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Design and Reconstruction of Regulatory Parts for Fast-Growing Vibrio natriegens Synthetic Biology
Abstract
The fast-growing Vibrio natriegens is an attractive robust chassis for diverse synthetic biology applications. However, V. natriegens lacks the suitable constitutive regulatory parts for precisely tuning gene expression, and thus recapitulating physiologically relevant changes in gene expression levels. In this study, we designed, constructed and screened the synthetic regulatory parts by varying the promoter region and ribosome binding site element for V. natriegens with different transcriptional or translational strengths, respectively. The fluorescence intensities of the cells with different synthetic regulatory parts could distribute evenly over a wide range of 5 orders of magnitude. The selected synthetic regulatory parts had good stability in both nutrient-rich and minimal media. The precise combinatorial modulation of galP and glk from Escherichia coli by using three synthetic regulatory parts with different strengths was confirmed in phosphoenolpyruvate:carbohydrate phosphotransferase system inactive V. natriegens strain to alter glucose transport. This work provides the simple, efficient and standardized constitutive regulatory parts for V. natriegens synthetic biology.
... These promoters do not require regulation and maintain constant activity, making them attractive in biotechnology. The constitutive promoter library for V. natriegens has been curated by direct transfer of wellknown bacterial constitutive promoters or by modification of existing promoters [7,12,13]. A synthetic promoter library was created by modifying the nucleotide sequences of the −35 and −10 boxes of the PJ23119 promoter [7] which has the perfect match of the bacterial consensus promoter sequence. ...
... Furthermore, a predictive model was developed to accurately predict promoter strength, suggesting possibilities for synthetic promoter design. In another study, a constitutive promoter library was generated by screening from a mutant promoter library containing fully-randomized -35 and -10 boxes [12]. Combined with a ribosome-binding site (RBS) library, the constitutively expressed parts offered varying gene expression levels spanning five orders of magnitude. ...
... Translation initiation is primarily governed by the interaction between the SD sequence and 16S rRNA within the 30S ribosomal subunit. Therefore, variations in the RBS region can significantly affect the translation efficiency [12,19,20]. The influence of RBS and 5'-UTR on gene expression in Vibrio species has been studied using few RBS variants [13,14] or randomized libraries [7,12]. ...
Synthetic biology aims to establish engineering principles for biological systems by leveraging the design-build-test-learn (DBTL) cycle. Central to the success of the DBTL cycle is the selection of a suitable chassis, which is the environment in which biological designs are tested. Every step of this cycle is strongly influenced by the properties of chassis. A successful chassis must meet various criteria, prompting ongoing research regarding new candidates. Recently, species within the Vibrio genus, notably Vibrio natriegens and related strains, have emerged as promising next-generation chassis due to their rapid growth rates, versatile substrate utilization, and biosafety level 1 classification. These properties make them highly attractive for accelerating the DBTL cycle with the potential for efficient protein and metabolite production. This review emphasizes the foundational requirements for efficient engineering in synthetic biology, including genetic parts, vectors, and genome engineering technologies tailored to Vibrio species. Practical applications, such as metabolic engineering and protein expression, have been discussed, offering a comprehensive summary of recent advances. This paper also outlines the future directions and suggestions for fully unlocking the potential of Vibrio species as next-generation chassis.
... It is feedstock flexible, and in particular is capable of growth on low-energy substrates such as formate (9) and acetate (10), and fixes nitrogen under anaerobic conditions (11). Like cloning strains of E. coli, it is BSL-1 and is genetically tractable, with a robust selection of promoters, terminators, and ribosomal binding sites (12,13) and has previously been engineered for production of numerous commercially relevant compounds, including the bioplastic polyhydroxybutyrate (PHB) (14), alanine (3), and 2,3-butanediol (2,3-BDO) (15,16). Non-sterile seawater can be used as the source of water in growth media without substantial losses in yield, which has implications for sustainable bioproduction (16). ...
... We successfully inserted a different version of the lacI/Vc tfoX construct, this time derived from pMMB67EH-tfoX (14) at the same genomic site (Vn NC3, Supplementary Fig. S1D) but observed that NPT could not be induced with IPTG in this strain. We then opted to embrace constitutive tfoX expression, deleting lacI in order to create Vn NC4 ( Supplementary Fig. S1E), and two versions using the optimized strong constitutive genomic promoter P23 from Wu et al. (13) (Vn NC5, NC6, Supplementary Fig. S1F) in lieu of Ptac. However, Vn NC4 unexpectedly also did not exhibit NPT, and we were unable to create strains Vn NC5 and NC6 because the genomic edit could not be inserted, leading us to speculate that tfoX expression from the P23 promoter may be lethal. ...
