Tandem Repeat Gene Strategy for High-Yield Production and Functional Evaluation of Bioactive Wheat Oligopeptides.

Improving Yield and Assessing Quality of Secreted Proteins Over-Expressed in Pichia Pastoris with Chaperones and Structural Mass Spectrometry

Pichia pastoris is a widely used host for recombinant protein production because it combines the advantages of microbial cultivation with eukaryotic protein folding and secretion. However, secretion efficiency is often limited by the folding capacity of the endoplasmic reticulum (ER), where recombinant proteins must be translocated, folded, and processed prior to export. When ER folding capacity is exceeded, proteins may be retained, degraded, or secreted in non-native conformations, reducing both yield and product quality. Chaperone engineering and codon optimization represent two promising strategies to address these limitations. Here, we generated stable Pichia strains expressing four model secreted proteins (human serum albumin, interleukin-2, thaumatin-I, and thaumatin-II) using either conventional codon optimization or Epi-MAX codon engineering, which adapts transgene codon usage to stress-responsive translational programs. We also engineered strains containing an additional chromosomal copy of either the ER Hsp70 chaperone Kar2 or protein disulfide isomerase (Pdi1). To assess protein quality, we applied limited proteolysis mass spectrometry (LiP-MS), a structural proteomics approach that can detect subtle conformational differences to secreted proteins. Increased Pdi1 levels improved secretion of all four proteins tested, whereas Kar2 overexpression generally reduced yield. For thaumatin-II, Pdi1 enhanced secretion but promoted release of a non-native conformation, which we could correct through codon engineering. Together, these results demonstrate that maximizing recombinant protein production requires optimization of both yield and structural quality and establish complementary strategies for improving secreted protein expression in Pichia.

Advances in Key Genetic Elements and Strategies for High-Yield Heterologous Protein Expression in Komagataella phaffii

This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.

Toward Rational Design of Precision-Fermented Milk Proteins: Integrating Cross-Species Selection, Post-Translational Modification, and AI Optimization.

Milk proteins deliver nutritional, functional, and bioactive properties that alternative protein sources cannot adequately replicate, yet conventional livestock-based production faces compounding constraints of scalability, resource intensity, and sustainability. Precision fermentation offers a structurally distinct solution, but existing reviews have addressed neither a systematic cross-species framework for target selection nor a treatment of post-translational modifications (PTMs) gap-bridging, leaving critical gaps in rational pipeline design. This review integrates four analyses: a cross-species comparison of sequence, structural, and PTMs characteristics across human, bovine, goat, and camel milk proteins to inform target prioritization; a consolidation of advances in host engineering, fermentation scale-up, and downstream purification; a structural-functional comparison of precision-fermented and native milk proteins encompassing phosphorylation, disulfide bond pairing, and glycosylation fidelity, alongside strategies for bridging identified PTMs gaps; and an evaluation of AI-driven optimization strategies for heterologous milk protein expression. Cross-species analysis favors human-derived sequences for infant nutrition and immunity, while ruminant proteins excel in expression compatibility and scalability. α-Lactalbumin and β-lactoglobulin are the most tractable targets given minimal PTM dependency; caseins and lactoferrin require intracellular phosphorylation and glycosylation unavailable in microbial hosts. AI accelerates process optimization but cannot yet co-optimize yield, folding fidelity, and PTM accuracy, a key frontier for next-generation engineering. Scale-up robustness, glycoengineering consistency, and regulatory definition remain underexplored.

A modular secretion platform in Bacillus subtilis enables scalable production of protein-glutamine glutaminase

Protein-glutamine glutaminase (PGG) is a promising enzyme for improving the functionality of plant proteins, but its industrial production is limited by low native yields and the need for proteolytic activation. Here, we developed a standardized modular Golden Gate-based secretion platform in Bacillus subtilis to screen promoter-signal peptide combinations for extracellular production of Chryseobacterium proteolyticum PGG ( Cp PGG) within a common construct architecture. Reporter and enzyme-based screening identified Pgrac100-amyQ* as the configuration that consistently supported robust secretion and functional Cp PGG production in B. subtilis 168, where the secreted proenzyme underwent host-mediated maturation. In 3-L batch bioreactor cultivation in rich medium, this construct reached 2.9 U/mL of supernatant (SN), whereas fed-batch cultivation in defined medium revealed extracellular proteolysis as a major limitation under high-cell-density conditions. To decouple secretion from activation, the construct was transferred to the protease-deficient strain KO7-S which enabled stable accumulation of non-processed Cp PGG. Subsequent controlled in vitro activation with a food-grade neutral protease yielded 12.8 ± 0.8 U/mL SN. Together, these results show that efficient PGG production in B. subtilis requires coordinated control of secretion, extracellular stability and proenzyme activation. • Modular screening identified Pgrac100-amyQ* for CpPGG secretion in B. subtilis. • Proteolysis limited high-cell-density fed-batch production in B. subtilis 168. • In Bs KO7-S, secretion and activation were decoupled yielding 12.8 ± 0.8 U/mL SN.

High-yield soluble expression and affinity-tag-free purification of growth hormone receptor antagonist B2036 in Escherichia coli

Bioactivities and Functional Potential of Soy-Derived Small Peptides (2–5 Amino Acids): A Comprehensive Review

Soy-derived small peptides (SSPs, 2–5 amino acids) are food-derived bioactive peptides with efficient intestinal absorption, primarily via peptide transporter 1 (PepT1), and greater digestive stability than longer peptides. This review systematically summarizes 175 SSPs reported between 1996 and 2026, covering their production, separation, characterization, absorption mechanisms, and biological activities. SSPs are obtained mainly by controlled enzymatic hydrolysis using alcalase, thermolysin, papain, and flavourzyme, or by fermentation with Lactobacillus plantarum, Bacillus subtilis, and Aspergillus oryzae. In vitro and in vivo studies demonstrate that SSPs lower blood pressure, reduce oxidative stress and inflammation, modulate glucose and lipid metabolism, and exert anticancer and neuroprotective activities. These effects are linked to peptide sequence, hydrophobicity, Pro/Tyr enrichment, digestive stability, and transporter-mediated or paracellular transport. However, clinical evidence, dose–response data, intact systemic availability, and standardized production remain scarce. This review identifies the critical validation, scalability, and formulation gaps that must be addressed before SSPs can advance into nutraceutical and functional food applications.