Metabolic engineering of Komagataella phaffii for enhanced production of heterologous proteins and small molecules: key strategies and future directions
This review provides an overview of advances in K. phaffii metabolic engineering and analyzes core optimization strategies, including metabolic flux control, metabolic dynamic control, application of system modeling, chassis cell optimization, and recombinant protein and small-molecule production.
Abstract
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.
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
This review provides the current state of PHAs production from wild yeast strains and the various approaches that have been used to improve yield, and discusses the performance, challenges, and limitations of various synthetic biology and metabolic engineering strategies in yeast strains for PHAs production.
K. Mohanrasu, R. Selvakumar, I. Grainge et al.· International Journal of Bio...· 0 citations
Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.
The heterologous production of terpene in microbial hosts is often limited by inefficient and unstable pathway expression, creating a major bottleneck for industrial-scale synthesis. While E. coli as a chassis offers significant advantages, such as rapid growth, ease of cultivation, and genetic tractability. Its endogenous supply of terpenoid precursors remains a critical constraint, fundamentally restricting high-yield production. To address this challenge, we developed a genomically integrated Mevalonate (MVA) pathway from Actinomycetota in E. coli BL21(DE3) to enhance terpene precursor supply. Our approach began with an in silico multi-layer global genome mining analysis of 25,261 Actinomycetota genomes to identify a series of MVA pathway enzymes with potentially high catalytic efficiency, created a high-efficiency chassis E. coli MVA platform (ecMVA-1 and ecMVA-2) for terpene precursor synthesis. Its functionality was validated by testing eight distinct TSs. Among them, the fermentation of artemisinin precursor amorphadiene using a 5-liter bioreactor yielded 947.80 mg/L. These results indicated that E. coli (MVA) is well-suited for TS studies in the laboratory as well as holding significant promise for industrial applications. In addition, this in silico approach offers a new perspective for metabolic engineering and provides potential reservoir of diverse chassis for the industrial production of terpenoid-derived compounds.
Wenchao Liu, Xueying Tian, W. Wong et al.· Metabolic Engineering· 0 citations
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.
Qing Zhang, Mengyuan Zhang, Zhihao Zhu et al.· Synthetic and Systems Biotec...· 0 citations
Sclareol, a plant-derived diterpene, holds significant industrial value in perfumery, pharmaceuticals, and agriculture. Traditional production from Salvia sclarea or chemical synthesis has faced challenges such as low yield, high cost, and sustainability concerns. Recent advances in synthetic biology and metabolic engineering have enabled sclareol biosynthesis in microbial chassis cells. This Review systematically examines synthetic biology-driven strategies for improving sclareol production, with particular emphasis on microbial chassis engineering, metabolic pathway optimization, and enzyme engineering. Emerging approaches such as AI-assisted prediction, multiomics analysis, high-throughput screening, and intelligent fermentation technologies are also highlighted. Achievements and challenges in microbial sclareol biosynthesis are critically analyzed, and strategic priorities for future development are proposed. This paper provides a comprehensive reference for science and practice, aiming to advance the sustainable biomanufacturing of high-value plant-derived terpenoids.
Yi-Wen Yu, Long-Qing Wang, Yan Zhang et al.· ACS Omega· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
