Target protein selection

α-Lactalbumin possesses a relatively flexible tertiary structure and is enriched in hydrophilic residues, features that render it highly susceptible to proteolytic attack and facilitate gastrointestinal digestion. Its digestibility exceeds 90%, markedly higher than that of most plant-derived proteins. Owing to its favorable digestibility and amino-acid profile, α-lactalbumin has high nutritional value and is widely incorporated into infant formula, sports nutrition products, and functional foods.

In the context of space missions, the advantages of α-lactalbumin are particularly salient. Its high content of branched-chain amino acids can help mitigate muscle atrophy and bone loss under microgravity. Its elevated tryptophan content may aid adaptation to the psychological stressors associated with long-duration missions. In addition, cysteine residues present in peptide hydrolysates contribute antioxidant capacity, potentially alleviating oxidative stress induced by the space environment.

Protein source: human vs. bovine/ovine

Human α-lactalbumin exhibits an amino-acid composition that more closely matches the requirements for human protein synthesis, with a favorable distribution of essential amino acids and relatively higher levels of tryptophan and cysteine. Compared with bovine/ovine α-lactalbumin, the human ortholog is associated with lower allergenicity, higher digestibility, and stronger calcium-binding properties. Taken together, human α-lactalbumin offers advantages across nutritional value, physiological compatibility, and safety.

Expression strategy: intracellular vs. secretory

We initially considered both intracellular expression and extracellular secretion. Although α-lactalbumin is naturally processed and secreted in mammalian systems—suggesting that a secretion strategy could simplify downstream purification—secretion efficiencies in Gram-negative bacteria are generally low, and secretory pathways impose stringent substrate requirements. Moreover, multiple engineering studies in Cupriavidus necator H16 indicate that intracellular expression typically affords higher yields and greater stability. Considering literature and our operational constraints, we judge intracellular expression to be the more feasible and robust option, particularly under the energy-limited conditions relevant to space applications. Accordingly, we adopt an intracellular expression strategy for α-lactalbumin and co-express the folding assistants hPDI, SLY1, and KAR2 to promote correct folding and enhance protein solubility.

We selected the Para-RBS1-eGFP plasmid as the expression backbone. This vector carries a kanamycin resistance marker (KanR) and the L-arabinose–inducible araBAD promoter (Para), enabling inducible and tunable expression of heterologous genes. The Para system has been extensively validated across bacterial hosts, providing a solid literature basis for employing Para-RBS1-eGFP as our foundational vector.

The coding sequence of human α-lactalbumin was codon-optimized for expression in Cupriavidus necator and synthesized with a C-terminal His tag (6×His). Primers bearing appropriate homology arms were designed in SnapGene, and the fragments were assembled via Gibson Assembly. The human LALBA (hLALBA) expression cassette was placed immediately downstream of the Para promoter, replacing the original eGFP reporter.

To enhance the folding of the target protein, we implemented chaperone co-expression in parallel strain sets: Group A harbors hPDI, whereas Group B carries KAR2–SLY1, both serving as folding assistants to improve the correctness of protein folding and increase soluble yield.

During plasmid construction, the synthesized gene fragment was amplified by PCR with primers incorporating homologous arms at both termini. The amplicons were examined by agarose gel electrophoresis and migrated at the expected sizes. The purified fragments were then ligated using DNA ligase to assemble the complete plasmid.

Chemically competent E. coli were transformed with the ligation products and incubated to allow colony formation. Single colonies were picked for liquid expansion, followed by sequence verification. Sequencing confirmed correct assembly for Group A, whereas Group B repeatedly exhibited junction errors and/or aberrant gel bands. Despite multiple rounds of colony PCR screening and re-ligation, a correctly assembled Group B plasmid could not be obtained. We hypothesized that the issue originated from one of the two chaperone inserts (KAR2 or SLY1). Subsequent subcloning and testing of the components individually revealed that constructs harboring SLY1 alone yielded normal sequencing results. Consequently, the Group B design was revised to a single-chaperone configuration retaining only SLY1.

Following verification of construct integrity, the two plasmid sets were electroporated into the hydrogenotrophic bacterium. After induction, cells were lysed and analyzed by SDS–PAGE and Western blotting. Under L-arabinose induction, a band corresponding to the expected molecular mass of the target protein was observed. Immunodetection with anti-His antibodies confirmed that the band contained the His tag. These results indicate successful production of His-tagged α-lactalbumin in the bacterial host.

1.Plasmid Construction Verification

•Successfully constructed a protein expression plasmid containing the kanamycin resistance gene (KanR), α-lactalbumin gene fragment, chaperone gene fragment, and molecular tags under the control of an L-arabinose inducible promoter (araBAD promoter, Para), laying a foundation for subsequent scale-up culture and batch validation.

