Cell Factories Research Articles

Towards engineering agaricomycete fungi for terpenoid production.

Since ancient times, humans have harnessed the vast metabolic abilities of fungi to produce food, beverages, and medicines. Biotechnology and genetic engineering have opened new avenues to tailor and enhance these abilities, transforming fungi into powerful industrial workhorses. In this minireview, we focus on the biotechnological potential of Agaricomycetes, a class of basidiomycete fungi that includes the so-called 'true' mushrooms. Although many species are widely used in the food sector, their broader potential in biotechnology remains largely untapped. These fungi naturally produce a diverse array of metabolites with promising applications across various industries. Here, we highlight their ability to synthesize a wide range of terpenoids, many unique to this taxon, and we present recent advancements in genomics and genetic engineering tools developed for Agaricomycetes. We anticipate that continued progress in tailored genetic engineering tools and improved cultivation technologies will facilitate the establishment of these fungi as robust cell factories for producing valuable terpenoids, with significant contributions to the food, biotech, and pharmaceutical sectors.

New Yarrowia lipolytica chassis strains for industrial enzyme production

BackgroundYarrowia lipolytica has emerged as a well-established platform for producing a wide range of biomolecules, including recombinant proteins (rProteins). Its robust metabolism and resistance to various environmental stressors make it particularly well-suited as a microbial cell factory. However, additional physiological modifications are still required to fully meet industrial demands. Over years of strain development, Y. lipolytica has been engineered to carry auxotrophic markers, streamline the secretory pathway via deletion of native secretory proteins, prevent filamentation, and enable inducible gene expression systems.ResultsIn this study, we continued the fine-tuning of Y. lipolytica as a platform for rProtein synthesis, building on previous work. Specifically, we: (i) introduced a third auxotrophy to facilitate more complex genetic engineering strategies, (ii) removed bacterial vector elements (including antibiotic resistance genes) from previous constructs, and (iii) carried out extensive deletions of extracellular proteases and a peroxidase gene. The newly constructed chassis strains, JMY9438 and JMY9451/9452, both bear triple auxotrophies. The latter strain additionally lacks proteolytic activity due to the deletion of five protease genes. We evaluated the rProtein production efficiency of these strains harboring one, two or three integrated copies of the target gene. rProtein expression levels increased with copy number up to two; however, no further improvement was observed with three copies. Notably, the strain with protease deletions and a single gene copy showed the highest rProtein production per cell, while the strain retaining proteases but harboring two copies yielded the highest absolute rProtein levels.ConclusionsWe present a new generation of Y. lipolytica chassis strains specifically optimized for recombinant protein production. Our results demonstrate that extensive protease deletions can provide a high-performance genetic background, enabling high-level rProtein production without relying on multi-copy expression strategies.

Constructing CRISPR-Cas9 system for metabolic reprogramming and cordycepin biomanufacturing in Pichia pastoris.

Constructing CRISPR-Cas9 system for metabolic reprogramming and cordycepin biomanufacturing in Pichia pastoris.

Yeast as C1 cell factory: Transforming methanol and Formate into high-value compounds.

Yeast as C1 cell factory: Transforming methanol and Formate into high-value compounds.

A Comprehensive Review of l-Theanine Production in Escherichia coli: The Recent Progress, Metabolic Engineering Strategies, and Future Prospects.

l-theanine is a nonproteinogenic amino acid widely utilized as a food additive and cosmetic ingredient, with promising potential in medical applications. Owing to its distinctive properties and well-established safety profile across the food, cosmetics, health supplement, and pharmaceutical sectors, global demand for l-theanine has been rising rapidly. Among available production methods, microbial fermentation, recognized for its environmental sustainability, has emerged as the preferred approach, gradually supplanting traditional techniques, such as plant extraction and chemical synthesis. Recent advances, particularly the development of ethylamine-free biosynthetic pathways and the successful de novo biosynthesis of l-theanine in Escherichia coli, a well-established microbial chassis with rapid growth and versatile genetic engineering tools, have opened new avenues for industrial-scale production. This review highlights the biosynthetic pathways of l-theanine and outlines targeted metabolic engineering strategies to enhance its yield. These include the identification and overexpression of key biosynthetic enzymes, enhancement of ATP regeneration, construction of endogenous ethylamine biosynthetic routes, redirection of metabolic flux toward l-theanine production, and suppression of competing pathways. Furthermore, current limitations in strain optimization are discussed along with perspectives on future directions for developing E. coli as a robust microbial cell factory for l-theanine production.

Third Generation Biorefineries: An Approach towards a Sustainable Future

Microalgae are the broad range of photosynthetic autotrophs that are found both in fresh and marine water bodies worldwide. Microalgae are the known for producing value added products via the sequestration of carbon dioxide. This production is supported by other salient features like higher light conversion efficiency, higher substrate conversion efficiency, higher biomass productivities and shorter generation time. Microalgae biomass can be contemplated as a microbial cell factory that potentially biosynthesize wide range of value-added biomolecules like biopigments, therapeutic compounds, bioenergy, food, food supplements, animal feeds, biofertilizers and cosmetics etc. Sustainable industrial and biorefinery implications becomes feasible because of the multifaceted utility of microalgal biomass. The ultimate aim of a biorefinery is to sustainably transform biomass into a wide range of significant chemicals, fuels, and materials. Bio-renewables industry is empowered by third-generation (3G) biorefinery whose principal components are algal feedstock i.e., microalgae and macroalgae. Still, algal feedstock are the chief producers of pharmaceuticals, nutraceuticals and cosmetics.

