Microbial Cell Factories最新文献

{"title":"Evaluation of recombinant trypsinogen and chymotrypsinogen production in Komagataella phaffii through co-expression of HAC1 and PDI1.","authors":"Aitor González-Titos, Belén Toledo, Sonakshi De, María José Grande, Antonio Gálvez, Juan Antonio Marchal, Diethard Mattanovich, Macarena Perán","doi":"10.1186/s12934-026-03019-5","DOIUrl":"10.1186/s12934-026-03019-5","url":null,"abstract":"<p><strong>Background: </strong>Trypsinogen and chymotrypsinogen are serine proteases with potential therapeutic applications in cancer. However, due to ethical, safety and variability concerns, their clinical use has been limited by the reliance on bovine or porcine pancreatic extracts. Recombinant expression in Komagataella phaffii (syn. Pichia pastoris) offers a scalable and controlled alternative. However, further optimisation is required to increase protein titer and secretion.</p><p><strong>Results: </strong>Recombinant strains were engineered to express human trypsinogen and chymotrypsinogen. The effect of medium supplementation and co-expression of HAC1 and PDI1 on protein production was evaluated. Co-expression of HAC1 increased trypsinogen production by 3.2-fold, while PDI1 significantly increased chymotrypsinogen titer by 8.5-fold compared to parental strains. Medium optimisation had minimal effect on protein expression. SDS-PAGE and Western blotting analyses confirmed successful expression and secretion of both enzymes.</p><p><strong>Conclusions: </strong>The combination of genetic modification and medium optimisation significantly improved the recombinant production of trypsinogen and chymotrypsinogen in K. phaffii. HAC1 proved to be more effective in increasing the production of trypsinogen, while PDI1 co-expression significantly increased chymotrypsinogen titers. These findings provide a promising strategy for improving the production of these proteases, supporting their potential applications in biotechnology and biomedical research.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13352816/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147973911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Utilization of potato peels waste for production, purification, and characterization of cold-adapted amylase under solid state fermentation by Penicillium goetzii AUMC 498.","authors":"Rabab Shobak Sakr, Ahmed M Moharram, Heba Atia Yassa, Bahaa E S Abd El-Fatah, Osama A M Al-Bedak","doi":"10.1186/s12934-026-03021-x","DOIUrl":"10.1186/s12934-026-03021-x","url":null,"abstract":"<p><p>This study explores the utilization of potato peel waste as a substrate for producing cold-adapted amylase under solid-state fermentation (SSF) using Penicillium goetzii AUMC 498. Optimization through Box-Behnken design identified pH, incubation period, and beef extract concentration as key factors, achieving a maximum activity of 4.21 U/g dry substrate at pH 7 and 10 °C after 6 days. The enzyme was purified via two-column chromatography (Trilite MA12 and Sephacryl S 200), resulting in a 63.6-fold increase in purity and a molecular weight of 51.91 kDa confirmed by SDS-PAGE. The purified amylase exhibited optimal activity at pH 5 and 25 °C, with sorghum starch as the preferred substrate (594.68 U/mg). Activity was significantly enhanced by MnSO₄ and ZnSO₄, while NiCl₂, SDS, and EDTA inhibited it. Kinetic constants for pure amylase were determined for six types of starch: soluble starch, sorghum starch, oat starch, maize starch, wheat starch, and rice starch. The Km values were 575.75 mM, 59.1 mM, 149.27 mM, 195.9 mM, 81.44 mM, and 37.48 mM, respectively. These findings demonstrate the effective conversion of potato peel waste into a value-added cold-adapted biocatalyst with desirable catalytic and stability characteristics. The ability of the enzyme to function efficiently at low temperatures highlights its strong potential for use in energy-efficient industrial processes, particularly in the food, detergent, textile, and starch-processing industries. Moreover, the utilization of agro-industrial waste as a fermentation substrate provides an environmentally sustainable and economically attractive approach, reinforcing the relevance of this study to waste valorisation strategies and green biotechnological applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":"25 1","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13185359/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147973932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosynthetic pathway construction and production enhancement of phenazine-1-carboxylic acid derivatives in Pseudomonas chlororaphis H18.","authors":"Sijia Xu, Chaozhi Wang, Chenxi Gong, Jinbei Shi, Xiao Zhang, Mohd Sadeeq, Wei Huang, Peng Xiong, Feifei Hou","doi":"10.1186/s12934-026-03023-9","DOIUrl":"10.1186/s12934-026-03023-9","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Phenazines represent a promising class of natural products with diverse biological activities, including antitumor, antiparasitic and antibacterial properties. Among them, phenazine-1-carboxylic acid (PCA)-a registered biopesticide-stands out for its broad-spectrum antifungal efficacy, low environmental toxicity and potential as a lead structure for developing anti-tumor and antituberculosis agents. However, the chemical synthesis of PCA derivatives is often hindered by harsh reaction conditions, toxic byproducts and poor selectivity, limiting their sustainable production. In contrast, biosynthesis emerges as a green and scalable alternative, yet efficient microbial platforms for structurally diverse PCA derivatives remain underdeveloped.