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AbstractAstaxanthin (AXT), a natural carotenoid, has strong antioxidant and anti-ageing effects and can reduce ultraviolet light-induced damage to cells and DNA, stimulate the immune system, and improve cardiovascular disease prognosis. Despite its wide applications in the: nutraceutical, cosmetic, aquaculture, and pharmaceutical industries, AXT industrial production and application are hindered by natural source scarcity, low production efficiency, and high requirements. This review compares the qualitative differences of AXT derived from different natural sources, evaluates the upstream procedures for AXT expression in different chassis organisms, and investigates synthetic biology- and cell factory-based strategies for the industrial production of natural AXT. Synthetic biology is a promising novel strategy for reprogramming plants or microorganisms to produce AXT. Additionally, genetic engineering using cell factories extends beyond terrestrial applications, as it may contribute to the long-term sustainability of human health during space exploration and migration endeavors. This review provides a theoretical basis for the efficient and accurate genetic engineering of AXT from the microalga Haematococcuspluvialis, providing a valuable reference for future research on the biomanufacturing of AXT and other biological metabolites. [ABSTRACT FROM AUTHOR]

D-glucaric acid is an important organic acid with numerous applications in therapy, food, and materials, contributing significantly to its substantial market value. The biosynthesis of D-glucaric acid (GA) from renewable sources such as glucose has garnered significant attention due to its potential for sustainable and cost-effective production. This review summarizes the current understanding of the cell factories for GA production in different chassis strains, from static to dynamic control strategies for regulating their metabolic networks. We highlight recent advances in the optimization of D-glucaric acid biosynthesis, including metabolic dynamic control, alternative feedstocks, metabolic compartments, and so on. Additionally, we compare the differences between different chassis strains and discuss the challenges that each chassis strain must overcome to achieve highly efficient GA productions. In this review, the processes of engineering a desirable cell factory for highly efficient GA production are just like an epitome of metabolic engineering of strains for chemical biosynthesis, inferring general trends for industrial chassis strain developments. [ABSTRACT FROM AUTHOR]

Thermophilic acetogens are gaining recognition as potent microbial cell factories, leveraging their unique metabolic capabilities to drive the development of sustainable biotechnological processes. These microorganisms, thriving at elevated temperatures, exhibit robust carbon fixation abilities via the linear Wood-Ljungdahl pathway to efficiently convert C1 substrates, including syngas (CO, CO2 and H2) from industrial waste gasses, into acetate and biomass via the central metabolite acetyl-CoA. This review summarizes recent advancements in metabolic engineering and synthetic biology efforts that have expanded the range of products derived from thermophilic acetogens after briefly discussing their autotrophic metabolic diversity. These discussions highlight their potential in the sustainable bioproduction of industrially relevant compounds. We further review the remaining challenges for implementing efficient and complex strain engineering strategies in thermophilic acetogens, significantly limiting their use in an industrial context. [ABSTRACT FROM AUTHOR]

Heme, as an essential cofactor and source of iron for cells, holds great promise in various areas, e.g., food and medicine. In this study, the model cyanobacteria Synechocystis sp. PCC6803 was used as a host for heme synthesis. The heme synthesis pathway and its competitive pathway were modified to obtain an engineered cyanobacteria with high heme production, and the total heme production of Synechocystis sp. PCC6803 was further enhanced by the optimization of the culture conditions and the enhancement of mixotrophic ability. The co-expression of hemC, hemF, hemH, and the knockout of pcyA, a key gene in the heme catabolic pathway, resulted in a 3.83-fold increase in the heme production of the wild type, while the knockout of chlH, a gene encoding a Mg-chelatase subunit and the key enzyme of the chlorophyll synthesis pathway, resulted in a 7.96-fold increase in the heme production of the wild type; further increased to 2.05 mg/L, its heme production was 10.25-fold that of the wild type under optimized mixotrophic culture conditions. Synechocystis sp. PCC6803 has shown great potential as a cell factory for photosynthetic carbon sequestration for heme production. This study provides novel engineering targets and research directions for constructing microbial cell factories for efficient heme production. [ABSTRACT FROM AUTHOR]

