Biodegradation: the best solution to the world problem of discarded polymers.

The widespread use of polymers has made our lives increasingly convenient by offering a more convenient and dependable material. However, the challenge of efficiently decomposing these materials has resulted in a surge of polymer waste, posing environment and health risk. Currently, landfill and incineration treatment approaches have notable shortcomings, prompting a shift towards more eco-friendly and sustainable biodegradation approaches. Biodegradation primarily relies on microorganisms, with research focusing on both solitary bacterial strain and multi-strain communities for polymer biodegradation. Furthermore, directed evolution and rational design of enzyme have significantly contributed to the polymer biodegradation process. However, previous reviews often undervaluing the role of multi-strain communities. In this review, we assess the current state of these three significant fields of research, provide practical solutions to issues with polymer biodegradation, and outline potential future directions for the subject. Ultimately, biodegradation, whether facilitated by single bacteria, multi-strain communities, or engineered enzymes, now represents the most effective method for managing waste polymers.

[Research progress in biosynthesis of arbutin].

Arbutin, a glycosylated compound of hydroquinone, exists in two forms of ß-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.

[Construction of microbial cell factories for synthesizing value-added chemicals with xylose].

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.

Microbial synthesis of gallic acid and its glucoside ß-glucogallin.

Gallic acid (GA) and ß-glucogallin (BGG) are natural products with diverse uses in pharmaceutical, food, chemical and cosmetic industries. They are valued for their wide-ranging properties such as antioxidant, antibacterial, antidiabetic, and anticancer properties. Despite their significant importance, microbial production of GA and BGG faces challenges such as limited titers and yields, along with the incomplete understanding of BGG biosynthesis pathways in microorganisms. To address these challenges, we developed a recombinant Escherichia coli strain capable of efficiently producing GA. Our approach involved screening efficient pathway enzymes, integrating biosynthetic pathway genes into the genome while balancing carbon flux via adjusting expression levels, and strengthening the shikimate pathway to remove bottlenecks. The resultant strain achieved impressive results, producing 51.57 g/L of GA with a carbon yield of 0.45 g/g glucose and a productivity of 1.07 g/L/h. Furthermore, we extended this microbial platform to biosynthesize BGG by screening GA 1-O-glucosyltransferase, leading to the de novo production of 92.42 mg/L of BGG. This work establishes an efficient chassis for producing GA at an industrial level and provides a microbial platform for generating GA derivatives.

Unlocking the potential of enzyme engineering via rational computational design strategies.

Enzymes play a pivotal role in various industries by enabling efficient, eco-friendly, and sustainable chemical processes. However, the low turnover rates and poor substrate selectivity of enzymes limit their large-scale applications. Rational computational enzyme design, facilitated by computational algorithms, offers a more targeted and less labor-intensive approach. There has been notable advancement in employing rational computational protein engineering strategies to overcome these issues, it has not been comprehensively reviewed so far. This article reviews recent developments in rational computational enzyme design, categorizing them into three types: structure-based, sequence-based, and data-driven machine learning computational design. Case studies are presented to demonstrate successful enhancements in catalytic activity, stability, and substrate selectivity. Lastly, the article provides a thorough analysis of these approaches, highlights existing challenges and potential solutions, and offers insights into future development directions.

Exploring the role of flavin-dependent monooxygenases in the biosynthesis of aromatic compounds.

Hydroxylated aromatic compounds exhibit exceptional biological activities. In the biosynthesis of these compounds, three types of hydroxylases are commonly employed: cytochrome P450 (CYP450), pterin-dependent monooxygenase (PDM), and flavin-dependent monooxygenase (FDM). Among these, FDM is a preferred choice due to its small molecular weight, stable expression in both prokaryotic and eukaryotic fermentation systems, and a relatively high concentration of necessary cofactors. However, the catalytic efficiency of many FDMs falls short of meeting the demands of large-scale production. Additionally, challenges arise from the limited availability of cofactors and compatibility issues among enzyme components. Recently, significant progress has been achieved in improving its catalytic efficiency, but have not yet detailed and informative viewed so far. Therefore, this review emphasizes the advancements in FDMs for the biosynthesis of hydroxylated aromatic compounds and presents a summary of three strategies aimed at enhancing their catalytic efficiency: (a) Developing efficient enzyme mutants through protein engineering; (b) enhancing the supply and rapid circulation of critical cofactors; (c) facilitating cofactors delivery for enhancing FDMs catalytic efficiency. Furthermore, the current challenges and further perspectives on improving catalytic efficiency of FDMs are also discussed.

