New Part

New Basic Part

placUV5-MB7(BBa_K4941056)

The provided sequence is an improved version of the wild-type lacUV5 promoter (PlacUV5, BBa_I732021), known as placUV5-MB. This is a naturally occurring strong inducible promoter in Escherichia coli. placUV5-MB is part of a series of derivatives obtained by mutating specific sites in the -35 and -10 regions of PlacUV5. As registries typically don't allow multiple sequences to be inputted, we are presenting the sequence of PlacUV5_MB7 as a representative (with the highest fluorescence intensity). For completeness and ease of use by other teams and researchers, the remaining sequences are also provided below.

Biology

The T7 RNA polymerase from the λ phage is controlled by the strong inducible lacUV5 promoter and can be induced by IPTG. The conventional host for the T7 expression system utilizing T7RNAP and the T7 promoter is BL21(DE3), a host naturally susceptible to λ phage infection. However, this has two major limitations: (1) the T7 expression system can only be applied in this specific host, and (2) different types of proteins or metabolic pathway gene expressions may require different intensities, and higher intensity is not necessarily better. Based on this observation, we drew inspiration from the book "Basic Molecular Biology (4th Edition)" written by Zheng Yonglian, published by China Higher Education Press. We discovered that the -35 and -10 regions of promoters have standard sequences. If both regions of a promoter are close to the consensus sequence, the expression strength is enhanced; conversely, if they deviate, the strength is weakened. Therefore, we performed base mutations on the PlacUV5 region (Table 1) based on the standard consensus and existing sequences of strong and weak promoters (Plac, PlacUV5, ParaBAD, PrecA) as references. Simultaneously, we used a plasmid to express GFP under the control of placUV5 to serve as a reporter protein, indicating changes in transcriptional intensity.

placUV5-MB sequences

The table below displays ten newly designed lacUV5 promoters. All promoters underwent mutations targeting the -35 and -10 region sequences with reference to the standard sequence and the known sequences of strong and weak promoters (Fig.1). The specific sequences are detailed in Table 2.

Experimental design of promoter strength

To assess the strength of the mutated lacUV5 promoters, we employed the Golden Gate assembly protocol. Ten mutant promoter variants were cloned, along with a weak RBS (B0031), the GFP reporter gene (BBa_E0040), and the T7 terminator (BBa_B0012), onto a plasmid vector simultaneously. The use of a weak RBS was chosen to minimize transcriptional level differences that might arise due to strong translation levels. Simultaneously, these fragments were assembled onto a low-copy-number plasmid (RK2 Broad Host Range Vector with Kanamycin Resistance, BBa_K2491030), aiming to avoid excessive host burden and reduce noise, as illustrated in Fig.2.

The mixture obtained through the Golden Gate assembly protocol was chemically transformed into Dh3α and plated on agar plates containing kanamycin for selection. Transformed colonies containing different lacUV5 promoter variants were inoculated into tubes containing 5 ml of Terrific Broth (TB) medium and cultured at 37°C with shaking at 220 rpm. When the OD600 reached 0.6-0.8, induction was initiated by adding IPTG to a final concentration of 0.5 mM, and fermentation continued for 24 hours. Samples were taken at 12 hours and 24 hours, and the fluorescence intensity was measured using a microplate reader on a 96-well plate.

Results

Through fluorescence intensity testing of different lacUV5 promoter mutants, we observed results consistent with our expectations. The lacUV5 variant (placUV5-MB7), identical to the standard sequence, exhibited the highest transcriptional expression intensity. Conversely, as deviations from the standard sequence increased, expression intensity exhibited a decreasing trend, particularly when the -35 region was replaced with a sequence from a weaker promoter (placUV5-MB8), resulting in a noticeable reduction in fluorescence intensity (Fig.3). These observations indicate that mutating the -35 and -10 regions of the promoter, especially when using sequences based on standard motifs and known promoter strengths, is feasible. This approach allows for the construction of variant libraries of the target promoter with different expression intensities.

Application

In order to verify the effect of three catalytic properties of the pET expression system on PsXR by modulating the intensity of T7RNAP expression and thus affecting the catalytic properties of PsXR, we transformed T7 RNAP expression plasmids and pet28a-PsXR containing different lacUV5 promoters into Escherichia coli BL21 hosts at the same time. Here is an example of transforming the T7RNAP plasmid driven by PlacUV5MB and the pet28a-PsXR plasmid. The transformants containing different lacUV5 promoters were screened out by transformation and colony PCR, and transfected into 96-well dishes containing LB+Kana+Cm liquid medium for culture and fermentation testing. The results showed that MB7-PsXR catalyzed the production of 6.8 g/L xylitol from the substrate 8 g/L xylose after 36h (Fig.4), which proved the feasibility of the strategy applied to optimize the pET expression system by constructing a promoter library expressing T7RNAP.