Abstract Background Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI. Results Here we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent. Conclusion The awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Coriolopsis trogii is a basidiomycete fungus which utilizes a large array of lignin-modifying enzymes to colonize and decompose dead wood. Its extracellular enzymatic arsenal includes laccases, i.e., polyphenol oxidases of relevant interest for different industrial applications thanks to their ability to oxidize a diverse range of natural and synthetic compounds. In this work, the production of laccases in C. trogii MUT3379 was explored and improved. From an initial production of ca. 10,000 U L−1, the fermentation process was gradually optimized, reaching a final yield of ca. 200,000 U L−1. An SDS-PAGE analysis of the secretome highlighted the presence of a main protein of ca. 60 kDa showing laccase activity, which was designated as Lac3379-1 once its primary sequence was established by tandem mass spectrometry. The characterization of Lac3379-1 revealed a remarkable enzymatic stability in the presence of surfactants and solvents and a diversified activity on a broad range of substrates, positioning it as an interesting tool for diverse biotechnological applications. The high-yield and robust production process indicates C. trogii MUT3379 as a promising cell factory for laccases, offering new perspectives for industrial applications of lignin-modifying enzymes. [ABSTRACT FROM AUTHOR]

Abstract The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

Abstract5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward. [ABSTRACT FROM AUTHOR]

β-Carotene is an orange fat-soluble compound, which has been widely used in fields such as food, medicine and cosmetics owing to its anticancer, antioxidant and cardiovascular disease prevention properties. Currently, natural β-carotene is mainly extracted from plants and algae, which cannot meet the growing market demand, while chemical synthesis of β-carotene cannot satisfy the pursuit for natural products of consumers. The β-carotene production through microbial fermentation has become a promising alternative owing to its high efficiency and environmental friendliness. With the rapid development of synthetic biology and in-depth study on the synthesis pathway of β-carotene, microbial fermentation has shown promising applications in the β-carotene synthesis. Accordingly, this review aims to summarize the research progress and strategies of natural carotenoid producing strain and metabolic engineering strategies in the heterologous synthesis of β-carotene by engineered microorganisms. Moreover, it also summarizes the adoption of inexpensive carbon sources to synthesize β-carotene as well as proposes new strategies that can further improve the β-carotene production. [ABSTRACT FROM AUTHOR]

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure. [ABSTRACT FROM AUTHOR]

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Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production. [ABSTRACT FROM AUTHOR]

In the twenty first century, biotechnology offers great opportunities and solutions to climate change mitigation, energy and food security and resource efficiency. The use of metabolic engineering to modify microorganisms for producing industrially significant chemicals is developing and becoming a trend. As a famous, generally recognized as a safe (GRAS) model microorganism, Saccharomyces cerevisiae is widely used due to its excellent operational convenience and high fermentation efficiency. This review summarizes recent advancements in the field of using metabolic engineering strategies to construct engineered S. cerevisiae over the past ten years. Five different types of compounds are classified by their metabolites, and the modified metabolic pathways and strategies are summarized and discussed independently. This review may provide guidance for future metabolic engineering efforts toward such compounds and analogues. Additionally, the limitations of S. cerevisiae as a cell factory and its future trends are comprehensively discussed. [ABSTRACT FROM AUTHOR]

Thermophilic acetogens are gaining recognition as potent microbial cell factories, leveraging their unique metabolic capabilities to drive the development of sustainable biotechnological processes. These microorganisms, thriving at elevated temperatures, exhibit robust carbon fixation abilities via the linear Wood-Ljungdahl pathway to efficiently convert C1 substrates, including syngas (CO, CO2 and H2) from industrial waste gasses, into acetate and biomass via the central metabolite acetyl-CoA. This review summarizes recent advancements in metabolic engineering and synthetic biology efforts that have expanded the range of products derived from thermophilic acetogens after briefly discussing their autotrophic metabolic diversity. These discussions highlight their potential in the sustainable bioproduction of industrially relevant compounds. We further review the remaining challenges for implementing efficient and complex strain engineering strategies in thermophilic acetogens, significantly limiting their use in an industrial context.