The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using an engineered strain with a genomic copy of the master competence regulator tfoX from Vibrio cholerae in combination with a new minimal competence media (MCM) that uses acetate as an energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely in the same media, without exchange or addition of fresh media. Cells are naturally competent to plasmids, recombination with linear DNA, and cotransformation of both to select for scarless and markerless genomic edits. The entire process is simple and inexpensive, requiring no capital equipment for an entirely room temperature process (zero capital protocol, 104 cfu/μg), or just an incubator (high-efficiency protocol, 105−6 cfu/μg). These cells retain their naturally competent state when frozen and are transformable immediately upon thawing like a typical chemical or electrochemical competent cell. Since the optimized transformation protocol requires only 50 min of hands-on time, and V. natriegens grows quickly even on plates, a transformation started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages of transformation occur in the same media, and the process can be arbitrarily scaled in volume, this natural competence strain and media could be ideal for automated directed evolution applications. As a result, naturally competent V. natriegens could compete with Escherichia coli as an excellent chassis for low-cost and highly scalable synthetic biology.
... Similarly, efficient production of 1,3-propanediol from glycerol was achieved using V. natriegens through systematic metabolic engineering, and the productivity reached 2.36 g/L/h [27]. In addition, the advanced synthetic biology methods and tools, such as the whole genome sequencing [29][30][31], genome editing [24,32], artificial regulatory elements [33,34] as well as metabolic flux analysis [35] are also paving the way for the industrial applications of V. natriegens. ...
... Next, in order to reduce the carbon flux entering the TCA cycle, the effects of gene deletion and down-regulation of aceE were examined for strain PYR11. The artificial regulatory part P2, including the promoter region and ribosome binding site (RBS) element, is a constitutive promoter with low expression activity [34]. Then, the regulatory part P2 and two rare start codons (GTG and TTG) were used to down-regulate the expression of aceE, generating strains PYR14 and PYR15, respectively. ...
Background: Pyruvate is a widely used value-added chemical which also serves as a hub of various metabolic pathways. The fastest-growing bacterium Vibrio natriegens is a promising chassis for synthetic biology applications with high substrate uptake rates. The aim of this study was to investigate if the high substrate uptake rates of V. natriegens enable pyruvate production at high productivities.
Results: Two prophage gene clusters and several essential genes for the biosynthesis of byproducts were first deleted. In order to promote pyruvate accumulation, the key gene aceE encoding pyruvate dehydrogenase complex E1 component was down-regulated to reduce the carbon flux into the tricarboxylic acid cycle. Afterwards, the expression of ppc gene encoding phosphoenolpyruvate carboxylase was fine-tuned to balance the cell growth and pyruvate synthesis. The resulting strain PYR32 was able to produce 54.22 g/L pyruvate from glucose within 16 h, with a yield of 1.17 mol/mol and an average productivity of 3.39 g/L/h. In addition, this strain was also able to efficiently convert sucrose or gluconate into pyruvate at high titers.
Conclusion: A novel strain of V. natriegens was engineered which was capable to provide higher productivity in pyruvate synthesis. This study lays the foundation for the biosynthesis of pyruvate and its derivatives in fast-growing V. natriegens.
... It is feedstock flexible, and in particular is capable of growth on low-energy substrates such as formate 9 and acetate, 10 and fixes nitrogen under anaerobic conditions. 11 Like cloning strains of E. coli, it is BSL-1 and is genetically tractable, with a robust selection of promoters, terminators, and ribosomal binding sites, 12,13 and has previously been engineered for production of numerous commercially-relevant compounds, including the bioplastic polyhydroxybutyrate (PHB), 14 alanine, 3 and 2,3-butanediol (2,3-BDO). 15,16 Non-sterile seawater can be used as the source of water in growth media without substantial losses in yield, which has implications for sustainable bioproduction. ...
... Subsequent editing was done in this ∆dns strain using a helper plasmid which Producing a strain with genomic expression of tfoX adequate to trigger measurable NPT proved to be unexpectedly difficult. Genomic insertion of two different versions of tfoX expression derived from pMMB-tfoX from Dalia et al. 14 did not induce natural competence and two versions using the optimized genomic promoter P23 from Wu et al.13 ...
The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using an engineered strain with a genomic copy of the master competence regulator tfoX from Vibrio cholera in combination with a new minimal competence media (MCM) that uses acetate as an energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely in the same media, without exchange or addition of new media. Cells are naturally competent to plasmids, recombination with linear DNA, and co-transformation of both to select for scarless and markerless genomic edits. The entire process is simple and inexpensive, requiring no capital equipment for an entirely room temperature process (Zero Capital protocol, 10^4 cfu/μg), or just an incubator (High Efficiency protocol, 10^5-6 cfu/μg). These cells retain their naturally competent state when frozen and are transformable immediately upon thawing like a typical chemical or electrochemical competent cell. Since the optimized transformation protocol requires only 50 minutes of hands-on time, and V. natriegens grows quickly even on plates, a transformation started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages of transformation occur in the same media, and the process can be arbitrarily scaled in volume, this natural competence strain and media could be ideal for automated directed evolution applications. As a result, naturally competent V. natriegens could compete with E. coli as an excellent chassis for low-cost and highly scalable synthetic biology.