2.Expression Verification

•No expression was detected under uninduced conditions, indicating minimal leakage and confirming the system's tight inducibility.

•Under arabinose induction, Western Blot analysis using the His tag confirmed target protein expression. The band size matched the design, demonstrating that the constructed plasmid can drive correct expression of foreign proteins in Hydrogenobacter.

3.Improvements and Next Steps

•While His tag detection provides a rapid and reliable verification method, it does not reflect protein folding status. Subsequent functional assays or structural analyses are required to confirm proper protein folding and activity.

•The combination strategy for KAR2 and SLY1 requires optimization of cloning methods (e.g., Golden Gate assembly or longer homologous arms) to enhance splicing success rates.

•Pan, H., Wang, J., Wu, H. et al. Synthetic biology toolkit for engineering Cupriviadus necator H16 as a platform for CO(2) valorization. Biotechnol Biofuels 14, 212 (2021). https://doi.org/10.1186/s13068-021-02063-0

•Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact. 2018 Mar 29;17(1):52. doi: 10.1186/s12934-018-0901-3. PMID: 29598818; PMCID: PMC5875014.

•Gruber S, Schwendenwein D, Magomedova Z, Thaler E, Hagen J, Schwab H, Heidinger P. Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains. J Biotechnol. 2016 Oct 10;235:92-9. doi: 10.1016/j.jbiotec.2016.04.026. Epub 2016 Apr 13. PMID: 27085887.

•Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD. Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. J Bacteriol. 2000 Dec;182(24):7029-34. doi: 10.1128/JB.182.24.7029-7034.2000. PMID: 11092865; PMCID: PMC94830.

•Ruohao Tang, Rui Xu, Xuemin Gao, Cunxi Dai, Xiaochun Qin, Jianming Yang, Production of α-amylase from gluconate and carbon dioxide by protein synthesis and secretion optimization in Cupriavidus necator H16, Bioresource Technology, Volume 416, 2025, 131744, ISSN 0960-8524, https://doi.org/10.1016/j.biortech.2024.131744.

In Cupriavidus necator H16, we sought to evaluate and compare the usability and performance of different promoter–inducer systems, with the goal of identifying optimal induction conditions characterized by low basal leakage, tunable expression, rapid response, high yield, and minimal metabolic burden, thereby establishing a parameter foundation for subsequent target protein expression. Based on a literature survey, we selected four inducible promoters whose regulatory mechanisms are largely orthogonal to the native metabolic and regulatory networks of C. necator H16—pAra, pIPTG, pCumate, and pRha—along with their corresponding inducers as candidate systems for comparative analysis. Given that C. necator H16 is not inherently a high-yield host for heterologous protein production, the choice of an inducer–promoter pair that enables rapid response and robust expression is of critical importance.

We constructed four plasmids, each harboring the red fluorescent protein (mRFP) reporter gene under the control of one of the inducible promoters—PBAD, Plac, Pcmt, or PrhaBAD. All constructs were generated on the same plasmid backbone, pBBR1MCS, to ensure comparability.

Following cultivation under four induction regimes, fluorescence was quantified at 24 and 48 h using a microplate reader. Fluorescence intensity–time plots were generated, with signals normalized to optical density (RFU/OD) to compare induced expression levels and basal expression (leakiness) in the absence of inducer.

Results：Among the four systems tested, arabinose (Para) exhibited the highest induction, reaching approximately 800-fold with no detectable basal expression in the absence of inducer; IPTG (Plac) achieved ~400-fold induction but exhibited substantial basal leakiness; rhamnose (PrhaBAD) produced a lower induction of ~30-fold with minimal leakiness; and cumate (Pcmt) gave ~50-fold induction with similarly low leakiness.

Based on the results above, we selected arabinose (PBAD) as the standard inducer for subsequent protein expression, as it exhibits the greatest dynamic range (≈800-fold) with no detectable basal expression in the absence of inducer, thereby simplifying downstream control and preventing undesired background expression.

•Mishra S, Perkovich PM, Mitchell WP, Venkataraman M, Pfleger BF. Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production. J Ind Microbiol Biotechnol. 2024 Jan 9;51:kuae008. doi: 10.1093/jimb/kuae008. PMID: 38366943; PMCID: PMC10926325.

•Alagesan S, Hanko EKR, Malys N, Ehsaan M, Winzer K, Minton NP. Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16. Appl Environ Microbiol. 2018 Sep 17;84(19):e00878-18. doi: 10.1128/AEM.00878-18. PMID: 30030234; PMCID: PMC6146998.

In the previous cycle, we benchmarked and validated multiple promoter–inducer systems under heterotrophic conditions in Cupriavidus necator H16 and identified the Ara (PBAD) system as superior with respect to dynamic range and control of basal leakiness. Concurrently, we observed substantial background expression with the IPTG (Plac) system, which impeded precise regulation; accordingly, IPTG was excluded from the present cycle. We, therefore, focused on evaluating the suitability of three systems—PBAD (Ara), Pcmt (cumate), and PrhaBAD (rhamnose)—under autotrophic conditions.