Acetogenic bacteria (acetogens) are a class of microorganisms with conserved Wood-Ljungdahl pathway that can utilize CO and CO2/H2 as carbon source for autotrophic growth and convert these substrates to acetate and ethanol. Acetogens have great potential for the sustainable production of biofuels and bulk biochemicals using C1 gases (CO and CO2) from industrial syngas and waste gases, which play an important role in achieving carbon neutrality. In recent years, with the development and improvement of gene editing methods, the metabolic engineering of acetogens is making rapid progress. With introduction of heterogeneous metabolic pathways, acetogens can improve the production capacity of native products or obtain the ability to synthesize non-native products. This paper reviews the recent application of metabolic engineering in acetogens. In addition, the challenges of metabolic engineering in acetogens are indicated, and strategies to address these challenges are also discussed.

With a better understanding of the health benefits of β-carotene, the precursor of vitamin A, as well as its coloring property, the need for this carotenoid has increased in various sectors. In order to meet the increasing demand efficiently, cheaply, and sustainably, interest in heterologous β-carotene production through metabolic engineering strategies has increased in recent years. In this context, although it is not a native producer of β-carotene, Yarrowia lipolytica yeast stands out due to its metabolic, physiological, and genomic properties. Successful results have been obtained by using a series of engineering strategies, including biosynthesis pathway engineering, morphological engineering, and fermentation engineering strategies, in the production of heterologous β-carotene from Y. lipolytica. However, these strategies have various strengths and weaknesses against each other, and there are also some points open to improvement. In this review, the engineering strategies that have been applied and have the potential to be applied for the production of β-carotene from Y. lipolytica have been examined in depth, including their advantages and disadvantages, and compared with each other. Moreover, a future perspective has been presented to increase the potential of using Y. lipolytica yeast as a cell factory in β-carotene production. [ABSTRACT FROM AUTHOR]

As the most widely distributed phenolic compounds in the plant kingdom, flavonoids play an integral role in plant reproduction and defense. Also, they represent many important quality traits of edible plants like color and antioxidants, and have a variety of biological activities beneficial to human health. To diversify the functions of synthesized flavonoids, plants have evolved various enzymes to perform structural modifications on different flavonoid backbones. One of these modifications is prenylation, which refers to the attachment of an isoprenoid moiety, most commonly a prenyl (C5) group. Numerous structure-activity analyses of prenylflavonoids have shown that isopentenyl substitutions at specific sites can significantly expand and enhance their chemical properties, bioactivities and potential health benefits. This review summarizes prenylflavonoids reported so far in all plant species and highlights the current knowledge on naturally occurring prenyltransferases from different biological sources that can act on plant flavonoids to synthesize prenylflavonoids. Most of them have strict flavonoid substrate- and regio-specificities, and they provide a valuable gene repository to facilitate the efficient scale-up production of flavonoids with specific prenylation patterns in cell factories. To truly achieve this goal, it is necessary to explore more diversified natural prenyltransferases, and to optimize the bioreactors system such as pathway regulation and modular co-culture engineering in the future. [ABSTRACT FROM AUTHOR]

β-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of β-carotene and α-carotene. Aiming to develop the microalgae with enhanced β-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of β-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant β-carotene), confirming the superior activity of CrtY on β-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased β-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae. [ABSTRACT FROM AUTHOR]

Abstract The use of marine microalgae in industrial systems is attractive for converting CO2 into value-added products using saline water and sunlight. The plant nature and demonstrated industrial potential facilitate Chlorella spp. as excellent model organisms for both basic research and commercial application. However, the transformation method has not been developed in marine Chlorella spp., thus genetic engineering is hindered in exploiting the industrial potentialities of these strains. In this study, we provided a transformation protocol for the marine Chlorella strain MEM25, which showed robust characteristics, including high production of proteins and polyunsaturated fatty acids in multiple cultivation systems over various spatial–temporal scales. We showed that transformants could be obtained in a dramatically time-saving manner (comparable to Saccharomyces cerevisiae) with four functional proteins expressed properly. The transgenes are integrated into the genome and can be successfully inherited for more than two years. The development of a marine Chlorella transformation method, in combination with the complete genome, will greatly facilitate more comprehensive mechanism studies and provide possibilities to use this species as chassis for synthetic biology to produce value-added compounds with mutual advantage in neutralization of CO2 in commercial scales.

