Advances In Metabolic Engineering Research Articles

Reconstitution of the Mevalonate Pathway for Improvement of Isoprenoid Production and Industrial Applicability in Escherichia coli.

Natural products, especially isoprenoids have many industrial applications, including medicine, fragrances, food additives, personal care and cosmetics, colorants, and even advanced biofuels. Recent advancements in metabolic engineering with synthetic biology and systems biology have drawn increased interest in microbial-based isoprenoid production. In order to engineer microorganisms to produce a large amount of value-added isoprenoids, great efforts have been made by employing various strategies from synthetic biology and systems biology. We also have engineered E. coli to produce various isoprenoids by targeting and engineering the isoprenoid biosynthetic pathways, methylerythritol phosphate (MEP), and mevalonate (MVA) pathways. Here, we introduced new combinations of the MVA pathway in E. coli with genes from biosafety level 1 (BSL 1) organisms. The reconstituted MVA pathway constructs (pSCS) are not only preferred to the living modified organism (LMO) regulation, but they also improved carotenoid production. In addition, the pSCS constructs resulted in enhanced lycopene production and cell-specific productivity compared to the previous MVA pathway combination (pSNA) in fed-batch fermentation. The pSCS constructs would not only bring an increase in isoprenoid production in E. coli, but they could be an efficient system to be applied for the industrial production of isoprenoids with industry-preferred genetic combinations.

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for itsstrong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application inmedicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longercompared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged asa promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use ofthe natural host Xanthophyllomyces dendrorhous, andthe heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

From pollutants to products: Microbial cell factories driving sustainable biomanufacturing and environmental conservation

Microbial cell factories are emerging as powerful tools in addressing pressing environmental challenges and promoting sustainable biomanufacturing. This paper highlights the key role of engineered microorganisms in mitigating pollution, converting waste and pollutants into valuable products, and reducing reliance on fossil fuels. Through advancements in metabolic engineering, gene editing technologies, and synthetic biology, microbial cell factories are being optimized to enhance their efficiency in breaking down pollutants and producing renewable chemicals, such as biofuels, bioplastics, and specialty chemicals. These processes contribute to environmental conservation, waste valorization, and the establishment of a circular economy.The study focuses on overcoming key barriers in microbial biotechnology, such as limited scalability, process inefficiency, and economic viability, by employing strategies like metabolic pathway optimization, enzyme overexpression, and tolerance enhancement. These strategies are applied to various microbial species, demonstrating how their metabolic capabilities can be fine-tuned for industrial applications. Detailed case studies illustrate successful implementations, such as the conversion of lignocellulosic biomass, CO2, and industrial waste into high-value products, underscoring the practical impact of microbial cell factories in diverse sectors, including energy, materials, and chemicals.Furthermore, this research addresses the challenges faced by microbial cell factories in industrial-scale operations, such as maintaining genetic stability and optimizing growth conditions, and offers insight into emerging technological solutions to these obstacles. By providing a comprehensive overview of recent developments and identifying future research directions, this paper offers actionable recommendations for unlocking the full potential of microbial biotechnology. These efforts aim to further integrate microbial processes into industrial systems, contributing to a more sustainable and resilient global economy, with the ultimate goal of fostering a circular bioeconomy.

Recent advances in metabolic engineering of microorganisms for production of tyrosol and its derivatives

Tyrosol is a natural phenolic compound with antioxidant, anti-inflammatory and other biological activities, serving as an important precursor of high-value products such as hydroxytyrosol and salidroside. Therefore, the green and efficient biosynthesis of tyrosol and its derivatives has become a research hotspot in recent years. Building cell factories by metabolic engineering of microorganisms is a potential industrial production way, which has low costs and environmental friendliness. This paper introduces the biosynthesis pathway of tyrosol and presents the key regulated nodes in the de novo synthesis of tyrosol in Escherichia coli and Saccharomyces cerevisiae. In addition, this paper reviews the recent advances in metabolic engineering for the production of hydroxytyrosol and salidroside. This review can provide a reference for engineering the strains for the high-yield production of tyrosol and its derivatives.

