Cell Factories Research Articles

Newly isolated halotolerant Gordonia terrae S-LD serves as a microbial cell factory for the bioconversion of used soybean oil into polyhydroxybutyrate

Polyhydroxybutyrate (PHB) is a class of biodegradable polymers generally used by prokaryotes as carbon sources and for energy storage. This study explored the feasibility of repurposing used soybean oil (USO) as a cost-effective carbon substrate for the production of PHB by the strain Gordonia terrae S-LD, marking the first report on PHB biosynthesis by this rare actinomycete species. This strain can grow under a broad range of temperatures (25–40 ℃), initial pH values (4–10), and salt concentrations (0–7%). The findings indicate that this strain can synthesize PHB at a level of 2.63 ± 0.6 g/L in a waste-containing medium containing 3% NaCl within a 3 L triangular flask, accounting for 66.97% of the cell dry weight. Furthermore, 1H NMR, 13C NMR, and GC–MS results confirmed that the polymer was PHB. The thermal properties of PHB, including its melting (Tm) and crystallization (Tc) temperatures of 176.34 °C and 56.12 °C respectively, were determined via differential scanning calorimetry analysis. The produced PHB was characterized by a weight-average molecular weight (Mw) of 5.43 × 105 g/mol, a number-average molecular weight (Mn) of 4.00 × 105 g/mol, and a polydispersity index (PDI) of 1.36. In addition, the whole genome was sequenced, and the PHB biosynthetic pathway and quantitative expression of key genes were delineated in the novel isolated strain. In conclusion, this research introduces the first instance of polyhydroxyalkanoate (PHA) production by Gordonia terrae using used soybean oil as the exclusive carbon source, which will enrich strain resources for future PHB biosynthesis.

De Novo Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in Yarrowia lipolytica.

Yarrowia lipolytica has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of Y. lipolytica metabolism challenging. Herein, a Cas9 system was established to redesign the Y. lipolytica homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the de novo synthesis of (S)-reticuline and (2S)-taxifolin. The developed platform strains and tools helped transform Y. lipolytica into an easy-to-operate model cell factory, similar to Saccharomyces cerevisiae.

Bioinspired Nanochitin-Based Porous Constructs for Light-Driven Whole-Cell Biotransformations.

Solid-state photosynthetic cell factories (SSPCFs) are a new production concept that leverages the innate photosynthetic abilities of microbes to drive the production of valuable chemicals. It addresses practical challenges such as high energy and water demand and improper light distribution associated with suspension-based culturing; however, these systems often face significant challenges related to mass transfer. The approach focuses on overcoming these limitations by carefully engineering the microstructure of the immobilization matrix through freeze-induced assembly of nanochitin building blocks. The use of nanochitins with optimized size distribution enabled the formation of macropores with lamellar spatial organization, which significantly improves light transmittance and distribution, crucial for maximizing the efficiency of photosynthetic reactions. The biomimetic crosslinking strategy, leveraging specific interactions between polyphosphate anions and primary amine groups featured on chitin fibers, produced mechanically robust and wet-resilient cryogels that maintained their functionality under operational conditions. Various model biotransformation reactions leading to value-added chemicals are performed in chitin-based matrix. It demonstrates superior or comparable performance to existing state-of-the-art matrices and suspension-based systems. The findings suggest that chitin-based cryogel approach holds significant promise for advancing the development of solid-state photosynthetic cell factories, offering a scalable solution to improve the efficiency and productivity of light-driven biotransformation.

Efficient Escherichia coli Platform for Cannabinoid Precursor Olivetolic Acid Biosynthesis from Inexpensive Inputs.

