Technological Innovations and Applications of Microbial Protein Production: From Genetic Engineering to Sustainable Manufacturing

Facing global climate change, resource shortages, and the urgent need for carbon neutrality goals, microbial protein production has demonstrated significant potential in the fields of food, pharmaceuticals, and industrial applications. With the emergence and integration of novel editing technologies, synthetic biology, and fermentation processes, researchers have achieved not only significant enhancement in protein yields but also efficient resource utilization and green production model innovations. The Special Issue “Research on Microbial Protein Synthesis” includes papers showcasing recent advancements in microbial protein production, which can be summarized across four dimensions: genetic engineering optimization, fermentation processes innovations, sustainable production strategies, and novel application development. Furthermore, these papers show the challenges and opportunities for future research.

The rapid development of genetic engineering technologies has provided precise regulation tools for microbial protein production. Rational gene editing of microbial strains enables accurate regulation to enhance production efficiency. Zheng et al. [1] established a systematic optimization strategy by integrating promoter and signal peptide screening, gene dosage optimization, molecular chaperone co-expression, and host engineering. They developed an antibiotic-free plasmid recombination technology to eliminate resistance gene-associated risks and leveraged high-density fermentation to obtain gram-scale production capacity of nanobodies. Similarly, Li et al. [2] achieved effective secretion of the swine fever virus E2-Spy antigen protein in Komagataella pastoris through a combined strategy of signal peptide engineering, multi-copy gene integration, and molecular chaperone co-expression. Notably, the innovative hybrid signal peptide cSP3 overcame the traditional secretion limitations of single signal peptides, and the synergistic effects of molecular chaperones like SSA1 and YDJ1 significantly improved protein folding efficiency. These two papers systematically analyzed the bottleneck issues in nanobody or antigen expression by K. pastoris and developed combinatorial optimization strategies to break through the yield limitations of conventional expression systems, providing an important reference framework for recombinant protein production in K. pastoris.

Besides pharmaceutical proteins, proteins also include enzymes that play catalytic roles in metabolic pathways. Zhong et al. [3] mined a high-efficiency hyaluronic acid synthase (SthasA) from the probiotic Streptococcus thermophilus and constructed a de novo hyaluronic acid synthesis pathway in the non-pathogenic Bacillus amyloliquefaciens, aiming to replace the traditional hyaluronic acid production system that relies on pathogenic bacteria. By enhancing precursor supply via heterologous hasB gene introduction and manipulating the global regulatory factor CcpA, they achieved a hyaluronic acid production of 5.57 g/L, demonstrating the feasibility of large-scale production. This work provides a methodological paradigm for the production of high-value products through enzymatic proteins.

The drawbacks of traditional batch fermentation have driven researchers to develop novel fermentation modes to achieve continuous protein production. Zhang et al. [4] constructed a genomically integrated human epidermal growth factor (hEGF) expression system in Escherichia coli. By overexpressing biofilm formation genes (dgcC, csgD) and cellulose synthesis genes (bcsA, bcsB), they created E. coli cells self-immobilized in a biofilm reactor. The biofilm formation ability of the engineered E. coli increased by 91%. As a result, a biofilm-based continuous secretion process was established for hEGF and the productivity was increased by 28% compared to that of the conventional planktonic cell system. Besides the prokaryotic biofilm system, the same team also engineered a continuous hEGF production process based on Saccharomyces cerevisiae’s biofilm system [5]. Through the overexpression of endogenous or heterologous adhesion proteins (FLO11, ALS3), S. cerevisiae cells could be effectively adhered onto carrier surfaces, facilitating a continuous (repeated batch) production mode. Ten consecutive fermentation cycles were performed with an average hEGF production of 300 mg/L, achieving a 28.6% increase in productivity. These two studies demonstrated the feasibility of using biofilms for continuous, secretory production of proteins. This breakthrough overcomes the limitation of applying biofilm predominantly in small molecules production, pioneering the application in larger molecule production and paving a new pathway for continuous manufacturing of diverse therapeutic proteins and other proteins.

Technological innovations in solid-state fermentation are also noteworthy, which are commonly used to produce mycelial proteins serving as enzymes or animal feed. Londoño-Hernández et al. [6] explored the production of protease from fish flour through solid-state fermentation using Aspergillus strains, focusing on the effects of glucose concentration and support (polyurethane) ratio on enzyme yield. By optimizing the ratio of fish flour to polyurethane foam and glucose concentration, A. oryzae achieved efficient production of neutral and alkaline proteases within 36 h, with a peak yield of 20.5 U/mL. They employed online CO₂ monitoring technology to track microbial growth in real time and established a model to elucidate the partial correlation mechanism between protease synthesis and microbial growth. This research provides a metabolic kinetics-based approach for intelligent control of solid-state fermentation processes. The development of aquaculture can also benefit from solid-state fermentation. Weng et al. [7] demonstrated that fermented soybean meal can replace high-price fishmeal in the feed of Micropterus salmoides. Through fermentation using a microbial consortium (lactic acid bacteria, yeast, and Bacillus), phytate content in the soybean was reduced while crude protein increased. The fermented soybean meal could replace 30% of fish meal without affecting growth performance. This study provides a solution for plant-based protein substitution in aquatic feeds.

Microbial fermentation, leveraging its diverse substrate catabolism and low-carbon production attributes, could transform agricultural waste and industrial by-products into high-value-added products. This “waste-to-resource” process reduces dependence on fossil fuels and lowers carbon footprint. Muniz et al. [8] reviewed the advances in sustainable, non-animal protein production, showing that new bioprocess strategies, enzyme hydrolysis, and precision fermentation can improve protein yield and nutritional quality. Microbial proteins produced from agro-industrial residues would result in cost reduction and enhanced feasibility. These technologies could mitigate environmental strain from livestock farming, meet growing nutritional needs, and advance functional food development. Key challenges regarding non-meat proteins include large-scale implementation, high production cost, and strict regulations. More research efforts should be devoted to process design, interdisciplinary collaboration, and market strategies to overcome these challenges.

Through novel application development and multidisciplinary integration, the application scenarios of microbial proteins are expanding from traditional fields to emerging fields such as healthcare and materials, redefining the boundaries of the protein industry. For instance, nattokinase is an enzyme that is included in traditional food natto. Fang et al. [9] reviewed nattokinase, highlighting its therapeutic potential beyond traditional cardiovascular field, like Alzheimer’s disease prevention and retinal disease treatment. Enhancing the stability and protection of nattokinase through encapsulation or immobilization techniques is important for its bioavailability. A deeper exploration of the biological mechanisms of nattokinase would facilitate its diverse applications.

In summary, this Special Issue of Fermentation compiles the representative advances in microbial protein production. Collectively, microbial protein production technology is undergoing a pivotal transition from empirical approaches to rational design. Moving forward, the synergistic application of gene editing, process innovation, and sustainability strategies will enable this field to not only meet global demands for foods, pharmaceuticals, and materials but also deliver bio-manufacturing solutions to address climate and resource issues. However, challenges and technical bottlenecks persist in microbial protein production. Technical and economic feasibility of new production strategies or processes need to be evaluated in scaled-up production. High production cost and regulatory barriers are often encountered by novel alternative proteins. Future development demands large-scale implementation and interdisciplinary integration. Emerging tools including artificial intelligence and multi-omics analyses are powerful for deciphering complex biological behaviors, thereby guiding protein production and process optimization. Collaboration across microbiology, materials science, food engineering, and beyond will further drive technological innovations and applications of microbial protein production.