It is a part that is responsible for expressing F/Xpk from Bifidobacterium adolescentis ATCC 15703 with Pthl promotor. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence (BBa_K103015), F/Xpk sequence (BBa_K4886000) and terminator sequence (BBa_K3585002). F/Xpk is a gene that encodes phosphoketolase. Phosphoketolase is an enzyme with both the Fpk and Xpk activity. It catalyzes the conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), and the conversion of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 808
1000
COMPATIBLE WITH RFC[1000]
Results
（1）Plasmid construction
We used Pthl-adhE2 from BBa_K4408008 as the template and X-pMTL-F and X-pMTL-R as primers to obtain a X-Pthl vector (5461bp) by amplification. We used the Bifidobacterium adolescentis ATCC 15703 genome as a template to amplify the phosphoketolase gene [FXpk(QS)] fragment (2478bp). The vector and fragment were confirmed by gel electrophoresis (Figure 2 and 3). The FXpk(QS) fragment was ligated to the X-Pthl linear vector, using Gibson assembly. The plasmid was then transformed into E.coli JM109. After colony PCR for the transformed bacterial colonies, positive colonies were inoculated, and plasmids were extracted. The recombinant plasmid pMTL-Pthl-FXpk(QS) obtained was confirmed by gene sequencing.
Figure 2 Verification of F/Xpk(QS) (2478 bp) by DNA gel electrophoresis
Figure 3 Verification of X-pMTL-Pthl vector (5461bp) by DNA gel electrophoresis
（2 ）Transfection of C. tyrobutyricum and its growth
By using E. coli CA434 as a donor strain, pMTL-Pthl-F/Xpk(QS) plasmid and pMTL-Pthl-F/Xpk(BD) plasmid were transferred to C. tyrobutyricum, noted as Ct(F/Xpk-QS) and Ct(F/Xpk-BD), respectively. Refer to BBa_K4886001 for the construction of pMTL-Pthl-F/Xpk(BD). The native C. tyrobutyricum was the control.
Fermentation experiment showed that the growth of Ct(F/Xpk-BD) is better than that of Ct(F/Xpk-QS) and that of the control (Figure 4).
Figure 4 Growth comparison of Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS)
(3)Product yield of the transfected strain
Acetyl phosphate (AcP) is the final product of NOG pathway. AcP assay showed that the levels of AcP in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) were both higher than the control, which was in accordance with the growth of the strains. This indicated that NOG pathway was open in the engineered strains, Figure 5.
Figure 5 Levels of acetyl phosphate in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS)
HPLC experiment showed that after fermentation for 26h, the yields of butyric acid were 3.35 g/L and 3.31 g/L in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS), both higher than the yield in the control. The yields of acetic acid were 1.36 g/L and 1.28 g/L in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS), both lower than that in the control. Glucose consumption was much higher in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) compared with the control (Table 1). Ct(Pthl F/Xpk-BD) showed higher glucose consumption and butyric acid yield than Ct(Pthl F/Xpk-QS).
Butyric acid is a 4-carbon molecule, while acetic acid is a 2-carbon molecule. The increase in the butyric acid production and glucose consumption and decrease in the by-product acetic acid yield suggested that Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) both have higher efficiency of using glucose and less carbon loss in glycosis compared with the native strain. In addition, Ct(Pthl F/Xpk-BD) was better in reducing carbon loss than Ct(Pthl F/Xpk-QS).
Table 1 Metabolite levels in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) after 26h fermentation
===(4) Carbon source selection for engineered C. tyrobutyricum===
Due to limited experimental conditions, it was not possible to directly measure specific emissions of CO2 We estimated the value of carbon dioxide being fixed from the yield of the obtained product butyric acid, based on the principle of carbon conservation. The experimental results show that the introduction of the NOG pathway increases the production of butyric acid by 9.5% compared to the control group. Therefore, assuming that the global demand for butyric acid production is 80,000 tons, the formula calculates that the carbon dioxide emissions can be reduced by approximately 6,941 tons. The detailed calculation is shown in the following PDF file.
CO2 emission reduction models.pdf
Target 3 (Test) Carbon source selection for engineered C. tyrobutyricum
To find the best carbon source to grow the engineered C. tyrobutyricum, we compared the growth of Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) on different carbon sources, including glucose, fructose and xylose. Fermentation experiment found that both strains had better growth than the native strain (control) on all the carbon sources, and fructose was the best carbon source for the growth of Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS), Figure 5.
HPLC was used to compare the product yields and carbon source consumption of the strains cultured on different carbon sources for 45h (Table 2). The yields of acetic acid in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) were both higher than the control when cultured on fructose, indicating a low flow in NOG pathway. In Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) cultured on xylose, the yields of acetic acid were both lower than the control, and the xylose consumption was higher than the control. Considering both the product yields of butyric acid and acetic acid and the consumption of carbon source, xylose was the best carbon source for NOG pathway in the engineered strains.
These experimental results of carbon source selection showed that engineered C. tyrobutyricum with NOG pathway had an enhanced ability to utilize xylose, which is more beneficial to the utilization of cheap substrates like plant straw in subsequent industrial applications of the strain.
Note: a) Glucose,b) Fructose, c) Xylose
Figure 5 Growth of Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) cultured on glucose, fructose and xylose
Table 2 Metabolite level in Ct(Pthl F/Xpk-BD) and Ct(Pthl F/Xpk-QS) cultured on different carbon sources for 45h
