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The current food production system is negatively impacting planetary and human health. A transition to a sustainable and fair food system is urgently needed. Microorganisms are likely enablers of this process, as they can produce delicious and healthy microbial foods with low environmental footprints. We review traditional and current approaches to microbial foods, such as fermented foods, microbial biomass, and food ingredients derived from microbial fermentations. We discuss how future advances in science-driven fermentation, synthetic biology, and sustainable feedstocks enable a new generation of microbial foods, potentially impacting the sustainability, resilience, and health effects of our food system.
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Alimentos Fermentados , Microbiología de Alimentos , Humanos , Fermentación , Alimentos , Crecimiento Sostenible , Conservación de los Recursos NaturalesRESUMEN
Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.
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Productos Biológicos/metabolismo , Descubrimiento de Drogas , Microbiología Industrial/métodos , Ingeniería Metabólica , Animales , Bacterias/clasificación , Bacterias/metabolismo , Vías Biosintéticas , Células CHO , Cricetulus , Escherichia coli/metabolismo , Hongos/clasificación , Hongos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Lipophilic compounds have a variety of positive effects on human physiological functions and exhibit good effects in the prevention and treatment of clinical diseases. This has led to significant interest in the technical applications of synthetic biology for the production of lipophilic compounds. However, the strict selective permeability of the cell membrane and the hydrophobic nature of lipophilic compounds pose significant challenges to their production. During fermentation, lipophilic compounds tend to accumulate within cell membrane compartments rather than being secreted extracellularly. The toxic effects of excessive lipophilic compound accumulation can threaten cell viability, while the limited space within the cell membrane restricts further increases in production yield. Consequently, to achieve efficient production of lipophilic compounds, research is increasingly focused on constructing robust and multifunctional microbial cell factories. Utilizing membrane engineering techniques to construct highly flexible cell membranes is considered an effective strategy to break through the upper limit of lipophilic compound production. Currently, there are two main approaches to cell membrane modification: constructing artificial storage compartments for lipophilic compounds and engineering the cell membrane structure to facilitate product outflow. This review summarizes recent cell membrane engineering strategies applied in microbial cell factories for the production of liposoluble compounds, discussing the challenges and future prospects. These strategies enhance membrane flexibility and effectively promote the production of liposoluble compounds.
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Membrana Celular , Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , HumanosRESUMEN
Parageobacillus thermoglucosidasius is a thermophilic and facultatively anaerobic microbe, which is emerging as one of the most promising thermophilic model organisms for metabolic engineering. The use of thermophilic microorganisms for industrial bioprocesses provides the advantages of increased reaction rates and reduced cooling costs for bioreactors compared to their mesophilic counterparts. Moreover, it enables starch or lignocellulose degradation and fermentation to occur at the same temperature in a Simultaneous Saccharification and Fermentation (SSF) or Consolidated Bioprocessing (CBP) approach. Its natural hemicellulolytic capabilities and its ability to convert CO to metabolic energy make P. thermoglucosidasius a potentially attractive host for bio-based processes. It can effectively degrade hemicellulose due to a number of hydrolytic enzymes, carbohydrate transporters, and regulatory elements coded from a genomic cluster named Hemicellulose Utilization (HUS) locus. The growing availability of effective genetic engineering tools in P. thermoglucosidasius further starts to open up its potential as a versatile thermophilic cell factory. A number of strain engineering examples showcasing the potential of P. thermoglucosidasius as a microbial chassis for the production of bulk and fine chemicals are presented along with current research bottlenecks. Ultimately, this review provides a holistic overview of the distinct metabolic characteristics of P. thermoglucosidasius and discusses research focused on expanding the native metabolic boundaries for the development of industrially relevant strains.
