Proof of Concept

Overview of Our POC

Our goals are developing efficiently enabling technology for high efficiency strain engineering and next-generation biotechnology based on Vibrio natriegens and seawater fermentation. In We achieve this in thiee different ways:

  1. Established a golden gate collection for cross-strain expression, we designed synthetic promoter for bacteria and yeast expression
  2. Established a platform strain for high level synthesis of FPP, and the production of our downstream production lycopene increased 13 times, meaning that our platform is very efficient
  3. Demonstrate the feasibility of seawater fermentation for Vibrio natriegens.

Golden gate collection that enables rapid production validation across strains

The Marburg Collection, proposed by the 2018 Marburg team, has well-established prokaryotic expression elements. To expand its cross-species functionality, we propose ONCE_Collection with the following modifications:

  • 1. introduce hybrid promoters compatible with prokaryotes and brewer's yeast to the Promoter module, we characterized these parts, demenstrated our concept;
  • 2. add short synthetic terminators for yeast to the terminator module. There is some data from reference, and this parts can work well;
  • 3. add elements for splice transfer and replication in yeast to the ORI module, we characterized these parts, demenstrated our concept;
  • 4. add enzymes for rapid genome integration to the CDS module, we characterized these parts, demenstrated our concept;
  • 5. add recombinase recognition module and yeast homologous recombination module to the connector, we characterized these parts, and there is some data from reference, those two demenstrate our concept;
  • 6. add nutritional defect screening tools to the antibiotic resistance module. There is some data from reference, and this parts can work well;
  • 7. introduce inducible promoters and corresponding regulators, we characterized these parts, demenstrated our concept.

Promoters and terminaors that are compatible with yeast and prokaryotic organisms

We crossed the constitutive promoters of the J23 family (J23119, J23100, J23104, J23113) in prokaryotes as well as plac with several artificially designed promoters (TE1, TE2, TE3, TE4, TE5) in yeast that have been recently reported in the literature. These data suggest that our design worked, and although these synthetic promoters may differ in intensity from when they work alone, at the functional level this can be left out of the discussion.

Figure 1 Plasmid profiles and results for testing synthetic promoters in E. coli and Saccharomyces cerevisiae.

ORI compatible with splice transfer, yeast shuttle

The combination of OriT with other ORIs allows ORIs of other copy numbers to shuttle between E. coli and non-model bacteria. Further addition of CEN/PK or 2u ORIs capable of replication in yeast to the backbone would enable shuttling in yeast.

Figure 2 ORI combinations that can be conjugated and work in yeast and E. coli.

We first constructed the three combinations as above and tested them with in DH5a. It can be found that for each ORI, together with OriT as well as CEN/PK replicas, clones can be successfully constructed and none of them affect the expression function in E. coli.

Figure 3 Characterization data of different ORI combinations in E. coli DH5α.

We next tested whether five OriT combination ORIs could be used for conjugation and the copy number changes in the host bacteria after conjugation. All OriT combinations were successfully transferred for splicing.The combination of these five ORIs, together with pBBR1, enables a broader spectrum of non-model organism gene expression.

Figure 4 Conjugation ORI working in Nissle 1917

Efficient strategies for rapid genome integration

During the actual efficiency test, we first recombined a segment of the CmR expression frame with a fragment with attB1-attB2 and lox66-lox71 in the UTR region by TnsABCD at MG1655 as well as the attTn7 site of V.nat, and a sequence with a bidirectional terminator was recombined, and the successfully recombined bacteria could not express CmR normally and then could not be grown on CmR plates. The specific efficiency can be obtained by counting CFU. Similarly, this expression frame was inserted at the attB site by PhiC31 and the attTn7 site was set for testing the recombination efficiency of TnsABCD.
We tested PhiC31 with different expression intensities and the same incubation time, the TnsABCE and Cre at different expression intensities and the same incubation time. We found that the recombination efficiency of recombinase was positively correlated with the expression intensity within a certain range, while the recombination efficiency decreased when the expression intensity continued to increase, which might be due to the cytotoxicity caused by recombinase. Among them, Cre has the lowest recombination efficiency, probably because it occurs as a reversible exchange.

