Construction and identification of plasmid pEZ15Asp-efe
A 1,050 bp fragment of efe gene was obtained using P1,P2 primers. With P3, P4 primers and P5, P6 primers, respectively a 300 bp fragment of pdc strong promotor and terminator were further amplified. A 720 bp fragment of gfp gene was obtained using P7,P8 primers. P9, P10 primers were used to obtain a 3009 bp fragment of pEZ15Asp.
These fragments were connected by Gibson assembly, and then transformed into E. coil NEB β10 and screened on LB agar plates with 100 (μg/mL) spectinomycin. The recombinant plasmid pEZ15Asp-efe was double-digested by BglI and EcoRI to obtain two fragments (4161 bp and 1200 bp). As expected, the recombinant plasmid pEZ15Asp-efe used for EFE expression was successfully obtained. The structure of the plasmid pEZ15Asp-efe is shown in the Fig. 1a.
Construction of recombinant strain ZM4-efe
The plasmid pEZ15Asp-efe was electroporated into competent cell of Z. mobilis. Tranformants were screened on RM agar plates with 100 (μg/mL) spectinomycin. The result of the identification is that a 2409 bp sequence was obtained when recombinant strain ZM4-efe was used as template and P4,P7 were used as primers, while genomic DNA of Z. mobilis was used as template at the same condition without amplified fragments. These results indicated that the plasmid pEZ15Asp-efe was successfully transferred into Z. mobilis. What’s more, green fluorescence was detected by Fluorescent Inverted microscope (Fig. 1b). The result indicated that the plasmid pEZ15Asp-efe was successfully expressed in Z. mobilis. Based on these results, the recombinant strain ZM4-efe was obtained.
Ethylene production of strain ZM4-efe
Experiments were conducted to test whether the strain ZM4-efe was capable of producing ethylene by cultivating in serum bottles with rubber stoppers and aluminum cap. The initial production of ethylene observed for strain ZM4-efe was 1.36 nmol/OD600/ml when 30 ml cultures with a start OD600=0.102 cultured for 18 h at 30℃ in serum bottles. While the wild-type strain ZM4 couldn’t produce ethylene under the same conditions. Subsequently, we attempted to reduce the culture volume to 20 ml and 10 ml, and the ethylene production for two different volumes cultured by the same condition was respectively 3.03 nmol/OD600/ml and 4.31 nmol/OD600/ml, which did not decrease as expected but increased than previously working volume. What’s more, the glucose concentration in the medium increased from 2 % to 5 %, and the ethylene production did not increase. This may suggest that there are other factors affecting ethylene production.
To investigate the causal links between cell density and ethylene production, a series of experiments were further conducted. More than three parallel experiments were set in each group. As shown in the Fig. 2, the initial OD600 was 0.1, and with the extension of the culture time, the OD600 value gradually increased. When cultured for 9 h, the OD600 reached the bottleneck period. The final OD600 value was 1.56. while the ethylene production did not change regularly. when OD600 was between 0.3-0.5, ethylene production reached its peak of 6.7 nmol/OD600/ml. And then with the prolongation of the culture time, there was no significant fluctuation in ethylene production.
Optimization of ethylene metabolic pathway
Based on our previous work, four single knockout strains of ZM1360 (Δpdc), ZM1570 (Δpfl), ZM1596 (ΔadhB), and ZM1237 (ΔldhA) were already constructed . The recombinant plasmid pEZ15Asp-efe was respectively electrotransformed into four single knockout strains, and four recombinant strains of ZM1360-efe, ZM1237-efe, ZM1570-efe, and ZM1596-efe were obtained. We tested the expression of GFP protein to reflect the expression of the target protein EFE. As shown in Fig. 3, there was no expression of GFP protein in wild-type ZM4, and there are two bands in all the five ethylene-producing strains. One band (28.8 kDa) was the size of the GFP protein, and the other band (70.8 kDa) was the size of protein of the fusion gene. Those indicated that efe gene was successfully expressed in all five strains.
