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

Metabolic engineering of Komagataella phaffii for enhanced production of heterologous proteins and small molecules: key strategies and future directions

ABSTRACT Komagataella phaffii (syn. Pichia pastoris) is an important host for the production of recombinant proteins and small molecules, often exhibiting superior performance compared to other heterologous hosts. With advancements in synthetic biology and computational biology, metabolic engineering has become a key strategy for achieving high-level heterologous proteins and small molecules in K. phaffii for industrial and commercial applications. In this review, we provide an overview of advances in K. phaffii metabolic engineering. We first discuss the development of the K. phaffii expression system. Subsequently, we analyze core optimization strategies, including metabolic flux control, metabolic dynamic control, application of system modeling, chassis cell optimization, and recombinant protein and small-molecule production, before finally summarizing the current challenges and future development directions.

System reconstruction of Bacillus licheniformis for efficient expression of alkaline protease

Industrial enzymes are widely used in diverse applications, but low productivity limits their further widespread utilization. This research aimed to develop high-performance alkaline protease (AprE) expression strains of Bacillus licheniformis through element optimization and modular engineering. Firstly, the aprE gene expression cassette was systematically optimized through element engineering. To minimize host background interference, five large gene fragments were deleted from the genome of B. licheniformis DW2. This expression cassette and genome-reduced strain resulted in 5.77-, 4.84- and 1.31-fold increases in the activities of alkaline protease, nattokinase and chitinase, respectively. Crucially, metabolomics analysis then served as the pivotal discovery tool, revealing that high expression of AprE was constrained by insufficient precursor amino acids and excessive metabolic overflow. Subsequently, the amino acid biosynthesis, energy metabolism, overflow metabolism, and cell membrane/wall modules of the strain were successively modified. The final AprE expression host DM6E10 achieved a remarkable enzyme activity of 34,343 U/mL, with a maximum activity of 107,100 U/mL in a 5-L bioreactor. This study built an efficient cell factory for AprE production and provided insights for the optimization of other protein expression hosts.

Transcriptional reprogramming of the stress response and translational-secretory machinery enhances recombinant secreted protein production in Yarrowia lipolytica.

The results of contrasting the Azf1- and Dep1-driven regulomes suggest that for efficient rProt secretion, upregulation in the vesicle-mediated transportation must co-occur, as in the case of Azf1-KO.

Precursor supply and translational machinery engineering of Saccharomyces cerevisiae for improving cellular protein content and biomass-based microbial protein bioproduction.

The sustainable production of biomass-based microbial protein (MP) requires efficient microbial cell factories for accumulating cellular protein with high content, which is beneficial both for improving protein production and downstream cellular protein isolation and purification. To overcome the limited protein content of the Saccharomyces cerevisiae, we designed a systematic multilevel metabolic engineering strategy. Initially, single-gene edits based on predictions using the genome-scale model Yeast 9.0.2 and the OptForce algorithm failed to increase protein content due to precursor supply limitations. Enhancing genes in nitrogen metabolic (GDH1, GDH2, GLN1, GLT1) and central carbon (CIT1, IDH1) pathways were implemented to synergistically enhance ammonium assimilation. Subsequently, overexpression of valyl-tRNA synthetase (VAS1) alleviated the translational bottleneck, increasing cellular protein content to 52.3 g/100 g dry cell weight (DCW). The ribosomal synthesis pathway was further enhanced via ribosomal regulator IFH1 and ribosomal protein gene overexpression, with cellular protein content reaching 57.3 g/100 g DCW. Finally, diploidization and global transcriptional regulator SUT1 integration in strain D3 achieved a protein content of 66.5 g/100 g DCW in shake flask culture. Under controlled 5 L bioreactor conditions, its protein content further increased to a peak of 75.2 g/100 g DCW, representing a 50.3% increase over the parental strain Y1. This study developed a multilevel engineering strategy to enhance yeast protein production by optimizing precursor supply, translation machinery, and diploid construction. Using marker-free editing and endogenous gene regulation, it provides both improved protein content and key targets for breeding high-protein microbial strains.

cerevisiae-based platform for rapid production and evaluation of Eukaryotic Nutrient transporters and transceptors for biochemical studies and crystallography

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.