Olivetolic acid (OLA), an initial precursor of cannabinoids, is catalyzed by type III polyketide synthase, which has a wide range of pharmacological activities, such as antimicrobial and cytotoxic effects. Here, we applied systematic metabolic engineering to develop a multienzyme cascade system to produce OLA via two low-cost inputs. The polyketide synthase (OLS) and cyclase enzymes (OAC), along with the best combination of hexanoyl-CoA and malonyl-CoA synthetases (AEE3 and MatB), were first introduced into the biocatalytic system to increase the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units. To drive the catalysis smoothly, an ATP regeneration system and a CoA-sufficient supply system were incorporated into the biocatalysts to provide enough cofactors. Furthermore, malonyl-CoA flux was redirected to OLA biosynthesis through delicate control of the fatty acid biosynthesis (FAB) pathway via promoter engineering. Collectively, these strategies have led us to produce OLA at a titer of 102.1 mg/L with a productivity of 25.5 mg/L/h by using malonate and hexanoate as direct substrates. Our biocatalytic system provides an effective platform for the production of the cannabinoid precursor OLA in Escherichia coli and may be a valuable reference for the development of microbial cell factories that use hexanoyl-CoA and malonyl-CoA as important intermediates.

Membrane transport engineering for efficient yeast biomanufacturing.

Yeast strains have been widely recognized as useful cell factories for biomanufacturing. To improve production efficiency, their biosynthetic pathways and regulatory strategies have been continuously optimized. However, commercial production using yeasts is still limited by low product yield and high production cost. Accumulating evidences have demonstrated the importance of metabolite transport processes in addressing these challenges. Engineering yeast membrane transporters for transporting precursors, substrates, intermediates, products and toxic inhibitors has been successful. In addition, membrane properties are also important for metabolite production. Here we propose membrane transport engineering (MTE) to integrate manipulation of both membrane transporters and membrane properties. We emphasize that systematic optimization of both transporters and membrane lipid bilayers benefits production efficiency. We also envision the potential of artificial intelligence and automation process in MTE for economic and sustainable bioproduction using yeast cell factories.

MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering.

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

Efficient production of spermidine from Bacillus amyloliquefaciens by enhancing synthesis pathway, blocking degradation pathway and increasing precursor supply.

Spermidine has broad application potential in food, medicine and other fields. In this study, a novel Bacillus amyloliquefaciens cell factory was constructed for production of spermidine from renewablebiomass resources. Firstly, the speB gene was found to be optimal for synthesis of spermidine, and the function of SpeB was explained by amino acid sequence analysis and molecular docking. By replacing the native promoter of the speEB operon with the P43, the synthesis of spermidine was significantly enhanced in B. amyloliquefaciens HSPM1-P43speEB. After knockout of the genes yobN and bltD associated with spermidine degradation, the spermidine titer of the strain HSPM2 was further improved to 115.96 mg/L, increased by 108 % compared to HSPM1-P43speEB. Subsequently, the titer of spermidine was further increased to 277.47 mg/L through enhancing the supply of the precursor methionine by overexpression of speD. Finally, the renewable biomass resources, xylose and feather meal were optimized to produce spermidine, and the maximum titer is up to 588.10 mg/L after optimization. In conclusion, an efficient spermidine producing B. amyloliquefaciens was constructed through combinatorial metabolic engineering strategies, and the sustainable production of spermidine was achieved using the biomass resources of xylose and feather meal.

Exploring the complexity of xylitol production in the fungal cell factory Aspergillus niger.

Production of xylitol from agricultural by-products offers a promising approach for the circular bioeconomy. This study investigates the roles of transcription factors XlnR and CreA in xylitol production from wheat bran in Aspergillus niger by generating strains with a constitutively active XlnR (XlnRc, V756F mutation) and/or deletion of creA, in a previously generated xylitol-producing strain. The XlnRc mutation increased the initial rate of xylitol production but lowered the overall accumulation. Deletion of creA in this strain significantly improved both the onset and rate of xylitol production, indicating an inhibitory role of CreA in the PCP. These results demonstrate the complexity of metabolic engineering to generate fungal cell factories for valuable biochemicals, such as xylitol, as not only metabolic but also multiple gene regulation aspects need to be considered.