Recent Advances in Metabolic Engineering for the Biosynthesis of Phosphoenol Pyruvate–Oxaloacetate–Pyruvate-Derived Amino Acids

The phosphoenol pyruvate–oxaloacetate–pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol pyruvate–oxaloacetate–pyruvate (POP) node is the switch point among the major metabolic pathways existing in most living organisms. POP-AAs have widespread applications in the nutrition, food, and pharmaceutical industries. These amino acids have been predominantly produced in Escherichia coli and Corynebacterium glutamicum through microbial fermentation. With the rapid increase in market requirements, along with the global food shortage situation, the industrial production capacity of these two bacteria has encountered two bottlenecks: low product conversion efficiency and high cost of raw materials. Aiming to push forward the update and upgrade of engineered strains with higher yield and productivity, this paper presents a comprehensive summarization of the fundamental strategy of metabolic engineering techniques around phosphoenol pyruvate–oxaloacetate–pyruvate node for POP-AA production, including L-tryptophan, L-tyrosine, L-phenylalanine, L-valine, L-lysine, L-threonine, and L-isoleucine. Novel heterologous routes and regulation methods regarding the carbon flux redistribution in the POP node and the formation of amino acids should be taken into consideration to improve POP-AA production to approach maximum theoretical values. Furthermore, an outlook for future strategies of low-cost feedstock and energy utilization for developing amino acid overproducers is proposed.

Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica.

Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.

Metabolic Engineering of Corynebacterium glutamicum for the Production of Flavonoids and Stilbenoids.

Flavonoids and stilbenoids, crucial secondary metabolites abundant in plants and fungi, display diverse biological and pharmaceutical activities, including potent antioxidant, anti-inflammatory, and antimicrobial effects. However, conventional production methods, such as chemical synthesis and plant extraction, face challenges in sustainability and yield. Hence, there is a notable shift towards biological production using microorganisms like Escherichia coli and yeast. Yet, the drawbacks of using E. coli and yeast as hosts for these compounds persist. For instance, yeast's complex glycosylation profile can lead to intricate protein production scenarios, including hyperglycosylation issues. Consequently, Corynebacterium glutamicum emerges as a promising alternative, given its adaptability and recent advances in metabolic engineering. Although extensively used in biotechnological applications, the potential production of flavonoid and stilbenoid in engineered C. glutamicum remains largely untapped compared to E. coli. This review explores the potential of metabolic engineering in C. glutamicum for biosynthesis, highlighting its versatility as a cell factory and assessing optimization strategies for these pathways. Additionally, various metabolic engineering methods, including genomic editing and biosensors, and cofactor regeneration are evaluated, with a focus on C. glutamicum. Through comprehensive discussion, the review offers insights into future perspectives in production, aiding researchers and industry professionals in the field.

Vibrio natriegens: Application of a Fast-Growing Halophilic Bacterium.

The fast growth accompanied with high substrate consumption rates and a versatile metabolism paved the way to exploit Vibrio natriegens as unconventional host for biotechnological applications. Meanwhile, a wealth of knowledge on the physiology, the metabolism, and the regulation in this halophilic marine bacterium has been gathered. Sophisticated genetic engineering tools and metabolic models are available and have been applied to engineer production strains and first chassis variants of V. natriegens. In this review, we update the current knowledge on the physiology and the progress in the development of synthetic biology tools and provide an overview of recent advances in metabolic engineering of this promising host. We further discuss future challenges to enhance the application range of V. natriegens.

Progresses and challenges of engineering thermophilic acetogenic cell factories.

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.

Engineered microbial consortia for next-generation feedstocks

Addressing urgent environmental challenges, this commentary emphasizes the need for green, bio-based solutions in chemical production from renewable feedstocks. It highlights advanced metabolic engineering of microbial strains and the use of microbial consortia as innovative approaches for efficient resource recovery. These strategies aim to enhance the conversion of diverse renewable feedstocks, including agricultural residues, industrial by-products, and greenhouse gases, into value-added chemicals. This article discusses cutting-edge techniques in renewable feedstock upcycling, utilizing both engineered unicellular and multicellular systems. It advocates a paradigm shift in sustainable biomanufacturing, focusing on transforming renewable resources into value-added products. This approach is crucial for developing a circular bioeconomy, aligning with global efforts to mitigate environmental impacts.

Advances in Metabolic Engineering of Pichia pastoris Strains as Powerful Cell Factories.

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.

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids.

Monoterpene indole alkaloids (MIAs) encompass a diverse family of over 3000 plant natural products with a wide range of medical applications. Further utilizations of these compounds, however, are hampered due to low levels of abundance in their natural sources, causing difficult isolation and complex multi-steps in uneconomical chemical syntheses. Metabolic engineering of MIA biosynthesis in heterologous hosts is attractive, particularly for increasing the yield of natural products of interest and expanding their chemical diversity. Here, we review recent advances and strategies which have been adopted to engineer microbial and plant systems for the purpose of generating MIAs and discuss the current issues and future developments of manufacturing MIAs by synthetic biology approaches.

