Vibrio Natriegens: an Ultrafast-Growing Marine Bacterium As Emerging Synthetic Biology Chassis

Vibrio natriegens: An ultrafast-growing marine bacterium as emerging synthetic biology chassis

Josef Hoff1,2,*, Benjamin Daniel2,3,*, Daniel Stukenberg2,*, Benjamin W. Thuronyi4, Torsten Waldminghaus5,6, and Georg Fritz1,#

1School of Molecular Sciences, The University of Western Australia, Perth, Australia.

2Center for Synthetic Microbiology, Philipps-Universität Marburg, Marburg, Germany,

3Institute of Microbiology, ETH Zurich, Zürich, Switzerland.

4Department of Chemistry, Williams College, Williamstown, MA, USA

5Centre for Synthetic Biology, Technische Universität Darmstadt, Darmstadt, Germany

6Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany

*These authors contributed equally to this work.

#For correspondence: [email protected]

Summary The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applications – from speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolism – key physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.15128

Introduction Biotechnology, synthetic biology and many life sciences rely on bacteria as tools for the construction and propagation of engineered DNA. While important advancements in DNA synthesis and sequencing have drastically decreased both the cost and the time required for molecular biology applications, the time it takes to grow the chassis organism becomes a major bottleneck in the attempt to accelerate progress in the life sciences. Likewise, as prokaryotic synthetic biology methods become increasingly well developed, the capabilities of the host bacterium (almost invariably Escherichia coli) begin to limit their scope and efficiency. Interestingly, members of the Vibrionaceae family have been recognized for their rapid growth, often featuring cell doubling times even below those of fast-growing, engineered Escherichia coli strains. For instance, Vibrio parahaemolyticus and Vibrio alginolyticus can double as fast as every 12- 14 minutes (Ulitzur, 1974), while Vibrio natriegens beats all records with a generation time of less than 10 min in bulk measurements (Eagon, 1962; Hoffart et al., 2017). With this, V. natriegens is the fastest growing and non-pathogenic organism known to date. While V. natriegens was already described in 1958 by William Payne (Payne, 1958), only recently a wave of efforts has focused on establishing V. natriegens as a new chassis for synthetic biology and biotechnology (Maida et al., 2013; Lee et al., 2016; Weinstock et al., 2016; Dalia et al., 2017b; Hoffart et al., 2017; Eichmann et al., 2019; Ellis et al., 2019; Lee et al., 2019; Tschirhart et al., 2019). Based on its remarkable speed and its ability to grow to significantly higher cell densities than E. coli, V. natriegens holds enormous potential as a time- saving chassis for molecular cloning and protein expression, with the potential to replace E. coli as the major workhorse in our laboratory routines. However, while a strong focus has been placed on development of methods and tools in recent years, many challenges remain for harnessing the full potential of V. natriegens. In this review we will give a broad overview about V. natriegens, starting with its discovery, followed by its early microbiological characterization. We will discuss the available knowledge about the intriguing biology of V. natriegens, including its ecology, metabolism and first insights into its gene regulation. Moreover, we summarize established molecular biological methods and discuss examples of successful applications of V. natriegens in synthetic biology and biotechnology in recent years. In the last section, we try to reveal major blind spots in our understanding of the biology of V. natriegens, as well as missing methods and tools which need to be established in order to exploit the full potential of this fascinating bacterium.

History and basic physiology In 1958, the group of William J. Payne (University of Georgia) conducted a series of studies concerning uronic acid utilization by diverse bacteria. In the course of this work, the Gram-negative bacterium V. natriegens was first isolated from marsh mud from the coast of Sapelo Island, Georgia (Payne, 1958). It tolerates a broad range of pH conditions with an optimum at pH 7.5, an optimal growth temperature at 37°C (Payne et al., 1961) and a remarkable doubling time of less than 10 min (Eagon, 1962; Hoffart et al., 2017). It strictly depends on Na+ ions for its metabolic activity and growth cannot be sustained under Na+-limiting conditions (Webb and Payne, 1971). Microscopy revealed it as rod-shaped with a single, polar flagellum (Austin et al., 1978). Recapitulating the history of V. natriegens in the literature is complicated by multiple names and strain identifiers used for the species. The strain was initially referred to as M11 until it was given the scientific name Pseudomonas natriegens in 1961, although Payne expressed doubts about this classification (Payne et al., 1961). The first major reclassification of V. natriegens was in 1971, where it was briefly placed in the genus Beneckea (Baumann et al., 1971). However, in the same year, Webb and Payne referred to it as Vibrio natriegens for the first time (Webb and Payne, 1971), but this proposal was widely ignored. Only in 1978 was it officially proposed to belong to the Vibrio genus (Austin et al., 1978), which became the valid name in 1980 and has remained so to date (Baumann et al., 1980; Skerman et al., 1980). An indication of its current position within the domain of bacteria is shown in Figure 1. The V. natriegens strain originally isolated by Payne in 1958 remains the strain used in most publications to date and is commonly referred to as ATCC14048. In 2016 this strain was described as the fastest-growing V. natriegens strain available, while other strains displayed doubling times ranging from 15 to 25 min (Weinstock et al., 2016). However, very recently some new isolates of V. natriegens were found to grow marginally faster than ATCC14048 in a minimal medium (Peng et al., 2020). The strain ATCC14048 is available from several culture collections under a confusing number of different identifiers including DSM 759, CCUG 16371 /CCUG 4980 A /CCUG 70547T, HUFP9204, NCCB 73019, BCCM/LMG:10935, IFO (now NBRC)15636, NCIMB:857, NCTC:11319, CECT 526, FIRDI 805, CIP 103193 and CAIM 12 Lee MV2190. In general, widespread adoption and characterization of V. natriegens will benefit from strain standardization, though it remains to be seen what that standard will be. Another key argument for the wider adoption of V. natriegens for molecular biology and biotechnology is that strain ATCC14048 is considered to be safe for laboratory work, meeting biosafety

