Meta-analysis Driven Strain Design for Mitigating Oxidative Stresses Important in Biomanufacturing

Abstract

As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 E. coli adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant oxyR, fur, iscR, and ygfZ were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into oxyR, fur, iscR, and ygfZ exhibited increased tolerance to H₂O₂ and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of ygfZ, an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.

Keywords: ALE mutations; SOS response; acid stress; iModulons; meta-analysis driven strain design; reactive oxygen species.

Figure 1

The parent strain has higher ROS stress sensitivity and SOS response compared to the wild type. (A) Growth of the wild-type strain and melatonin production strain (parent strain) with or without H₂O₂. The wild-type strain could tolerate 10 mM H₂O₂, but the parent strain cannot. (B) SOS response of the wild-type and parent strain measured by a GFP-based SOS sensor. The parent strain has a slightly higher SOS response when growing in the LB medium and about 50% higher SOS response level in the presence of H₂O₂ (*p < 0.05, **p < 0.001). See Materials and Methods for details.

Figure 2

Meta-analysis of mutated genetic features and their experimental conditions in ALEdb samples. (A) Public ALEdb mutated genetic features statistically associated with sources of ROS stress in ALEdb (Fisher’s exact test, p value < 0.01, Bonferroni corrected). (B) The features sorted according to the sum of their unique mutations per independent ALE replicate across paraquat and FeSO4 ALE experiments. The total amount of ALE experiments per feature also displayed. (C) The mutation count of genes of interest in samples for ALE experiments explicitly involving ROS stresses. Slight differences in totals exist between B and C and Figures 3 through 6 due to different filtering methods applied (see the ALEdb Mutations section).

Figure 3

AQALEdb mutations, their effects on OxyR, and to iModulons. (A) Mutation needle plot demonstrating the effect and position of ALEdb mutations to oxyR. Slight differences in totals exist between Figure 2B,C and Figures 3 through 6 due to different filtering methods applied (see the ALEdb Mutations section). (B) OxyR’s 3D structure and mutated residues from mutations. The residue chain and transparent surfaces are colored according to the legend of the corresponding mutation needle plot. Mutations are represented by a small opaque sphere with a value representing their amino acid position on the corresponding mutation needle plot. The color of the mutation’s sphere corresponds to the mutation’s predicted effect as described by the legend on the corresponding mutation needle plot. The transparent sphere centered on the mutations’ opaque sphere represents the number of mutations with a specific predicted effect on that position. (C) OxyR iModulon activities for all available samples (1035 from iModulonDB and 12 new samples from this study), where the experiments and strains with oxyR mutations are differentiated from the rest of the distribution. The oxyR mutant strains were from E. coli ALE experiments that manifested oxyR mutations as well as mutations to other genes.

Figure 4

ALEdb mutations and their effects to Fur. (A) Mutation needle plot demonstrating the effect and position of ALEdb mutations to fur. Slight differences in totals exist between Figures 2B,C and Figures 3 through 6 due to different filtering methods applied (see the ALEdb Mutations section). (B) Fur’s 3D structure and mutated residues from mutations. The residue chain and transparent surfaces are colored according to the legend of the corresponding mutation needle plot. Mutations are represented by a small opaque sphere with a value representing their amino acid position on the corresponding mutation needle plot. The color of the mutation’s sphere corresponds to the mutation’s predicted effect as described by the legend on the corresponding mutation needle plot. The transparent sphere centered on the mutations’ opaque sphere represents the number of mutations with a specific predicted effect on that position. (C) Fur-1 and Fur-2 iModulon activities for all available samples (1035 from iModulonDB and 12 new samples from this study), where the experiments and strains with fur mutations are differentiated from the rest of the distribution. The fur mutant strains were from E. coli ALE experiments that manifested fur mutations as well as mutations to other genes.