... igem.org/) [28][29][30], the region of two replaced promoters (P als , P trc , P tac , P lac , P J23110 , P J23100 , and P 16 ) and polyclonal sites were synthesized with ClaI and XhoI at the fragments' two ends, respectively. Then, the fragments were inserted in pACYCDuet-1 (digested with ClaI and XhoI), respectively. ...
... To further optimize the expression levels of glmS and gna1, as well as increase the titer of GlcNAc, we fine-tuned the promoter strength of these two genes. In addition to the P als promoter, three inducible promoters (P trc , P lac , and P tac ) and three constitutive promoters (P J23110 , P J23100 , and P 16 ) with varying strengths were selected [28][29][30]. Promoter replacement was conducted, followed by the insertion of EcglmS and Ltgna1 in these plasmids, respectively (Fig. 4A). Then, the resulting plasmids were transformed into FA2-7, and the fermentation results of six recombinant strains were shown in Fig. 4B. ...
Members of the Vibrionaceae family are predominantly fast-growing and halophilic microorganisms that have captured the attention of researchers owing to their potential applications in rapid biotechnology. Among them, Vibrio alginolyticus FA2 is a particularly noteworthy halophilic bacterium that exhibits superior growth capability. It has the potential to serve as a biotechnological platform for sustainable and eco-friendly open fermentation with seawater. To evaluate this hypothesis, we integrated the N-acetylglucosamine (GlcNAc) pathway into V. alginolyticus FA2. Seven nag genes were knocked out to obstruct the utilization of GlcNAc, and then 16 exogenous gna1s co-expressing with EcglmS were introduced to strengthen the flux of GlcNAc pathway, respectively. To further enhance GlcNAc production, we fine-tuned promoter strength of the two genes and inactivated two genes alsS and alsD to prevent the production of acetoin. Furthermore, unsterile open fermentation was carried out using simulated seawater and a chemically defined medium, resulting in the production of 9.2 g/L GlcNAc in 14 h. This is the first report for de-novo synthesizing GlcNAc with a Vibrio strain, facilitated by an unsterile open fermentation process employing seawater as a substitute for fresh water. This development establishes a basis for production of diverse valuable chemicals using Vibrio strains and provides insights into biomanufacture.
... We conclude that it is possible to randomise the regulatory region and drive gene expression in V. natriegens. However, targeting core regulatory region elements strongly affects expression, which has also been found by Wu et al. (Wu et al., 2020). Wu et al. reported similar work by randomising either the −10 motif and −35 motif, the spacer in between the two motifs, or parts of the RBS. ...
... V. natriegens plates were stored at room temperature since the bacteria are sensitive to refrigeration (Weinstock et al., 2016). M9N minimal medium consisted of [per litre (Wu et al., 2020)]: 200 ml of sterile 1X M9N minimal medium (5x M9N medium: KH 2 PO 4 , 15 g/L, NaCl, 2.5 g/L, Na 2 HPO 4 , 33.9 g/ L, NH 4 Cl, 5 g/L) supplemented with 2 mM MgSO 4 , 0.1 mM CaCl 2 , and 15 g/L NaCl. The carbon source was supplemented in a concentration of 20 g/L. ...
Vibrio natriegens has recently gained attention as a novel fast-growing bacterium in synthetic biology applications. Currently, a limited set of genetic elements optimised for Escherichia coli are used in V. natriegens due to the lack of DNA parts characterised in this novel host. In this study, we report the identification and cross-characterisation of artificial promoters and 5′ untranslated regions (artificial regulatory sequence, ARES) that lead to production of fluorescent proteins with a wide-range of expression levels. We identify and cross-characterise 52 constructs in V. natriegens and E. coli. Furthermore, we report the DNA sequence and motif analysis of the ARESs using various algorithms. With this study, we expand the pool of characterised genetic DNA parts that can be used for different biotechnological applications using V. natriegens as a host microorganism.
... To drive adjusted gene expression in V. natriegens, synthetic promoter libraries have been constructed and characterized [28,31]. Permutation of promoter elements (−35, −10, and UP element), spacer sequences, ribosomal-binding sites, and terminator sequences yielded a library which varies up to five orders of magnitude in gfp expression. ...
... Examples include several reporter constructs (e.g. GFP, sfGFP, mCherry, mSCFP3, and LacZ, worked nicely, whereas functionality of RFP, YFP, and eBFP2 was poor) as well as proteins, that are largely insoluble, typically yielded at low concentrations in E. coli, isotopically labeled proteins or even multimeric membrane protein complexes [6,7,20,28,31,[36][37][38]. ...