level 1 standards according to the US Public Health and Safety Guidelines laid out in the 5th edition of Biosafety in Microbiological and Biomedical Laboratories (Chosewood and Wilson, 2009). While the organism has been in use for more than 70 years, to our knowledge not a single human infection with V. natriegens has been reported. However, some reports concerning infection and disease in marine animals by some V. natriegens strains exist (Rivera-Posada et al., 2011; Bi et al., 2016), but unfortunately, they do not detail how the pathogen was identified, nor do they characterize the particular strains in any detail. Therefore, it is advisable that any new isolates are thoroughly screened for potential pathogenicity factors before they are put to use in the laboratory. Natural isolates of V. natriegens indeed vary greatly in their genome composition, with some reportedly even encoding for genes known to be involved in pathogenicity (Lee et al., 2016). The type strain ATCC14048 was also screened for potential pathogenicity factors, toxins, as well as the possibility to propagate the phage CTXΦ responsible for the toxin production in Vibrio cholerae, yet no factors indicating any safety risks to humans or animals were identified (Lee et al., 2016). Nevertheless, to improve biosafety of V. natriegens ATCC14048 in the future, it will be important to further study any potential risk factors and to explore, e.g., whether mutants auxotrophic for particular amino acids will retain high growth rates under amino acid supplementation, while experiencing severe selective disadvantages when accidentally released into the environment. Such strain modifications might become key for V. natriegens to attain GRAS (generally recognized as safe) status from the United States Food and Drug Administration (FDA). Based on its uncomplicated handling, safety, and rapid growth, from the 1970’s V. natriegens was used to demonstrate basic microbiology techniques to students (Mullenger and R. Gill, 1973; Delpech, 2001). Its growth rate shortened experiments so that they could take place over the course of high- school classes. Likewise, in recent years at least 5 student teams have adopted the use of V. natriegens in the International Genetically Engineered Machine Competition (iGEM). Members of the medical community became interested in V. natriegens in the 1980’s for more serious reasons (Bannatyne and Cheung, 1981). Tiracillin is a β-lactam antibiotic essential for the treatment of Pseudomonas aeruginosa infections. Since it is toxic to humans in high doses, there was a need to reliably and quickly determine Tiracillin concentrations in patients. To this end, a Tiracillin plate diffusion bioassay was established, in which a droplet of patient's blood was deposited on agar plates seeded with V. natriegens cells, and within 4 hours of incubation the Tiracillin concentration in the blood could be assessed from the size of the growth inhibition zone (Bannatyne and Cheung, 1981). In the early 2010s, the number of publications about V. natriegens began to increase slowly, with the first draft genomes published in 2013 (Maida et al., 2013; Wang et al., 2013). The major breakthrough came in 2016, when two papers

were independently published by the Church and Venter groups (Lee et al., 2016; Weinstock et al., 2016), generating wide publicity for the organism that sparked strong interest in the Synthetic Biology and Biotechnology communities. In 2019, researchers of the German aerospace center working with NASA proposed the use of this mesophilic organism for microbial research in outer space (Garschagen et al., 2019). By reducing person hours and room requirements due to its rapid doubling, V. natriegens could help to make microbial research in low to zero gravity more affordable. Experiments under microgravity showed a 60-fold increase in cell density after 24 hours compared to 1G conditions, indicating that in the absence of gravity V. natriegens extended its exponential growth phase significantly (Garschagen et al., 2019). Most of these remarkable studies were inspired by V. natriegens’ most outstanding feature: its unparalleled capability (for a mesophilic organism) for rapid cell doubling. In the next sections, we highlight some research into possible causes of this ability, as well as look into other intriguing aspects of V. natriegens, starting off with the ecological niche that led to the evolution of this rapidly growing organism.

Ecology Originally, V. natriegens was only known to live in salt marsh mud and estuarine environments (Payne, 1958). Gradually, as more research became public, it became clear that V. natriegens is ubiquitous and widespread in most of our oceans, inhabiting various niches within them (Payne, 1958; Benbouzid- Rollet et al., 1991; Mehbub et al., 2018; Kim et al., 2019; Peng et al., 2020). Based on its ability to survive in a low metabolic state for extended periods of time when the nutrient pool is depleted (Nazly et al., 1980) and its capacity to utilize a wide range of substrates to outgrow competitors with its fast replication (Nazly et al., 1980; Benbouzid-Rollet et al., 1991), it is unsurprising that it was found to be an important founding member in marine biofilms (Nivens et al., 1986). Biofilms offer several advantages to microorganisms, especially since submerged surfaces provide significantly higher nutrient availability due to a combination of factors (Dang and Lovell, 2016). These biofilms provide almost O2-free microhabitats and often turn into multi-species consortia, harboring complementing bacteria (Dowling et al., 1988; Benbouzid-Rollet et al., 1991). In fact, V. natriegens can fix atmospheric

N2 in low oxygen environments (Coyer et al., 1996), possibly making it an important provider of this limiting nutrient in the ocean, as was postulated for its close relative V. parahaemolyticus (Criminger et al., 2007). V. natriegens also promotes the growth of the unicellular algae Euglena gracilis (Kim et al., 2019), which may be related to the presence of an auxin-producing gene cluster in V. natriegens