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;To address this gap, we engineered Pseudomonas chlororaphis H18 into a versatile cell factory for PCA derivative biosynthesis. We first characterized the substrate promiscuity of the S-adenosylmethionine (SAM)-dependent methyltransferase Pcm2, demonstrating its capacity to catalyze methylation and ethylation of many aromatic carboxylic acids. This catalytic versatility broadens the scope of PCA derivatives that can be accessed through biosynthesis. By integrating the pcm2 gene into the P. chlororaphis H18 genome, we established de novo pathways for two key derivatives: phenazine-1-carboxymethyl (PCM) and 2-hydroxy-phenazine-1-carboxymethyl (2-OH-PCM) for the first time, with PCM having been reported to exhibit superior fungicidal activity against the tobacco brown spot pathogen, Alternaria alternata, compared to PCA. To enhance titers of PCM and 2-OH-PCM, we implemented a systematic metabolic engineering strategy comprising: (1) increasing intracellular SAM supply, (2) overexpressing key genes, (3) knocking out the negative regulators and (4) disrupting competitive pathway. The engineered strain H18A4 produced 124.6 mg/L PCM and 130.8 mg/L 2-OH-PCM in KB medium. To further increase production of PCM and prevent PCA hydroxylation, we disrupted the phzO gene to block conversion to hydroxylated derivatives, increased pcm2 gene copy number and optimized the fermentation medium. The resulting engineered strain H18A6 produced 1.37 g/L PCM in fed-batch fermentation-a marked advance toward scalable, bio-based production of this high-value compound.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions: &lt;/strong&gt;This study fills a technical gap by establishing a novel biosynthetic route for diverse PCA derivatives, overcoming the limitations of chemical synthesis and expanding the scope of sustainable natural product production. And the integrated metabolic engineering strategy (spanning enzyme characterization, pathway construction, metabolic flux optimization and process refinement) validated herein offers a reusable template for engineering microbial cell factories for complex high-value natural products. Ultimately, this study contributes to the green production of functionally com","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13352866/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147964199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering Streptococcus thermophilus for heterologous gene expression of cell envelope proteases from lactic acid bacteria.","authors":"Joanna Ivy Irorita Fugaban, Emilie Munk, Saria Otani, Pascal Fourcassié, Claus Heiner Bang-Berthelsen, Egon Bech Hansen","doi":"10.1186/s12934-026-03030-w","DOIUrl":"10.1186/s12934-026-03030-w","url":null,"abstract":"<p><strong>Background: </strong>Cell envelope proteinases have long played a pivotal role in dairy science. However, as demand for alternative food sources grows, their application in plant-based food matrices is scarcely investigated. A deeper physiological and technological understanding of these enzymes requires efficient and effective tools tailored to this emerging sector. To date, plasmid-based recombination in Lactococcus spp. remains the most widely used and effective method.</p><p><strong>Results: </strong>In this study, we engineered S. thermophilus LMD-9 as a host for heterologous expression of protease from Lc. cremoris. S. thermophilus LMD-9 offers several advantages, including its GRAS status, efficient chromosomal gene integration via natural competence, and native machinery for functional protease production, making it a highly versatile host. Previous attempts to employ this strain yielded inactive protease due to unresolved bottlenecks; here, we characterize and overcome those challenges to establish LMD-9 as a robust system for protease expression. Exploration of potential bottlenecks highlighted the availability of intrinsic peptidyl-prolyl cis/trans isomerase (PPIase; prtM/prsA) as a key factor influencing successful enzyme expression. Three recombinant strains with genotypes: LMD-9 ΔprtS::ermR, LMD-9 ΔprtS::ermR-ΩprtP, and LMD-9 ΔprtS::ermR-Ω (prtM-prtP), were generated to test the role of prtP-associated PPIase. Results demonstrated that inclusion of PrtP-specific PPIase from L. cremoris markedly enhances protease activity. In its absence, although partially compensated by the pleiotropic PPIase in S. thermophilus, we observed slower growth and reduced proteolytic activity.