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We engineered a specialized actinomycete cell factory, 306A, optimized for the efficient production of PoTeMs. By introducing the heterologous biosynthetic gene cluster fla from marine‐derived Streptomyces flavogriseus SCSIO 40032—akin to a factory receiving a production order—the biosynthetic machinery functions like an assembly line. Flavin oxidase and P450 enzymes function as key "machines" on this line, catalyzing the cyclization of PoTeMs precursors, hydroxylation, and the formation of a C14 ketone group, ultimately yielding mature PoTeMs such as 10‐epi‐xanthobaccin C. More details are discussed in the article by Zhang et al. on pages 483—490. image

Metabolic engineering of Escherichia coli for improved cofactor regeneration in lactate to acetoin via whole-cell conversion

Background: Acetoin is a crucial intermediate in asymmetric syntheses of high-value chemicals and pharmaceuticals. However, its production still relies on traditional fossil-based processes. Developing efficient microbial cell factories for green and low-cost acetoin production is urgently needed. Methods: Acetoin was produced from inexpensive and shortcut lactate substrate using whole-cell Escherichia coli through overexpression of highly active α-acetolactate synthetase and decarboxylase from Bacillus subtilis (annotated as SD). Precise stepwise optimization of pathway and enzymatic reaction was executed by (1) harboring the most efficient cofactor-regenerating system, (2) tuning expression design, (3) disrupting byproduct pathway, and (4) optimizing a series of biotransformation parameters. Significant Findings: The recombinant E. coli successfully produced acetoin. The titer was gradually increased by expressing a pyruvate-producing gene from NAD+ dependent or independent system and its cofactor regeneration systems. Co-expressing lactate oxidase (lox) and catalase (cat) achieved a conversion efficiency of 50% and eliminated NAD+ usage. The conversion efficiency was further pulled by knocking out acetate-generating genes (pta and poxB), thus boosting acetoin conversion to 92.4%. Under optimized whole-cell biotransformation parameters, the highest acetoin titer reached 20.6 g/L within 30 h. This work provides an economical biomanufacturing process for acetoin from lactate via whole-cell bioconversion with remarkable yield.

Unleashing potential of microbial biosynthesis of monoterpenes via enzyme and metabolic engineering.

Monoterpenes (MTPs) are valuable isoprenoids widely used in cosmetics, food flavorings, pharmaceuticals, etc. Compared to plant extraction and chemical synthesis, microbial biosynthesis offers superior sustainability and efficiency in producing natural MTPs, overcoming the limitations of raw material dependency, environmental impact, and racemic mixtures inherent in these methods. This review comprehensively discusses the development of natural or non-natural biosynthetic pathways for producing regular and irregular MTPs, emphasizing the importance of enzyme and metabolic engineering to optimize monoterpene synthases (MTPSs) in various engineered microbial cell factories (MCFs). The advances in functional expression of MTPS to enhance enzyme activity, substrate channeling of MTPS with critical biosynthesis enzymes, protein engineering of MTPS, targeted localization of MTPS in the subcellular organelle, and other favorable engineering strategies are discussed in detail. Leveraging these technologies, the engineered microbes will achieve the production of the defined product profile with higher titer/yield/productivity and improved industrial adaptability. Furthermore, we highlight the important development direction for optimizing MTPS performance and biosynthetic pathways, ensuring the microbial production of natural MTPs in a more efficient and application-specific manner.

