Abstract:Microbial chemical factories utilize engineering design principles to re-build natural production pathways, enabling the precise, quantitative, and efficient synthesis of chemicals. This is achieved through the optimization of synthetic pathways, the reconstruction of biochemical networks, the development of novel components, and the integration of pathways with cellular and environmental contexts. As a transformative approach to chemical production, microbial chemical factories play a critical role in establishing renewable raw material pathways for industrial economic development and advancing sustainable growth. This innovative model has become a strategic priority for technological advancement and industrial competitiveness in developed nations. This industry has expanded into diverse sectors, including pharmaceuticals, food production, chemicals, and energy. To showcase the most recent scientific advancements in the field of microbial chemical production and to promote the evolution of the bio-manufacturing industry, we organized a special issue entitled “Microbial Chemical Factories”. This edition features the latest research conducted by domestic scientists, focusing on areas such as the synthesis of material monomers, pharmaceutical intermediates, functional food ingredients, organic acid biosynthesis, and the development and utilization of non-food feedstock. It provides reference and guidance for the further development of microbial chemical factories.

Abstract:1,3-propanediol (1,3-PDO) is an important diol with wide applications in the pharmaceutical, food, and cosmetics industries. In addition, 1,3-PDO serves as a crucial monomer in the synthesis of polytrimethylene terephthalate, an important synthetic fiber material. Microbial conversion of renewable resources such as glucose into 1,3-PDO has been industrialized due to its environmentally friendly, energy-efficient, safe, and sustainable characteristics. It serves as a successful case in the design and application of microbial cell factories for biochemicals. However, concerns such as food scarcity and climate change are driving the exploration of non-food, low-cost, and sustainable alternatives as biomanufacturing feedstocks. The biosynthesis of 1,3-PDO from the C3 feedstock glycerol by microorganisms has been well studied. In recent years, increasing attention has been paid to the synthesis of 1,3-PDO from C1 feedstocks such as methanol, which has higher energy density than glucose and glycerol. Several new artificial biosynthetic pathways have been proposed and validated, laying a foundation for the sustainable bioproduction of 1,3-PDO. This article reviews the feedstock transition from C6 to C3 and C1 carbon sources for the microbial synthesis of 1,3-PDO and discusses the strategies for reprogramming metabolic pathway to enhance 1,3-PDO biosynthesis from different feedstocks. Finally, the development prospects of 1,3-PDO bioproduction from C1 feedstocks are forecasted.

Abstract:1,3-propanediol is an important monomer for the production of polytrimethylene terephthalate (PTT). Currently, it is mainly produced by microbial fermentation, which, however, has low production efficiency. To address this problem, this study employed atmospheric room temperature plasma (ARTP) mutagenesis technology and high-throughput screening to obtain a strain with high tolerance to osmotic pressure, which achieved a 1,3-propanediol titer of 87 g/L. Furthermore, the gene expression elements suitable for Klebsiella pneumoniae were screened, and metabolic engineering was employed to block redundant metabolic pathways (deletion of ldhA, budA, and aldA) and enhance the synthesis pathway (overexpression of dhaB and yqhD). The titer of 1,3-propanediol produced by the engineered strain increased to 107 g/L. Finally, in a 5 L fermenter, the optimal strain KP-FMME-6 achieved a 1,3-propanediol titer of 118 g/L, with a glycerol conversion rate of 42% and productivity of 2.46 g/(h·L), after optimization of the fermentation parameters. This study provides a reference for the industrial production of 1,3-propanediol.

Abstract:Cadaverine is a fundamental C5 building block in the production of polyamides. Due to the limited regeneration efficiency of intracellular pyridoxal 5′-phosphate (PLP), the current fermentation-based production of cadaverine exhibits low efficiency. In this study, we developed an Escherichia coli strain L01 by introducing lysine decarboxylase (lysine decarboxylase, LDC, a key enzyme in the synthesis of cadaverine) into a lysine-producing strain E. coli LY-4, achieving a cadaverine tier of 1.07 g/L in shake flask fermentation. Subsequently, a dual metabolic pathway enhancement strategy was proposed to synergistically strengthen both endogenous and exogenous PLP synthesis modules, thereby improving intracellular PLP synthesis. The optimized strain L11 achieved a cadaverine titer of 9.23 g/L in shake flask fermentation. Finally, the fermentation process for cadaverine production by strain L11 was optimized in a 5 L fermenter. After 48 h of fed-batch fermentation, the engineered strain L11 achieved the cadaverine titer, yield, and productivity of 54.43 g/L, 0.22 g/g, and 1.13 g/(L·h), respectively. This study provides a theoretical and technical foundation for establishing microbial cell factories for bioamine production.

