Expression of Cellulases
We obtained the sequence of β-1, 4-endoglucanase (CenA) and β-1,4-exoglucanase (Cex) from team UESTC-China, and the gene encoding β-glucosidase is amplified from Streptomyces coelicolor`s genomic DNA. We linked the genes into the pET30a(+) backbone respectively and induced these genes to express by IPTG in the E.coli strain BL21(DE3). After a few trials, we determined the optimal inducting conditions, which is with 0.08 mM IPTG at 16℃ overnight (about 15h). After confirming the expression and determined the activities of the original cellulases, we added INP-N (N terminus of ice-nucleation protein) sequence to the N terminus of each cellulase and conducted the same procedures to INP-N fused cellulases. Due to the time limit, we only created INPN-CenA and INPN-Cex successfully.
Figure 1 cenA, cex and β-glucosidase genes acquisition and expression. (a) three genes are obtained by PCR amplification. (b) The expression of Cex, CenA and fused INPN-Cellulases are confirmed by SDS-PAGE assay.
Surface Display of Cellulases
To determine if the enzymes were anchored on the surface of the cell, we detected the presence of the fusion protein by immunofluorescence staining, then employed the undisrupted cells expressing fused enzymes to the enzyme activity assays.
6His tag was added to present after the original cellulases (CenA and Cex) sequence and the fused cellulases (INPN-CenA and INPN-Cex) sequence as the antigen to be targeted by the primary antibody. Since the Cellulase-6His is originally expressed in the interior of the cell, we would not detect the fluorescent signals in the sample of CenA and Cex, while the fluorescent signals are detectable for INPN-Cellulase-6His due to the cell outer membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INPN-CenA and INPN-Cex)under the fluorescence microscopy`s 20X objective (Fig 2)
Figure 2 E.coli cell immunofluorescence staining observation via fluorescence microscopy 20x objective
Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the unfused cellulases field are dimmer than those of the fused cellulases field on average. To examine it more clearly, we observed the slices with the confocal fluorescence microscopy (Fig 3). The field of fusion protein samples showed that some foci are located on the borders of the cells, while this phenomenon was not observed in the field of the unfused protein samples. But due to the minuscule size of E.coli cells, our equipment falls short when trying to determine whether the fluorescent dot on a single cell is located on the outer membrane surface or not.
Figure 3 E.coli cell immunofluorescence staining observation via confocal fluorescence microscopy, 100x objective (images are locally zoomed)
Activity Assay
After confirming the expression of the cellulases, the recombinant cells are disrupted by ultrasonication to obtain the crude enzyme. We measured the enzyme activity by the method of CMC-Na (sodium carboxymethyl cellulose) assay. The activity of unfused and fused cellulases are determined respectively. We determined the activities of cellulases based on the standard curve of glucose concentrations. Considering the relatively weak cellulose degradation capacity of β-glucosidase and relatively low activity of CenA we measured, we also mixed these crude enzymes and determined the mixed cellulases activity as well as the activity of the single cellulase Cex. The data and results are shown on Table 1 to 4 and Fig.4.
Figure 4.Enzyme activity assay. (a)The standard curve of glucose concentration. (b)Mixed cellulases (CenA and beta-glucosidase) activity assay. (c) Cex activity assay. (d) Fusion cellulases activity assay. The activities are determined under both cell-undisrupted and cell-disrupted conditions.
We also measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INP-CenA and INP-Cex.
From the data above, we summarized that the cellulases` activities were not affected remarkably with the presence of INP-N. Also, the difference of enzyme activities between the ultrasonic-disrupted samples and undisrupted samples may also provide evidence of the anchoring effect of INP-N. Since the fusion protein is anchored in the outer membrane surface, which would appear in the sediments after centrifugation, the samples of suspension with fused cellulases showed the relatively low level of the activity, compared with samples of unfused ones.
The acquisition of individual genes
CrtE, CrtB, CrtI, CrtY, CrtZ and BKT are crucial genes in the astaxanthin synthesis pathway. CrtE and CrtB can be amplified by PCR using Rhodobacter sphaeroides's genomic DNA as templete, while CrtI and CrtY can be amplified using Rhodospirillum rubrum’s and Pantoea agglomerans’s genomic DNA respectively. Figure 1 shows the agarose gel electrophoresis test results.
Figure 5 Astaxanthin synthesis genes were amplified from several bacteria's genomic sequences.(a)The CrtE gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR. The length of CrtE was 879 bp;(b)The CrtB gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR. The length of CrtB was 1068 bp;(c)The CrtI gene was amplified from Rhodospirillum rubrum's genomic sequence by PCR. The length of CrtI was 1524 bp；（d）The CrtY gene was amplified from Pantoea agglomerans 's genomic sequence by PCR. The length of CrtY was 1161 bp.