Vibrio natriegens is emerging as a promising host for biotechnology which is basically due to the remarkable intrinsic properties such as the exceptionally high growth and substrate consumption rates. The facultatively anaerobic marine bacterium possesses a versatile metabolism, is able to utilize a variety of substrates as carbon and energy sources and is easy to handle in the lab. These features initiated the rapid development of genetic tools and resulted in extensive engineering of production strains in the past years. Although recent examples illustrate the potential of V. natriegens for biotechnology, a comprehensive understanding of the metabolism and its regulation is still lacking but essential to exploit the full potential of this bacterium. In this review, we summarize the current knowledge on the physiological traits and the genomic organization, provide an overview of the available genetic engineering tools and recent advances in metabolic engineering of V. natriegens. Finally, we discuss the obstacles which have to be overcome in order to establish V. natriegens as industrial production host.
... [28] The establishment of procedures for transformation and conjugation of plasmids and the development of the first genetic tools for this bacterium paved the way for a rapid succession of studies that raised V. natriegens to the status of an emerging chassis organism for biotechnology. [29] Several labs developed and benchmarked extensive genetic toolkits that include promoters, ribosome binding sites, and resistance markers, [8] numerous regulatory parts, [30] and synthetic promoters and 5'-UTRs. [31] Furthermore, V. natriegens was also at the center of the Grand Prize-winning project of the iGEM competition 2018, [32] which later resulted in a collection of 191 genetic parts for Golden Gate assembly. ...
Oceans cover 71 % of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions, and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example of the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed.
... V. natriegens is an emerging industrial biotechnology chassis, and the nonpathogenic nature [25], genetic tractability [26,27], high substrate uptake rates [28,29], remarkably short metabolic prowess, and efficient protein expression [30] of V. natriegens make it a promising next-generation industrial microorganism [25,26,31,32] (Figure 1). Genetically engineered V. natriegens strains have been used to produce diverse chemicals such as 1,3-propanediol [33], 2,3-butanediol [31,34], PHB [, [28], [35]], lycopene [31], succinic acid [36], alanine [29], and ethanol [31]. ...
The development of MFC using electroactive industrial microorganisms has seen a surge of interest because of the co-generation for bioproduct and electricity production. Vibrio natriegens as a promising next-generation industrial microorganism chassis and its application for microbial fuel cells (MFC) was first studied. Mediated electron transfer was found in V. natriegens MFC (VMFC), but V. natriegens cannot secrete sufficient electron mediators to transfer electrons to the anode. All seven electron mediators supplemented are capable of improving the electronic transfer efficiency of VMFC. The media and carbon sources switching study reveals that VMFCs have excellent bioelectricity generation performance with feedstock flexibility and high salt-tolerance. Among them, 1% glycerol as the sole carbon source produced the highest power density of 111.9 ± 6.7 mW/cm2. The insight of the endogenous electronic mediators found that phenazine-1-carboxamide, phenazine-1-carboxylic acid, and 1-hydroxyphenazine are synthesized by V. natriegens via the shikimate pathway and the phenazine synthesis and modification pathways. This work provides the first proof for emerging industrial biotechnology chassis V. natriegens as a novel high salt-tolerant and feedstock flexibility electroactive microorganism for MFC, and giving insight into the endogenous electron mediator biosynthesis of VMFC, paving the way for the application of V. natriegens in MFC and even microbial electrofermentation (EF).
... Increasing RBS strength of heterologous mRNA transcripts has been modelled to reduce the free ribosome pool in a cell, thus inhibiting cellular growth [21]. Hence, RBS strength plays an important role in determining the success of synthetic gene circuits within cells, where an uninhibited metabolism is important for a healthy cell and for a well-behaving circuit [24,[40][41][42][43]. pLit gIII is constitutively expressed from a high-copy plasmid under a medium strength promoter (BBa_J23106) and translated under a strong RBS (BBa_B0034), the combination of which is likely to result in very high expression levels and burden ( Figure 2) [2,3]. ...
Orthogonal or non‐cross‐reacting transcription factors are used in synthetic biology as components of genetic circuits. Brödel et al. (2016) engineered 12 such cIλ transcription factor variants using a directed evolution ‘PACEmid’ system. The variants operate as dual activator/repressors and expand gene circuit construction possibilities. However, the high‐copy phagemid vectors carrying the cIλ variants imposed high metabolic burden upon cells. Here, the authors ‘remaster’ the phagemid backbones to relieve their burden substantially, exhibited by a recovery in Escherichia coli growth. The remastered phagemids' ability to function within the PACEmid evolver system is maintained, as is the cIλ transcription factors' activity within these vectors. The low‐burden phagemid versions are more suitable for use in PACEmid experiments and synthetic gene circuits; the authors have, therefore, replaced the original high‐burden phagemids on the Addgene repository. The authors’ work emphasises the importance of understanding metabolic burden and incorporating it into design steps in future synthetic biology ventures.
