Systems-level Analysis of Oxygen Exposure in Zymomonas mobilis: Implications for Isoprenoid Production
Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial biofuel production. Here, we examine the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis, likely caused by oxidative damage to the iron-sulfur co-factors of the final two enzymes of the pathway. This bottleneck was resolved with minimal changes in expression level of enzymes in the pathway but pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e. flavodoxin reductase and the suf operon). We also detected prominent changes in glucose utilization under aerobic conditions. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake after 24 hours of aerobic growth. Our results suggest that under aerobic conditions, electrons from the oxidation of glucose to gluconate are delivered to the electron transport chain to minimize oxidative damage by reducing reactive oxygen species such as H2O2. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome c peroxidase, and a cytochrome bd terminal oxidase.
Sample Processing Protocol
Experimental design. After inoculation of experimental cultures as described above, cultures were grown up until they reached an OD600 of 0.3-0.45, at which point the time zero time-point extractions were taken. Immediately following the time-zero time-point, aerobic (O2) cultures were removed from the anaerobic glove bag and placed in a 30°C water bath under atmospheric conditions where they were shaken at 250 rpm for the duration of the two-hour time-course. Anaerobic (An) cultures remained in the glove bag. Extractions were taken for metabolomics, proteomics, or transcriptomics analysis at t = 0, 1, 5, 10, 15, 30, 45, 60, and 120 minutes after O2 cultures were transferred. This experimental design was followed four separate times. The iterations differed in both replicate number and exact time-point sampling scheme, however each iteration lasted two hours and included at least 3 O2 replicates and 2 An replicates. The first three iterations were performed to collect mRNA samples, proteomics samples, and extracellular metabolite samples. Intracellular metabolites were extracted alongside all four iterations in order to ensure a reproducible physiological response. Intracellular metabolite values matched well from iteration to iteration and the log2 fold-change values were averaged together for all metabolites except HMBDP and IDP/DMADP. The aforementioned metabolites produced low-quality signals for the first three iterations, so a fourth iteration was conducted specifically to better quantify these metabolites using a slightly modified LC-MS method. The fold-change values for HMBDP and IDP/DMADP presented here are therefore an average of only the replicates of the fourth iteration. A modified experimental design was also conducted, which differed from the experiment described above in duration and time of transfer to oxygen. For this experiment, O2 cultures were transferred to aerobic conditions at an OD600nm of approximately 0.1, after only one hour of aerobic growth. Extracellular metabolite samples were then taken every two hours for eight hours following the transfer. A final sample was taken 24 hours after inoculation. Protein extraction and sample preparation for proteomics. At the time of extraction, 4mL of culture was collected and cells were pelleted by centrifugation for 2.5 minutes at 16,000 × g. Supernatant was discarded and pellets were flash frozen in liquid nitrogen, and stored at -80°C until further analysis. To prepare proteomics samples for analysis by LC-MS/MS, pellets were thawed and cells were lysed by suspension in 6 M GnHCl, followed by addition of MeOH to 90%. Samples were centrifuged at 15,000 × g for 5 min. Supernatant was discarded and pellets were allowed to dry for ~5 min. Pellets were resuspended in 200 µL 8 M urea, 100 mM Tris pH 8.0, 10 mM TCEP, and 40 mM chloroacetamide, then diluted to 2 M urea in 50 mM tris pH 8. Trypsin was added at an estimated 50:1 ratio, and samples were incubated overnight at ambient temperature. Each sample was desalted over a PS-DVB solid phase extraction cartridge and dried down. Peptide mass was assayed with the peptide colorimetric assay. Proteomics LC-MS/MS method. For each analysis, 2 µg of peptides were loaded onto a 75 µm i.d. 30 cm long capillary with an imbedded electrospray emitter and packed with 1.7 µm C18 BEH stationary phase. The mobile phases used were A: 0.2% formic acid and B: 0.2% formic acid in 70% acetonitrile. Peptides were eluted with in increasing gradient of acetonitrile from 0% to 53% B over 100 minutes followed by a 5 minute 100% B wash and a 10 minute equilibration in 0% B. Eluting peptides were analyzed with an Orbitrap Fusion Lumos. Survey scans were performed at R = 2400,000 with wide isolation analysis of 300-1,350 mz. Data dependent top speed (1 seconds) MS/MS sampling of peptide precursors was enabled with dynamic exclusion set to 20 seconds on precursors with charge states 2 to 4. MS/MS sampling was performed with 0.7 Da quadrupole isolation, fragmentation by HCD with NCE of 30, analysis in the ion trap using the “rapid” scan speed, with a max inject time of 18 msec, and AGC target set to 3 x 104.
Data Processing Protocol
Proteomics computational analysis. Raw files were analyzed using MaxQuant 188.8.131.52. Spectra were searched using the Andromeda search engine against a target decoy database. Label free quantitation and match between runs were toggled on, ms/ms tolerance was set to 0.4 Da, and numbers of measurements for each protein was set to 1. Default parameters were used for all other analysis parameters. Peptides were grouped into subsumable protein groups and filtered to 1% FDR, based on target decoy approach. Log2 transformed label-free quantitation intensities were further processed to obtain log2 fold change values relative to the time-zero time-point for O2 and An samples separately.
Alexander Hebert, UW madison
Joshua Jacques Coon, Genome Center of Wisconsin,Department of Chemistry, Department of Biomolecular Chemistry, and DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA Morgridge Institute for Research, Madison, WI 53706, USA ( lab head )
Martien JI, Hebert AS, Stevenson DM, Regner MR, Khana DB, Coon JJ, Amador-Noguez D. Systems-Level Analysis of Oxygen Exposure in Zymomonas mobilis: Implications for Isoprenoid Production. mSystems. 2019 4(1) PubMed: 30801024