Screening promoters and construction of probe-vector
C. ammoniagenes ATCC 6871 cells grown in LB medium and fermentation medium to the exponential phase (OD600nm≈2.0) were collected and sent to Mega genomics (Beijing, China) for RNA-seq. A total of 2411 genes were identified from the transcriptomic data. Considering that there are many cases in which genes are in the same operon, genetic loci analysis was performed and the results showed that 1508 genes in 547 operons were expressed. In general, genes in the same operons share a common promoter, so the first gene in each operon was selected. Thus, the total number of remaining genes was 1450 (2411-1508+547). Transcription abundance analysis was used to select 71 genes with transcription levels in the top 100 in both RNA-seq profiles. Sequences upstream of the top 20 genes were predicted and scored using the online promoter prediction tool as described below. The annotations and RPKM values for these genes are shown in Table 1. A transcriptomic data analysis flow chart and the transcription levels of genes are shown in Figure 2.
Previous studies have shown that most promoters of molecular chaperones exhibit high transcription levels in prokaryotes, so all 6 annotated molecular chaperones in the C. ammoniagenes ATCC 6871 genome (Accession Number: NZ_CP009244) (two GroEL, two DnaJ, one DnaK, and one Co-chaperone GroES) were identified and their promoters were predicted as described in materials and methods. The information of these genes and transcriptomic RPKM values are also listed in Table 1. By predicting the promoter regions, 26 promoters were constructed according to the rules described below and their corresponding RFP expression plasmids were named as pXMJ190-Pn. The sequences of the 26 promoters are listed in Table S3.
For a comprehensive comparison, two conserved homologous promoters CJ1 and IJ59 that are active in C. ammoniagenes ATCC 6872 were aligned against the genome of C. ammoniagenes ATCC 6871 by BLAST and were cloned into pXMJ190 to give pXMJ190-CJ1 and pXMJ190-IJ59 [7, 15], respectively. Moreover, previous studies have shown promoters that work well in C. glutamicum may also be active in C. ammoniagenes . Thus, a strong endogenous C. glutamicum promoter named Pgro (the promoter of groES gene) was cloned as pXMJ190-Pgro . In addition, the most widely used tac promoter and conserved SD sequence was also selected and cloned into pXMJ190 as pXMJ190-Ptac . The pXMJ190 was used as negative control. All of the above constructed plasmids were validated by sequencing and then transformed into C. ammoniagenes ATCC 6871.
Analysis and comparison of promoters in C. ammoniagenes ATCC 6871
To analyze the activities of our cloned promoters, we first made a visual assessment of all C. ammoniagenes ATCC 6871 strains harboring pXMJ190, pXMJ190-CJ1, pXMJ190-IJ59, pXMJ190-Pgro, pXMJ19-Ptac and pXMJ190-Pn series plasmids on LB plates containing 20 μg/ml chloramphenicol. Red fluorescence was observed by microscopy, with different intensities in different colonies, indicating that there was variable expression of RFP from the 26 putative promoters (Figure 3a). Among these promoters, Prpl21, Prpl10, PgroELB, and PdnaK exhibited strong activity in C. ammoniagenes ATCC 6871, with Prpl21 being the strongest promoter. The remaining promoters had very low or non-existent red fluorescence.
To quantify the activities of the cloned promoters, growth-normalized fluorescence intensities were measured using a microplate assay and the data are shown in Figure 3b and Table S2, the results showed that we successfully isolated 20 endogenous promoters with different activities. Among the 20 promoters (PrrlA-Psbp) from genes with high transcription levels, Ptmr, Pnat, PatpG, PgpdⅠ, Pacn are essentially inactive, while PgroELA from the 6 molecular chaperone promoters was also inactive. Meanwhile, four strong promoters, including the Prpl21 with 43433 RFU/OD intensity and followed by PdnaK (19125 RFU/OD), PgroELB (8166 RFU/OD) and Prpl10 (6490 RFU/OD) were identified whose fluorescence levels were consistent with those observed on plates. Thus, a range of promoters with different transcriptional activities, including four strong promoters, were identified and can be used directly in further applications. According to the obtained data, only two promoters (Prpl21 and Prpl10) from the 20 genes with high levels of transcription had strong activities, while two promoters (PgroELB and PdnaK) sourced from the six molecular chaperone genes exhibited relatively high activities, which may indicate that isolating promoters from molecular chaperones may be a more efficient strategy. Although most of the promoters identified from the RNA-seq profiles exhibited low level expression or were silent, the red fluorescence intensity of Prpl21 was almost 2.3 times that of the highest promoter (PdnaK) derived from upstream regions of the molecular chaperones. This suggests isolating promoters from genes with high transcription levels may yield strong promoters. However, it is necessary to point out that the RPKM value of the gene downstream of the Prpl21 promoter was not the highest, which might be ascribed to multiple factors, such as the codon usage of genes, copy number of genes, the half-lives of mRNA and limitations inherent in transcriptomics [22-24].
When it comes to the known promoters, the promoter CJ1 had some certain activity, the IJ59 promoter had almost no activity, and the activity of Pgro was slightly higher than the Ptac promoter but still lower than promoter Prpl10. This demonstrates the promoters that work well in one species or homologous strains may not possess the same characteristics in another.
Application of Prpl21 in C. ammoniagenes for improving the production of CoA
It has been reported that CoA, a ubiquitous and essential cofactor in biochemical reactions, can be produced by C. ammoniagenes ATCC 6871 . In the CoA biosynthetic pathway, the committed step catalyzed by pantothenate kinase (coaA) is subject to feedback inhibition by CoA and acyl-CoAs . Bacterial coaA proteins are categorized based on their amino acid sequences into three types, namely type I, II, and III. C. ammoniagenes carries a type I coaA which is highly regulated by CoA and its derivatives. In contrast, type II and III enzymes are insensitive to CoA and its thioesters [27, 28], therefore, in order to reduce feedback inhibition and increase the production of CoA, the type III pantothenate kinase from Pseudomonas putida (PpcoaA) was selected and overexpressed by the control of the strongest promoter Prpl21 in C. ammoniagenes ATCC 6871 [29, 30]. Considering there are no direct enzyme assays for type III pantothenate kinase, RFP was co-expressed with PpcoaA. As shown in Figure 4a, bright red fluorescence was observed in the tube with cells containing pXMJ190-Prpl21-PpcoaA-RFP, suggesting that RFP and PpcoaA were successfully co-expressed. By measuring the red fluorescence intensity, the strain harboring pXMJ190-Prpl21-PpcoaA-RFP had up to 7560 RFU/OD. This value was lower than that observed when was expressed RFP alone under Prpl21, possibly due to the long distance from the transcription initiation site and the increased cellular burden caused by co-expression of two proteins. The expression of RFP and PpcoaA was also be observed by SDS-PAGE. As shown in Fig 4b, significant bands of approximately 27 kDa and 32 kDa were observed indicating that RFP and PpcoaA were overexpressed and that the Prpl21 promoter functioned well in C. ammoniagenes. Furthermore, three crucial substrates (pantothenic acid (2 mM), L-cysteine (2 mM), ATP (6 mM)) were added to the reaction mixture containing a certain number of bacterial cells (OD600 nm≈40) at 39 ℃. Coenzyme A production increased to a satisfactory yield of approximately 315 U/mL in 6 h (Figure 4c), which was almost 4.1 times higher than the same conditions without pXMJ190-Prpl21-PpcoaA-RFP in the cells (76 U/mL). The results indicated that PpcoaA was successfully overexpressed under the control of promoter Prpl21 and increased the anabolic flow of CoA.