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Ingeniería Metabólica , Polisacáridos/metabolismo , Polisacáridos/genética , Bacillaceae/genética , Bacillaceae/metabolismoRESUMEN
Tanshinone and phenolic acids are the two main chemical constituents in Salvia miltiorrhiza, which are used clinically for the treatment of hypertension, coronary heart disease, atherosclerosis, and many other diseases, and have broad medicinal value. The efficient synthesis of the target products of these two metabolites in isolated plant tissues cannot be achieved without the regulation and optimization of metabolic pathways, and transcription factors play an important role as common regulatory elements in plant tissue metabolic engineering. However, most of the regulatory effects are specific to one class of metabolites, or an opposing regulation of two classes of metabolites exists. In this study, an artificially modified transcription factor, SmMYB36-VP16, was constructed to enhance tanshinone and phenolic acids in Salvia miltiorrhiza hair roots simultaneously. Further in combination with the elicitor dual-screening technique, by applying the optimal elicitors screened, the tanshinone content in the transgenic hairy roots of Salvia miltiorrhiza reached 6.44 mg/g DW, which was theoretically 6.08-fold that of the controls without any treatment, and the content of phenolic acids reached 141.03 mg/g DW, which was theoretically 5.05-fold that of the controls without any treatment. The combination of artificially modified transcriptional regulatory and elicitors dual-screening techniques has facilitated the ability of plant isolated tissue cell factories to produce targeted medicinal metabolites. This strategy could be applied to other species, laying the foundation for the production of potential natural products for the medicinal industry.
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ß-Carotene is one kind of the most important carotenoids. The major functions of ß-carotene include the antioxidant and anti-cardiovascular properties, which make it a growing market. Recently, the use of metabolic engineering to construct microbial cell factories to synthesize ß-carotene has become the latest model for its industrial production. Among these cell factories, yeasts including Saccharomyces cerevisiae and Yarrowia lipolytica have attracted the most attention because of the: security, mature genetic manipulation tools, high flux toward carotenoids using the native mevalonate pathway and robustness for large-scale fermentation. In this review, the latest strategies for ß-carotene biosynthesis, including protein engineering, promoters engineering and morphological engineering are summarized in detail. Finally, perspectives for future engineering approaches are proposed to improve ß-carotene production.
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Ingeniería Metabólica , Yarrowia , beta Caroteno/genética , beta Caroteno/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Saccharomyces cerevisiae/genética , Regiones Promotoras GenéticasRESUMEN
Anthocyanins (ACNs) are secondary metabolites found in plants. Due to their impressive biological activities, ACNs have gained significant popularity and extensive application within the food, pharmaceutical, and nutraceutical industries. A derivative of ACNs: pyranoanthocyanins (PACNs) possesses more stable properties and interesting biological activities. However, conventional methods for the production of ACNs, including chemical synthesis and plant extraction, involve organic solvents. Microbial synthesis of ACNs from renewable biomass, such as amino acids or flavonoids, is considered a sustainable and environmentally friendly method for large-scale production of ACNs. Recently, the construction of microbial cell factories (MCFs) for the efficient biosynthesis of ACNs and PACNs has attracted much attention. In this review, we summarize the cases of microbial synthesis of ACNs, and analyze the bottlenecks in reconstructing the metabolic pathways for synthesizing PACNs in microorganisms. Consequently, there is an urgent need to investigate the mechanisms behind the development of MCFs for PACNs synthesis. Such research also holds significant promise for advancing the production of food pigments. Meanwhile, we propose potential solutions to the bottleneck problem based on metabolic engineering and enzyme engineering. Finally, the development prospects of natural food and biotechnology are discussed.
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Microbial cell factories serve as pivotal platforms for the production of high-value natural products, which tend to accumulate on the cell membrane due to their hydrophobic properties. However, the limited space of the cell membrane presents a bottleneck for the accumulation of these products. To enhance the production of intracellular natural products and alleviate the burden on the cell membrane caused by product accumulation, researchers have implemented various membrane engineering strategies. These strategies involve modifying the membrane components and structures of microbial cell factories to achieve efficient accumulation of target products. This review summarizes recent advances in the application of membrane engineering technologies in microbial cell factories, providing case studies involving Escherichia coli and yeast. Through these strategies, researchers have not only improved the tolerance of cells but also optimized intracellular storage space, significantly enhancing the production efficiency of natural products. This article aims to provide scientific evidence and references for further enhancing the efficiency of similar cell factories.