... These strains were incubated at 37 °C and 200 rpm in CGXII medium containing 20 g/l glucose in test tubes, with or without the addition of 1 mg/ml streptomycin. The sample preparation and fluorescence assay were conducted according to the procedures modified from reported methods [12,13]. After 12 h of incubation, an appropriate amount of the cells for each strain were harvested by centrifugation at 5000 rpm for 5 min. ...
Bacterial resistance to streptomycin is often acquired as a consequence of mutations in rpsL, the gene encoding ribosomal protein S12. Corynebacterium glutamicum is a non-pathogenic Gram-positive soil bacterium that has been widely used in industry. In a previous study, we screened several streptomycin-resistant rpsL K43 mutants of C. glutamicum, and surprisingly found that two of them also confer chloramphenicol and/or kanamycin resistance. In order to understand whether or not a single mutation of rpsLK43 could confer resistance to multiple antibiotics, in this study we attempted to construct saturation mutagenesis of rpsL K43 by rational genetic manipulation. Despite many efforts had been made, only nine mutants were successfully constructed. They were indeed resistant to streptomycin, but not to other antibiotics. This suggested that other mutations should be acquired, contributing to multiple antibiotics in the screened strains. The growth and enhanced green fluorescent protein (eGFP) expression of these nine mutants were then investigated. The results showed that they grew differently in CGXII minimal medium, but not in BHI medium. When cultured in the absence of streptomycin, the expression of eGFP was positively proportional to the growth, approximately, while in the presence of streptomycin, the expression of eGFP was proportional to the ability of streptomycin resistance.
... Interestingly, the relative maximum output did not necessarily correspond to the promoter strength measured in previous studies (Stukenberg et al., 2021;Tschirhart et al., 2019), but this could be due to the different reporter or media used. Cultivation media were shown to have an influence on promoter functioning in V. natriegens (Wu et al., 2020). Another explanation could be the lack of promoter insulation, since the Anderson promoter sequences are relatively short (35bp), and only a short RBS was added (21bp). ...
The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse.
In this work, the potential of V. natriegens as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from Corynebacterium glutamicum, was adapted for expression in V. natriegens to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, i.e. potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producer cells could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the production metabolism of V. natriegens.
This work demonstrates the successful expression and application of transcription factor-based biosensors in V. natriegens and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe.
... Certainly, 'dormant' (non-replicating) cells can be quite active metabolically [527][528][529][530]. Consequently, although not usually a focus of biotechnology, it remains the case that the more time cells spend in a fermentor non-productively the less good the process. This has led to the consideration of hosts such as Vibrio natriegens [50,[531][532][533][534][535][536][537][538][539][540][541], whose optimal doubling time can be as little as 7 min, some threefold quicker than the widely quoted 20 min for E. coli in rich media. Whether or not organisms such as V. natriegens turn out to be valuable production hosts, there is no doubt that understanding how to make cell growth quicker might help enhance the rates of recombinant protein production. ...
Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.
... Recently, several strategies for genome engineering have been published 5,15,16 and different transformation protocols for plasmids have been established. 15,17,18 In addition, several genetic parts from E. coli 15,17,18 and from synthetic libraries 19 have been functionally characterized in V. natriegens. However, to date, there exists no simple, one-stop solution for quickly building multigene constructs from a library of V. natriegens-tested DNA parts. ...
Achieving cost-competitive bio-based processes requires development of stable and selective biocatalysts. Their realization through in vitro enzyme characterization and engineering is mostly low throughput and labor-intensive. Therefore, strategies for increasing throughput while diminishing manual labor are gaining momentum, such as in vivo screening and evolution campaigns. Computational tools like machine learning further support enzyme engineering efforts by widening the explorable design space. Here, we propose an integrated solution to enzyme engineering challenges whereby ML-guided, automated workflows (including library generation, implementation of hypermutation systems, adapted laboratory evolution, and in vivo growth-coupled selection) could be realized to accelerate pipelines towards superior biocatalysts.
Vibrio natriegens is a Gram-negative bacterium with an exceptional growth rate that has the potential to become a standard biotechnological host for laboratory and industrial bioproduction. Despite this burgeoning interest, the current lack of organism-specific qualitative and quantitative computational tools has hampered the community's ability to rationally engineer this bacterium. In this study, we present the first genome-scale metabolic model (GSMM) of V. natriegens. The GSMM (iLC858) was developed using an automated draft assembly and extensive manual curation and was validated by comparing predicted yields, central metabolic fluxes, viable carbon substrates, and essential genes with empirical data. Mass spectrometry-based proteomics data confirmed the translation of at least 76% of the enzyme-encoding genes predicted to be expressed by the model during aerobic growth in a minimal medium. iLC858 was subsequently used to carry out a metabolic comparison between the model organism Escherichia coli and V. natriegens, leading to an analysis of the model architecture of V. natriegens' respiratory and ATP-generating system and the discovery of a role for a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data were further used to investigate additional halophilic adaptations of V. natriegens. Finally, iLC858 was utilized to create a Resource Balance Analysis model to study the allocation of carbon resources. Taken together, the models presented provide useful computational tools to guide metabolic engineering efforts in V. natriegens.