Fig 5 Integration efficiency of different recombinant enzymes at different expression intensities

inducible promoter and the corresponding regulator

As we mentioned in the Human practice module, we systematically characterized the leaked expression of the marionette promoter for better metabolic engineering applications in V.nat. First, we mounted the device of the inducible promoter on a plasmid with ORI pBBR1 and transferred it into V.nat. We found that the leakage expression of PbetI was moderate, and we judged that it was more suitable for Plac for the leakage expression of metabolic pathways in V.nat.

On the other hand, some of the inducers in the marionette collection are probiotic small molecules that can be used in the genetic circuit in the probiotic Nissle 1917. We tested the response of the food additive vanillic acid response promoter PvanCC and found that its activation ploidy was low compared to the original literature, and to further increase its activation ploidy, we used the literature-reported, RNA-coordinated THS strategy to increase the activation ploidy from approximately 20-fold to 34-fold.

Fig 7 Inducer response of PVan_CC and PVan_CC_THS

A general strategy for the construction of high FPP production and downstream terpenoid chassis strains

We plan to improve FPP precursor synthesis as well as downstream product production by exogenously expressing the MVA pathway, incorporating an artificially designed IUP pathway, and through a modular strategy of increasing the intracellular hydrophobic environment.
We characterized the supply of FPP precursors by lycopene synthesis. Lycopene is inherently colorful and can be used for rapid characterization, with a good linear relationship between its concentration and absorbance. We tested the feasibility as well as the efficiency of our strategy in E. coli DH5a.
First we tested the effect of adding the MVA pathway on lycopene production. We found that the MVA pathway significantly enhanced the production of lycopene. Next, we added the artificially designed IUP pathway, selecting two enzymes previously reported in the literature, PhoN and IPK, and added DMAOH and IOH to the medium, which significantly increased lycopene production at appropriate concentrations.
Finally, we tested the strategy of increasing the intracellular hydrophobic environment, which again increased the lycopene production. The lycopene production was increased approximately 13-fold compared to our most primitive strain.

Fig 8 Validation of the strategy for high yield of FPP and downstream terpenoids

The strategy we designed has great potential for efficient synthesis of terpenoids.

FPP sensing system based on split fluorescent protein

Although we used lycopene synthesis to characterize FPP supply, given the complex FPP metabolic network within bacteria, we further developed a sensing system that directly binds FPP and emits fluorescence (FPR) and validated the feasibility of this system in E. coli DH5α as well as the optimal detection time.

Limited by the lack of available FPP monomers, we used DH5α that had been characterized with the MVA pathway transferred as a positive control and DH5α with only the MVA pathway as a negative control. As shown in the result graph, FPPR has only a small background fluorescence intensity in wild-type DH5α, which is due to the fact that E. coli naturally contains the MEP pathway for FPP synthesis and low levels of FPP concentration exist in the bacteria. In contrast, the addition of the MVA pathway greatly increased the fluorescence signal intensity of FPPR (~3.6-fold), demonstrating the responsiveness of our reporter system to the concentration of FPP.

Validation of strategy for high yield FPP and downstream terpenoids

We next further validated the delay of the system by testing the variation of the fluorescence intensity of DH5α containing MVA as well as FPPR with bacterial growth, and the results showed that the fluorescence intensity started to increase at approximately K/2, with a significant fluorescence signal appearing after the bacterial growth entered a stable phase, and eventually stabilized as the bacteria entered a plateau phase. This is similar to the concentration change of FPP secondary metabolism, further demonstrating the usability and stability of our sensor in the secondary metabolism phase.

Figure 10 The delay of the FPP sensing system

Production of (-)-α-bisabolol

Further, to verify that our FPP high-yielding strain could be used correctly for the production of our intended product, (-)-α-bisabolol, we tested its yield after 60 h of batch fermentation and optimized the fermentation conditions, using wild-type DH5α containing only the endogenous MEP pathway as a control. After several attempts, we identified the strong solvent signal in the n-dodecane used for extraction, and after changing the loading volume and removing the solvent peak, we observed a 2.8-fold increase in the yield of DH5α containing MVA compared to the wild type.