A series of experiments were further conducted to test the ethylene production of strain ZM4-efe, ZM1360-efe, ZM1237-efe, ZM1570-efe, and ZM1596-efe cultured in RM (2% glucose). As expected, the ethylene production of four strains all improved production compared with strain ZM4-efe. The strains ZM1360-efe and ZM1596-efe significantly increased by 51.6 % and 41.3 %, were 8.3 nmol/OD600/ml and 7.73 nmol/OD600/ml, respectively (Fig. 4c). Similarly, ZM1570-efe and ZM1237-efe also showed higher ethylene production compared with the control strain ZM4-efe, were 8.13 nmol/OD600/ml and 7.18 nmol/OD600/ml respectively (Fig. 4c).
The expression of efe at the transcriptional level was explored when OD600 value of five ethylene-producing strains was about 0.4. As shown in Fig. 5. With the ZM4-efe as the control strain，the value of expression of efe for ZM1360-efe, ZM1237-efe, ZM1596-efe, and ZM1570-efe were 2.65, 2.07, 2.14 and1.668. The efe expression of the four single-gene knockout strains was higher than that of ZM4-efe. Combined with the ethylene production of four strains in Fig. 4c, we found that the strain ZM1360-efe with the highest efe expression had the highest ethylene production. However, the trend of expression of efe gene for other single-gene knockout strains was different from that of ethylene production.
Experiments of cell growth, glucose consumption and ethanol production for six strains were also perfomed. As shown in Fig. 4 a, b and d. When cultured for 9 h, the vaule of OD600 for wild-type strains ZM4 is 1.46, and for five recombinant strains was around 1.25. Among recombinant strains, the cell growth of ZM1360-efe is slightly superior to other recombinant strains. At this time, glucose consumption of the control group ZM4 was almost consumed, and the ethanol production reached 92% of the theoretical output value. After culturing to 12 hours, the vaule of OD600 for five recombinant strains is about 1.47, and the glucose consumption of five recombinant strain was exhausted, and the average ethanol production reached 92% of the theoretical output value. When cultured for 9 h 14 hours, the wild-type strain ZM4 and the five recombinant strains basically reached the same in terms of cell growth, glucose consumption and ethanol production, without significant differences.
Effect of additional additives on ethylene production
In addition to supplementing arginine and AKG, which was essential substrates for ethylene production, we also measured the effects of glutamate, glutamine, and proline on ethylene production. As glutamate and glutamine can be convented to AKG via oxidative deamination during the metabolism of amino acids. We deduced that the addition of glutamate and glutamine to the culture medium may increase ethylene production by increasing intracellular AKG levels.
The strain ZM1360-efe which showed the comparable ethylene production among the knockout strains was used to measure the effect of substrate availability on ethylene production. Ethylene production with 5 mM proline, 5 mM arginine and 5 mM glutamic acid was 6.58 nmol /OD600/mL, 10.15 nmol /OD600/mL and 8.94 nmol /OD600/mL. The greatest improvement in production was observed via the addition of AKG and glutamine to the growth media, with 5 mM AKG and 5 mM glutamine compared to cultures grown in standard RM media (2 % glucose): 12.8 nmol/ /OD600/mL and 11.6 nmol /OD600/mL (Fig. 7), respectively.
Ethylene production from straw enzymatic hydrolysate
In our laboratory, there is currently a strain of ZM532, which can withstand 5.0 g/L furanaldehyde and 3.0 g/L acetic acid (Unpublished data, ZM532 has been deposited at Guangdong Microbial Culture Center under the Accession Number GDMCC60527). Therefore, in order to produce ethylene form cellulosic hydrolysate, we introduced pEZ15Asp-efe plasmid in stress tolerant strain ZM532, and successfully obtained engineered strain ZM532-efe.
Cellulsoic hydrolysate obtained from pretreated straw contained 22.6 g/L glucose and 25.3 g/L xylose in this study. But its nitrogen content was only 0.9 g/L. Nutrient has great impact on sugar utilization as the ability of strains to achieve a high level of metabolite. So, we prepared RM medium with straw enzymatic hydrolysate as the sole carbon source, and tested the ethylene yield of strain ZM532-efe. Finally, we reached the ethylene production of 5.8 nmol/OD600/ml.