Abstract:Glycolic acid is an important chemical product widely used in various fields, including cosmetics, detergents, textiles, and more. Currently, microbial production of glycolic acid has disadvantages such as poor genetic stability, low yield, and high cost. Additionally, whole-cell catalytic production of glycolic acid typically requires the addition of relatively expensive sorbitol as a carbon source, which limits its industrial production. To develop an industrially applicable method for glycolic acid production, this study used ethylene glycol as a substrate to screen the glycolic acid-producing strains through whole-cell catalysis, obtaining a Rhodotorula sp. capable of producing glycolic acid. The strain was then subjected to UV mutagenesis and high throughput screening, and the positive mutant strain RMGly-20 was obtained. After optimization in shake flasks, the glycolic acid titer of RMGly-20 reached 17.8 g/L, a 10.1-fold increase compared to the original strain. Using glucose as the carbon source and employing a fed-batch culture in a 5 L fermenter, strain RMGly-20 produced 61.1 g/L of the glycolic acid. This achievement marks the preliminary breeding of a genetically stable glycolic acid-producing strain using a cheap carbon source, providing a new host for the biosynthesis of glycolic acid and promoting further progress toward industrial production.

Abstract:Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E. coli. Firstly, deoA, tdk, udp, rihA, rihB, and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E. coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5′-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Abstract:Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, EcTnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, MaFMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM-IND01 was obtained. The beneficial mutant MaFMO^D197E was obtained by protein engineering of the rate-limiting enzyme MaFMO. MaFMO^D197E showed the specific activity and k_cat/K_m value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, MaFMO^D197E was introduced into the strain EM-IND01 to construct the strain EM-IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased MaFMO activity in this study, which laid a foundation for industrial production of indigo.

Abstract:Arbutin, a glycosylated compound of hydroquinone, exists in two forms of β-arbutin and α-arbutin based on the configuration of the glycosidic bond. As a safe and stable whitening agent, arbutin is widely used in cosmetics, and it has antioxidant, antimicrobial, anti-inflammatory, and anti-tumor activities. The production of arbutin by plant extraction faces challenges such as long plant growth periods, complex extraction processes, and low yields. The chemical synthesis of arbutin suffers from harsh reaction conditions, poor stereo-selectivity, and low yields. In recent years, biosynthesis emerges as the most popular method to produce arbutin because of the simple and mild reaction conditions, low costs, and environmental friendliness. This review summarizes the research progress in four biosynthetic strategies for arbutin, including plant conversion, enzyme catalysis, whole-cell catalysis, and microbial fermentation. The advantages and limitations of these biosynthetic strategies are discussed, and future research directions are proposed.

Abstract:Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Abstract:With the rapid development of the medical beauty industry, functional skin care products become increasingly popular. The functions of cosmetics mainly depend on the active ingredients, which are mainly proteins, peptides, polysaccharides, phenolic acids, terpenes, vitamins, and amino acids. These active ingredients endow cosmetics with skin repairing, moistening, whitening, UV protecting, and anti-aging effects. They are mainly obtained through biological extraction and chemical synthesis. In recent years, with the development of biomanufacturing, microbial synthesis of active ingredients in cosmetics has been widely studied and applied. This article reviews the research progresses in the production of natural products including collagens, peptides, hyaluronic acid, polyphenols, terpenes, and vitamins by microbial synthetic biotechnology. Moreover, this article highlighted the synthetic pathways, metabolic regulation, and prospects of the natural products, providing a reference for subsequent microbial synthesis of active ingredients in cosmetics.

Abstract:L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Abstract:Vitamins are a class of organic substances essential for maintaining the normal physiological function of organisms. Most vitamins cannot be synthesized by the human body, and a small number of vitamins can only be synthesized in a limited manner, which cannot meet the body needs. Therefore, people need to take food or drugs containing vitamins to meet the body needs. Nowadays, vitamins are widely used in medicine, food or feed additives, cosmetics and other industries, and the demand for vitamins is growing. Vitamins are mainly produced by chemical synthesis and biosynthesis. Compared with chemical synthesis, biosynthesis of vitamins is praised for the environmental friendliness, high safety, and low costs. Therefore, it is of great practical significance to study the biosynthesis methods of vitamins. This paper reviews the research progress in the methods and summarizes the research results in the biosynthesis of water-soluble vitamins (B vitamins and vitamin C) in recent years and then makes an outlook on the future development in this field.