EPA and DHA in microalgae: Health benefits, biosynthesis, and metabolic engineering advances

AbstractEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω‐3 very long‐chain polyunsaturated fatty acids (VLC‐PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti‐inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini‐review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC‐PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.

Sooting tendency of isopropanol-butanol-ethanol (IBE)/diesel surrogate blends in laminar diffusion flames

Recent advancements in metabolic engineering have demonstrated the cost-effective production of isopropanol-butanol-ethanol (IBE) from switchgrass biomass. As such, IBE is seen as a viable future fuel to transition the transportation sector towards renewable biofuels. In this study, an atmospheric co-flow diffusion flame burner was used to examine the sooting tendency of IBE when mixed with a diesel surrogate for blend ratios varying from 0% to 100% by volume. Experimental measurements of soot volume fraction were obtained by implementing a color-ratio pyrometry technique and reporting the variation of sooting tendency in terms of the Yield Sooting Index (YSI). Measurements demonstrated soot formation and hence YSI decreased nonlinearly with increasing IBE content. To gain further insight into the kinetics affecting soot formation, numerical modeling studies were carried out using laminarSMOKE++ and a comprehensive kinetic mechanism with soot chemistry. From a reaction path and rate of production analysis, IBE demonstrated that it can perturb OH and HO2 radical pools and enhance CO and CO2 reactive channels from IBE oxygenated intermediates to suppress the formation of soot precursors. Studies were also extended to examine the role of CO2 dilution on soot formation. It was shown that further soot reduction can be attained by lowering the rate of overall soot formation kinetics, perturbing reaction intermediates leading to soot formation by the depletion of H-atom radical pool, while enhancing the oxidation through enhanced OH-radical production.

Biotechnology in Future Food Lipids: Opportunities and Challenges.

Lipids are a large group of essential nutrients in daily diets that provide energy and maintain various physiological functions. As the global population is rapidly expanding, there is an urgent need to enhance the production and quality of food lipids. The development of modern biotechnology allows the manipulation of oil production in plants and microorganisms and the improvement of the nutritional value of food lipids. Various metabolic engineering strategies have been exploited to increase oil production and produce value-added oils in traditional oil crops and other novel lipid sources (e.g., plant vegetative tissues, microalgae, and oleaginous microorganisms). Furthermore, natural lipid structures can be modified by lipases to prepare functional lipids, e.g., diacylglycerols, medium-long-medium-type structured triacylglycerols, human milk-fat substitutes, and structuralphospholipids, for specific nutritional demands. In this review, we focus on the recent advances in metabolic engineering of lipid production in plants and microorganisms, and the preparation of functional lipids via biocatalysis.

Recent Advances in Yeast Recombinant Biosynthesis of the Triterpenoid Protopanaxadiol and Glycosylated Derivatives Thereof.

Plant natural products are a seemingly endless resource for novel chemical structures. However, their extraction often results in high prices, fluctuation in both quantity and quality, and negative environmental impact. The latter might result from the extraction procedure but more often from the high amount of plant biomass required. With the advent of synthetic biology, producing natural plant products in large quantities using yeasts as hosts has become possible. Here, we focus on the recent advances in metabolic engineering of the yeasts species Saccharomyces cerevisiae and Yarrowia lipolytica for the synthesis of ginsenoside triterpenoids, namely, dammarenediol-II, protopanaxadiol, protopanaxatriol, compound K, ginsenoside Rh1, ginsenoside Rh2, ginsenoside Rg3, and ginsenoside F1. A discussion is provided on advanced synthetic biology, bioprocess strategies, and current challenges for the biosynthesis of ginsenoside triterpenoids. Finally, future directions in metabolic and process engineering are summarized and may help reify sustainable ginsenoside production.

Metabolic Engineering: Methodologies and Applications.

Metabolic engineering aims to improve the production of economically valuable molecules through the genetic manipulation of microbial metabolism. While the discipline is a little over 30 years old, advancements in metabolic engineering have given way to industrial-level molecule production benefitting multiple industries such as chemical, agriculture, food, pharmaceutical, and energy industries. This review describes the design, build, test, and learn steps necessary for leading a successful metabolic engineering campaign. Moreover, we highlight major applications of metabolic engineering, including synthesizing chemicals and fuels, broadening substrate utilization, and improving host robustness with a focus on specific case studies. Finally, we conclude with a discussion on perspectives and future challenges related to metabolic engineering.

Coenzyme self-sufficiency system-recent advances in microbial production of high-value chemical phenyllactic acid.