Heme, as an essential cofactor and source of iron for cells, holds great promise in various areas, e.g., food and medicine. In this study, the model cyanobacteria Synechocystis sp. PCC6803 was used as a host for heme synthesis. The heme synthesis pathway and its competitive pathway were modified to obtain an engineered cyanobacteria with high heme production, and the total heme production of Synechocystis sp. PCC6803 was further enhanced by the optimization of the culture conditions and the enhancement of mixotrophic ability. The co-expression of hemC, hemF, hemH, and the knockout of pcyA, a key gene in the heme catabolic pathway, resulted in a 3.83-fold increase in the heme production of the wild type, while the knockout of chlH, a gene encoding a Mg-chelatase subunit and the key enzyme of the chlorophyll synthesis pathway, resulted in a 7.96-fold increase in the heme production of the wild type; further increased to 2.05 mg/L, its heme production was 10.25-fold that of the wild type under optimized mixotrophic culture conditions. Synechocystis sp. PCC6803 has shown great potential as a cell factory for photosynthetic carbon sequestration for heme production. This study provides novel engineering targets and research directions for constructing microbial cell factories for efficient heme production.

Coriolopsis trogii is a basidiomycete fungus which utilizes a large array of lignin-modifying enzymes to colonize and decompose dead wood. Its extracellular enzymatic arsenal includes laccases, i.e., polyphenol oxidases of relevant interest for different industrial applications thanks to their ability to oxidize a diverse range of natural and synthetic compounds. In this work, the production of laccases in C. trogii MUT3379 was explored and improved. From an initial production of ca. 10,000 U L−1, the fermentation process was gradually optimized, reaching a final yield of ca. 200,000 U L−1. An SDS-PAGE analysis of the secretome highlighted the presence of a main protein of ca. 60 kDa showing laccase activity, which was designated as Lac3379-1 once its primary sequence was established by tandem mass spectrometry. The characterization of Lac3379-1 revealed a remarkable enzymatic stability in the presence of surfactants and solvents and a diversified activity on a broad range of substrates, positioning it as an interesting tool for diverse biotechnological applications. The high-yield and robust production process indicates C. trogii MUT3379 as a promising cell factory for laccases, offering new perspectives for industrial applications of lignin-modifying enzymes.

Fully understanding the environmental implications of engineered nanomaterials is crucial for their safe and sustainable use. Cyanobacteria, as the pioneers of the planet earth, play important roles in global carbon and nitrogen cycling. Here, we evaluated the biological effects of molybdenum disulfide (MoS2) nanosheets on a N2-fixation cyanobacteria (Nostoc sphaeroides) by monitoring growth and metabolome changes. MoS2 nanosheets did not exert overt toxicity to Nostoc at the tested doses (0.1 and 1 mg/L). On the contrary, the intrinsic enzyme-like activities and semiconducting properties of MoS2 nanosheets promoted the metabolic processes of Nostoc, including enhancing CO2-fixation-related Calvin cycle metabolic pathway. Meanwhile, MoS2 boosted the production of a range of biochemicals, including sugars, fatty acids, amino acids, and other valuable end products. The altered carbon metabolism subsequently drove proportional changes in nitrogen metabolism in Nostoc. These intracellular metabolic changes could potentially alter global C and N cycles. The findings of this study shed light on the nature and underlying mechanisms of bio-nanoparticle interactions, and offer the prospect of utilization bio-nanomaterials for efficient CO2 sequestration and sustainable biochemical production.

Abstract β-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of β-carotene and α-carotene. Aiming to develop the microalgae with enhanced β-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of β-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant β-carotene), confirming the superior activity of CrtY on β-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased β-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β-carotene) is a type of C40 carotenoid with remarkable antioxidant characteristics, showing significant application prospects in many fields. Traditionally, the astaxanthin is mainly obtained from chemical synthesis and natural acquisition, with both approaches having many limitations and not capable of meeting the growing market demand. In order to cope with these challenges, novel techniques, e.g., the innovative cell engineering strategies, have been developed to increase the astaxanthin production. In this review, we first elaborated the biosynthetic pathway of astaxanthin, with the key enzymes and their functions discussed in the metabolic process. Then, we summarized the conventional, non-genetic strategies to promote the production of astaxanthin, including the methods of exogenous additives, mutagenesis, and adaptive evolution. Lastly, we reviewed comprehensively the latest studies on the synthesis of astaxanthin in various recombinant microorganisms based on the concept of microbial cell factory. Furthermore, we have proposed several novel technologies for improving the astaxanthin accumulation in several model species of microorganisms. [ABSTRACT FROM AUTHOR]