(Lee et al., 2019). In fact, in V. parahaemolythicus production of auxin was implied in growth stimulation of higher plants, such as sedges on the seashore or mangroves (Criminger et al., 2007) suggesting that this might also be the case for V. natriegens (Kim et al., 2019). While direct evidence for such growth-promoting interactions between V. natriegens and plants has yet to be identified, several interactions with marine animals are well-described. For instance, it was shown that the sponge Aplysilla rosea develops a more diverse microbiome when exposed to V. natriegens (Mehbub et al., 2018). This might help to protect the sponge from colonization by pathogenic microbes via niche exclusion. V. natriegens has also been found on bivalves, but here its effect is less clear, ranging from mutualistic to pathogenic interactions (Castro et al., 2002; Garnier et al., 2007). As mentioned above, for many marine animals, some V. natriegens strains act as a true or at least opportunistic pathogens, causing disease in several marine crustacea, most notably in the farmed swimming crab Portunus trituberculatus, in which V. natriegens has led to mortalities up to 85% (Bi et al., 2016). It has also been linked to deaths in echinoderms (Rivera-Posada et al., 2011). However, no infections of humans or other vertebrates have been reported to date, and it has been shown that the CTXΦ vibriophage carrying the toxins associated with V. cholerae could not efficiently replicate in V. natriegens (Lee et al., 2016). Other bacteriophages, on the other hand, have specialized on V. natriegens (Zachary, 1974; Clark et al., 2019). Some phages show a strong specialization towards a narrow range of environmental conditions (Zachary, 1978) and display preference for a certain osmolarity or temperature range (Zachary, 1976). These specializations may be exploited for the creation of phage- derived inducible promoters in the future, continuing a paradigm that has been extremely useful for E. coli in the past. Two chromosomally integrated prophages were recently reported in the V. natriegens type strain ATCC14048 (Pfeifer et al., 2019). The factor activating the lytic cycle was shown to be the induction of V. natriegens SOS response in hypo-osmotic conditions. This suggested that the dependence of the wild type (WT) strain on high Na+ concentrations in the media was mainly related to the fact that this prevents prophage induction. Indeed, after the removal of these phages from the chromosome, the new V. natriegens strain Δvnp12 was able to grow in reduced salt media, as well as out-compete the WT under several conditions (Pfeifer et al., 2019), suggesting this strain as a promising host for future biotechnological applications. Besides the interaction with plants and higher organisms, V. natriegens also grows on anthropogenic substrates where it can cause problems. For instance, V. natriegens colonizes several metal surfaces including steel and copper (Benbouzid-Rollet et al., 1991; Cheng et al., 2009) and a number of studies showed that V. natriegens biofilms on metallic surfaces increase their corrosion rates (Dowling et al.,

1988; Benbouzid-Rollet et al., 1991; Guo et al., 2019). Although it remains unclear which reactions exactly cause metal corrosion, it seems linked to anaerobic growth, as patches of corrosion co-localize with anaerobic zones in the biofilm (Yin et al., 2009). As copper was often used as an anti-corrosive plating for the submerged hull of ships and is still part of many antifouling paints, this V. natriegens’ corrosive properties are problematic for commercial shipping (Anisimov et al., 2019). Similarly, V. natriegens’ growth in large-scale steel bioreactors might lead to similar issues, calling for strain domestication efforts to mitigate corrosion in biotechnological applications. Another interesting route is suggested by recent observations showing that Bacillus subtilis growing in seawater can mitigate V. natriegens-induced corrosion of metals via a process called “bio-mineralization” – a thin film composed of extracellular polymeric substances and calcite, exhibiting stable anti-corrosion activity (Guo et al., 2019). These studies show that more research into the lifestyle, ecology and phage predation spectrum of V. natriegens are warranted, especially in view of the prospect of its large-scale application in bioreactors.

Metabolism As mentioned above, V. natriegens can utilize a wide range of substrates (Hoffart et al., 2017; Ellis et al., 2019) and according to API-stripe based tests performed by the German culture collection (DMSZ), V. natriegens can grow on various carbon sources, such as citrate, D-glucose, D-mannitol, fructose, glycerol, L-rhammnose, sucrose, amygdalin, L-arabinose, D-mannitol, N-acetyl-glucosamine, maltose, gluconate, caparic acid, malic acid, citric acid and phenyl-acetic acid. It also displays a range of enzymatic activities, such as gelatinase, cytochrome oxygenase, cytochrome oxidase and β-glucosidase activity, some of which might be important to thrive in their natural habitats. At the same time, it is capable of nitrate reduction (Rehr and Klemme, 1986), and, as stated above, possesses the ability to fix atmospheric nitrogen under anaerobic, nitrogen-limited conditions (Coyer et al., 1996). Notably, even in the absence of oxygen it still grows considerably faster than other bacteria under anaerobic conditions (Hoffart et al., 2017). When growing anaerobically, V. natriegens produces acids that lead to a drop in the pH of the medium, but when grown under strong aeration, the pH can rise in some media - most likely due to formation of ammonia (Payne et al., 1961; Eagon, 1962). V. natriegens’ uptake rates of many carbon sources were shown to be significantly higher than in other comparable species of bacteria (Hoffart et al., 2017). Long et al. (2017) applied 13C-labeled carbon tracking to elucidate the central carbon metabolism in minimal media with labeled glucose as carbon source. This analysis showed that the absolute carbon fluxes through the central carbon metabolic

pathways are much higher in V. natriegens compared to E. coli, with approximately 2.5-fold higher glucose uptake rates. However, in terms of relative fluxes the core carbon metabolism turned out to be very similar to that of E. coli (Figure 2), with the notable addition of one enzyme for the decarboxylation of oxalate (Long et al., 2017). The most striking difference however was a 33% reduced flux through the oxidative pentose phosphate pathway, but the cause and consequences of this remain to be elucidated (Long et al., 2017).

While high concentrations of dissolved O2 are required for the high oxygen uptake during rapid growth of V. natriegens (Lee et al., 2019), it is also reported to be more vulnerable to reactive oxygen species (ROS) stress than E. coli (Anand et al., 2020). Adaptive evolution has been used to create a strain that is more resilient to ROS, which is caused by a point mutation in oxyR encoding a widely conserved peroxide-sensing transcriptional regulator (Anand et al., 2020). Like other species in the genus Vibrio, V. natriegens cannot survive storage at 4°C for extended periods (Martinez et al., 2010; Mudoh et al., 2014; Weinstock et al., 2016). Weinstock et al. (2016) linked this to a reduced activity of its catalase at lower temperatures leading to a toxic buildup of ROS from the still active metabolism. They also showed that overexpression of the E. coli-derived katG can improve survival and thus long- term storage of V. natriegens at 4°C (Weinstock et al., 2016).