Conclusion

In summary, by referencing standard sequences and the -35 and -10 regions of different strength promoters, we have successfully achieved precise modification of the placUV5 expression intensity. This optimization strategy has not only been successfully applied to the construction of the T7RNAP expression library but has also yielded significant results in optimizing the expression of the EutS protein. We are confident that this library will be of great assistance to other research teams in future studies.

Reference

[1] Sun, X. M., Zhang, Z. X., Wang, L. R., Wang, J. G., Liang, Y., Yang, H. F., ... & Yang, S. (2021). Downregulation of T7 RNA polymerase transcription enhances pET‐based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis. Biotechnology and Bioengineering, 118(1), 153-163.

[2] Romano, E., Baumschlager, A., Akmeric, E. B., Palanisamy, N., Houmani, M., Schmidt, G., ... & Di Ventura, B. (2021). Engineering AraC to make it responsive to light instead of arabinose. Nature Chemical Biology, 17(7), 817-827.

New Composite Part

Description

The erythritol biosynthesis pathway is consist of erythrose-4P phosphatase (encoding by Yida) and erythritose reductase (encoding by ER), and different sources of Yida could affect the production of erythritol. Thus, it is essential to establish a system for rapid screening of different sources of erythrose-4P phosphatase. As a proof of concept, the erythrose-4P phosphatases derived from Escherichia coli, Lactobacillus helveticus and Streptococcus thermophilus were selected for the next, and they were christened as EcYida (BBa_K4941018), LhYida (BBa_K4941020), StYida (BBa_K4941028) and ylER (BBa_K4941019). Besides, the erythrose reductase from Y. lipolytica (ylER) was used for co-expression with erythrose-4P phosphatases to convert erythrose-4-phosphate into erythrose, and both of them were expressed by the promoter of pTEF (BBa_K4941012). Importantly, the gene Nluc (BBa_K4941022)code the NanoLuc-furimazine was expressed by an erythritol-inducible promoter pEYK1, and then was introduced into Y. lipolytica for rapid screening of the best Yida. Specifically, when the engineered strain produce more erythritol, the stronger the fluorescence will be detected (Fig. 1).

Characterization of the fluorescent reporter system

The Nluc (BBa_K4941022) expressed by pEYK1 was transferred into Y. lipolytica to assess the feasibility of the fluorescent reporter system. And the combination of luciferase substrate addition and kinetic assays can be used to compare the fluorescence intensity of fermentation broths from different engineered strains. The result in Fig. 2 showed that significant differences in fluorescence intensity of fermentation broths of Y. lipolytica after addition of different concentrations of erythritol. Therefore, the fluorescent expression system for characterizing erythritol production was thought to be constructed successfully which can visualize the concentration of erythritol in fermentation broth by fluorescence intensity.

Application of the fluorescent reporter system for screening the best Yida

Construction：

Parts of EcYida (BBa_K4941018), LhYida (BBa_K4941020), StYida (BBa_K4941028) and ylER (BBa_K4941019) were created separated, as stated on their own page. And then erythrose-4P phosphatase (Yida) from different sources and erythrose reductase (ER) mentioned above were combined two by two. These plasmids were transformed in E. coli DH5α, and cloning was verified by a restriction profile. Then combination of them were placed into the appropriate vector to be integrated into the genome of Y. lipolytica in the subsequent step. The part Nluc (BBa_K4941022) were created as described in the Basic Part, and it was placed into the appropriate vector.

Testing

Erythrose-4P phosphatase derived from different sources and erythrose reductase derived from Y. lipolytica (ylER) were used to be integrated into the genome of Y. lipolytica, and then several positive inverters were selected for fermentation experiment and fluorescence intensity was measured. It was found that Erythrose-4P phosphatase derived from E. coli (EcYida) showed the highest fluorescence (Fig.3). Furthermore, the highest erythritol titer of 5.9 g/L was obtained when EcYida and ylER was used in combination (please see cycle 5 in the Engineering success section).

Conclusion

In summary, the fluorescent reporter system for erythritol production we used in the composition part has been proved. And it was illustrated that the highest content of erythritol of the positive inverters was 5.9 g/L.