We analyzed that this might be due to the lack of carbon source in the medium at the later stage of the fermentation, so we further replaced LB medium with carbon rich TB medium and this time we observed a 3.8-fold increase compared to the wild type.

In order to produce (-)-α-bisabolol more efficiently, we verified the expression of bacterial ccBOS using Coomassie Blue staining and found that although ccBOS expression was higher at 30°C compared to bacteria grown at 25°C, it was mostly concentrated in the cellular sediment, which we hypothesized was due to too rapid synthesis of ccBOS and failure to fold correctly. We therefore lowered the fermentation temperature to 25°C, and this time we achieved a 5.7-fold increase in yield, demonstrating that our optimized strain as well as fermentation conditions can be used for efficient (-)-α-bisabolol production.

Figure 11 Coomassie Blue staining of ccBOS Figure 12 Yield of (-)-α-bisabolol under different fermentation conditions

Sea water cultivation of V.nat

Sustainable and environment-friendly microbial fermentation processes have been developed to produce numerous chemicals. However, the high energy input required for sterilization and substantial fresh water consumption restrict the economic feasibility of traditional fermentation processes, It also runs counter to the goal of carbon neutrality.


To address these problems, Vibrio natriegens, a promising microbial chassis with low nutritional requirements, high salt tolerance and rapid growth rate can be selected as the host for chemical production. This bacterium is non-pathogenic and belongs to the biosafety level 1.

We use non-sterilized fermentation of Vibrio natriegens with seawater, to systematically verify the feasibility of the project, we plan to collect seawater from China Major sea areas and two major marine economic species farming wastewater。for non-nterilized fermentation.

This experiment has been submitted to check-in form and approved.We ensure that the obtained seawater does not violate local laws and regulations, the water source does not involve the epidemic area. The water intake of each water source shall not exceed four liters and the intaking will not affect the local natural ecology. We guarantee that the obtained seawater is safely and properly stored and used in the laboratory and treated in a biosafety manner after the experiment.

Sampling name Sampling locations longitude and latitude
Hainan Baishamen Park, Meilan District, Haikou City 110.341546,20.076778
Xiamen White City Beach, Siming District, Xiamen 118.109729,24.438016
Shenzhen Shenzhen Bay, Nanshan District, Shenzhen 114.003481,22.52837
Yancheng Moon Bay, Binhai County, Yancheng City 120.274268,34.316129
H.americanus; P.trituberculatus aquaculture wastewater Yangpu District, Shanghai 121.479965,31.238646
Dalian Dayaowan Port, Jinzhou District, Dalian 121.85914,39.029128
Figure12 Sampling site location

To systematically validate the potential of non-sterile fermentation of sodium-demanding Vibrio seawater, we used seven natural seawater or marine aquaculture wastewater as substrates, adding carbon sources under normal culture conditions without additional sodium chloride. Depending on whether the formulated medium was added with or without kana, the autoclaved steam was non-sterile or non-sterile divided into four groups. Bacterial growth is monitored in real time by testing OD600.

Fig13A General overview of seawater fermentation growth curves Figure13B Culture curve of Shenzhen seawater Figure13C Culture curve of Hainan seawater Figure13D Culture curve of P.trituberculatus farming wastewater Figure13E: Culture curve of H.americans farming wastewater Figure13F: Culture curve of Shenzhen seawater Figure13G: Culture curve of Xiamen seawater Figure13H: Culture curve of Yancheng seawater Figure13I: Culture curve of Selected seawater compared with LBV2

The growth curves of water samples from different regions were significantly different, and we speculate that this may be due to differences in the physicochemical properties of seawater in different regions. To further investigate the reason, we examined the salinity, pH, nitrite, phosphate, Ca2+, Mg2+, total hardness, total alkalinity of all water samples, and the results are summarized below, which in turn facilitate subsequent reference for the selection of seawater non-sterilized fermentation water sources.

 Figure14 The physicochemical properties (salinity, pH, nitrite, phosphate, Ca2+, Mg2+, total hardness, total alkalinity) of different seawater