We selected the Para-RBS1-eGFP plasmid as our expression backbone and used kanamycin resistance for selection. The target gene hLALBA was inserted into the plasmid, with a C-terminal His-tag added to facilitate protein purification. A T7 strong promoter was introduced to enhance transcription efficiency.

To prevent misfolding and inclusion body formation of the target protein, and to improve its activity, we co-expressed molecular chaperones for comparison. Two main plasmids were constructed: Group A incorporated the chaperone hPDIA3, while Group B incorporated KAR2-SLY1 (Figure 1).

Agarose gel electrophoresis of Group A showed that the fragment sizes matched the expected results, confirming successful plasmid construction (Figure 2).

Unfortunately, Group B underwent multiple rounds of colony PCR and re-ligation, but the expected bands were never observed. We hypothesized that one of the two chaperones, KAR2 or SLY1, might be causing the issue. To verify this, we cloned KAR2 and SLY1 individually into the plasmid. The electrophoresis result for the SLY1-only plasmid showed the expected band (Figure 3). Consequently, Group B was modified to retain only SLY1 as a single-chaperone construct.

Based on the same plasmid backbone (pBBR1MCS), we compared four inducible promoters — pAra, pIPTG, pCumate, and pRha — using mRFP as a reporter gene. Cultures were grown in LB medium, and fluorescence intensity was measured at 24 and 48 hours using a microplate reader.

The fluorescence intensity was normalized as RFU/OD to compare expression strength and promoter leakage in the absence of inducers (Figure 4).

The results showed that Ara induction achieved the strongest expression intensity with no detectable leakage in the absence of inducer.The IPTG induction system also showed strong expression, but exhibited high background leakage without induction.Both Rha and Cumate systems demonstrated lower expression intensity, with minimal leakage in the absence of inducers.Therefore, we selected the Ara (pBAD) system as the standard inducer for subsequent protein expression experiments.

Due to the significant background leakage observed in the IPTG induction system, it was unsuitable for precise regulation. Under autotrophic conditions, we compared only the other three systems — Ara, Cumate, and Rha (Figure 5).

To simulate resource recycling in a space-station-like environment, we tested urea as a nitrogen source. In the ISM medium, ammonium sulfate and urea (coexisting with NH₄⁺) were used as the main nitrogen sources. Optical density (OD₆₀₀) was measured every 24 hours to generate growth curves of C. necator H16 under different nitrogen conditions (Figure 6).The results showed that the engineered strain exhibited minimal growth in ISM medium containing only ammonium sulfate, whereas when urea served as the nitrogen source, the cells maintained stable and significant growth.

Furthermore, we observed that the strain expressing chaperone hPDI (Figure 7, A12) utilized urea more efficiently than the strain expressing SLY1 (Figure 7, B10). Group A exhibited a faster growth rate and higher final biomass than Group B.

To verify whether α-lactalbumin (His-tagged) was successfully expressed in the engineered strains, Western blot analysis was performed (Figure 8).

The results showed that the strain induced with L-arabinose displayed a strong band corresponding to the target protein, while almost no band was observed in the uninduced sample. This indicates that α-lactalbumin was strongly expressed upon arabinose induction.

The protein was then purified using a Ni-NTA affinity column, and the eluate was analyzed by SDS-PAGE (Figure 9).

A distinct band around 14.2 kDa was observed in the elution fraction, confirming that the plasmid hLALBA-SLY1 successfully expressed α-lactalbumin, with a molecular weight of approximately 14.2 kDa.

We then measured the concentration of the eluted proteins using a BCA Protein Assay Kit (Table 1, Figure 10). The purified protein eluted with 250 mM imidazole showed a concentration of 197.41 μg/mL, while that eluted with 500 mM imidazole measured 139.92 μg/mL in strain hLALBA-hPDI. The purified protein eluted with 250 mM imidazole showed a concentration of 182.82 μg/mL, while that eluted with 500 mM imidazole measured 135.63 μg/mL in strain hLALBA-SLY1.