Pichia pastoris is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell factory for the production of chemicals and natural products. In this review, the general physiological properties of P. pastoris are summarized and the readily available genetic tools and elements are described, including strains, expression vectors, promoters, gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and adaptive laboratory evolution. Moreover, the recent achievements in P. pastoris-based biosynthesis of proteins, natural products, and other compounds are highlighted. The existing issues and possible solutions are also discussed for the construction of efficient P. pastoris cell factories. [ABSTRACT FROM AUTHOR]

The use of marine microalgae in industrial systems is attractive for converting CO2 into value-added products using saline water and sunlight. The plant nature and demonstrated industrial potential facilitate Chlorella spp. as excellent model organisms for both basic research and commercial application. However, the transformation method has not been developed in marine Chlorella spp., thus genetic engineering is hindered in exploiting the industrial potentialities of these strains. In this study, we provided a transformation protocol for the marine Chlorella strain MEM25, which showed robust characteristics, including high production of proteins and polyunsaturated fatty acids in multiple cultivation systems over various spatial–temporal scales. We showed that transformants could be obtained in a dramatically time-saving manner (comparable to Saccharomyces cerevisiae) with four functional proteins expressed properly. The transgenes are integrated into the genome and can be successfully inherited for more than two years. The development of a marine Chlorella transformation method, in combination with the complete genome, will greatly facilitate more comprehensive mechanism studies and provide possibilities to use this species as chassis for synthetic biology to produce value-added compounds with mutual advantage in neutralization of CO2 in commercial scales. [ABSTRACT FROM AUTHOR]

Abstract Background Expression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering. Results An in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively. Conclusions Expression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production.

The large consumption of non-renewable fossil fuels has brought about energy depletion and environmental pollution, spawning the production of renewable biofuels, an important alternative to alleviate the energy crisis effectively. As one of the ideal types of biofuel, bioethanol synthesis in Saccharomyces cerevisiae has attracted much attention. S. cerevisiae has been developed as essential chassis cells with high efficiency for bioethanol synthesis on account of many advantages. This study systematically summarized the preponderance of S. cerevisiae in biosynthesis. It objectively stated the research strategies of bioethanol synthesis based on S. cerevisiae and the existing bottleneck problems. This study further proposed reasonable prospects for bioethanol synthesis by S. cerevisiae, attempting to provide alternative research strategies. [ABSTRACT FROM AUTHOR]

The demand for robust microbial cell factories that produce valuable biomaterials while resisting stresses imposed by current bioprocesses is rapidly growing. Rhodosporidium toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. To explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 and 42°C) and two ethanol concentrations (2 and 4%), while the other used the inexpensive and abundant sugarcane juice as substrate. Differential expression and functional analysis were implemented using transcriptomic data to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in studying gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA) is a natural linear polysaccharide extensively used in many fields, including the food, medicine, and cosmetics industries. Currently, species that produce HA synthetase (HAS) from microbial sources are relatively small and mainly pathogenic, such as Streptococcus pyogenes and Pasteurella multicide. Moreover, there is limited research on the safe microbial sources of HAS. Thus, we characterized SthasA, a HAS derived from the probiotic Streptococcus thermophilus, and used it for the de novo synthesis of HA in a chassis strain of Bacillus amyloliquefaciens. Metabolic engineering of the precursor supply modules suggested that hasB (encoding UDPG dehydrogenase), which was derived from Corynebacterium glutamicum ATCC 13032, effectively promoted the accumulation of HA products. Furthermore, by combining the expression of the global regulatory factor CcpA, HA yield from the recombinant strain reached 3.20 g/L. Finally, we obtained a yield of 5.57 g/L HA with a molecular weight of 1.7 × 106 Da using various process optimization strategies in a 5 L bioreactor. This study enriches our understanding of obtaining HAS from non-pathogenic bacteria and provides a safe and effective process for producing HA, which has the potential to promote the industrial applications of HA further. [ABSTRACT FROM AUTHOR]

Abstract Background Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. Results Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. Conclusion The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production.