Metabolic engineering of Bacillus subtilis for the production of active hemoglobins and myoglobins by improving heme supply

Hemoglobins (Hb) and myoglobins (Mb) are important hemoproteins with broad applications in food and medicine. Microbial cell factory is a promising approach for the green and sustainable production of hemoproteins. However, current microbial hosts face the challenges of safety and insufficient heme supply. Here, we report a global regulation strategy, "push-restrain-pull-block," to enhance heme supply for producing various active Hb and Mb in food-grade Bacillus subtilis. Initially, the insufficient supply of the precursor 5-aminolevulinate was overcome by relieving feedback inhibition and mitigating the negative effects of HemX on HemA. Next, HemD was identified as the primary uroporphyrinogen III synthase and self-assembled with HemC to minimize the formation of the uroporphyrinogen I by-product. Additionally, the coproporphyrin-dependent pathway was selected as the superior downstream route for heme synthesis, and crucial rate-limiting steps were subsequently enhanced. Moreover, heme consumption was blocked by eliminating protoheme IX farnesyltransferase. Finally, through the combination and fine-tuned expression of key genes, a 221-fold improvement of heme supply was achieved in the engineered strain. Using this stable prokaryotic chassis, we achieved production of 0.81, 0.82, 1.11, and 1.01 g L-1 of soybean Hb, clover Hb (C-Hb), bovine Mb (B-Mb), and porcine Mb, respectively, through fermentation, marking the highest reported titers in prokaryotic systems. These hemoproteins exhibit properties similar to natural standards. Furthermore, the synthesized C-Hb and B-Mb demonstrated superior effects for preparing plant-based meat analogs as colorants and flavoring agents. This work provides a universal platform for producing other high-value hemoproteins, promising future advancements in food processing and biocatalysis.

Engineering a novel adenine-sulfotransferase for efficient synthesis of PAPS and chondroitin sulfate in microbial cells.

Engineering a novel adenine-sulfotransferase for efficient synthesis of PAPS and chondroitin sulfate in microbial cells.

Multiplexed engineering of cytochrome P450 enzymes for promoting terpenoid synthesis in Saccharomyces cerevisiae cell factories: A review.

Multiplexed engineering of cytochrome P450 enzymes for promoting terpenoid synthesis in Saccharomyces cerevisiae cell factories: A review.

Rhodotorula sp. as a promising host for microbial cell factories.

Rhodotorula sp. as a promising host for microbial cell factories.

Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory.

Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory.

Engineering Komagataella phaffii cell factories for the production of chondroitin sulfate A with high sulfation degree

Engineering Komagataella phaffii cell factories for the production of chondroitin sulfate A with high sulfation degree

Reprogramming Schizochytrium cell factory for High-Yield production of astaxanthin via mutagenesis and metabolic engineering

Reprogramming Schizochytrium cell factory for High-Yield production of astaxanthin via mutagenesis and metabolic engineering

Recent advancements in the evolution, production, and degradation of biodegradable mulch films: A review.

Recent advancements in the evolution, production, and degradation of biodegradable mulch films: A review.

Developing Bacillus subtilis as cell factory for the production of the natural biocontrol compound pulcherrimin.

Developing Bacillus subtilis as cell factory for the production of the natural biocontrol compound pulcherrimin.

Rewiring the methanol assimilation pathway in the methylotrophic yeast Pichia pastoris for high-level production of erythritol.

Rewiring the methanol assimilation pathway in the methylotrophic yeast Pichia pastoris for high-level production of erythritol.

SCALING COWPOX VACCINE PRODUCTION TECHNOLOGY: FROM DEVELOPMENT TO GMP STANDARDS

This paper presents a comprehensive analysis and systematization of the key stages of scaling the technological chain of production of cowpox vaccine, with an emphasis on the full-scale adaptation of production processes to strict criteria of good manufacturing practice (GMP). Special attention is paid to the detailed validation of all key stages, including virus cultivation, vaccine formation, lyophilization process and comprehensive quality control of the final product. Validation of key processes was carried out with careful elaboration of virus culture protocols, which included the use of the Nunc Cell Factory, ensuring high stability and reproducibility of the procedure. The introduction of improved methods of microbiological purity control at all stages of production, including regular checks for the presence of microorganisms and monitoring of environmental parameters, contributed to improving the reliability of the production process. Operational standardization was achieved through the integration of real-time systems for data monitoring and analysis, which helped minimize human error and improve production accuracy. As a result, key optimization strategies have been identified and implemented to improve production efficiency, including improving automation processes and digitalizing production lines. The results of the study represent a significant step forward in the development of veterinary vaccinology, providing a reliable basis for future developments and innovations in the field of prevention and treatment of infectious diseases in farm animals. The work highlights the importance of an interdisciplinary approach combining biological, engineering, and management competencies to achieve high standards of vaccine quality and safety. In light of the emerging challenges associated with changes in legislation and the development of new technologies, this work offers a platform for continuing research in the field of biotechnology and vaccine production aimed at adapting to the dynamic conditions of the veterinary industry.

Hydroxybenzoic acids: Microbial metabolism, pathway engineering and products.

Hydroxybenzoic acids: Microbial metabolism, pathway engineering and products.

Enhancing nicotinamide mononucleotide production in Escherichia coli through systematic metabolic engineering.

Enhancing nicotinamide mononucleotide production in Escherichia coli through systematic metabolic engineering.

Production of phosphorylated and functional αs1-casein in Escherichia coli.

Production of phosphorylated and functional αs1-casein in Escherichia coli.