Results

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;
• 2. add short synthetic terminators for yeast to the terminator module;
• 3. add elements for splice transfer and replication in yeast to the ORI module;
• 4. add enzymes for rapid genome integration to the CDS module;
• 5. add recombinase recognition module and yeast homologous recombination module to the connector module;
• 6. add nutritional defect screening tools to the antibiotic resistance module;
• 7. introduce inducible promoters and corresponding regulators.

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. The bacterial constitutive promoter is loaded upstream of the yeast promoter to form a hybrid promoter. We tested these combinations in brewer's yeast as well as bacteria and found that these promoters stably drove gene expression in both yeast and bacteria, and showed a decreasing trend consistent with data 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.

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.

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.

All OriT combinations were successfully transferred into Nissle 1917. The combination of these five ORIs, together with pBBR1, enables a broader spectrum of non-model organism gene expression.

Efficient strategies for rapid genome integration

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.

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.

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, The lycopene production was increased approximately 13-fold compared to our most primitive strain.

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.

Production of (-)-α-bisabolol

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.

Sea water cultivation of V.nat

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.

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.

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.