Abstract:Vitamins are the essential organic substances to ensure the normal life activities of the human body. At present, vitamins are widely used in the pharmaceutical, food, animal farming, beauty and other industries, appearing in increasing application scenarios. Accordingly, the global demand for vitamins has also increased greatly. The current methods of vitamin production mainly include chemical synthesis and biosynthesis, with the latter being greener, more environmentally friendly, safer, and lower in energy consumption. Establishing the method for the biosynthesis of vitamins is of great scientific significance for achieving the goals of low carbon, energy saving, and emission reduction, as well as carbon emission peak and carbon neutrality in China. This paper reviews the research progress in the biosynthesis of vitamins, especially fat-soluble vitamins (vitamins A, D, E, and K), in recent years.

Abstract:Vitamins, as indispensable organic compounds in life activities, demonstrate a complex and refined metabolic network in organisms. This network involves the coordination of multiple enzymes and the integration of various metabolic pathways. Despite the achievements in metabolic engineering and catalytic mechanism research, the lack of studies regarding detailed enzymatic properties for a large number of key enzymes limits the enhancement of vitamin production efficiency and hinders the in-depth understanding and optimization of vitamin synthesis mechanisms. Such limitations not only restrict the industrial application of vitamins but also impede the development of related bio-technologies. This study comprehensively reviews the research progress in the enzymes involved in vitamin biosynthesis and details the current status of research on the enzymes of 13 vitamin synthesis pathways, including their catalytic mechanisms, kinetic properties, and applications in biology. In addition, this study compares the properties of enzymes involved in vitamin metabolic pathways and the glycolysis pathway, and reveals the characteristics of catalytic efficiency and substrate affinity of enzymes in vitamin synthesis pathways. Furthermore, this study discusses the potential and prospects of applying deep learning methods to the research on properties of enzymes associated with vitamin biosynthesis, giving new insights into the production and optimization of vitamins.

Abstract: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.

Abstract:d-mannitol is a six-carbon sugar alcohol and one of the most abundant polyols in the nature. With antioxidant and osmotic pressure-regulating effects and non-metabolism by the human body, d-mannitol has been widely used in functional food and pharmaceutical industries. At present, a major way for industrial production of d-mannitol is chemical hydrogenation. In addition, d-Mannitol can be produced by microbial metabolism or catalysis. Compared with the chemical hydrogenation, the microbial methods for synthesizing mannitol do not produce sorbitol as a by-product and have the advantages of mild reaction conditions, strong specificity, and high conversion rate. Microbial fermentation is praised for easy access of strains and raw materials and simple separation of the product. Microbial catalysis usually adopts a multi-enzyme coupling strategy, which uses enzymes produced by engineered bacteria for whole-cell catalysis, and the cofactor recycling pathway is introduced to replenish expensive cofactor. This method can achieve high yields with cheap substrates under mild conditions without the formation of by-products. However, the application of microbial methods in the industrial production of d-mannitol is limited by the high costs of fermentation media and substrates and the long reaction time. This article reviews the reported microbial methods for producing d-mannitol, including the use of high-yielding strains and their fermentation processes, the utilization of low-cost substrates, whole-cell catalytic strategies, and the process control for high productivity. The biosynthesis of mannitol is not only of great significance for promoting industrial upgrading and realizing green manufacturing, but also provides strong support for the development of new bio-based products to meet the growing market demand. With the continuous improvement of technological innovation and industrial chain, it is expected to become one of the main ways of mannitol production in the future.

Abstract:Succinic acid is an important C4 platform compound that serves as a raw material for the production of 1,4-butanediol, tetrahydrofuran, and biodegradable plastics such as polybutylene succinate (PBS). Compared to the traditional petrochemical-based route that uses maleic anhydride as a raw material, the microbial fermentation method for producing succinic acid offers more sustainable economic value and environmental friendliness. Yeasts with good acid tolerance can achieve low-pH fermentation of succinic acid, significantly reducing the cost of product extraction. Therefore, constructing high-yield succinic acid yeast strains through metabolic engineering has garnered increasing attention. This paper systematically introduced the application value and market size of succinic acid, summarized the pathways and key enzymes involved in succinic acid synthesis in microorganisms, and elaborated on the latest research progress in the synthesis of succinic acid using yeast cell factories. It also presented the current status of succinic acid synthesis using non-food raw materials such as glycerol, acetic acid, lignocellulosic hydrolysate, and others as substrates by engineered yeast strains. Finally, the paper provided a prospect for low-pH succinic acid biomanufacturing based on yeast cell factories.