The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.
Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens -produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.
A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources (“feedstock flexibility”). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.
The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.
Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.
Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative - 10 (NNTANANT) and - 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.
The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.
Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, Pbs, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli–B. subtilis–S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters Pbs1, Pbs2, and Pbs3, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.
The productivity of industrial fermentation processes is essentially limited by the biomass specific substrate consumption rate (q S ) of the applied microbial production system. Since q S depends on the growth rate (μ), we highlight the potential of the fastest growing non-pathogenic bacterium, Vibrio natriegens , as novel candidate for future biotechnological processes. V. natriegens grows rapidly in BHIN complex medium with a μ of up to 4.43 h ⁻¹ (doubling time of 9.4 min) as well as in minimal medium supplemented with various industrially relevant substrates. Bioreactor cultivations in minimal medium with glucose showed that V. natriegens possesses an exceptionally high q S under aerobic (3.90 ± 0.08 g g ⁻¹ h ⁻¹ ) and anaerobic (7.81 ± 0.71 g g ⁻¹ h ⁻¹ ) conditions. Fermentations with resting cells of genetically engineered V. natriegens under anaerobic conditions yielded an overall volumetric productivity of 0.56 ± 0.10 g alanine L ⁻¹ min ⁻¹ (i.e. 34 g L ⁻¹ h ⁻¹ ). These inherent properties render V. natriegens a promising new microbial platform for future industrial fermentation processes operating with high productivity.
Importance Low conversion rates are one major challenge to realize microbial fermentation processes for the production of commodities operating competitively to existing petrochemical approaches. For this reason, we screened for a novel platform organism possessing superior characteristics to traditionally employed microbial systems. We identified the fast growing Vibrio natriegens which exhibits a versatile metabolism and shows striking growth and conversion rates, as a solid candidate to reach outstanding productivities. Due to these inherent characteristics V. natriegens can speed up common laboratory routines, is suitable for already existing production procedures, and forms an excellent foundation to engineer next generation bioprocesses.
Background
Plasmid expression is a popular method in studies of MVA pathway for isoprenoid production in Escherichia coli. However, heterologous gene expression with plasmid is often not stable and might burden growth of host cells, decreases cell mass and product yield. In this study, MVA pathway was divided into three modules, and two heterologous modules were integrated into the E. coli chromosome. These modules were individually modulated with regulatory parts to optimize efficiency of the pathway in terms of downstream isoprenoid production. ResultsMVA pathway modules Hmg1-erg12 operon and mvaS-mvaA-mavD1 operon were integrated into E. coli chromosome followed by modulation with promoters with varied strength. Along with activation of atoB, a 26% increase of β-carotene production with no effect on cell growth was obtained. With a combinatory modulation of two key enzymes mvas and Hmg1 with degenerate RBS library, β-carotene showed a further increase of 51%. Conclusions
Our study provides a novel strategy for improving production of a target compound through integration and modulation of heterologous pathways in both transcription and translation level. In addition, a genetically hard-coded chassis with both efficient MEP and MVA pathways for isoprenoid precursor supply was constructed in this work.
Background
Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. ResultsAltering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. Conclusions
In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.
The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.
During exponential growth some cells of E. coli undergo senescence mediated by asymmetric segregation of damaged components, particularly protein aggregates. We showed previously that functional cell division asymmetry in E. coli was responsive to the nutritional environment. Short term exposure as well as long term selection in low calorie environments led to greater cell division symmetry and decreased frequency of senescent cells as compared to high calorie environments. We show here that long term selection in low nutrient environment decreased protein aggregation as revealed by fluorescence microscopy and proportion of insoluble proteins. Across selection lines protein aggregation was correlated significantly positively with the RNA content, presumably indicating metabolic rate. This suggests that the effects of caloric restriction on cell division symmetry and aging in E. coli may work via altered protein handling mechanisms. The demonstrable effects of long term selection on protein aggregation suggest that protein aggregation is an evolvable phenomenon rather than being a passive inevitable process. The aggregated proteins progressively disappeared on facing starvation indicating degradation and recycling demonstrating that protein aggregation is a reversible process in E. coli.
Vibrio natriegens is a Gram-negative bacterium known for its extremely short doubling time. Here we present the annotated draft genome sequence
of Vibrio natriegens strain DSMZ 759, with the aim of providing insights about its high growth rate.
Vibrio natriegens bacteria are Gram-negative aquatic microorganisms that are found primarily in coastal seawater and sediments and are perhaps
best known for their high growth rates (generation time of <10 min). In this study, we report the first sequenced genome of
this species, that of the type strain Vibrio natriegens ATCC 14048, a salt marsh mud isolate from Sapelo Island, GA.