Identifying and charactering a 4-aminobutyryl-CoA ligase for the production of butyrolactam.

Butyrolactam, a crucial four-carbon molecule, serves as building block in synthesis of polyamides. While biosynthesis of butyrolactam from renewable carbon sources offers a more sustainable approach, it has faced challenges in achieving high product titer and yield. Here, an efficient microbial platform for butyrolactam production was constructed by elimination of rate-limiting step and systematic pathway optimization. Initially, a superior 4-aminobutyryl-CoA ligase was discovered and characterized among six acyl-CoA ligases from different sources, which greatly improved the pathway efficiency. Subsequent optimizations were implemented to further enhance butyrolactam production, including promoter engineering, the elimination of competing pathways, transporter engineering and improving the availability of precursors. There efforts resulted in achieving approximately 2 g/L butyrolactam in shake flask experiments. Finally, the biosynthesis of butyrolactam was scaled up in a 3-L bioreactor in 84 hours, resulting in a significantly increased production of 45.2 g/L, with a carbon yield of 0.34 g/g glucose. This study highlights the construction of a microbial platform with the capability to achieve elevated levels of butyrolactam production and unlocks its potential in sustainable manufacturing processes.

Rational protein engineering of a ketoacids decarboxylase for efficient production of 1,2,4-butanetriol from arabinose.

BACKGROUND: Lignocellulose, the most abundant non-edible feedstock on Earth, holds substantial potential for eco-friendly chemicals, fuels, and pharmaceuticals production. Glucose, xylose, and arabinose are primary components in lignocellulose, and their efficient conversion into high-value products is vital for economic viability. While glucose and xylose have been explored for such purpose, arabinose has been relatively overlooked. RESULTS: This study demonstrates a microbial platform for producing 1,2,4-butanetriol (BTO) from arabinose, a versatile compound with diverse applications in military, polymer, rubber and pharmaceutical industries. The screening of the key pathway enzyme, keto acids decarboxylase, facilitated the production of 276.7 mg/L of BTO from arabinose in Escherichia coli. Through protein engineering of the rate-limiting enzyme KivD, which involved reducing the size of the binding pocket to accommodate a smaller substrate, its activity improved threefold, resulting in an increase in the BTO titer to 475.1 mg/L. Additionally, modular optimization was employed to adjust the expression levels of pathway genes, further enhancing BTO production to 705.1 mg/L. CONCLUSION: The present study showcases a promising microbial platform for sustainable BTO production from arabinose. These works widen the spectrum of potential lignocellulosic products and lays the foundation for comprehensive utilization of lignocellulosic components.

Characterization of the horse chestnut genome reveals the evolution of aescin and aesculin biosynthesis.

Horse chestnut (Aesculus chinensis) is an important medicinal tree that contains various bioactive compounds, such as aescin, barrigenol-type triterpenoid saponins (BAT), and aesculin, a glycosylated coumarin. Herein, we report a 470.02 Mb genome assembly and characterize an Aesculus-specific whole-genome duplication event, which leads to the formation and duplication of two triterpenoid biosynthesis-related gene clusters (BGCs). We also show that AcOCS6, AcCYP716A278, AcCYP716A275, and AcCSL1 genes within these two BGCs along with a seed-specific expressed AcBAHD6 are responsible for the formation of aescin. Furthermore, we identify seven Aesculus-originated coumarin glycoside biosynthetic genes and achieve the de novo synthesis of aesculin in E. coli. Collinearity analysis shows that the collinear BGC segments can be traced back to early-diverging angiosperms, and the essential gene-encoding enzymes necessary for BAT biosynthesis are recruited before the splitting of Aesculus, Acer, and Xanthoceras. These findings provide insight on the evolution of gene clusters associated with medicinal tree metabolites.

High-yield production of ß-arbutin by identifying and eliminating byproducts formation.

