Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development
Abstract:The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and… Show more
“…Consistent with previous stress response transcriptomic studies in Z. mobilis [ 42 – 44 ], xylose as a stressor inhibited the cellular biosynthesis process with many biosynthesis genes down-regulated in the sugars comparison (xylose versus glucose) in all three strains (Additional file 5 : Table S4). However, among these genes, 15 ribosomal protein-related genes (such as rplO , rplQ , rplJ , rplL , and rpsI ) and 6 amino acid biosynthesis-related genes ( glnB , glnA , hisC3 , glyA , hisG , hisD ) were significantly up-regulated more than twofold in strain 8b-S38 compared with 8b in RMX5 and RMX15 media (Additional file 6 : Table S8).…”
Section: Resultssupporting
confidence: 87%
“…This result suggested that despite of improved xylose-utilization capability, adapted strains remained physiologically challenged by the xylose culture conditions, which could be due to the stressful environment of xylose media for Z. mobilis cells as reported before [ 42 , 43 ] . Such morphological disturbances have been described previously in Z. mobilis when exposed to different stresses, such as high temperature [ 44 ] and inhibitory lignocellulosic hydrolysate [ 18 ]. Fig.…”
Section: Resultsmentioning
confidence: 54%
“…Z. mobilis can regulate universal stress-response genes to protect macromolecules including proteins and DNA from the damage caused by the stressful environments [ 12 , 42 – 44 , 50 ]. Gene expression in three strains demonstrated that the transcriptional level of ZMO0246 ( hslV ), ZMO0247 ( hslU ), ZMO0405 ( clpA ), ZMO1424 ( clpB ), and ZMO1704 ( lon2 ) involved in protein remodeling and reactivation, as well as ZMO1231 ( recJ ) and ZMO1588 ( uvrA ) involved in DNA repair were significantly up-regulated in response to the variable of sugar (glucose versus xylose) media (Additional file 6 : Table S8).…”
Background
Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.
Results
Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.
Conclusions
This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.
“…Consistent with previous stress response transcriptomic studies in Z. mobilis [ 42 – 44 ], xylose as a stressor inhibited the cellular biosynthesis process with many biosynthesis genes down-regulated in the sugars comparison (xylose versus glucose) in all three strains (Additional file 5 : Table S4). However, among these genes, 15 ribosomal protein-related genes (such as rplO , rplQ , rplJ , rplL , and rpsI ) and 6 amino acid biosynthesis-related genes ( glnB , glnA , hisC3 , glyA , hisG , hisD ) were significantly up-regulated more than twofold in strain 8b-S38 compared with 8b in RMX5 and RMX15 media (Additional file 6 : Table S8).…”
Section: Resultssupporting
confidence: 87%
“…This result suggested that despite of improved xylose-utilization capability, adapted strains remained physiologically challenged by the xylose culture conditions, which could be due to the stressful environment of xylose media for Z. mobilis cells as reported before [ 42 , 43 ] . Such morphological disturbances have been described previously in Z. mobilis when exposed to different stresses, such as high temperature [ 44 ] and inhibitory lignocellulosic hydrolysate [ 18 ]. Fig.…”
Section: Resultsmentioning
confidence: 54%
“…Z. mobilis can regulate universal stress-response genes to protect macromolecules including proteins and DNA from the damage caused by the stressful environments [ 12 , 42 – 44 , 50 ]. Gene expression in three strains demonstrated that the transcriptional level of ZMO0246 ( hslV ), ZMO0247 ( hslU ), ZMO0405 ( clpA ), ZMO1424 ( clpB ), and ZMO1704 ( lon2 ) involved in protein remodeling and reactivation, as well as ZMO1231 ( recJ ) and ZMO1588 ( uvrA ) involved in DNA repair were significantly up-regulated in response to the variable of sugar (glucose versus xylose) media (Additional file 6 : Table S8).…”
Background
Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.
Results
Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.
Conclusions
This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.
“…This demonstrated that ampicillin was more stressful to ZM4∆0103 and ZM4∆ARs during cell growth, and ZM4∆0103 and ZM4∆ARs were more sensitive to ampicillin than ZM4. Similar morphological changes of Z. mobilis have been previously described when the cells were exposed to different stresses, such as high temperature [ 30 ], lignocellulosic hydrolysate inhibitory [ 31 ], high concentration of xylose [ 32 ], and salt conditions [ 33 ].…”
Section: Resultssupporting
confidence: 65%
“…The previous study reported that ZMO0103 was a β−lactamase gene, which contains a 55% amino acid sequence identity with class C β−lactamase genes [ 20 ]. A higher expression level [ 30 , 34 , 35 , 36 ] of ZMO0103 in ZM4 may be ascribed to the absence of the AmpR that can inhibit the expression of ZMO0103 . The result of multiple sequence alignment shows that the homologous protein of β−lactamase ZMO0103 was only found in Sphingomonas ( Supplementary Materials Figures S7 and S8 ).…”
Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. Zymomonas mobilis is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a−based genome−editing technology. Six ampicillin−resistant genes (ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four−gene mutant ZM4∆ARs was constructed by knocking out ZMO0103, ZMO0893, ZMO1094, and ZMO1650 continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 μg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in Z. mobilis, and other ampicillin−resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in Z. mobilis and laid a foundation for further study of other antibiotic resistance mechanisms.
Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner–Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self‐flocculated, the bacterial cells are more productive, since they can self‐immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self‐flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c‐di‐GMP) on self‐flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c‐di‐GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c‐di‐GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c‐di‐GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self‐flocculating morphology to boost production but also understanding mechanism underlying c‐di‐GMP biosynthesis and degradation in the bacterium.
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