In addition, given nitrogen management constraints in spaceflight, we considered replacing ammonium sulfate in the medium with urea as the sole nitrogen source to approximate the operational environment of a spacecraft. In parallel, we performed a preliminary balance between spacecraft gas supply and cellular production demand to assess feasibility for future life-support integrations, with the aim of coupling to a CO₂/H₂ loop and achieving closed-cycle utilization of carbon, hydrogen, and nitrogen resources.

•The construction of inducible expression systems followed the design of the previous cycle. Three inducible plasmids were built on the pBBR1MCS backbone and subsequently introduced into Cupriavidus necator H16. The engineered strains were cultivated under autotrophic conditions in ISM medium, with periodic gas exchange (H₂, O₂, CO₂) every 24 hours. Optical density and fluorescence intensity were measured at each interval.

•To evaluate the feasibility of urea as a nitrogen source for C. necator H16, we established cultivation systems in ISM medium supplemented with either ammonium sulfate (NH₄⁺), urea (as the sole nitrogen source), or a combination of both. Representative clones from lineages A and B (e.g. A12 and B10), all carrying the same plasmid backbone, were selected to eliminate potential vector-related variability. This design allows for the comparative assessment of different nitrogen sources on growth rate, maximum biomass yield, and physiological adaptability, thereby providing critical parameters for subsequent protein expression experiments.

•Validation of inducers

Under autotrophic conditions, a comparative analysis of the three inducible systems demonstrated that Ara (PBAD) achieved the highest induction efficiency together with a well-defined dose–response relationship, thereby representing the most effective regulatory option. In contrast, Rha (PrhaBAD) exhibited a more limited dynamic range but maintained exceptionally low basal expression, rendering it advantageous in scenarios where minimal background activity is required. Cumate (Pcmt) provided an intermediate level of induction with a relatively insensitive dose–response, favoring stable high-level expression rather than fine-tuned regulation. Collectively, these findings highlight Ara as the most suitable standard inducible system under autotrophic growth conditions, with Rha and Cumate offering valuable complementary alternatives depending on the specific experimental requirements.

•Nitrogen source validation

Experimental results indicated that C. necator H16 exhibited no appreciable growth in ISM medium supplemented solely with ammonium sulfate, whereas cells cultured with urea as the nitrogen source maintained stable and pronounced growth, with OD values increasing significantly over a 10-day period. This finding suggests that the efficiency of urea utilization as a nitrogen source is markedly higher than that of ammonium sulfate. Moreover, strain-specific differences were observed under urea conditions; for instance, A12 displayed a faster growth rate and achieved a higher final biomass compared to B10, implying that urea-driven growth may be influenced by the repertoire of chaperone proteins, which in turn modulates biomass accumulation.

Inducer system:

Under autotrophic conditions, the Ara (PBAD) system consistently exhibited the widest dynamic range and the most distinct dose–response relationship, while showing virtually no basal leakage in the absence of inducer. It thus remains the most suitable standard regulatory tool for C. necator H16, and was therefore selected for subsequent expression experiments.

Nitrogen source utilization:

The efficiency of urea utilization was found to be markedly higher than that of ammonium sulfate. Strain-specific differences in growth performance under urea conditions suggest that future optimization of urea-based cultivation systems may require consideration of different chaperone protein profiles.

Implications for space applications: The combined results of the Ara (PBAD) system and urea as a nitrogen source demonstrate that C. necator H16 can achieve both efficient inducible expression and sustained growth under autotrophic conditions. Since urea is a metabolic by-product of astronauts and can be recycled, while the Ara system enables efficient and tightly controlled heterologous protein expression, their integration provides experimental evidence supporting the feasibility of closed-loop carbon–hydrogen–nitrogen resource utilization in spacecraft life-support systems.

•Weldon M, Euler C. Physiology-informed use of Cupriavidus necator in biomanufacturing: a review of advances and challenges. Microb Cell Fact. 2025 Jan 22;24(1):30. doi: 10.1186/s12934-025-02643-x. PMID: 39844200; PMCID: PMC11755831.

•Siwar ISMAIL, Géraldine GIACINTI, Christine DELAGADO RAYNAUD, Sandrine ALFENORE, Stéphane E. GUILLOUET, Nathalie GORRET, Characterization of Single Cell Protein produced by Cupriavidus necator grown on various nitrogen and carbon sources, Journal of Biotechnology, 2025, ISSN 0168-1656,https://doi.org/10.1016/j.jbiotec.2025.08.007.