There are bright bands in Figure 1a. The size of those bands is between 750 bp and 1 kb, which coincides with the size of the CrtE gene (879 bp). No obvious non-specific bands were observed. CrtB is 1068 bp in length while CrtY is 1161 bp. Bright bands between 1 kb and 1.5 kb can be seen in Figure 1b and Figure 1d, so CrtB and CrtY are amplified successfully. Some non-specific bands can be seen in the first three lanes in Figure 1c, but the destination bands are bright and their length is in the correct range so CrtI is also amplified successfully.
The last two genes in the pathway, CrtZ and BKT, were optimized and synthesized by the company, so the gene sequences used in the pathway have been all obtained.
Successfully induced each protein
In order to determine the activity of individual enzymes in the astaxanthin synthesis pathway, we induced individual enzymes separately. In the experiment, we used gradient IPTG concentration and gradient temperature to induce each protein and explore the optimal IPTG concentration and temperature of single protein expression. Although low temperature induction is not conducive to protein expression, it is beneficial to avoid the formation of inclusion bodies and the subsequent determination of enzyme activity. Therefore, gradient IPTG concentration from 0 mM to 1 mM was used to induce the proteins at 25 °C, 30 °C and 37 °C respectively.
In this part, we successfully induced genes CrtE, CrtB, CrtI, CrtY (Fig. 2) and BKT (FIg. 3), and recorded the optimal conditions (chart 1).
Figure 6 SDS-PAGE assay for CrtE, CrtB, CrtI and CrtY proteins.
Figure 7 SDS-PAGE assay for BKT protein.
Chart 1 The optimal induction condition of each protain.
However, we induced CrtZ with gradient IPTG concentration (0 mM-1 mM), under 25℃, 30℃ and 37℃ respectively, still didn't see the target band. Subsequently, plasmid ptrc99A-M-Z with gene CrtZ and recombinant plasmid pACYC184-M-EBI-Y were co-transformed into E. coli BL21, and the yellow strain was successfully obtained. However, whether the strain did produce zeaxanthin exactly still remained further verification.
Produced lycopene successfully
In the actual process of construction of lycopene producing strain, we successfully use overlap PCR to connect the first two genes (CrtE, CrtB，figure 4), but the CrtI gene has been unable to overlap with the CrtE-CrtB junction product. Therefore, we use the seamless cloning kit to connect CrtE - CrtB junction product and CrtI, thus successfully build the lycopene production strains. Figure 5a shows the atlas of successfully constructed pACYC184-M-EBI plasmid. Figure 5b shows our construction results: the colonies (2, 3, 4, 8) with the successfully constructed plasmid turned red significantly, while those colonies (1, 5, 6, 7) with misconnected plasmid remained white. Then we extracted the plasmid of strain 8 and sequenced it. The sequencing results confirmed that the plasmid we constructed is correct.
Figure 8 CrtE gene was linked with CrtB by overlap PCR. (a) The linked band appeared after 7 overlap PCR cycles;(b) The linked sequence was amplified after 30 cycles.
Figure 9 The first three genes (CrtE, CrtB and CrtI) were cloned into plasmid pACYC184-M. (a) The constructed plasmid map. (b) The colonies (2, 3, 4, 8) with the successfully constructed plasmid turned red significantly, while those colonies (1, 5, 6, 7) with misconnected plasmid remained white.
In order to further confirm that our engineering bacteria produced lycopene, we transferred the constructed plasmid (184M-EBI) into E. coli BL21 and induced it with IPTG, using E. coli BL21 with pACYC184-M empty plasmid as control. After induced by 0.1 mM IPTG for 10 hours, we could see that E. coli cells with the constructed plasmid (184M-EBI) turned red significantly, while those with empty vectors remained white (see Figure 6a). Then we extracted lycopene with acetone and measured its absorbance at its maximum absorption peak at 473 nm and the yield of lycopene was calibrated using a standard curve (see Figure 6b and Figure 6c).
Figure 10 Induce the engineered E.coli cells to produce lycopene. (a) E. coli cells with the constructed plasmid (184M-EBI) turned red significantly; (b) Lycopene production of engineered E.coli cells after being induced by 0.1 mM IPTG for 10 hours; (c) Standard curve of lycopene concentration.
