets for strain improvement (Binder et al 2012;Mahr et al 2015;Mustafi et al 2012). However, our study revealed that several parameters require careful consideration to set up a screening procedure for effector-responsive promoters, including the adaption to growth medium, the uptake and catabolism of effector molecules (Luo et al 2014;Payne 1977), the activation of the gene expression machinery (Bintu et al 2005b;Carey et al 2013;Fritz et al 2014;Mäkelä et al 2013), the maturation of fluorescent proteins (Craggs 2009;Iizuka et al 2011), and last but not least, the composition of the particular growth medium.…”

Section: Discussionmentioning

confidence: 99%

“…To reduce the amount of false positive isolates, we included two iterative enrichment steps in the FACS HT-screening (Fig. 4a) (Mahr et al 2015). Remarkably, about 30 % of 90 isolated and characterized mutant strains showed at least 2-to 4-fold increased L-phenylalanine titers (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…As precision mode, four-way purity was used for cell sorting with a threshold rate of up to 10, 000 events/s. If not stated differently, cells were sorted on agar plates or on MultiScreen HTS 96-well filter plates (Millipore, Billerica, USA) to separate cells from the FACSFlow™ buffer (Becton Dickinson, San Jose, USA) as already described in Mahr et al (2015). The filter containing the sorted cells was excised with single-use scalpels (Braun, Melsungen, Germany) and inoculated in fresh medium.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Therefore, mutant cells were sorted and re-cultivated again before a second sorting step was conducted (Fig. 4b) (Mahr et al 2015). The two enrichment steps resulted in an increase of top fluorescent cells from 2 % (gate P1) to 53.8 % (gate P2, Fig.…”

Section: Biosensor-based High-throughput Screening Of Randomly Mutagementioning

confidence: 99%

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Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Mahr

Boeselager

Wiechert

et al. 2016

Appl Microbiol Biotechnol

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In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

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In Vivo Biosensors for Directed Protein Evolution

Tay

Ang

2021

Protein Engineering

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Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum

Cited by 142 publications

References 73 publications

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

In Vivo Biosensors for Directed Protein Evolution

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