Genome organization and DNA repair Another intriguing question is how V. natriegens’ genome organization may contribute to the rapid growth phenotype, and how genome integrity is maintained under extreme cell doubling times below 10 min. The first genome sequences of V. natriegens became available in 2013 (Maida et al., 2013; Wang et al., 2013) and the first closed genome sequence was reported in 2016 (Lee et al., 2016). Automatic annotations resulted in 4578 CDS, 11 rRNA operons and 129 tRNA encoding genes (Lee et al., 2019). V. natriegens’ genome size of 5.16 Mbp is larger than the genome sequence of some other Vibrio species with a longer generation time (V. cholerae ~ 4 Mbp (Heidelberg et al., 2000), Vibrio fischeri ~ 4.2 Mbp (Ruby et al., 2005)). Given that both V. natriegens (Eagon, 1962) and the second- fastest growing species of the Vibrio genus, V. parahaemolyticus (Katoh, 1965), have rather large genomes (Jangam et al., 2018; Lee et al., 2019), the rapid growth phenotype thus cannot be attributed to a small, streamlined genome with a low number of protein-encoding genes. This is in accordance with the general finding that no positive correlation can be found between genome size and growth rate when comparing many species from distant taxa (Mira et al., 2001; Touchon and Rocha, 2007; Vieira- Silva and Rocha, 2010).

Species of the Vibrio genus almost exclusively have a genome contained on two circular chromosomes (Okada et al., 2005), with only rare exceptions (Xie et al., 2017; Yamamoto et al., 2018). Because bacterial chromosomes carry only single replication origins, the time needed for replication is directly dependent on the size of the respective chromosome. It takes for example about 40 min to replicate the 4.6 Mbp E. coli chromosome, corresponding to a replication speed of almost 1000 bps/sec, so replication cycles must overlap to allow for generation times faster than 40 min (Stokke et al., 2012). To allow for generation times of 10 min in V. natriegens, it thus seems plausible that splitting its 5.16 Mbp genome into two chromosomes, replicating in parallel, might help to accelerate genome duplication. However, when fusing both chromosomes of V. cholerae into a single replicon, only a minor reduction in growth rate was observed (Val et al., 2012), suggesting that genome duplication is not the bottleneck for rapid growth in this organism. However, it has not been tested if this is also true for V. natriegens, which grows considerably faster and has a larger genome, leaving room for speculation that a split genome could still convey selective advantages to this organism. The V. natriegens genome is indeed organized into a larger chromosome (Chr1) comprising 3.24 Mbp and 63% of all CDS, while the smaller chromosome (Chr2) has a size of 1.92 Mbp (Lee et al., 2016). To synchronize replication of the two chromosomes in Vibrio species, typically the primary chromosome dictates the replication start of the secondary chromosome through a Chr2 replication triggering site (crtS). Replication of crtS located on Chr1 will trigger the start of Chr2 replication (Baek and Chattoraj, 2014; Val et al., 2016). For V. natriegens, a crtS site matching the established consensus sequence is located about 640 kbps downstream of the replication origin (Kemter et al., 2018). This crtS location suggests that replication of the two V. natriegens chromosomes are regulated to terminate in synchrony as prevalent in Vibrionaceae (Kemter et al., 2018). Functional genomics based on a CRISPRi screen revealed 278 essential genes, with 272 located on Chr1, leaving only six essential genes on Chr2 (Lee et al., 2019). An additional 309 genes, termed growth-supporting genes, were identified as required for rapid growth in rich medium (Lee et al., 2019). One intriguing finding of the automatically generated gene annotation was that V. natriegens possesses many gene duplicates of essential and growth-supporting genes. These duplicates tend to be located on Chr2, while the essential or growth-supporting copy of the gene is mostly located on Chr1 (Lee et al., 2019). The low number of essential genes and the presence of gene duplicates suggest Chr2 as an “evolutionary test bed”, as has been hypothesized for other Vibrio species and more distant taxa (Cooper et al., 2010). Interestingly, while an initial transposon mutagenesis approach did not result in mutants with faster growth than the wild type (Lee et al., 2016), a competition assay during the CRISPRi screen

(Lee et al., 2019) revealed a small number of genes with positives beta scores, indicating that their dCas9-induced knock-down led to faster growth, shorter lag phase or higher final cell density compared to cells not expressing dCas9. While these genes might add to our foundational understanding of the rapid growth phenotype and serve as starting points for the development of an even faster-growing V. natriegens strain, it has to be carefully investigated if this observation is limited to repression by CRISPRi, or if similar results can be obtained for a strain carrying a clean deletion of the respective genes. Some studies were carried out to investigate V. natriegens’ DNA repair mechanisms. It was found that V. natriegens possesses both the “light repair mechanism”, as well as the “dark repair mechanisms” which are well understood in E. coli (see (Weber, 2005; Kisker et al., 2013) for reviews about DNA repair mechanisms in E. coli). The studies carried out with V. natriegens suggest that DNA repair mechanisms function qualitatively similar as in E. coli, and V. natriegens’ genes can complement homologous genes of E. coli mutants (Joux et al., 1999; Booth et al., 2001a; Booth et al., 2001b; Delpech, 2001). However, in case of uvrB, which encodes a protein involved in nucleotide excision repair, it was found that the gene is constitutively expressed in V. natriegens and not induced by UV irradiation as in E. coli (Simons et al., 2010). Thus, it is tempting to speculate that the constitutive expression of uvrB could be related to a higher demand of DNA repair during ultrafast growth of V. natriegens, but further experiments are needed to shed light on this.