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Membrana Celular , Escherichia coli , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Productos Biológicos/metabolismo , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.
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Glicerol , Levaduras , Glicerol/química , Ácidos Grasos/química , Carbono , BiocombustiblesRESUMEN
BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
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Edición Génica , Saccharomyces cerevisiae , Xilanos , Saccharomyces cerevisiae/metabolismo , Fermentación , Hidrólisis , Sistemas CRISPR-Cas , Etanol/metabolismo , Polímeros/metabolismo , Glucuronidasa , Xilosa/metabolismoRESUMEN
Class IIa bacteriocins produced in lactic acid bacteria are short cationic peptides with antimicrobial activity. In the search for new biopreservation agents, class IIa bacteriocins are considered to be the best potential candidates, not only due to their large abundance but also because of their high biological activity and excellent thermal stability. However, regulated by the biosynthetic regulatory system, the natural class IIa bacteriocin yield is low, and the extraction process is complicated. The biotechnological production of class IIa bacteriocins in various cell factories has been attempted to improve this situation. In this review, we focus on the application of biotechnological routes for class IIa bacteriocin production. The drawbacks and improvements in the production of class IIa bacteriocins in various cell factories are discussed. Furthermore, we present the main challenge of class IIa bacteriocins, focusing on increasing their production by constructing suitable cell factories. Recombinant bacteriocins have made considerable progress from inclusion body formation, dissolved form and low antibacterial activity to yield recovery. The development of prospective cell factories for the biotechnological production of bacteriocins is still required, which may facilitate the application of bacteriocins in the food industry.
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Bacteriocinas , Biotecnología , Bacteriocinas/biosíntesis , Biotecnología/métodos , Antibacterianos/biosíntesis , Antibacterianos/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Lactobacillales/metabolismoRESUMEN
Odd-chain fatty acids (OcFAs) and their derivatives have attracted great interest due to their wide applications in the food, pharmaceutical and petrochemical industries. Microorganisms can naturally de novo produce fatty acids (FAs), where mainly, even-chain with acetyl-CoA instead of odd-chain with propionyl-CoA is used as the primer. Usually, the absence of the precursor propionyl-CoA is considered the main reason that limits the efficient production of OcFAs. It is thus crucial to explore/evaluate/identify promising propionyl-CoA biosynthetic pathways to achieve large-scale biosynthesis of OcFAs. This review discusses the latest advances in microbial metabolism engineering toward producing propionyl-CoA and considers future research directions and challenges toward optimized production of OcFAs.
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Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.
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The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.
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Ingeniería Celular , Biología Sintética , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Procesamiento Proteico-Postraduccional , Ingeniería Metabólica , Mamíferos/metabolismoRESUMEN
BACKGROUND: Advanced DNA synthesis, biosensor assembly, and genetic circuit development in synthetic biology and metabolic engineering have reinforced the application of filamentous bacteria, yeasts, and fungi as promising chassis cells for chemical production, but their industrial application remains a major challenge that needs to be solved. RESULTS: As important chassis strains, filamentous microorganisms can synthesize important enzymes, chemicals, and niche pharmaceutical products through microbial fermentation. With the aid of metabolic engineering and synthetic biology, filamentous bacteria, yeasts, and fungi can be developed into efficient microbial cell factories through genome engineering, pathway engineering, tolerance engineering, and microbial engineering. Mutant screening and metabolic engineering can be used in filamentous bacteria, filamentous yeasts (Candida glabrata, Candida utilis), and filamentous fungi (Aspergillus sp., Rhizopus sp.) to greatly increase their capacity for chemical production. This review highlights the potential of using biotechnology to further develop filamentous bacteria, yeasts, and fungi as alternative chassis strains. CONCLUSIONS: In this review, we recapitulate the recent progress in the application of filamentous bacteria, yeasts, and fungi as microbial cell factories. Furthermore, emphasis on metabolic engineering strategies involved in cellular tolerance, metabolic engineering, and screening are discussed. Finally, we offer an outlook on advanced techniques for the engineering of filamentous bacteria, yeasts, and fungi.