</p><p><strong>Conclusions: </strong>These findings establish S. thermophilus LMD-9 as a robust chassis and alternative host for heterologous expression of cell envelope proteinases. To our knowledge, this is the first work to heterologously express active CEP enzyme in this host, and it highlights the role of PPIase availability as a key determinant of successful enzyme expression. This then provides a suitable and robust host for studying the application of CEPs in both dairy and emerging plant-based food applications.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13374181/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147960102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toxic and useful: harnessing yeast killer toxins for product recovery.","authors":"Ankita Suri, Michael Norman, David Wollborn, Thomas D Loan, Jian-Wei Liu, Shoko Okada","doi":"10.1186/s12934-026-03024-8","DOIUrl":"10.1186/s12934-026-03024-8","url":null,"abstract":"<p><strong>Background: </strong>Saccharomyces cerevisiae is widely used in biotechnology to produce proteins and other biomolecules. Recovery of intracellular products, however, remains a key bottleneck for industrial applications. The robust cell wall demands energy-intensive chemical or mechanical disruption that can damage sensitive targets and result in limited product recovery.</p><p><strong>Results: </strong>In this study, we investigated yeast killer toxins as a genetically encoded tool to enhance membrane permeability and facilitate intracellular product recovery. Five killer toxins with distinct modes of action were expressed intracellularly and their effects on cell survival, membrane permeability, susceptibility to lysis and enhancement of intracellular product recovery were examined. In combination with common mechanical disruption methods, toxin expression enhanced total intracellular protein recovery up to 6-fold relative to the control strain without a killer toxin expressed.</p><p><strong>Conclusion: </strong>These results establish killer toxin expression as a tuneable strategy for enhanced product recovery from yeast, that complements and augment conventional bioprocessing techniques.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13343598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147948911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Vibrio natriegens as a fast-growing alternative host for plasmid DNA production.","authors":"Lara S Möller, Gabriel A Monteiro, Duarte M F Prazeres, A Rita Silva-Santos","doi":"10.1186/s12934-026-03026-6","DOIUrl":"10.1186/s12934-026-03026-6","url":null,"abstract":"<p><strong>Background: </strong>Plasmid DNA (pDNA) is a key biomolecule for gene therapies, DNA vaccines, and mRNA-based applications. Although Escherichia coli is the established host for pDNA production, limitations such as moderate growth rates and endotoxin-associated challenges motivate the exploration of alternative production hosts. Vibrio natriegens is a fast-growing, non-pathogenic bacterium with a doubling time below 10 min and high metabolic versatility. While it has been adopted for cloning and protein expression, its potential for pDNA production remains largely unexplored.</p><p><strong>Results: </strong>We evaluated pDNA production by V. natriegens in comparison to E. coli in shake flask cultivations across four complex media (LB, BHI, 2xYT, TB) and one defined minimal medium (MSM). Under all tested conditions, V. natriegens exhibited faster biomass accumulation and enabled earlier pDNA isolation. Quantitative analysis of triplicate experiments revealed that V. natriegens achieved comparable or higher volumetric and specific pDNA yields in shorter cultivation times than E. coli. Agarose gel electrophoresis confirmed plasmid integrity, and functional validation using a cell-free expression system demonstrated efficient eGFP reporter expression from V. natriegens-derived plasmids.</p><p><strong>Conclusions: </strong>This proof-of-concept study demonstrates that V. natriegens is a promising alternative host for pDNA production. Its combination of accelerated growth, robust pDNA yields, and functional plasmid quality highlights its potential for further development as a pDNA production platform.