We also detected the yield of lycopene in engineered bacteria under different IPTG concentration conditions, and the change of lycopene production with time after IPTG induction (see Figure 7a and Figure 7b). We found that lycopene production peaked at an IPTG concentration of 0.3 mM. But to our surprise, leaky expression of the three lycopene synthesis genes was observed when there is no inducer. This suggested that the switch that controlled the synthesis of lycopene may not completely shut down the expression of all the three genes. Figure 7b was measured under 0.1 mM IPTG concentration and 30℃, lycopene production increased in the first 700 minutes and remained stable in the following period.
Figure 11 The yield of lycopene in engineered bacteria under different IPTG concentration conditions. (a) The changes of lycopene production with IPTG concentration; (b) The changes of lycopene production with induction time.
Successfully produced β-carotene
On the basis of pACYC184-M-EBI plasmid, we inserted the fourth gene CrtY between BamHI and HindIII, and then obtained the constructed plasmid pACYC184-M-EBI-Y for β-carotene production (see Figure 8a for plasmid atlas ).
s expected, we obtained yellow colonies. Colony PCR was carried out to verify the existence of the CrtY gene in the engineered bacteria. (Figure 8b, 8c). Then we extracted the plasmid of strain 7 and sequenced it. The sequencing results confirmed that the plasmid we constructed is correct.
Figure 12 The construction of pACYC184-M-EBI-Y plasmid. (a) Recombinant plasmid atlas for β-carotene production; (b) Colonies with the constructed plasmid turned yellow; (c) Colony PCR verified that CrtY did exist in colony 1 to 7 and colony 9, 10, 12, 13, 14, 15.
In order to further confirm that our engineering bacteria produced β-carotene, we transferred the constructed plasmid (pACYC184-M-EBI-Y) into E. coli BL21 and induced it with IPTG, using E. coli BL21 with pACYC184-M empty plasmid as control. After induced by 0.1 mM IPTG for 10 hours, we could see that E. coli cells with the constructed plasmid (pACYC184-M-EBI-Y) turned orange significantly, while those with empty vectors remained white (see Figure 9a). Then we extracted β-carotene with acetone and measured its absorbance at its maximum absorption peak at 453 nm and the yield of β-carotene was calibrated using a standard curve (see Figure 9b and Figure 9c).
Figure 13 Induce the engineered E.coli cells to produce lycopene. (a) E. coli cells with the constructed plasmid (184M-EBI-Y) turned orange significantly; (b) β-carotene production of engineered E.coli cells after being induced by 0.1 mM IPTG for 10 hours; (c) Standard curve of β-carotene concentration.
After successfully obtaining the strain producing β-carotene, we also explored the relationship between the β-carotene production level and the IPTG concentration (Figure 14a). The result was similar to that of lycopene production, the β-carotene production increased firstly and peaked at an IPTG concentration of approximately 0.3 mM. And the optimal induced time for β-carotene producing is 550 min (Figure 14b).
Figure 14 The yield of β-carotene in engineered bacteria under different IPTG concentration conditions. (a) The changes ofβ-carotene production with IPTG concentration; (b) The changes ofβ-carotene production with induction time.
Co-transformation of pACYC184-M-EBI-Y and ptrc99A-M-CrtZ
In subsequent experiments, we cloned CrtZ into ptrc99A-M plasmid and carried out a colony PCR assay to verify the success of ligation (Figure 12a). We also sequenced the constructed plasmid and result furtherly verified that the CrtZ gene had been cloned into ptrc99A-M. Then the two plasmids pACYC184-M-EBI-Y and ptrc99A-M-CrtZ were co-transformation into E. coli BL21. Colonies
Figure 15Atlas of constructed plasmid ptrc99A-M-Z.
with these two plasmids could grow both on chloramphenicol plate and ampicillin plate (Figure 12b, 12c). The engineered E.coli cells seemed to have different color comparing with lycopene producing E.coli and β-carotene producing E.coli cells (Figure 12d). But whether the colonies produced zeaxanthin still needs further verification.
Figure 16 Construct E.coli cells to produce zeaxanthin. (a) Colony PCR verified the CrtZ gene had been cloned into ptrc99A-M；(b) Colonies with the two constructed plasmids pACYC184-M-EBI-Y and ptrc99A-M-CrtZ could grow on chloramphenicol plate；(c) Colonies with the two constructed plasmids could grow on ampicillin plate; (d) E.coli cells with plasmids pACYC184-M-EBI-Y and ptrc99A-M-CrtZ appeared different color comparing with lycopene producing E.coli and β-carotene producing E.coli cells.
It is a pity that we have not built an astaxanthin-producing engineered bacteria before the deadline, but we are willing to work on our project continuously and make it finally be put into production practice.