L‐Tyrosine derivatives are widely applied in the pharmaceutical, food, and chemical industries. Their production is mainly confined to chemical synthesis and plant extract. Microorganisms, as cell factories, exhibit promising advantages for valuable chemical production to fulfill the increase in the demand of global markets. Yeast has been used to produce natural products owing to its robustness and genetic maneuverability. Focusing on the progress of yeast cell factories for the production of L‐tyrosine derivatives, we summarized the emerging metabolic engineering approaches in building L‐tyrosoine‐overproducing yeast and constructing cell factories of three typical chemicals and their derivatives: tyrosol, p‐coumaric acid, and L‐DOPA. Finally, the challenges and opportunities of L‐tyrosine derivatives production in yeast cell factories were also discussed. Take away: Methods for constructing L‐tyrosine‐producing chassis.Enzyme discovery and screening for rate‐limiting steps.Pathway engineering for remodeling L‐tyrosine derivatives biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Expression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering. Results: An in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively. Conclusions: Expression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications. [ABSTRACT FROM AUTHOR]

Recent advances in synthetic biology have transformed mushroom farming from a focus on traditional cultivation to comprehensive applications based on cutting-edge biotechnologies. Synthetic biology has promising applications in this field, including precision breeding, mining biosynthetic gene clusters, developing mushroom chassis cells, and constructing cell factories for high value-added products. [ABSTRACT FROM AUTHOR]

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has a long-standing history of biotechnological applications even before the dawn of modern biotechnology. The field is undergoing accelerated advancement with the recent systems and synthetic biology approaches. In this review, we highlight the recent findings in the field with a focus on omics studies of S. cerevisiae to investigate its stress tolerance in different industries. The latest advancements in S. cerevisiae systems and synthetic biology approaches for the development of genome-scale metabolic models (GEMs) and molecular tools such as multiplex Cas9, Cas12a, Cpf1, and Csy4 genome editing tools, modular expression cassette with optimal transcription factors, promoters, and terminator libraries as well as metabolic engineering. Omics data analysis is key to the identification of exploitable native genes/proteins/pathways in S. cerevisiae with the optimization of heterologous pathway implementation and fermentation conditions. Through systems and synthetic biology, various heterologous compound productions that require non-native biosynthetic pathways in a cell factory have been established via different strategies of metabolic engineering integrated with machine learning. [Display omitted] [ABSTRACT FROM AUTHOR]

Rhodotorula sp. are well-known for their ability to biosynthesize a diverse range of valuable biomolecules, including carotenoids, lipids, enzymes, and polysaccharides. Despite the high number of studies conducted using Rhodotorula sp. at the laboratory scale, most of these do not address all processual aspects necessary for scaling up these processes for industrial applications. This chapter explores the potential of Rhodotorula sp. as a cell factory for the production of distinct biomolecules, with a particular emphasis on exploring their use from a biorefinery perspective. Through in-depth discussions of the latest research and insights into non-conventional applications, we aim to provide a comprehensive understanding of Rhodotorula sp.'s ability to produce biofuels, bioplastics, pharmaceuticals, and other valuable biochemicals. This book chapter also examines the fundamentals and challenges associated with the optimizing upstream and downstream processing of Rhodotorula sp-based processes. We believe that through this chapter, readers with different levels of expertise will gain insights into strategies for enhancing the sustainability, efficiency, and effectiveness of producing biomolecules using Rhodotorula sp. [ABSTRACT FROM AUTHOR]

Aspergillus is widely distributed in nature and occupies a crucial ecological niche, which has complex and diverse metabolic pathways and can produce a variety of metabolites. With the deepening of genomics exploration, more Aspergillus genomic informations have been elucidated, which not only help us understand the basic mechanism of various life activities, but also further realize the ideal functional transformation. Available genetic engineering tools include homologous recombinant systems, specific nuclease based systems, and RNA techniques, combined with transformation methods, and screening based on selective labeling. Precise editing of target genes can not only prevent and control the production of mycotoxin pollutants, but also realize the construction of economical and efficient fungal cell factories. This paper reviewed the establishment and optimization process of genome technologies, hoping to provide the theoretical basis of experiments, and summarized the recent progress and application in genetic technology, analyzes the challenges and the possibility of future development with regard to Aspergillus. [ABSTRACT FROM AUTHOR]