Abstract:Itaconic acid (IA) is one of the twelve high value-added platform compounds applied in various fields including coatings, adhesives, plastics, resins, and biofuels. In this study, we established a one-pot catalytic synthesis system for IA from citric acid based on the engineered salt-tolerant bacterial strain Halomonas bluephagenesis TDZI-08 after investigating factors that hindered the process and optimizing the carbon source, nitrogen source, inducer addition time, and surfactant dosage. The open, non-sterile, one-pot synthesis with TDZI-08 in a 5 L fermenter achieved the highest IA titer of 40.50 g/L, with a catalytic yield of 0.68 g IA/g citric acid during the catalytic stage and a total yield of 0.42 g IA/g (citric acid+gluconic acid). The one-pot synthesis system established in this study is simple and does not need sterilization or aseptic operations. The findings indicate the potential of H. bluephagenesis for industrial production of IA.

Abstract:Propionic acid as an important C3 platform chemical has been widely used in food, pharmaceutical, and chemical fields. The chemical synthesis of propionic acid from petroleum and other chemical products has serious environmental pollution and is not sustainable. In recent years, the production of propionic acid by microbial transformation of renewable resources has received extensive attention. Focusing on the biomanufacturing of propionic acid, this paper firstly reviews the studies about the metabolic engineering of Propionibacterium and the pathway reconstruction in heterogeneous hosts such as Escherichia coli and Saccharomyces cerevisiae. Secondly, this paper reviews the recent progress in the synthesis of high-purity propionic acid from L-threonine or bio-based 1,2-propanediol by the design and modification of the pathway of Pseudomonas putida KT2440 based on synthetic biology.

Abstract:Lignocellulose is the most abundant renewable resource on earth. Constructing microbial cell factories for synthesizing value-added chemicals with lignocellulose is the key to realize green biomanufacturing. Xylose is the second most fermentable sugar in lignocellulose after glucose. Building microbial cell factories that can efficiently metabolize xylose is of great significance to achieve full utilization of lignocellulose. However, the lower metabolism efficiency of xylose than that of glucose in most microorganisms limits the application of xylose. In recent years, the deepening understanding of microbial metabolic mechanisms and the continuous advancement of synthetic biology have greatly improved the efficiency of microbial metabolism of xylose and expanded the spectrum of xylose-derived products. This article introduces several xylose metabolic pathways that exist in the nature and the derived products, summarizes the strategies for constructing recombinant strains that can co-utilize xylose and glucose, and reviews the research progress in the application of lignocellulose hydrolysates in the synthesis of target products. Finally, this article discusses the current technical bottlenecks and prospects the future development directions in this field.

Abstract:Microbial production of chemicals from renewable biomass has emerged as a crucial route for sustainable bio-manufacturing. Lignocellulose with a renewable property and wide sources is supposed to be a promising feedstock for the second-generation biorefinery. The efficient co-utilization of mixed sugars from lignocellulosic hydrolysates represents one of the key challenges in reducing the production cost. However, most microorganisms prefer glucose over xylose due to carbon catabolite repression, which constrains the efficiency of lignocellulosic conversion. Therefore, developing the microbial platforms capable of simultaneously utilizing glucose and xylose is paramount for economically viable industrial-scale production. This article reviews the key strategies and studies of metabolic engineering for promoting efficient co-utilization of glucose and xylose by microorganisms. The representative strategies include relieving glucose repression, enhancing xylose transport, constructing xylose metabolic pathways, and directed evolution.

Abstract:The construction and optimization of microbial cell factories are crucial steps and key technologies in achieving green biomanufacturing. As concern has been aroused regarding the excessive carbon dioxide (CO₂) emissions and food security, a new and promising research field, microbial conversion of CO₂ into food compounds, has emerged. The research in this field not only holds significant implications for achieving the carbon peaking and carbon neutrality goals but also plays a role in maintaining food security. This paper provides a comprehensive review and outlook of the research on utilizing CO₂ and its derived low-carbon chemicals for the production of food compounds, focusing on the production of glucose, sugar derivatives, and single-cell proteins and the development of artificial CO₂ fixation pathways.

Abstract:Methanol has been considered one of the most important alternative carbon sources for the next-generation biomanufacturing due to its low price, mature production processes, and potential sustainability. Constructing microbial cell factories for methanol to chemical biotransformation has become a research hotspot in the green biomanufacturing industry. Focusing on the microorganisms that can naturally use methanol, we compare them with non-natural cell factories for chemical production from methanol. We discuss the key issues and challenges associated with natural cell factories for chemical production from methanol, summarize recent research progress surrounding these issues, and propose possible solutions to these challenges. This review helps to generate feasible guidelines and research strategies for the modification of natural cell factories for efficient methanol to chemical production in the future.