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.
The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: -59 nnAAA(A/T) (A/T)T(A/T)TTTTnnAAAAnnn -38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the alpha C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the alpha-DNA interaction. Based on the evolutionary conservation of the residues in alpha responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.
Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS(-) strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.
Orthogonal ribosomes (o-ribosomes), also known as specialized ribosomes, are able to selectively translate mRNA not recognized
by host ribosomes. As a result, they are powerful tools for investigating translational regulation and probing ribosome structure.
To date, efforts directed towards engineering o-ribosomes have involved random mutagenesis-based approaches. As an alternative,
we present here a computational method for rationally designing o-ribosomes in bacteria. Working under the assumption that
base-pair interactions between the 16S rRNA and mRNA serve as the primary mode for ribosome binding and translational initiation,
the algorithm enumerates all possible extended recognition sequences for 16S rRNA and then chooses those candidates that:
(i) have a similar binding strength to their target mRNA as the canonical, wild-type ribosome/mRNA pair; (ii) do not bind
mRNA with the wild-type, canonical Shine-Dalgarno (SD) sequence and (iii) minimally interact with host mRNA irrespective of
whether a recognizable SD sequence is present. In order to test the algorithm, we experimentally characterized a number of
computationally designed o-ribosomes in Escherichia coli.
Microbial engineering often requires fine control over protein expression--for example, to connect genetic circuits or control flux through a metabolic pathway. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Experimental validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.
One-hundred-and-forty-five isolates of marine origin were submitted to an extensive physiological, nutritional, and morphological characterization. All strains were gram-negative, facultatively anaerobic, straight or curved rods which were motile by means of flagella. Glucose was fermented with the production of acid but no gas. Sodium but no organic growth factors were required. None of the strains were able to denitrify or fix molecular nitrogen. The results of nutritional and physiological tests were submitted to a numerical analysis. On the basis of phenotypic similarity, nine groups were established. These groups could be distinguished from one another by multiple, unrelated, phenotypic traits. Six groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea. All of the strains in this genus, when grown in liquid medium, had a single, polar flagellum. When grown on a solid medium, many strains had peritrichous flagella. Two groups were similar to previously described species and were designated B. alginolytica and B. natriegens. The remaining four groups were designated B. campbellii, B. neptuna, B. nereida, and B. pelagia. An additional group of phenotypically similar strains having the properties of the genus Beneckea was not included in the numerical analysis. These strains were readily separable from species of this genus and were designated B. parahaemolytica. Of the remaining groups, one was identified as Photobacterium fischeri. The other group (B-2) which had about 41 moles% GC content in its DNA could not be placed into existing genera.
The DNA sequence of 168 promoter regions (−;50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter
had been defined by genetic (promoter mutations) or biochemical (5′ end determination) criteria. A consensus promoter sequence
based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations.
In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following
general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.
Glucose is the preferred substrate for certain fermentation processes. During its internalization and concomitant formation of glucose-6-phosphate through the glucose phosphotransferase system (PTS), one molecule of phosphoenolpyruvate (PEP) is consumed. Together with erythrose 4-phosphate (E4P), PEP is condensed to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route, several commercially important aromatic compounds can be obtained. We have selected Escherichia coli mutants that can transport glucose efficiently by a non-PTS uptake system. In theory, this process should increase the availability of PEP for other biosynthetic reactions. Using these mutants, in a background where the DAHP synthase (the enzyme that catalyzes the condensation of PEP and E4P into DAHP) was amplified, we were able to show that at least some of the PEP saved during glucose transport, can be redirected into the aromatic pathway. This increased carbon commitment to the aromatic pathway was enhanced still further upon amplification of the E. coli tktA gene that encodes for a transketolase involved in the biosynthesis of E4P.
The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires
an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been
found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part,
from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.
In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from
one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor,
we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in
the amount of aggregate protein over time, (ii) a proportional relationship between the amount of aggregate protein and the
level of dead cells, (iii) a larger amount in dead cells than in culturable cells, (iv) a heterogeneous distribution of different
amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein
within a culturable population conditioned the death rate of the culture. Together, the results presented in this study suggest
that protein aggregates indeed represent one aging factor leading to bacterial cell death.
Recombinant DNA technology has revolutionized biomedical research with continual innovations advancing the speed and throughput of molecular biology. Nearly all these tools, however, are reliant on Escherichia coli as a host organism, and its lengthy growth rate increasingly dominates experimental time. Here we report the development of Vibrio natriegens , a free-living bacteria with the fastest generation time known, into a genetically tractable host organism. We systematically characterize its growth properties to establish basic laboratory culturing conditions. We provide the first complete Vibrio natriegens genome, consisting of two chromosomes of 3,248,023 bp and 1,927,310 bp that together encode 4,578 open reading frames. We reveal genetic tools and techniques for working with Vibrio natriegens . These foundational resources will usher in an era of advanced genomics to accelerate biological, biotechnological, and medical discoveries.