Figure 5

ALEdb mutations and their effects to IscR. (A) Mutation needle plot demonstrating the effect and position of ALEdb mutations to iscR. Slight differences in totals exist between Figures 2B,C and Figures 3 through 6 due to different filtering methods applied (see the ALEdb Mutations section). (B) IscR’s 3D structure and mutated residues from mutations. The residue chain and transparent surfaces are colored according to the legend of the corresponding mutation needle plot. Mutations are represented by a small opaque sphere with a value representing their amino acid position on the corresponding mutation needle plot. The color of the mutation’s sphere corresponds to the mutation’s predicted effect as described by the legend on the corresponding mutation needle plot. The transparent sphere centered on the mutations’ opaque sphere represents the number of mutations with a specific predicted effect on that position. (C) Suf and Isc iModulon activities for all available samples (1035 from iModulonDB and 12 new samples from this study), where the experiments and strains with iscR mutations are differentiated from the rest of the distribution. The iscR mutant strains were from E. coli ALE experiments that manifested iscR mutations as well as mutations to other genes.

Figure 6

ALEdb mutations and their effects to YgfZ. (A) Mutation needle plot demonstrating the effect and position of ALEdb mutations to ygfZ. Slight differences in totals exist between Figures 2B,C and Figures 3 through 6 due to different filtering methods applied (see the ALEdb Mutations section). (B) YgfZ’s 3D structure and mutated residues from mutations. The residue chain and transparent surfaces are colored according to the legend of the corresponding mutation needle plot. Mutations are represented by a small opaque sphere with a value representing their amino acid position on the corresponding mutation needle plot. The color of the mutation’s sphere corresponds to the mutation’s predicted effect as described by the legend on the corresponding mutation needle plot. The transparent sphere centered on the mutations’ opaque sphere represents the number of mutations with a specific predicted effect on that position. (C) Heatmap of iModulon activities for samples from a ygfZ mutant characterization experiment using the melatonin production strain as the parent strain. An iModulon activity of ≥5 or ≤−5 is meant to represent a very large change in activity relative to the baseline. (D) CP4-44 iModulon activities for all available samples (1035 from iModulonDB and 12 new samples from this study), where the experiments and strains with ygfZ mutations are differentiated from the rest of the distribution. The ygfZ mutant strains annotated with “paraquat ALE” were from E. coli ALE experiments that manifested ygfZ mutations as well as mutations to other genes.

Figure 7

Comparison of growth and stress response between the parent strain (control) and the mutant strains in H₂O₂ and acid stress. (A) Biomass represented by OD (600 nm) of the parent strain and strains with ALE mutations implemented with 10 mM H₂O₂ treatment (blue) or without (yellow) after 72 h cultivation (Materials and Methods). The dots represent the OD values of each replicate. Whereas the parent strain cannot grow in 10 mM H₂O₂, some mutants like OxyR A213P reached the same OD with or without H₂O₂. (B) SOS response of wildtype, parent strain, and ALE mutants with or without 10 mM H₂O₂ treatment. Data represent the average of three replicates. Error bars indicate standard deviation. Asterisks indicate that the difference is significant (p < 0.05) compared to the control (parent strain). (C) Tolerance of the parent strain and ALE mutants in acid stress. Cultures of neutral pH were diluted into pH 4.5. OD (600 nm) was monitored after 1, 3, and 5 h (Materials and Methods). The height of the bars indicates the average concentration of three biological replicates, and error bars indicate the standard deviations. Single asterisk (*) indicates p < 0.05, and double asterisk (**) indicates p < 0.001, all compared to the control (parent strain). (D) SOS response of the parent strain and ALE mutants in acid stress. Cultures of neutral pH were diluted to pH 4.0. SOS response was monitored after 1, 3, and 5 h using a GFP sensor (Materials and Methods). The height of the bars indicates the average of the three biological replicates. The error bars indicate the standard deviations. Asterisks indicate that the difference is significant (p < 0.05) compared to the control (parent strain). (E) Small-scale batch cultivation of the parent strain (control) and strains with one of the three mutations incorporated: Fur P18T, YgfZ T108P, or OxyR A213P. Growth curves represented by integral carbon dioxide transfer rate (CTR) measured online. Data represent the average of three replicates. Error bars indicate standard deviation. (F) Melatonin final titers measured by HPLC after 48 h. (G) Specific melatonin production of four strains normalized by biomass. No statistically significant improvement (p < 0.05) in mutant strains compared to the parent.