Production of HMF-derivatives from wine residues using Saccharomyces cerevisiae as whole-cell biocatalyst

BackgroundThere is an urgent need to develop bioprocesses independent of fossil resources to address resource depletion and mitigate environmental harm. Transitioning to a bio-based economy requires prioritizing chemical production processes that utilize renewable resources, ensuring sustainability and environmental responsibility. 5-Hydroxymethylfurfural (HMF) and its derivatives are promising building blocks, ranked among the top 12 bio-based molecules derived from biomass. This study investigates the potential of wine residues as substrates for HMF production and explores the yeast Saccharomyces cerevisiae, a robust industrial microbial cell factory, as a whole-cell biocatalyst for converting HMF into high-value compounds, offering an alternative to chemical synthesis.FindingsSeveral S. cerevisiae strains were compared for their ability to convert HMF, demonstrating varying capacities for oxidation or reduction. For the first time, HMF derivatives with potential industrial applications were produced using an HMF-rich hydrolysate obtained from sustainable processing of wine-growing waste, such as grape pomace and must surplus. The selected yeast strain was engineered to express the oxidoreductase enzyme of HMF/Furfural from Cupriavidua basilensis strain HMF14, resulting in a 15-fold increase in the accumulation of oxidized derivatives such as 2,5-furandicarboxylic acid (FDCA).ConclusionsThese findings highlight the potential of leveraging wine residues and engineered S. cerevisiae strains to develop sustainable bioprocesses for producing valuable HMF derivatives, thereby contributing to the advancement of bio-based chemical production.Graphical

Considerations for Domestication of Novel Strains of Filamentous Fungi.

Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.

Recent advances in the biosynthesis of polysaccharide-based antimicrobial glycoconjugate vaccines

Glycoconjugate vaccines are a vital category of effective and safe commercial vaccines that have significantly reduced the global prevalence of drug-resistant bacterial infections. These vaccines are synthesized by covalently linking bacterial polysaccharide antigens to a carrier protein. Given that they produce a stronger and longer-lasting immune response than pure polysaccharides that activate only B cells, glycoconjugate vaccines have become one of the most promising vaccine types. However, the chemical synthesis of glycoconjugate vaccines is complex, costly, and labor-intensive. Therefore, the efficient preparation of biosynthetic glycoconjugates using microbial cell factories has emerged as a highly desirable manufacturing alternative. This review focuses on advancements in the recombinant microbial biosynthesis of glycoconjugate vaccines and summarizes various strategies to optimize their production. It is based on three key aspects: the selection of oligosaccharyltransferase (OST), the use of different vaccine carrier proteins, and the enhancement of key concentrations in the uridine diphosphate (UDP)-sugar supply. Finally, the review highlights technical challenges and discusses future directions for the recombinant synthesis of glycoconjugate vaccines.

Engineering yeast to produce fraxetin from ferulic acid and lignin

Lignin, the most abundant renewable source of aromatic compounds on earth, remains underexploited in traditional biorefining. Fraxetin, a naturally occurring flavonoid, has garnered considerable attention in the scientific community due to its diverse and potent biological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory, and neurological protective actions. To enhance the green and value-added utilization of lignin, Saccharomyces cerevisiae was engineered as a cell factory to transform lignin derivatives to produce fraxetin. The expression of scopoletin 8-hydroxylase (S8H) and coumarin synthase (COSY) enabled S. cerevisiae to produce fraxetin from ferulic acid, one of the three principal monomers. The optimized fermentation strategies produced 19.1 mg/L fraxetin from ferulic acid by engineered S. cerevisiae. Additionally, the engineered cell factory achieved a fraxetin titer of 7.7 mg/L in lignin hydrolysate. This study successfully demonstrates the biotransformation of lignin monomers and lignin hydrolysate into fraxetin using a S. cerevisiae cell factory, thereby providing a viable strategy for the valorization of lignin.Key points• AtS8H showed substance specificity in the hydroxylation of scopoletin.• AtCOSY and AtS8H were key enzymes for converting ferulic acid into fraxetin.• Yeast was engineered to produce fraxetin from lignin hydrolysate.

CRISPR-Cas9 Cytidine-Base-Editor Mediated Continuous In Vivo Evolution in Aspergillus nidulans.

Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted in vivo evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in Aspergillus nidulans NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated in vivo evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.

Metabolic engineering of Escherichia coli for efficient biosynthesis of L-citrulline

L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.