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13343645/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147948936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Do seconds make a difference? Investigating strain-specific behavior of yeast in dynamic glucose environments.","authors":"Luisa Blöbaum, Markus Bünker, Julian Schmitz","doi":"10.1186/s12934-026-03029-3","DOIUrl":"10.1186/s12934-026-03029-3","url":null,"abstract":"<p><strong>Background: </strong>When applied for industrial-scale bioproduction, cells are subjected to ever-changing cultivation environments due to bioreactor heterogeneities, which can significantly influence their growth and production behavior. Cultivating and comparing cells and strains under conditions representative of an actual bioprocess instead of laboratory environments is therefore necessary for developing more reliable production strains. Scale-down bioreactors provide an experimental approach for this endeavor, yet they are limited in their temporal resolution and flexibility when it comes to environmental dynamics. Thus, the impact of second-scale differences in dynamic environments on microbial producers remains unexplored. This study uses the advantages of microfluidic single-cell cultivation to compare the growth behavior and intracellular parameters of three yeast strains under dynamic cultivation conditions with alternating phases of glucose excess and limitation at the timescale of seconds aiming to uncover strain-specific performance under as well as adaptation to fluctuating, bioprocess-relevant environments.</p><p><strong>Results: </strong>Across all three strains, a consistent trend was observed: decreasing glucose availability resulted in reduced growth rates, lower ATP levels, diminished glycolytic flux, and smaller cell sizes. Notably, cells already reached approximately 50% of their maximal growth rate when exposed to growth-promoting glucose conditions for only 10% of the time. Importantly, both growth rate and cell size exhibited an adaptation phase rather than an immediate response to oscillatory glucose supply. Longer exposure to favorable conditions shortened the adaptation phase and resulted in higher adapted growth rates and larger cell sizes.</p><p><strong>Conclusion: </strong>By leveraging microfluidic single-cell cultivation, this study provides unprecedented temporal resolution of environmental dynamics, enabling direct, strain-specific comparison of cellular responses and adaptation to rapidly changing cultivation environments. Seconds do make a difference. Our findings demonstrate that growth rate is a highly conserved trait under environmental perturbations, which points to an intrinsic robustness of the investigated strains and highlights adaptation as a key determinant of performance in dynamic environments. Together, these insights have important implications for the design of future single-cell experiments and for the development of robust bioprocesses operating under dynamic conditions.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13179603/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147931062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient vitamin A production in Lipomyces starkeyi through metabolic engineering of the β-carotene and retinoid pathways.","authors":"Akari Kinoshita, Rikako Sato, Hibiki Higuchi, Shunichi Kobayashi, Kento Koketsu, Taro Watanabe, Hiroaki Takaku","doi":"10.1186/s12934-026-03020-y","DOIUrl":"10.1186/s12934-026-03020-y","url":null,"abstract":"<p><strong>Background: </strong>Vitamin A, an essential micronutrient, is critical for vision, immune function, and cellular differentiation. Traditional vitamin A production methods, primarily based on chemical synthesis, pose significant environmental challenges. Microbial fermentation offers a sustainable alternative, but microbial production of vitamin A has yet to match chemical synthesis in terms of yield and cost-effectiveness. Oleaginous yeasts, such as Lipomyces starkeyi, which can synthesize high levels of acetyl-CoA and lipids, represent promising platforms for producing high-value lipophilic compounds, such as vitamin A.</p><p><strong>Results: </strong>L. starkeyi was engineered for the first time to produce vitamin A by introducing key enzymes in the β-carotene and retinoid biosynthetic pathways. This approach included integrating genes encoding lycopene cyclase/phytoene synthase (McCarRP), phytoene desaturase (McCarB), and β-carotene 15,15'-dioxygenase (MbBlh). Further optimization of the mevalonate pathway enabled the production of over 600 mg/L vitamin A in a 3-L fed-batch fermentation. Fe²⁺ supplementation improved yield, while a two-phase culture system using dodecane and butylated hydroxytoluene enhanced vitamin A recovery, with over 90% recovered in the extracellular phase.</p><p><strong>Conclusions: </strong>This study establishes L. starkeyi as a promising host for sustainable vitamin A production. Although significant improvements in yield were achieved, further optimization of pathway regulation and fermentation conditions is needed to reach economically competitive levels. These findings provide a foundation for developing L. starkeyi as a platform for large-scale production of vitamin A and other valuable terpenoids.