Translational machinery Besides its genome organization, another possible explanation for V. natriegens’ rapid growth could be its high number of ribosomes (Aiyar et al., 2002). In fact, the abundance of ribosomes and the growth rate of a bacterium are known to correlate linearly (Scott et al., 2014). V. natriegens was estimated to contain 115,000 ribosomes per cell when growing exponentially with a generation time of 15 minutes, compared to E. coli with 70,000 ribosomes at a generation time of 25 minutes (Aiyar et al., 2002). The high number of ribosomes may be due to 11 rRNA operons (10 on Chr1, 1 on Chr2), which are located close to the chromosomal origins of replication (ori) on both Chr1 and Chr2 (Lee et al., 2019). We note that 12 rRNA operons were reported in Weinstock et al. (2016). During multifork-replication the high copy-number of the rRNA encoding operons will even be further amplified, which may be key to provide sufficient rRNAs for ribosome biogenesis. Some rRNA promoters from V. natriegens were characterized in vitro and in vivo in E. coli (Aiyar et al., 2002), showing that their strength is at least as high as the rRNA promoters of E. coli and that they also possess upstream promoter elements which

strongly contribute to the strength of rRNA promoters. Thus, it seems that 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 (Aiyar et al., 2002), but in V. natriegens the number of active rRNA operons – and thus ribosomes – are higher than in E. coli.

Cell-free expression systems and protein production in vivo The high abundance of ribosomes of V. natriegens suggests a high capability for protein production and therefore tremendous potential for the expression of heterologous proteins. Within a time-frame of just five months, three groups published cell-free expression systems based on V. natriegens (Des Soye et al., 2018; Failmezger et al., 2018; Wiegand et al., 2018). These studies focused on different parameters for protocol optimization, including cultivation medium, growth phase for cell harvesting, reaction temperature, buffer concentrations, supplementing amino acids, energy source and the origin of the DNA template (organism used for plasmid amplification, linear or circular DNA). With regard to the growth phase for harvesting cells for the preparation of the expression extracts, contradictory observations were made. While Wiegand et al. (2018) reported highest yields with cells from mid- exponential phase, Des Soye et al. (2018) found best performance when extracts were prepared from cells in stationary phase. The theoretically achievable yield from cell-free expression extracts was calculated based on the abundance of rRNAs, however the experimentally obtained yields remained lower (Failmezger et al., 2018). It was speculated that this is due to a fraction of ribosomes which remains inactive during the reaction (Failmezger et al., 2018). Thus, it might be conceivable that the conflicting results regarding the timepoint for cell harvesting are not only due to different abundance, but also different activity of ribosomes in the different growth phases. A first attempt to optimize the expression strain itself was made by deleting genes which have been targets for the optimization of cell-free expression systems based on E. coli lysates (Des Soye et al., 2018). Unfortunately, no significant improvement could be observed, leaving room for future investigations (Des Soye et al., 2018). The cell-free expression platform was utilized to test for promoter sequences (Failmezger et al., 2018) which could allow for rapid screening for regulatory sequences and thereby help to extend the range of available tools for V. natriegens (Des Soye et al., 2018). While variants of the green fluorescent proteins were expressed during the process of protocol optimization (Des Soye et al., 2018; Failmezger et al., 2018; Wiegand et al., 2018), the production of antimicrobial peptides was shown as well (Des Soye et al., 2018).

In addition to cell free approaches, V. natriegens was also utilized for the production of proteins in vivo. Multi-subunit membrane proteins of V. cholerae were successfully expressed in V. natriegens, suggesting the latter as a safer expression host for studying proteins from phylogenetically related pathogens (Schleicher et al., 2018). In addition, “difficult proteins”, which are insoluble or only yield low quantities in other expression systems, have been successfully expressed in the commercial V. natriegens strain VmaxTM express (Weinstock et al., 2016; Eichmann et al., 2019). Furthermore, cytosolic proteins were shown to secrete to the periplasm by adding the N-terminal secretion tag ssYahJ from E. coli, which might enhance the applicability of V. natriegens for large scale applications (Eichmann et al., 2019).

Genetic tools and tractability A number of tools for genetic manipulation of V. natriegens have been established in recent years (Figure 3). To date the two most commonly used methods for genome engineering rely on homologous recombination, but differ in the DNA transformation method and the selection procedure used. The first method uses conjugation to transform plasmid DNA from an E. coli donor in V. natriegens, followed by allelic exchange via double crossover recombination (Lee et al., 2016; Weinstock et al., 2016). This method currently requires extended screening by negative selection against a plasmid-encoded ccdB toxin gene (Lee et al., 2016; Weinstock et al., 2016; Lee et al., 2017), but has enabled the deletion of large genomic regions (up to 194 kb through sequential rounds (Weinstock et al., 2016)) and the integration of a ~6 kb transcription unit expressing an inducible T7 RNA polymerase (Weinstock et al., 2016). The second method is called multiplex genome editing by natural transformation (MuGENT) (Dalia et al., 2014; Dalia et al., 2017b; Dalia et al., 2017a), which, as the name suggests, exploits V. natriegens’ natural competence to take up DNA from the environment. Although the mechanism of natural competence (Lorenz and Wackernagel, 1994; Dubnau and Lovett, 2002; Johnston et al., 2014) has not yet been clarified in V. natriegens, its reliance on the competence master-regulator TfoX (Meibom et al., 2005; Dalia et al., 2017b) suggests a system analogous to that of V. cholerae (Meibom et al., 2004; Meibom et al., 2005; Matthey and Blokesch, 2016). The MuGENT method operates under the premise that during competence-inducing conditions only a subpopulation of cells is transformable, but able to take up DNA with a high affinity (Dalia et al., 2017b). In fact, by co-incubating cells with multiple transforming DNA (tDNA) fragments, where each tDNA fragment contains 3 kb flanks of homology on both sides of a desired mutation, but only one mutation introduces a kanamycin resistance marker (Δdns::KanR), multiplexed editing was faithfully achieved for several loci simultaneously (Dalia