Effects of exogenous additives on growth and high-value bioproducts accumulation of microalgae

With the rapid development of synthetic biology, genetic engineering, and molecular manipulation methods in recent years, microalgae, as representatives of microbial cell factories, have been widely used as hosts in the production of high-value bioproducts, such as oils, pigments, proteins, and biofuels, demonstrating promising prospects of application in biochemical energy, food and drugs, and environmental protection. Despite these advancements, the low production efficiency of microalgae limits their industrial application. In addition to strain improvement and culture condition optimization, the regulation by exogenous chemical additives serves as a promising optimization strategy. This method relies on straightforward phenotypic screening and circumvents the necessity for intricate understanding of molecular targets in the metabolic and catabolic pathways involved in the synthesis of bioproducts. It enables rapid yield increasing of high-value bioproducts from microalgae and obtaining the required phenotypes. Although studies have reported the use of alternatives means such as exogenous additives to improve the growth of microalgae and the yield of high-value bioproducts, the classification and summarization of the types, applications, targeted strains, and molecular mechanisms of these additives are not comprehensive. Here, we review the studies using chemical inducers or enhancers to improve cell growth and high-value bioproduct accumulation in microalgae in recent years. This paper focuses on the types of exogenous additives, the effects of exogenous additives and their combinations on microalgae growth and high-value bioproduct accumulation, and the molecular mechanisms of related effects. We aim to provide information for researchers to use methods of synthetic biology to develop suitable cell chassis and harness microalgae for industrial production.

Construction and application of an inducible transcriptional regulatory tool from Medicago truncatula in Saccharomyces cerevisiae

Transcriptional regulation based on transcription factors is an effective regulatory method widely used in microbial cell factories. Currently, few naturally transcriptional regulatory elements have been discovered from Saccharomyces cerevisiae and applied. Moreover, the discovered elements cannot meet the demand for specific metabolic regulation of exogenous compounds due to the high background expression or narrow dynamic ranges. There are abundant transcriptional regulatory elements in plants. However, the sequences and functions of most elements have not been fully characterized and optimized. Particularly, the applications of these elements in microbial cell factories are still in the infancy stage. In this study, natural regulatory elements from Medicago truncatula were selected, including the transcription factors MtTASR2 and MtTASR3, along with their associated promoter ProHMGR1, for functional characterization and engineering modification. We constructed an inducible transcriptional regulation tool and applied it in the regulation of heterologous β-carotene synthesis in S. cerevisiae, which increased the β-carotene production by 7.31 folds compared with the original strain. This study demonstrates that plant-derived transcriptional regulatory elements can be used to regulate the expression of multiple genes in S. cerevisiae, providing new strategies and ideas for the specific regulation and application of these elements in microbial cell factories.

Cell-free systems: A synthetic biology tool for rapid prototyping in metabolic engineering.

Microbial cell factories provide sustainable alternatives to petroleum-based chemical production using cost-effective substrates. A deep understanding of their metabolism is essential to harness their potential along with continuous efforts to improve productivity and yield. However, the construction and evaluation of numerous genetic variants are time-consuming and labor-intensive. Cell-free systems (CFSs) serve as powerful platforms for rapid prototyping of genetic circuits, metabolic pathways, and enzyme functionality. They offer numerous advantages, including minimizing unwanted metabolic interference, precise control of reaction conditions, reduced labor, and shorter Design-Build-Test-Learn cycles. Additionally, the introduction of in vitro compartmentalization strategies in CFSs enables ultra-high-throughput screening in physically separated spaces, which significantly enhances prototyping efficiency. This review highlights the latest examples of using CFS to overcome prototyping limitations in living cells with a focus on rapid prototyping, particularly regarding gene regulation, enzymes, and multienzymatic reactions in bacteria. Finally, this review evaluates CFSs as a versatile prototyping platform and discusses its future applications, emphasizing its potential for producing high-value chemicals through microbial biosynthesis.