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13344046/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147931068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deletion of low-essentiality, secretion-associated genes enhances recombinant protein production in Komagataella phaffii.","authors":"Hayley Ford, Neil C Dalvie, Timothy R Lorgeree, Rachel M Barry, Anaisa N Sibel, Rachel A Ahlmark, Harini Narayanan, Brittney C Sunday, Charles A Whittaker, Raghav Acharya, Carmen M Elenberger, J Christopher Love","doi":"10.1186/s12934-026-03009-7","DOIUrl":"10.1186/s12934-026-03009-7","url":null,"abstract":"<p><strong>Background: </strong>Komagataella phaffii (K. phaffii) is used to manufacture biologic medicines, food proteins, reagents, and materials. Despite its increasing prevalence, further improvements to its productivity would enhance its economic and operational benefits. Genomic engineering represents one approach to increase its cell-specific productivity. We hypothesized that combining the metrics for the relative essentiality of genes with biological inference for relevance to protein secretion could identify genes that, when disrupted, would improve specific productivity in the resulting strains.</p><p><strong>Results: </strong>The essentiality of genes in K. phaffii (NRRL Y-11430) were predicted through a genome-wide knockout screen using CRISPR-Cas9. Based on the results from this screen, we selected and subsequently disrupted the least essential genes from two gene groups heavily associated with secretion, namely those relating to the cell wall and vacuolar transport. Strains of K. phaffii with single gene disruptions from these gene sets showed significantly improved production of a monoclonal antibody (mAb). These strains exhibited no discernible differences in growth or apparent profiles of host cell proteins when compared to the parental strain. The best-performing strains consistently showed 2-3x enhancements in specific productivity and titers across scales (3-150 mL), culture formats (plates, flasks, bioreactors), and processing operations (batch and fed-batch).</p><p><strong>Conclusions: </strong>This study demonstrates how combining data on gene essentiality and prior knowledge of biological pathways related to a phenotypic trait of interest (here protein secretion) can inform strain engineering to enhance the trait. This study expands the catalog of genetically engineered strains of K. phaffii with improved productivity. These strains support the long-term goal of achieving low-cost, high-volume production of recombinant proteins using this host. Further engineering of these strains and optimization of fermentation processes could enable volumetric productivities comparable to those of other established hosts used to produce mAbs and other complex recombinant proteins.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13343958/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147874247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combinatorial optimization engineering for the high-level production of shikimic acid in Escherichia coli.","authors":"Changyang Yang, Yang Li, Mingxiong Liu, Hongxin Fu, Jufang Wang","doi":"10.1186/s12934-026-03016-8","DOIUrl":"10.1186/s12934-026-03016-8","url":null,"abstract":"<p><p>Shikimic acid is a crucial precursor for the production of indole derivatives and chiral drugs, and the biomanufacturing offers reduced cost and simplified process compared to traditional plant extraction. Current research on industrial-scale production of shikimic acid achieves notable progress, yet the limitations associated with high production cost, complex operation process, and excessive byproduct formation still persist. To tackle these difficulties, this study aimed at establishing a high-efficient shikimic acid-producer through systematic modification and fermentation optimization in Escherichia coli. First, the route for the synthesis of shikimic acid was divided into three modules (i.e., sugar uptake, bioproduct synthesis, and metabolite catabolism), of which the disruption of glucose uptake system, reinforcement of bioproduct synthesis, and block of its catabolism rewired metabolic fluxes and provided the possibility for efficient synthesis. Next, series of systematic engineering involving in deleting pyruvate bypass, reinforcing the pentose phosphate (PP) pathway, and adjusting promoter strength promoted precursor supply and avoided metabolic overflow. The best strain EC022 generated 5.27 g/L of shikimic acid, highlighting the significance of precursor availability. Then, fermentation conditions including substrate concentration, inoculation size, cultivation temperature, induction time, and C/N ratio were comprehensively optimized, which boosted the bioproduction to 8.70 g/L in the optimized conditions. Finally, a final titer of 61.87 g/L shikimic acid was achieved in a 7 L bioreactor. This study developed an efficient shikimic acid-producer with industrial potential through metabolic engineering and bioprocess optimization.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":" ","pages":""},"PeriodicalIF":5.8,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13326388/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147856648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}