et al., 2017b). With this technology the Dalia group successfully increased the production rate of poly- β-hydroxybutyrate (PHB) by ~100 times in only one week, targeting nine involved genes (Dalia et al., 2017b). Further genome engineering is expected to increase the recombination efficiency and to reduce the length of the required homologous flanks (Marvig and Blokesch, 2010; Dalia et al., 2017a). A third method for genome engineering in V. natriegens is based on the combination of the λ-phage system (Datta et al., 2008) with proteins of the SXT mobile element from V. cholerae (Albert et al., 1993; Waldor et al., 1996; Chen et al., 2011), which are homologous to the λ-phage proteins (Lee et al., 2017). When upregulated, λ-Red, SXT-Beta and SXT-Exo were shown to potentiate allelic exchange due to homologous recombination between tDNA and a target gene sequence by ~10,000-fold (Lee et al., 2017). An interesting alternative to enable the generation of directed mutations could become available through further development of a CRISPR-Cas9 system (Jinek et al., 2012; Hsu et al., 2014) for V. natriegens. Previously, Lee et al. (2019) could not detect any transformants following electroporation of a plasmid encoding for the Cas9 nuclease and with a GFP-targeting gRNA, even without induction of cas9 expression, indicating significant toxicity due to double-stranded breaks and undetectable levels of non-homologous end joining (NHEJ). Therefore, they concluded that the current CRISPR–Cas9 system alone cannot be used for efficient generation of mutants by NHEJ (Lee et al., 2019). In studies with E. coli, λ Red-based recombineering with CRISPR-Cas9 counterselection (Reisch and Prather, 2015) has resulted in a powerful genome engineering tool, suggesting that future efforts in V. natriegens might yield a similar range of applications. Another missing capability is the convenient integration of whole plasmids into the genome. A common strategy for this task in E. coli uses phage- recombinase mediated site-specific recombination (Haldimann and Wanner, 2001; Vecchione and Fritz, 2019). Again, trying to transfer well-established methods developed for E. coli into V. natriegens may represent a promising starting point. Positive selection for genomic integration or the replacement of endogenous sequences was achieved with the help of antibiotic resistance cassettes (Weinstock et al., 2016; Dalia et al., 2017b; Lee et al., 2017). In a subsequent step, the Cre-loxP system (Sternberg and Hamilton, 1981; Grindley et al., 2006) was applied for excision of the marker, allowing recycling and going for multiple rounds of genome engineering (Weinstock et al., 2016). SacB and CcdB have been applied as markers for negative selection (Weinstock et al., 2016; Hoffart et al., 2017; Pfeifer et al., 2019). However, an early study indicates that the presence of sodium ions, which are highly abundant in V. natriegens growth media, greatly diminishes the toxicity of sucrose toward SacB-expressing cells (Blomfield et al., 1991). Thus,

even though the use of SacB has been successfully used as a negative selection marker (Hoffart et al., 2017; Pfeifer et al., 2019) it might not be the ideal strategy for negative selection in V. natriegens. During the development of genome engineering methods, several strains have been constructed, typically starting with the deletion of the exonuclease dns as proof of concept (Weinstock et al., 2016; Dalia et al., 2017b). As an alternative to ATCC 14048, the company SGI-DNA provides the commercially available strain VmaxTM express, optimized for highly efficient transformation and protein expression (Weinstock et al., 2020). This strain lacks genes predicted to be mobile elements or to be involved in restriction modification systems (Weinstock et al., 2016; Weinstock et al., 2020). In total over 150 genes have been deleted, 59 of which encode hypothetical proteins of unknown function (Weinstock et al., 2020). Because the genome of VmaxTM express is not published and its further modification is counter-indicated by the terms of sale, it is difficult to further develop VmaxTM into a widely accessible synthetic biology platform. However, this strain seems to perform very well for plasmid transformation and protein production (Eichmann et al., 2019). Transformation of V. natriegens with plasmid DNA has been successful using several methods (Figure 3). Many commonly used plasmids are stably maintained in V. natriegens (Lee et al., 2016; Dalia et al., 2017b; Tschirhart et al., 2019), including the broad host-range replicon RSF1010 (Jain and Srivastava, 2013; Lee et al., 2016), as well as p15A and pMB1 derivatives (Weinstock et al., 2016; Tschirhart et al., 2019) and even large plasmids with ~100 kb were successfully transformed (Weinstock et al., 2020). Currently, the most efficient transformation method in V. natriegens is via electroporation, with efficiencies ranging between 2x105 and 107 CFU/µg DNA, depending on the protocol used (Lee et al., 2016; Weinstock et al., 2016). Thus, as little as 10 ng of DNA is sufficient for successful transformation (Lee et al., 2016), thus also enabling the direct transformation of Gibson Assembly mixes (Weinstock et al., 2016). A more convenient, but less effective method is heat shock transformation with chemically competent cells, leading to efficiencies between 105 and 106 CFU/µg (Weinstock et al., 2016; Tschirhart et al., 2019). While the protocol of Tschirhart et al. (2019) enables heat shock transformation in the V. natriegens WT strain ATCC14048, Weinstock et al. (2016) showed that deletion of the gene encoding exonuclease Dns (homologous to the EndA nuclease in E. coli (Lin, 1992)) increases the transformation efficiency drastically. The last option is transferring plasmids via conjugation, which is effective, but also the most time-consuming of the available methods (Lee et al., 2016; Weinstock et al., 2016; Tschirhart et al., 2019). Evaluating the relative efficiencies of these methods and protocols will require further replication from diverse workers under standardized conditions. Additionally, the use of different sizes of genetic constructs for characterization can have

