Single-cell microfluidics: opportunity for bioprocess development

Grünberger, Alexander; Wiechert, Wolfgang; Kohlheyer, Dietrich

doi:10.1016/j.copbio.2014.02.008

Cited by 164 publications

(130 citation statements)

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“…Noninvasive and gradual upregulation of gene expression by light, moreover, provides a fast and easy option to screen for optimized expression conditions, rendering time-consuming and invasive inducer supplementation obsolete. The established optogenetic expression modules can thus be applied in novel photomicrobioreactors (53) and single-cell cultivation platforms (19) to precisely control the expression of target genes and thereby fully automatize the optimization of microbial production processes in a highthroughput fashion. Notably, cIPTG-based light induction requires elevated expenditures of costs and labor and is further unsuited for large-scale fermentations where light exposure poses an additional challenge.…”

Section: Discussionmentioning

confidence: 99%

“…However, currently applied expression setups show only moderate performance regarding both precise control and expression homogeneity. Population heterogeneity affecting both growth and expression may strongly impact biotechnological processes (19). For instance, a distinct population heterogeneity has recently been described for C. glutamicum cultures producing L-valine (20,21).…”

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confidence: 99%

“…For instance, a distinct population heterogeneity has recently been described for C. glutamicum cultures producing L-valine (20,21). While cell-to-cell variations within isogenic populations may constitute an overall fitness advantage over cohabiting competitors (22,23) in natural environments, such heterogeneity is highly unfavorable in biotechnological production processes (19).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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“…The gap between screening in batch culture mode (in shake flasks or deep-well plates) and the subsequently performed, labour-intensive, bioreactor culture in fedbatch mode is currently addressed by small-scale bioreactors and new feeding techniques (Barnard et al, 2010, Lattermann and Buchs, 2014, Wilming et al, 2014. Downscaled techniques of substrate addition include pump-controlled feeding (Barnard et al, 2010), release of glucose from a polymer (Hemmerich et al, 2014) or by an enzyme (PanulaPerala et al, 2008), and microfluidic systems (Grunberger et al, 2014). These fedbatch-like screening small-scale techniques better reflect conditions in the bioreactor to be used later in production scale.…”

Section: Overcoming Drawbacks Of Current Screening Conceptsmentioning

confidence: 99%

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

295

268

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Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

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“…Miniaturized high-throughput operation systems including microwell plates [1][2][3], microfluidic devices [4], Lab-on-Chip (LOC) devices [5,6] and low-volume bioreactors [7] have become increasingly important in modern biotechnology. These systems enable rapid, highly parallelized and automated screening and analyses of large numbers of samples.…”

Section: Introductionmentioning

confidence: 99%