Comparison of C. necator H16 and CMS
When the organic carbon source in the environment is lacking, C. necator can grow rapidly under the conditions of CO2, H2 and O2. It is commendable that unlike other sources of hydrogenases, the hydrogenase derived from C. necator is resistant to oxygen. C. necator H16 produces four distinct [NiFe]-hydrogenases that each serves unique physiological roles (Burgdorf et al. 2005; Cramm 2009). For example, membrane-bound hydrogenase (MBH) is composed of HoxG (PHG002, 68.8 kD) and HoxK (PHG001, 39.5 kD) structural subunits, which are anchored to the membrane by HoxZ. Soluble hydrogenase (SH) consists of four subunits: HoxH (PHG091, 54.9 kD), HoxY (PHG090, 22.9 kD), HoxU (PHG089, 59.6 kD), HoxF (PHG088, 66.7 kD), which deliver protons and electrons to NAD+ to synthesize NADH for cell growth and biosynthetic reactions. Compared with H16, CMS strengthens the promoter of MBH and SH gene clusters (Li et al. 2020). After expression in H16 and CMS, the homogeneity of these enzymes was examined by SDS-PAGE (Fig. 1A). The sample of CMS showed two significant peaks marked with red arrows, while the control H16 did not. This indicates that hydrogenase expression is greatly enhanced in CMS.
The metabolic features of autotrophic HOB allow them to grow on H2 and O2, while fixing CO2 into target products. For SCP accumulation, a completely stirred tank reactor or a sequencing batch reactor was used during the batch as well as continuous experiment (Matassa et al. 2016). Notably, the gas mixture of H2, CO2 and O2 was optimized with a ratio of 7:1:1 (Dou et al. 2019; Ruuskanen et al. 2021). The flow rate of the gas mixture was controlled at 0.5 VVM. The H2 was supplied by a generator, and the CO2 and O2 were provided from gas tanks. The effect of filling 40 mL of liquid was greater than 60 ml, and we speculated that a sufficient head space for gas may be needed for bacteria to grow better (Fig. 1B). The result also indicates that growth of CMS is better than H16, which should be attributed to the improvement in the expression of MBH and SH of the former (Li et al. 2020).
Selection of surface displayed enzymes
Although ROS are cytotoxic, their conversion to water can occur when protective antioxidant enzymes like SOD and CAT are present (Torella et al. 2015). Herein, we validated the effects of adding SOD and CAT to the cathodic chamber of a three-electrode system on the growth of C. necator CMS (Fig. 2). It clearly shows that the growth rate of the strain with exogenous SOD only or both SOD and CAT improves significantly compared to others. The impact of CAT, in contrast, appears to be very limited. These findings point to the conclusion that SOD protects cells from ROS damage much more effectively than CAT, at least in our case. Therefore, we assumed that cathodically generated O2·− was more cytotoxic in BES, suggesting the necessity to display SOD on the cell surface.
Influences of promoters and signal peptides on surface displayed SOD
Consist with previous studies, autotransporters which have distinct functions in their native hosts play the most prominent role in the anchor proteins in many Gram-negative bacteria (Tozakidis et al. 2015). FhuA, OmpA, Lpp-OmpA and IgA were compared to function in cell surface display of C. necator, and quantitative research demonstrated that IgA was a more effective anchor protein than other. (Tsai et al. 2015). Thus, IgA protease autotransporter was used in this study. In order to analyze the influence of the promoter and signal peptide on protein secretion by the autotransporter pathway, the C. necator CMS strains were transformed with plasmids BBa_J23100-SOD-E-tag-IgAβ, pBAD-pelB-SOD-E-tag-IgAβ, T7-SOD-E-tag-IgAβ and pBAD-pelB-SOD-E-tag-IgAβ.
Initially, we used three promoters with different expression strengths to find the one with the strongest displayed SOD activity. The result indicates that T7 expresses higher enzyme activity than BBa_J23100 or pBAD does (Fig. 3A). After three C. necator strains were cultured in BES, it was found that from the pBAD-driven strains were more resistant to O2·− and had a higher survival rate in terms of seven-day growth pattern (Fig. 3B). This result is slightly different from the activity assay (Fig. 3A), possibly due to a lower cell survival rate under the strong promoter. Similar result was also found in E. coli (Dong et al. 1995; Kurland and Dong 1996). These results suggest that the combination of IgAβ and pBAD promoter is the best choice for SOD display on C. necator CMS. Next, we tested the effect of signal peptides on the strain growth. Previous study indicated that pelB was essential for the IgAβ-anchored system (Valls et al. 2000). However, our experiment result shows that enzymatic activity of displayed SOD is the same with or without the presence of pelB (Fig. 3C), and the strain containing pelB performs even worse than that without pelB (Fig. 3D). The cause of this result may be low secretion, which was confirmed in the fluorescence analysis.
Taken together, these results suggest that pBAD-SOD-E-tag-IgAβ is the best recombinant strain with an enzymatic activity of 21 U/mg. The growth rate of C. necator CMS displayed by SOD can achieve 4.9 ± 1.0 of OD600 by 7 days, equivalent to 1.7 ± 0.3 g/L DCW, and the production rate is 0.24 ± 0.04 g/L/d DCW, which shows around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD600).
Fluorescence analysis
Microbial cell-surface display systems allow target protein or peptides to be displayed on the surface of microbes through the fusion with a anchor protein. Because of its decreased background for intracellular imaging, the mCherry was chosen as a reporter to replace SOD for the fluorescence microscopy study. As shown in Fig. 4A, there is a fluorescent halo in fluorescence microscopy on C. necator CMS when mCherry is anchored to the outer membrane, compared to the strain with mCherry but without the anchor protein. This suggests that the protein is displayed uniformly across the cell surface. Instead, the expression of unanchored mCherry is distributed diffusely throughout the cell. This further suggests the important role of IgAβ as an anchor protein module.
To confirm whether the fusion proteins were properly translocated to the surface, the different CMS strains harboring SOD-E-tag plasmids were further analyzed by immunofluorescence (Fig. 4B). The primary antibody used was rabbit anti-E antibody and the secondary antibody was goat anti-rabbit IgG with Alexa Fluor 488 labeling, and the original CMS strain was used as a control. It was found that only the green fluorescence-labeled anchoring protein E-tag-IgAβ appeared on the cell surface on fluorescence images. These indicate that the anchor module, IgAβ, is displayed on the cell surface and the fusion protein is expressed in a non-degraded and active form. Moreover, the fluorescence intensity of the pelB-containing strain is noticeably lower, showing that pelB is inefficient in this case.
Validation of surface displayed SOD
We took advantage of the ability that SOD may convert O2·− to hydrogen peroxide as a way in tackling the issue of the existence of ROS in BES. For comparison, extracellular secretion of SOD and intracellular overexpression of SOD were also conducted using the strains containing the plasmids of pBAD-SOD, pBAD-pelB-SOD. Both enzyme activity assay and the growth results reveal that the strain with the surface displayed SOD outperforms significantly to the other two in BES (Fig. 5). The effects of extracellular secretion or intracellular overexpression of SOD seem to be the same. The reason might be that extracellular secretion released into the solution or intracellular overexpression restrained in the cytoplasm of SOD are insignificant, which cannot prevent O2·− from breaking the outer membrane. In contrast, it is clear that SOD proteins are evenly displayed on the outer membrane of C. necator from the fluorescence experiments, which form a shield to protect the strain as expected.