an impact on transformation efficiency (Chan et al., 2002). However, it is clear that transformation in V. natriegens is not reaching efficiencies of E. coli yet, but most methods work and genome engineering or further optimization of protocols may further increase efficiencies (Weinstock et al., 2016). Highly efficient cloning strains and optimized protocols will be key for V. natriegens to replace E. coli as routine cloning host. An important consideration for switching from an established model organism to V. natriegens is the transferability of genetic constructs. One decisive factor for this transferability is the codon usage of the organism, which influences the expression levels and the folding of encoded proteins (Quax et al., 2015). Based on the codon usage table published in Lee et al., 2016, we compared the codon usage frequencies (occurrence of one particular codon per 1000 codons) between V. natriegens and E. coli (Figure 4), which we consider one of the most widely used sources for transferring genetic constructs to V. natriegens. While the strong correlation suggest that constructs built for E. coli are usually functional in V. natriegens, codon usage frequencies can deviate up to 2-fold for some codons (Figure 4) and we have observed significant improvements in protein production levels in V. natriegens when using codon-optimized sequences (D. Stukenberg et al., unpublished). Initial experiments have been made to characterize the behavior of individual genetic parts in V. natriegens and therefore provide quantitative data for the rational design of complex genetic circuits (Lee et al., 2016; Weinstock et al., 2016; Tschirhart et al., 2019). Until now, a number of inducible

promoters have been established, including Ptet, PBAD, a number of IPTG-inducible parts and the temperature-inducible λ PR (Lee et al., 2016; Weinstock et al., 2016; Tschirhart et al., 2019). Additional genetic features, such as short constitutive promoters, ribosomal binding sites, the RiboJ ribozyme, transcriptional terminators and protein degradation tags have been shown to be functional in V. natriegens (Tschirhart et al., 2019). Many frequently-used antibiotic resistance cassettes have been successfully tested, e.g. for chloramphenicol, kanamycin, ampicillin, carbenicillin and tetracycline (Lee et al., 2016; Weinstock et al., 2016; Dalia et al., 2017b; Tschirhart et al., 2019). However, drastically different working concentrations have been reported (Figure 5), probably due to the wide range of copy numbers between genomic integrations (Dalia et al., 2017b) and high-copy plasmid origins (Tschirhart et al., 2019).

Industrial Potential The ability of V. natriegens to utilize a wide range of substrates, its high substrate uptake rate (Hoffart et al., 2017) and an increasing number of tools for its genetic manipulation have sparked ambitions to

turn V. natriegens into a new industrial bioproduction workhorse (see Figure 6 for an overview). However, while its ultra-rapid growth may indeed streamline strain optimization – by minimizing the cycle times of metabolic engineering – its high specific growth rate might be disadvantageous for bioproduction purposes. In fact, theoretical and empirical studies agree that the biomass yield of a bacterial culture is often optimal at intermediate growth rates and that rapid growth can lead to overflow metabolism, futile metabolic cycles and high protein synthesis costs, reducing the attained biomass yield (Lipson, 2015). To explore the capacity of V. natriegens for bioproduction purposes, Hoffart et al. (2017) aimed at turning the organism into a highly effective alanine producer. They demonstrated that four deletions were sufficient to redirect the metabolism in a way that highly engineered and specialized E. coli and C. glutamicum alanine-producing strains were outperformed by a factor of 9 and 13, respectively. Similarly, a recent publication described how the heterologous expression of a tyrosinase gene was used to produce melanin, reporting faster melanin production than in established expression systems (Wang et al., 2020). Also, V. natriegens naturally prepares for starvation by storing carbon in polyhydroxyalkanoate (PHA) granules, allowing isolation of PHA as raw material for the production of biodegradable plastics (Chien et al., 2007). Co-cultivation of Euglena gracilis, an algal production host for value added compounds, with V. natriegens ATCC14048 increased algal paramylon content and biomass by 35% and 20%, respectively. This was induced by the production and secretion of indole- 3-acetic acid by V. natriegens, an auxin hormone which stimulates growth in E. gracilis (Kim et al., 2019). Over the last centuries, the number of soils contaminated by various chemicals and metals as a result of mining, heavy industry and urbanization has increased dramatically (Khalid et al., 2017). Remediation of affected soils is often very expensive and labor- and energy-intensive (Gong et al., 2018). One common pollutant is selenium, which is toxic to many species of bacteria in high concentrations (Gong et al., 2018). V. natriegens has one of the highest tolerances to selenium contamination so far reported (Fernández-Llamosas et al., 2017). It achieves this by reducing the dissolved selenite in a two-step process to metallic selenium and sequestering it in selenium nanoparticles with a size of 100-400 nm (Fernández-Llamosas et al., 2017). With this, V. natriegens is the most effective producer of biogenic selenium nano-particles known to date. Apart from the potential for the removal of polluting selenite, these particles have many industrial and medicinal applications, for instance in chemotherapy (Fernández-Llamosas et al., 2017).

Perhaps the most compelling arguments to consider V. natriegens for large scale adaptation in industry are those described in the sections above. The rapid growth rate combined with simple, fast methods for genetic tractability have the potential to shorten the design-build-test cycles of synthetic biology and bioengineering (Eichmann et al., 2019). The fast growth could also be exploited to shorten the timespan for adaptive laboratory evolution to take place, and in one case a strain with a higher tolerance towards reactive oxygen species was engineered (Anand et al., 2020). This ability allows fully optimized V. natriegens production strains to become available much more quickly than for competing production strains. These and future efforts in strain engineering will help realize the full potential of V. natriegens for industrial applications.