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Hongos , Levaduras , Hongos/genética , Hongos/metabolismo , Levaduras/metabolismo , Biotecnología/métodos , Candida/genética , Ingeniería Metabólica/métodos , Bacterias/genética , Bacterias/metabolismo , Biología Sintética/métodosRESUMEN
Nutraceuticals are defined as food or food components with therapeutic capabilities that have few side effects and are regarded as a natural therapy for preventing the onset of several life-threatening illnesses. The use of microbial cell factories to produce nutraceuticals is considered to be sustainable and promising for meeting market demand. Among the diverse strategies for optimizing microbial cell factories, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has emerged as a valuable tool for gene integration, deletion, activation, and downregulation. With the advent of multiplexed and precise CRISPR strategies, optimized microbial cell factories are revolutionizing the yield of nutraceuticals. This review focuses on the development of highly adaptable CRISPR strategies to optimize the production in microbial cell factories of some important nutraceuticals (belonging to the class of carotenoids, flavonoids, stilbenoids, polysaccharides, and nonprotein amino acids). Further, we highlighted current challenges related to the efficiency of CRISPR strategies and addressed potential future directions to fully harness CRISPR strategies to make nutraceutical synthesis in microbial cell factories an industrially favorable method.
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Bioingeniería , Ingeniería Metabólica , Biología Sintética , Suplementos DietéticosRESUMEN
Filamentous fungi are able to produce a wide range of valuable proteins and enzymes for many industrial applications. Recent advances in fungal genomics and experimental technologies are rapidly changing the approaches for the development and use of filamentous fungi as hosts for the production of both homologous and heterologous proteins. In this review, we highlight the benefits and challenges of using filamentous fungi for the production of heterologous proteins. We review various techniques commonly employed to improve the heterologous protein production in filamentous fungi, such as strong and inducible promoters, codon optimization, more efficient signal peptides for secretion, carrier proteins, engineering of glycosylation sites, regulation of the unfolded protein response and endoplasmic reticulum associated protein degradation, optimization of the intracellular transport process, regulation of unconventional protein secretion, and construction of protease-deficient strains. KEY POINTS: ⢠This review updates the knowledge on heterologous protein production in filamentous fungi. ⢠Several fungal cell factories and potential candidates are discussed. ⢠Insights into improving heterologous gene expression are given.
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Proteínas Portadoras , Hongos , Hongos/genética , Hongos/metabolismo , Transporte de Proteínas , Proteínas Portadoras/genética , Señales de Clasificación de Proteína/genética , Codón/metabolismoRESUMEN
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose.
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Sistemas CRISPR-Cas , Saccharomyces cerevisiae , Sistemas CRISPR-Cas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Edición Génica/métodos , Biotecnología , BioingenieríaRESUMEN
High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-ß peptides (Aß42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aß42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
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Enfermedad de Alzheimer , Proteínas de Saccharomyces cerevisiae , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Humanos , Estrés Oxidativo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Microbial cell factories offer a promising strategy for the sustainable production of industrial chemicals from renewable biomass feedstock. However, their performance is often limited by poor microbial cell viability (MCV). Here, MCV was engineered to enhance chemical production by optimizing the regulation of lifespan-specific genes to reduce the accumulation of reactive oxygen species (ROS). In Escherichia coli, MCV was improved by reducing ROS accumulation using second codon engineering to regulate hypoxia-inducible transcription factor (arcA), resulting in lysine production up to 213 g L-1 with its productivity 5.90 g L-1·h-1. In Saccharomyces cerevisiae, MCV was increased by decreasing ROS accumulation using second codon engineering to fine-tune ceramide synthase (lag1), leading to glucaric acid production up to 9.50 g L-1 with its productivity 0.057 g L-1·h-1. These results demonstrate that engineering MCV is a potential strategy to boost the performance of microbial cell factories in industrial processes.