Part:BBa_K5300037
PnuoA-pfa biosynthetic gene cluster, composite part composed of PnuoA and pfa.
This composite element consists of two main components: the nuoA promoter and the pfa biosynthetic gene cluster, which includes seven key elements: RBS1-4, pfa1-3, and ppt. PnuoA is a constitutive promoter that maintains continuous transcription of downstream genes. The biosynthetic gene cluster encodes PUFA synthase and consists of pfa1-3 and ppt from Aetherobacter fasciculatus SBSr002, along with RBS sequences from Sinorhizobium fredii CCBAU45436, derived from the genes NifD, NifK, NifE, and NifN. We hope that this gene cluster express key enzymes responsible for the production of PUFA, namely poly unsaturated fatty acids.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11381
Illegal NheI site found at 15545 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 706
Illegal BglII site found at 1619
Illegal BglII site found at 5458
Illegal BglII site found at 6608
Illegal BglII site found at 7480
Illegal BglII site found at 10753
Illegal BglII site found at 13285 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 268
Illegal NgoMIV site found at 766
Illegal NgoMIV site found at 2647
Illegal NgoMIV site found at 3705
Illegal NgoMIV site found at 4262
Illegal NgoMIV site found at 4338
Illegal NgoMIV site found at 4622
Illegal AgeI site found at 5
Illegal AgeI site found at 967
Illegal AgeI site found at 2810
Illegal AgeI site found at 5309
Illegal AgeI site found at 6009
Illegal AgeI site found at 8205
Illegal AgeI site found at 8532 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 11482
Illegal SapI site found at 13453
Illegal SapI site found at 14653
Illegal SapI site found at 16208
Illegal SapI.rc site found at 8777
Illegal SapI.rc site found at 9682
Illegal SapI.rc site found at 17002
Plasmid construction and validation
We first seamlessly cloned the regulation module and each two fragments of PUFA1-5 with the PCR linearized pUC19 vector in order to amplify three large fragments (i.e., regulation-PUFA1, PUFA2-3, and PUFA4-5) using the recombinant plasmid as a template. The three large fragments were seamlessly cloned with the PCR linearized pBBR1MCS-2 plasmid, and the recombinant plasmids were transformed into E. coli (DH5α) (Figure 3-4-1).
The sequencing results of this plasmid were disappointing, as it showed several point mutations and even nonsense mutations, making our experiment impossible. We were unable to repeat the experiment again due to time constraints. And in order to validate our synthesis module, we therefore designed a synthesis circuit that utilizes a constitutive promoter for expression. We repaired the point mutation in the pfa biosynthetic gene cluster and seamlessly cloned and ligated it to the nuoA promoter to pBBR1MCS-2. (Figure 3-4-2)
Triparental mating and validation
The verified correct colonies were inoculated into kanamycin-resistant LB liquid medium under shaking condition. Then we conducted triparental mating using the correct colony we obtained above, Helper and Sinorhizobium fredii CCBAU45436. Then colony PCR was performed on colonies grown on TY/NA/Kan solid medium incubated at 28°C for 24 h using the universal primer M13F/R.
RT-qPCR results
We used RT-qPCR to quantify gene transcription levels. We extracted RNA from the bacterial solution of the +pfa strain and the wild-type strain respectively and conducted reverse transcription. qPCR was performed using cDNA as a template. 16S rRNA was used as an internal reference to compare the expression of the target genes in the +pfa strain relative to the endogenous nuoA gene. We designed three pairs of primers (pfa1, pfa2, pfa3) of the three core enzyme genes of the pfa gene to characterize the amount of transcription in different segments of the gene in terms of the transcript levels of the three fragments and performed three biological replicates (Figure 3-6-1).
Obviously, we found that the expression of the pfa gene decreased following the increase of its distance from the promoter. The transcription levels of all three segments of pfa gene were up-regulated compared to the expression of the endogenous nuoA gene, but the overall trend was a gradual decrease. We hypothesized that the decrease in transcription levels in the second half of the gene may be due to the fact that the gene is long and the mRNA is prone to degradation or forms secondary structure during transcription, which leads to post-transcriptional instability in complete translation.
Total lipid TLC analysis
In order to pre-analyze the changes in fatty acid content in the +pfa strain, we analyzed the amount of triacylglycerol (TAG) in the +pfa strain using thin-layer chromatography to represent the fatty acid content (Figure 3-7-1).
Both △phaC2 and WT+pfa strains were able to show a significant increase in the content of TAG compared to the wild type. Whereas, the TAG bands of the WT+pfa strain deviated significantly from the other two, which we believed is due to the altered composition of the fatty acid content. This to some extent corroborates that the expression of the pfa biosynthetic gene cluster was successful.
Gas Chromatography Results
In order to directly verify that the gene cluster was actually successfully expressed and synthesized the target product, we used gas chromatography to isolate, purify and characterize the fatty acid content above ten carbons. We sent the wild-type strain and the +pfa strain for gas chromatographic identification (Figure 3-8-1).
It's a pity that we did not identify the presence of DHA and EPA. But with the bad news came good news. We identified a fatty acid in the +pfa strain that was not present in the wild-type strain - a long-chain saturated fatty acid with fourteen carbons. And the content of this fatty acid varied dramatically, from nonexistent to 5% of the total. We read the references and found that the expression of gene cluster is very complex. pfa biosynthetic gene clusters are capable of expressing several enzymes, each plays a different role (Figure 3-8-2).
Since new fatty acids that never appeared in the wild type appeared in our +pfa strain, it indicates that the expression of the gene cluster was successful to some extent. Based on the results of real-time fluorescence quantitative PCR, we analyzed that the failure to produce the target product was due to the greatly reduced transcription of the second half of the cluster. In Figure 3-8-2, we found that the oxidase was encoded only by pfa3, which is capable of forming unsaturated bonds. Also, the gas chromatography we used for time reasons was food grade and less accurate. Therefore there is a high probability that pfa3 failed to express the oxidase in large quantities thus DHA and EPA were not detected with the low precision of the assay.
To verify our speculation, we organized the data and reanalyzed it. pfa1 expresses the product of ER, the reductase, which is able to change unsaturated bonds to saturated bonds. We therefore counted the proportion of unsaturated fatty acids among the eighteen-carbon fatty acids and the changes in the content of different types of fatty acids (Fig. 3-8-3).
Obviously, we found that the content of unsaturated fatty acids in the +pfa strain decreased significantly compared to the wild type strain, which is in line with our speculation. Therefore, it can be proved to a certain extent that our synthesis module is successful and the related genes are able to be expressed normally. And in order to obtain the final target product as well as to increase the yield, it is necessary to optimize the subsequent experiments.
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