Conclusions and outlook The last 60 years of research have revealed exciting insights into the basic physiology and ecology of the ultrafast-growing bacterium V. natriegens. This has culminated in the establishment of a number of genetic tools and methods that transformed this marine mud bacterium into a rising star in the synthetic biology arena (Lee et al., 2016; Dalia et al., 2017b). Also, the close taxonomic distance to the human pathogens V. parahaemolyticus and V. cholerae make V. natriegens an ideal candidate as safe and convenient model organism for the Vibrio genus, which might provide important insights transferable to these human pathogens and therefore help in the prevention and treatment of diseases (Sawabe et al., 2013; Schleicher et al., 2018). Based on its remarkable growth speed V. natriegens may not only become the new chassis organism for molecular cloning, but also an important host for biotechnological applications. Although it has been proposed that V. natriegens might “dethrone” E. coli as cloning and protein production host in the future, it should not be forgotten that V. natriegens is more than just a new, fast-growing version of E. coli. Many gaps still exist in our knowledge about its unique biology and many more methods have to be established and optimized to fully leverage the potential of V. natriegens as novel laboratory workhorse. One of the most prominent open questions is related to the factors that allow V. natriegens to grow so rapidly. It appears as if individual lines of research, e.g. into the ribosome count (Aiyar et al., 2002) or the metabolism of V. natriegens (Hoffart et al., 2017) cannot provide a conclusive answer to this question, suggesting that the rapid growth phenotype could be an emergent, “systems-level” property that requires many factors playing together, including rapid nutrient influx rates, high fluxes through central metabolism, high ribosome content, etc. To close some of these knowledge gaps, it will be important to perform comprehensive proteomics and metabolomics analyses, which would constitute a

valuable resource for the emerging V. natriegens research community. Also, studying the genome organisation under these ultrafast growth rates and to investigate the regulation of the translational machinery under varying growth conditions will be highly interesting. For the purpose of using V. natriegens as molecular cloning host, further optimization and acceleration of transformation protocols will be beneficial. Given that to date the engineered strains of E. coli still feature higher transformation efficiencies than V. natriegens, optimizing this parameter is key to clone more difficult DNA assemblies in V. natriegens. In the future, V. natriegens also holds enormous potential to speed up directed evolution protocols as well as methods like multiplexed automated genome editing (MAGE) (Wang et al., 2009), since both of are highly time consuming due to repetitive cycles of exponential cell growth and dilution. Clearly, V. natriegens could reduce the cycle time drastically, thereby accelerating the optimization of synthetic pathways and protein expression.

Acknowledgements This work was supported via the German Federal Ministry of Education and Research (grant no. 031L0010B) funded in the framework of the ERASynBio initiative (G.F.), by the International Max Planck Research School for Environmental, Cellular and Molecular Microbiology (D.S.), and a grant of the Deutsche Forschungsgemeinschaft (grant no. WA2713/4-2) (T.W.). The authors declare that there are no conflicts of interest.

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Figure legends

Figure 1. Cladogram indicating the position of V. natriegens in the tree of life. V. natriegens belongs to the Vibrio genus in the family of the Vibrionaceae, which is the only family in the order of the Vibrionales (Sawabe et al., 2013). Within the Vibrio genus, it belongs to the Harveyi clade, a group of mostly marine bacteria, typically with short doubling times (Sawabe et al., 2013). V. parahaemolyticus is the most closely related species. The Vibrionales belong to the class of the Gammaproteobacteria, which contains many species of economical and scientific importance (Ciccarelli et al., 2006).

Figure 2. Carbon flux in the central carbon metabolism of V. natriegens. The flux for each reaction is indicated through the thickness of the arrows. Fluxes were normalized to a glucose uptake rate of 100. The relative change in flux between V. natriegens and E. coli is indicated through the colours and is given next to each reaction in % change. Only changes of >5% were coloured. The underlying data is based on (Gonzalez et al., 2017; Long et al., 2017).

Figure 3. Currently established molecular biology techniques in V. natriegens. These methods comprise DNA transformation methods, such as electroporation, heat-shock transformation and conjugation, as well as genome engineering techniques, such as recombineering, multiplexed genome editing via natural transformation (MuGENT) and the use of the CRISPR-Cas9 system. Please refer to the main text for details.

Figure 4. Comparison of codon usage between V. natriegens and E. coli. Each circle represents the frequency of one codon in V. natriegens and E. coli. Frequency is defined as the occurrence of one particular codon per 1000 codons in protein-coding sequences within the genome. The codon usage table for V. natriegens ATCC14048 was taken from (Lee et al., 2016). The codon usage table for E. coli was obtained from https://kazusa.or.jp/codon/.

Figure 5. Overview of various antibiotic concentrations (µg/mL) used in different papers for the selection of V. natriegens after transformation. *Eichmann et al. (2019) used the VmaxTM express strain, while the other work cited in this table is based on V. natriegens ATCC14048. **Gentamicin is not recommended for selection since more than 30 µg/mL are required (Tschirhart et al., 2019).

Figure 6. Biotechnological applications of V. natriegens as a cell factory. These applications comprise the production of a range of small molecules, proteins, biopolymers and nanoparticles from inexpensive carbon sources. Please refer to the main text for details.

Figure 1. Cladogram indicating the position of V. natriegens in the tree of life. V. natriegens belongs to the Vibrio genus in the family of the Vibrionaceae, which is the only family in the order of the Vibrionales (Sawabe et al., 2013). Within the Vibrio genus, it belongs to the Harveyi clade, a group of mostly marine bacteria, typically with short doubling times (Sawabe et al., 2013). V. parahaemolyticus is the most closely Article related species. The Vibrionales belong to the class of the Gammaproteobacteria, which contains many species of economical and scientific importance (Ciccarelli et al., 2006). Accepted

For Peer Review Only Article

Figure 2. Carbon flux in the central carbon metabolism of V. natriegens. The flux for each reaction is normalized to the total glucose influx and indicated through the thickness of the arrows. Fluxes were normalized to a glucose uptake rate of 100. The relative change in flux between V. natriegens and E. coli is indicated through the colours and is given next to each reaction in % change. Only changes of >5% were coloured. The underlying data is based on (Gonzalez et al., 2017; Long et al., 2017). Accepted

Article

Figure 6. Biotechnological applications of V. natriegens as a cell factory. These applications comprise the Article production of a range of small molecules, proteins, biopolymers and nanoparticles from inexpensive carbon sources. Please refer to the main text for details. Accepted