Phenyllactic acid (PLA), a natural antimicrobial substance, has many potential applications in the food, animal feed, pharmaceutical and cosmetic industries. However, its production is limited by the complex reaction steps involved in its chemical synthesis. Through advances in metabolic engineering and synthetic biology strategies, enzymatic or whole-cell catalysis was developed as an alternative method for PLA production. Herein, we review recent developments in metabolic engineering and synthetic biology strategies that promote the microbial production of high-value PLA. Specially, the advantages and disadvantages of the using of the three kinds of substrates, which includes phenylpyruvate, phenylalanine and glucose as starting materials by natural or engineered microbes is summarized. Notably, the bio-conversion of PLA often requires the consumption of expensive coenzyme NADH. To overcome the issues of NADH regeneration, efficiently internal cofactor regeneration systems constructed by co-expressing different enzyme combinations composed of lactate dehydrogenase with others for enhancing the PLA production, as well as their possible improvements, are discussed. In particular, the construction of fusion proteins with different linkers can achieve higher PLA yield and more efficient cofactor regeneration than that of multi-enzyme co-expression. Overall, this review provides a comprehensive overview of PLA biosynthesis pathways and strategies for increasing PLA yield through biotechnology, providing future directions for the large-scale commercial production of PLA and the expansion of downstream applications.

Utilizing Alcohol for Alkane Biosynthesis by Introducing a Fatty Alcohol Dehydrogenase.

Alkanes produced by microorganisms are expected to be an alternative to fossil fuels as an energy source. Microbial synthesis of alkanes involves the formation of fatty aldehydes via fatty acyl coenzyme A (acyl-CoA) intermediates derived from fatty acid metabolism, followed by aldehyde decarbonylation to generate alkanes. Advancements in metabolic engineering have enabled the construction of such pathways in various microorganisms, including Escherichia coli. However, endogenous aldehyde reductases in the host microorganisms are highly active in converting fatty aldehydes to fatty alcohols, limiting the substrate pool for alkane production. To reuse the alcohol by-product, a screening of fatty alcohol-assimilating microorganisms was conducted, and a bacterial strain, Pantoea sp. strain 7-4, was found to convert 1-tetradecanol to tetradecanal. From this strain, an alcohol dehydrogenase, PsADH, was purified and found to be involved in 1-tetradecanol-oxidizing reaction. Subsequent heterologous expression of the PsADH gene in E. coli was conducted, and recombinant PsADH was purified for a series of biochemical characterizations, including cofactors, optimal reaction conditions, and kinetic parameters. Furthermore, direct alkane production from alcohol was achieved in E. coli by coexpressing PsADH with a cyanobacterial aldehyde-deformylating oxygenase and a reducing system, including ferredoxin and ferredoxin reductase, from Nostoc punctiforme PCC73102. The alcohol-aldehyde-alkane synthetic route established in this study will provide a new approach to utilizing fatty alcohols for the production of alkane biofuel. IMPORTANCE Alcohol dehydrogenases are a group of enzymes found in many organisms. Unfortunately, studies on these enzymes mainly focus on their activities toward short-chain alcohols. In this study, we discovered an alcohol dehydrogenase, PsADH, from the bacterium Pantoea sp. 7-4, which can oxidize 1-tetradecanol to tetradecanal. The medium-chain aldehyde products generated by this enzyme can serve as the substrate of aldehyde-deformylating oxygenase to produce alkanes. The enzyme found in this study can be applied to the biosynthetic pathway involving the formation of medium-chain aldehydes to produce alkanes and other valuable compounds.

Antioxidant Potential and Capacity of Microorganism-Sourced C30 Carotenoids-A Review.

Carotenoids are lipophilic tetraterpenoid pigments produced by plants, algae, arthropods, and certain bacteria and fungi. These biologically active compounds are used in the food, feed, and nutraceutical industries for their coloring and the physiological benefits imparted by their antioxidant properties. The current global carotenoid market is dominated by synthetic carotenoids; however, the rising consumer demand for natural products has led to increasing research and development in the mass production of carotenoids from alternative natural sources, including microbial synthesis and plant extraction, which holds a significant market share. To date, microbial research has focused on C40 carotenoids, but studies have shown that C30 carotenoids contain similar—and in some microbial strains, greater—antioxidant activity in both the physical and chemical quenching of reactive oxygen species. The discovery of carotenoid biosynthetic pathways in different microorganisms and advances in metabolic engineering are driving the discovery of novel C30 carotenoid compounds. This review highlights the C30 carotenoids from microbial sources, showcasing their antioxidant properties and the technologies emerging for their enhanced production. Industrial applications and tactics, as well as biotechnological strategies for their optimized synthesis, are also discussed.