L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high throughput screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products. • Free lipoic acid produced in E. coli using heterologous Lipoamidase activity. • Metagenomic selection of improved heterologous lipoic acid biosynthetic enzymes. • >3000 combinatorial pathway enzyme expression levels tested to optimize balancing. • Lipoic acid cell factory produced 87/mg/L in two-stage fed-batch fermentation. [ABSTRACT FROM AUTHOR]

Covering 2009–2022. Phenylnaphthacenoid polyketides have gained significant interest in recent years owing to their potent antibacterial and anticancer activities. Notably, more than 100 members of this class of natural products have been discovered from various Streptomyces species by different research groups including ours over the last 13 years. This review summarizes the current knowledge of the discovery, chemical diversity, and bioactivity of phenylnaphthacenoid polyketides. The current review also highlights the cell factory for phenylnaphthacenoid production: (1) native strains, (2) mutant strains, (3) heterologous expression, and (4) biocatalytic halogenations. Furthermore, current challenges and future opportunities are also presented as a guide for researchers to explore them more purposefully. [ABSTRACT FROM AUTHOR]

The “big food view” has attracted widespread attention due to the view of sustainable nutrition and human health as part of sustainable development. The “big food view” starts from better meeting the people's needs for a better life. While ensuring the supply of grain, the effective supply of meat, vegetables, fruits, aquatic products and other foods also should be guaranteed. Using cell factories to replace the traditional food acquisition methods, establishing a new model of sustainable food manufacturing, will greatly reduce the demand for resources in food production, and improve the controllability of food production and manufacturing, and effectively avoid potential food safety and health risks. Cell factories can provide key technologies and supporting methods for the biological manufacturing of important food components, functional food ingredients and important functional nutritional factors, realizing a safer, nutritious, healthy and sustainable way of food acquisition. The combination of cell factory technology and other technologies meets the people's new dietary demand, and also supports that sustainable nutrition and human health as part of sustainable development. This paper focuses on the big food view and human health from the prospect of bio-manufacturing and future food, which aims to better meet people's dietary needs for increasingly diversified, refined, nutritious and ecological food through diversified food manufacturing.

The enzyme L-asparaginase (L-asparagine amidohydrolase) catalyzes the breakdown of L-asparagine into aspartate and ammonia, which leads to an anti-neoplastic activity stemming from its capacity to deplete L-asparagine concentrations in the bloodstream, and it is therefore used in cases of acute lymphoblastic leukemia (ALL) to inhibit malignant cell growth. Nowadays, this anti-cancer enzyme, largely produced by Escherichia coli, is well established on the market. However, E. coli L-asparaginase therapy has side effects such as anaphylaxis, coagulation abnormality, low plasma half-life, hepatotoxicity, pancreatitis, protease action, hyperglycemia, and cerebral dysfunction. This review provides a perspective on the use of filamentous fungi as alternative cell factories for L-asparaginase production. Filamentous fungi, such as various Aspergillus species, have superior protein secretion capacity compared to yeast and bacteria and studies show their potential for the future production of proteins with humanized N-linked glycans. This article explores the past and present applications of this important enzyme and discusses the prospects for using filamentous fungi to produce safe eukaryotic asparaginases with high production yields. [ABSTRACT FROM AUTHOR]

Background: Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. Results: Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. Conclusion: The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production. [ABSTRACT FROM AUTHOR]

Cordyceps militaris , widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks, expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to further research on C. militaris across various fields, including computational biology, existing DNA elements, and genome editing approaches. This review aims to describe specific and promising opportunities for the in-depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic biology development are illustrated. • C. militaris is a medicinal and edible mushroom. • C. militaris has the potential and possibility to be built as a cell factory. • Computational and advanced CRISPR technique were applied in C. militaris. • Metabolic engineering instructs in producing high-value compounds. • Synthetic biology facilitates the development of cell factory construction. [ABSTRACT FROM AUTHOR]

Pichia pastoris is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell factory for the production of chemicals and natural products. In this review, the general physiological properties of P. pastoris are summarized and the readily available genetic tools and elements are described, including strains, expression vectors, promoters, gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and adaptive laboratory evolution. Moreover, the recent achievements in P. pastoris-based biosynthesis of proteins, natural products, and other compounds are highlighted. The existing issues and possible solutions are also discussed for the construction of efficient P. pastoris cell factories.