The fast-growing non-model marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly-used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this non-model chassis.
Isotopic labeling of recombinant proteins is crucial for studying proteins by liquid state NMR spectroscopy. Nowadays, conventional E. coli-based expression systems like BL21 (DE3) are typically used to express recombinant proteins. Still, the production of isotopically labeled proteins is often costly and time-consuming, and yields are not sufficient enough for structural studies. Here, we present Vibrio natriegens (Vmax) as an alternative expression system in M9 minimal medium. Due to our optimized M9 minimal medium and conditions and the early time point of induction, we obtained a 2- to 4-fold higher protein yield for two test proteins, FKBP and EYFP, compared to E. coli BL21 (DE3). Production of proteins in V. natriegens in minimal medium is not only more cost-effective and convenient but also less time-consuming than in E. coli. Comparing 15N HSQC spectra of FKBP and EYFP expressed in Vmax and BL21 (DE3) revealed correct folding during expression.
Biosensors for target metabolites provide powerful high-throughput screening tools to obtain high-performing strains. However, well-characterized metabolite-sensing modules are often unavailable and limit rapid access to the robust biosensors with successful applications. In this study, we developed a strategy of transcriptome-assisted metabolite-sensing (TAMES) to identify the target metabolite-sensing module based on selectively comparative transcriptome analysis between the target metabolite producing and non-producing strains and a subsequent RT-qRCR evaluation. The strategy was applied to identify the sensing module cusR that responds positively to the metabolite 3-dehydroshikimate (DHS) and proved it was effective to narrow down the candidates. We further constructed the cusR-based synthetic biosensor and established the DHS biosensor-based high-throughput screening (HTS) platform to screen higher DHS-producing strains and successfully increased DHS production by more than 90%. This study demonstrated that the TAMES strategy was effective at exploiting the metabolite-sensing transcriptional regulator, and this could likely be extended to develop the biosensor-based HTS platforms for other molecules.
Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.
A rapidly growing bacterial host would be desirable for a range of routine applications in molecular biology and biotechnology. The bacterium Vibrio natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min. We report the development of genetic tools and methods to engineer V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.
We have compiled and analyzed 263 promoters vith known transcriptional start points for E. coll genes. Promoter elements (-35
hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent
with the start point and statistically most homologous to a reference list of promoters. The initial reference list was that
of Hawley and McClure (Nucl. Acids Res. 11, 2237–2255, 1983). Alignment of the complete list was used for reference until
successive analyses did not alter the structure of the list. In the final compilation, all bases in the -35 (TTGACA) and -10
(TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17±1 bp, and 75% of the uniquely defined
start points initiated 7±1 bases downstream of the -10 region. The consensus sequence of promoters with inter-region spacing
of 16, 17, or 18 bp did not differ. This compilation and analysis should be usaful for studies of promoter structure and function
and for programs which identify potential promoter sequences
In natural environments, bacteria are often challenged by nutrient starvation and other stresses. As a consequence, cell growth is arrested and bacteria enter stationary phase. In this report, we demonstrate that during stationary phase, Escherichia coli cells accumulate aggregates of misfolded proteins and complexes of Dps (starvation-induced protein) with chromosomal DNA. We found that the formation of multicomponent protein aggregates and insoluble Dps-DNA complexes depended on growth conditions and was influenced by the availability of oxygen and glucose in a medium. Aerobic stationary cells grown in unbuffered medium supplemented with glucose contained insoluble Dps-DNA, whereas multicomponent protein aggregates were accumulated under glucose starvation. On the contrary, under oxygen depletion, Dps-DNA complexes were formed in the absence of glucose, whereas multicomponent protein aggregates appeared in the presence of glucose. The mechanisms responsible for this phenomenon remain to be elucidated; however, we demonstrated that in MOPS-buffered cultures the level of insoluble Dps and protein aggregates was decreased.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.
A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], the vectors carry a strong hybrid trp/lac promoter, the lacZ ribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacIq allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.
The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the
Shine – Dalgarno (SD) sequence found just 5′ to the translation initiation codon. It is now clear that the SD sequence is
important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing
between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether
there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have
obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD
sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene.
Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned
spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3′-end of the 16s
rRNA and the P-site, we conclude that there is a unique optimal aligned SD– AUG spacing in the absence of
other complicating issues.
In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.
Eagon, R. G. (University of Georgia, Athens). Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol.
83
736–737. 1962.—Pseudomonas natriegens, a marine microorganism, was demonstrated to have a generation time of 9.8 min. This is the shortest generation time reported to date. Optimal growth occurred at 37 C in brain heart infusion broth supplemented with 1.5% sea salt.
Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.