Part:BBa_K4941036
pylxp-CEN1-1-pEYK1- Nluc-XPR2_terminator-ORI1001-Rep Origin (pMB1)-AmpR-Leu2 marker
Plasmid for Nluc expression.The plasmid carries an AmpR screening marker with Amp resistance, and the erythritol-inducible promoter pEYK1 responds to erythritol induction and initiates Nluc expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 4468
Illegal XhoI site found at 4497 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 724
Illegal NgoMIV site found at 5354
Illegal AgeI site found at 1308
Illegal AgeI site found at 3883 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3062
Illegal BsaI site found at 4639
Illegal BsaI.rc site found at 325
Illegal BsaI.rc site found at 4554
Illegal BsaI.rc site found at 4837
Illegal SapI.rc site found at 4085
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).
- Fig.1: Construction of the fluorescent expression system for characterizing erythritol production
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.
- fig.2 : Relative fluorescence after addition of different concentrations of erythritol in Y. lipolytica
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).
- Fig.3: Relative fluorescence of several positive inverters obtained by integration into the genome of Y. lipolytica
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.
| None |