In Cycle 1, bacterial strains with successful plasmid transfection were obtained; in Cycle 2, the optimal culture and induction conditions for these strains were further determined. In the current cycle (Note: referring to the subsequent experimental cycle), the target strains will first undergo scale-up cultivation. After harvesting the bacterial cells, soluble lysate will be prepared via ultrasonic disruption. Subsequently, His-tag affinity purification will be carried out using Ni-NTA (or similar nickel-chelating media), with chromatographic analysis technology supplemented simultaneously to verify the purity and yield of the target protein (α-lactalbumin), and basic verification of its structure and folding state will also be performed.

Based on the confirmed expression strains and induction conditions, this cycle conducts scale-up cultivation (from shake flasks to 1 L scale) and His-tag protein purification, with the specific procedure as follows: First, single clones verified to express α-lactalbumin were picked for shake-flask precultivation. Subsequently, the precultured bacterial solution was inoculated into 1 L medium containing corresponding antibiotics and the established induction protocol at an inoculation ratio of 1–10% (v/v), and cultured until the preset harvest point. In the subsequent operations, bacterial cells were first harvested and lysed by ultrasonic treatment under ice bath conditions, and the soluble supernatant was obtained by centrifugation. Then, pre-elution washing was completed via Ni-NTA affinity chromatography, and the target protein was eluted with 250 mM imidazole. After collecting the target protein fractions, buffer exchange was performed to meet the requirements of subsequent structural tests. The detection process includes three core items: SDS-PAGE was used to verify the band specificity and purity of the target protein; the combination of A280 absorbance method and Bradford method was applied to determine and calculate the protein yield (unit: mg/L); circular dichroism (CD, detection wavelength 190–260 nm) was employed to evaluate the secondary structure of the protein. Finally, linked baseline data of "yield–purity–structure" was formed, providing a reference basis for chassis cell modification and process parameter optimization in the next cycle.

Purified target protein samples were subjected to SDS-PAGE analysis, which revealed a distinct band corresponding to the target protein, confirming that α-lactalbumin was obtained at a relatively high yield. Coomassie staining was performed on lysate, flow-through, supernatant, and elution fractions to assess purity, and the final production level was calculated in mg/L.

B1E	B1H	A7E	A7H
1084.65532	908.75097	2266.21771	1046.90024
202.55936	197.41094	332.12793	182.82375
139.06218	139.92025	139.92025	135.6299

For plasmids in Group A (carrying the hPD1A3 chaperone) and Group B (carrying the SLY1 chaperone), protein expression was evaluated in both C. necator (abbreviated as H in the figure) and E. coli (abbreviated as E), resulting in four sets of fermentation assays.

His-tag purification analysis indicated that the target protein, α-lactalbumin, was recovered primarily in the 250 mM imidazole elution fraction. Quantitative results were as follows:

•B1E: 202.56 μg/mL, representing a relatively high protein concentration.

•A7E: 332.13 μg/mL, the highest among all four groups, with a clear advantage in protein yield.

•B1H: 197.41 μg/mL, slightly lower than B1E.

•A7H: 182.82 μg/mL, the lowest concentration among the four, below both B1E and B1H.

Overall, fermentation in E. coli yielded protein concentrations of 332.13 mg/mL and 202.56 mg/mL, whereas C. necator produced 182.82 mg/mL and 197.41 mg/mL, indicating that protein yield in C. necator was comparatively lower than in E. coli. Literature reports on α-lactalbumin production across microbial hosts show that Pichia pastoris achieves the highest yields of approximately 600 mg/L, while Kluyveromyces lactis can reach 279.5 mg/L through tandem gene fusion strategies, and Bacillus subtilis yields about 120 mg/L after signal peptide and promoter optimization. Compared with these data, the yields obtained in C. necator were lower than those in eukaryotic hosts such as P. pastoris and K. lactis, but were broadly comparable to other prokaryotic hosts such as E. coli and B. subtilis. Notably, although the preliminary fermentation data suggest that C. necator may achieve slightly higher yields than B. subtilis, this observation has not yet been structurally validated, and the actual fermentation performance remains to be confirmed. Future work should focus on validating these results with structural characterization, optimizing fermentation strategies, and exploring genetic or metabolic engineering

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•YUQI ZHU, PENGDONG SUN, CHUNJIAN LI, et al. Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening[J]. Food Science and Human Wellness, 2024,13(4):2310-2316. DOI:10.26599/FSHW.2022.9250192.

•Xinyi Wang, Xuguang Zhang, Yuejian Mao, Yaokang Wu, Xueqin Lv, Long Liu, Weiwei Han, Shenming Yin, Ruonan Wu, Jian Chen, and Yanfeng Liu. Ethanol-Inducible Bioproduction of Human α-Lactalbumin in Komagataella phaffii. Journal of Agricultural and Food Chemistry 2025 73 (15), 9246-9260. DOI: 10.1021/acs.jafc.5c0133