Precursor supply and translational machinery engineering of Saccharomyces cerevisiae for improving cellular protein content and biomass-based microbial protein bioproduction.
A multilevel engineering strategy to enhance yeast protein production by optimizing precursor supply, translation machinery, and diploid construction provides both improved protein content and key targets for breeding high-protein microbial strains.
Abstract
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.
Recent advances and future potential in engineering methylotrophic yeasts for food protein production, with particular focus on Komagataella phaffii are reviewed, addressing the dual goals of improving both yield and quality.
Bing-Yin Peng, Masahiro Tominaga, Jun Ishii et al.· Metabolic Engineering· 1 citation
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.
Ru-Yue Han, Ruizheng Hu, An-Ran Liu et al.· Journal of Fungi· 1 citation
Abstract Industrial adoption of microbial cell-free protein synthesis (CFPS), an in vitro protein production platform that uses transcription and translation machinery from lysed cells, remains limited despite sustained technical progress across the field. This perspective positions microbial CFPS within a manufacturing context, focusing on its current capabilities, the limits of those capabilities and the concrete steps required for industrial adoption. In CFPS, the absence of cell growth constraints supports rapid protein production, simplified upstream workflows and direct control over reaction conditions, while also lending itself to formats such as lyophilized reagents for storage and transport. Beyond basic protein expression, microbial CFPS is increasingly used as an adaptable production environment in which reaction composition can be tuned, accessory enzymatic functions can be introduced and post-translational modification strategies can be engineered to better align with product requirements. The expanding range of microbial chassis further broadens the design space, allowing platform choice to be guided by functional, regulatory and deployment considerations rather than yield alone. However, increased capability has not yet translated into routine manufacturing use, and persistent barriers include reproducibility, vulnerable supply chains, limited scale-up practice, downstream purification suitable for therapeutic products and the absence of CFPS-specific quality and regulatory expectations. Overall, microbial CFPS is best evaluated not as a replacement for existing biomanufacturing but as a complementary manufacturing paradigm whose industrial relevance will depend on establishing standardization, economics and regulatory readiness.
Methanol is a promising renewable C1 feedstock for sustainable single‐cell protein (SCP) production. However, its inherent cytotoxicity and metabolic trade‐offs between cell growth and protein synthesis remain significant bottlenecks. Here, we established an “evolutionary‐rational” dual‐driven paradigm to construct a high‐yield Pichia pastoris chassis. Through UV mutagenesis and adaptive laboratory evolution, we developed a highly tolerant strain A40, capable of growing in 70 g/L methanol. Notably, at 30 g/L methanol, A40 achieved a 3.4‐fold higher maximum biomass than the wild‐type. Whole‐genome resequencing and reverse genetics revealed that this superior performance stems from a multi‐gene synergistic network rather than a single dominant mutation. To further optimize SCP production, we rationally co‐overexpressed nitrogen assimilation genes (GLN1, GDH1) and a translation elongation factor (PpeEF3) in the A40 background. This targeted metabolic engineering effectively redirected carbon flux toward protein biosynthesis. The engineered strain A40‐2Ge3 achieved a peak intracellular crude protein content of 67.9% and a 51.3 g/L total titer in a 5‐L bioreactor, representing a 23.1% increase over the wild‐type strain. Collectively, this study provides deep insights into the synergistic mechanisms of methanol adaptation and establishes an efficient, scalable strategy for sustainable SCP production from C1 feedstocks.
Chong Xie, Cheng-Chao Zhu, Jun-Ze Liu et al.· Biotechnology Journal· 0 citations
Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.
Xiao-Xu Chen, Wentao Shen, Xian-Qing Chen et al.· Synthetic and Systems Biotec...· 0 citations
Chinese hamster ovary (CHO) cells serve as the predominant platform for producing recombinant therapeutic proteins in biopharmaceutical manufacturing, the production capacity of which relies heavily on efficient protein synthesis, folding, and secretion pathways. However, during high-density and prolonged cultivation, these cells frequently encounter bottlenecks—including excessive lactate and ammonia accumulation, redox imbalance, and endoplasmic reticulum (ER) stress—which ultimately constrain both the yield and quality of target protein. To overcome these limitations, metabolic engineering has emerged as a key strategy; through systematic modification of the CHO cellular metabolic network, it enhances recombinant protein yield, optimizes critical product qualities such as glycosylation, and improves overall process robustness. This review summarizes recent advances in CHO cell metabolic engineering, encompassing the regulation of central metabolic pathways, glycosylation engineering, cell cycle and metabolic reprogramming, culture condition optimization, byproduct accumulation control, and the application of systems biology and artificial intelligence technologies, including genome-scale metabolic modeling, machine learning-guided target prediction, and dynamic process control. These advances have significantly reduced biopharmaceutical production costs, improved scalability, and shortened time-to-market for monoclonal antibodies and other complex biologics. As the field transitions from single-gene manipulation toward multi-target, dynamic, and system-level rational design, metabolic engineering is advancing CHO cells into more efficient and intelligent “cell factories”, thereby providing sustained momentum for the industrial production of biologics.
Lu Hou, Weidong Li, Ziyan Li et al.· Frontiers in Bioengineering...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
