Establishing a Cell-Free <i>Vibrio natriegens</i> Expression System

Wiegand, Daniel J.; Lee, Henry H.; Ostrov, Nili; Church, George M.

doi:10.1021/acssynbio.8b00222

Cited by 70 publications

(79 citation statements)

References 35 publications

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“…Moreover, Garschagen et al (2019) recently showed that V. natriegens biomass increases remarkably after 24 h at 37 • C under conditions of SMG, and the final cell population can reach 60-fold that of cells grown at normal gravity (1 g). V. natriegens is a non-pathogenic, fast-growing marine bacterium doubling twice as fast as Escherichia coli, and several studies have documented that it would be the next-generation microbial chassis for the normal molecular biology and biotechnology applications, including molecular cloning (Weinstock et al, 2016;Dalia et al, 2017;Lee et al, 2019), protein synthesis (Schleicher et al, 2018;Wiegand et al, 2018;Eichmann et al, 2019), and small molecule production (Hoffart et al, 2017;Long et al, 2017), etc. The rapid growth rate of V. natriegens makes it a more promising engineering strain as an alternative workhorse to Escherichia coli (Hoff et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Changes in Vibrio natriegens Growth Under Simulated Microgravity

Yin¹,

Jin³

et al. 2020

Front. Microbiol.

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The growth rate of bacteria increases under simulated microgravity (SMG) with lowshear force. The next-generation microbial chassis Vibrio natriegens (V. natriegens) is a fast-growing Gram-negative, non-pathogenic bacterium with a generation time of less than 10 min. Screening of a V. natriegens strain with faster growth rate was attempted by 2-week continuous long-term culturing under SMG. However, the rapid growth rate of this strain made it difficult to obtain the desired mutant strain with even more rapid growth. Thus, a mutant with slower growth rate emerged. Multi-omics integration analysis was conducted to explore why this mutant grew more slowly, which might inform us about the molecular mechanisms of rapid growth of V. natriegens instead. The transcriptome data revealed that whereas genes related to mechanical signal transduction and flagellin biogenesis were up-regulated, those involved in adaptive responses, anaerobic and nitrogen metabolism, chromosome segregation and cell vitality were down-regulated. Moreover, genome-wide chromosome conformation capture (Hi-C) results of the slower growth mutant and wide type indicated that SMG-induced great changes of genome 3D organization were highly correlated with differentially expressed genes (DEGs). Meanwhile, whole genome re-sequencing found a significant number of structure variations (SVs) were enriched in regions with lower interaction frequency and down-regulated genes in the slower growth mutant compared with wild type (WT), which might represent a prophage region. Additionally, there was also a decreased interaction frequency in regions associated with well-orchestrated chromosomes replication. These results suggested that SMG might regulate local gene expression by sensing stress changes through conformation changes in the genome region of genes involved in flagellin, adaptability and chromosome segregation, thus followed by alteration of some physiological characteristics and affecting the growth rate and metabolic capacity.

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Section: Introductionmentioning

confidence: 99%

Changes in Vibrio natriegens Growth Under Simulated Microgravity

Yin¹,

Jin³

et al. 2020

Front. Microbiol.

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“…In order to tackle these problems, several new CFPS systems have recently been developed to better mimic the physicochemical environment of native hosts for synthetic biology and biotechnology applications. However, the newly developed CFPS systems are mainly derived from prokaryotic microorganisms, including some from Streptomyces species (Li et al, 2017(Li et al, , 2018aMoore et al, 2017), Bacillus subtilis (Kelwick et al, 2016), Pseudomonas putida (Wang et al, 2018), and Vibrio natriegens (Des Soye et al, 2018;Failmezger et al, 2018;Wiegand et al, 2018). Although a couple of eukaryote-based CFPS systems are available, they are mostly prepared from plant (e.g., wheat germ), insect (e.g., Spodoptera frugiperda), and mammalian (e.g., Chinese hamster ovary, CHO) cells (Tarui et al, 2001;Takai et al, 2010;Brödel et al, 2014), which often need laborious and expensive cell extract preparation approaches.…”

Section: Introductionmentioning

confidence: 99%

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

Zhang

Liu

2020

Front. Bioeng. Biotechnol.

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In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 µg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.

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“…Third, this approach does not rely on time-consuming enzyme purification procedures but rapidly builds and tests metabolic pathways directly in cell extracts by synthesizing required enzymes in vitro (Karim et al, 2018;Karim and Jewett, 2016). Given these advantages, cell-free systems have emerged as an important approach for accelerating biological design, especially with the advent of new extract based systems from non-model organisms: Bacillus (Moore et al, 2018), Streptomyces (Li et al, , 2017, Vibrio (Des Soye et al, 2018;Failmezger et al, 2018;Wiegand et al, 2018), and Pseudomonas among others. However, no clostridia cell-free system exists that produces protein yields sufficient for prototyping genetic parts and metabolic pathways.…”

Section: Introductionmentioning

confidence: 99%

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Krüger¹,

Mueller

Rybnicky³

et al. 2020

Preprint

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Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste.Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

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Establishing a Cell-Free Vibrio natriegens Expression System

Cited by 70 publications

References 35 publications

Changes in Vibrio natriegens Growth Under Simulated Microgravity

Changes in Vibrio natriegens Growth Under Simulated Microgravity

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

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