ß-Arbutin is a plant-derived glycoside and widely used in cosmetic and pharmaceutical industries because of its safe and effective skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. In recent years, microbial fermentation has become a highly promising method for the production of ß-arbutin. However, this method suffers from low titer and low yield, which has become the bottleneck for its widely industrial application. In this study, we used ß-arbutin to demonstrate methods for improving yields for industrial-scale production in Escherichia coli. First, the supply of precursors phosphoenolpyruvate and uridine diphosphate glucose was improved, leading to a 4.6-fold increase in ß-arbutin production in shaking flasks. The engineered strain produced 36.12 g/L ß-arbutin with a yield of 0.11 g/g glucose in a 3-L bioreactor. Next, based on the substrate and product's structural similarity, an endogenous O-acetyltransferase was identified as responsible for 6-O-acetylarbutin formation for the first time. Eliminating the formation of byproducts, including 6-O-acetylarbutin, tyrosine, and acetate, resulted in an engineered strain producing 43.79 g/L ß-arbutin with a yield of 0.22 g/g glucose in fed-batch fermentation. Thus, the yield increased twofold by eliminating byproducts formation. To the best of our knowledge, this is the highest titer and yield of ß-arbutin ever reported, paving the way for the industrial production of ß-arbutin. This study demonstrated a systematic strategy to alleviate undesirable byproduct accumulation and improve the titer and yield of target products. KEY POINTS: • A systematic strategy to improve titer and yield was showed • Genes responsible for 6-O-acetylarbutin formation were firstly identified • 43.79 g/L ß-arbutin was produced in bioreactor, which is the highest titer so far.

Functional expansion of the natural inorganic phosphorus starvation response system in Escherichia coli.

Phosphorus, an indispensable nutrient, plays an essential role in cell composition, metabolism, and signal transduction. When inorganic phosphorus (Pi) is scarce, the Pi starvation response in E. coli is activated to increase phosphorus acquisition and drive the cells into a non-growing state to reduce phosphorus consumption. In the six decades of research history, the initiation, output, and shutdown processes of the Pi starvation response have been extensively studied. Simultaneously, Pi starvation has been used in biosensor development, recombinant protein production, and natural product biosynthesis. In this review, we focus on the output process and the applications of the Pi starvation response that have not been summarized before. Meanwhile, based on the current status of mechanistic studies and applications, we propose practical strategies to develop the natural Pi starvation response into a multifunctional and standardized regulatory system in four aspects, including response threshold, temporal expression, intensity range, and bifunctional regulation, which will contribute to its broader application in more fields such as industrial production, medical analysis, and environmental protection.

Establishing biosynthetic pathway for the production of p-hydroxyacetophenone and its glucoside in Escherichia coli.

p-Hydroxyacetophenone (p-HAP) and its glucoside picein are plant-derived natural products that have been extensively used in chemical, pharmaceutical and cosmetic industries owing to their antioxidant, antibacterial and antiseptic activities. However, the natural biosynthetic pathways for p-HAP and picein have yet been resolved so far, limiting their biosynthesis in microorganisms. In this study, we design and construct a biosynthetic pathway for de novo production of p-HAP and picein from glucose in E. coli. First, screening and characterizing pathway enzymes enable us to successfully establish functional biosynthetic pathway for p-HAP production. Then, the rate-limiting step in the pathway caused by a reversible alcohol dehydrogenase is completely eliminated by modulating intracellular redox cofactors. Subsequent host strain engineering via systematic increase of precursor supplies enables production enhancement of p-HAP with a titer of 1445.3 mg/L under fed-batch conditions. Finally, a novel p-HAP glucosyltransferase capable of generating picein from p-HAP is identified and characterized from a series of glycosyltransferases. On this basis, de novo biosynthesis of picein from glucose is achieved with a titer of 210.7 mg/L under fed-batch conditions. This work not only demonstrates a microbial platform for p-HAP and picein synthesis, but also represents a generalizable pathway design strategy to produce value-added compounds.

Engineering Escherichia coli native metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli.

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.

Establishing a growth-coupled mechanism for high-yield production of ß-arbutin from glycerol in Escherichia coli.

Biodiesel production has increased significantly in recent years, leading to an increase in the production of crude glycerol. In this study, a novel growth-coupled erythrose 4-phosphate (E4P) formation strategy that can be used to produce high levels of ß-arbutin using engineered Escherichia coli and glycerol as the carbon source was developed. In the strategy, E4P formation was coupled with cell growth, and a growth-driving force made the E4P formation efficient. By applying this strategy, efficient microbial synthesis of ß-arbutin was achieved, with 7.91 g/L ß-arbutin produced in shaking flask, and 28.1 g/L produced in a fed batch fermentation with a yield of 0.20 g/g glycerol and a productivity of 0.39 g/L/h. This is the highest ß-arbutin production through microbial fermentation ever reported to date. This study may have significant implications in the large-scale production of ß-arbutin as well as other aromatic compounds of importance.

Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli.

Vanillyl alcohol is a precursor of vanillin, which is one of the most widely used flavor compounds. Currently, vanillyl alcohol biosynthesis still encounters the problem of low efficiency. In this study, coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E. coli. First, two pathways were compared for biosynthesis of the immediate precursor 3, 4-dihydroxybenzyl alcohol in monocultures, and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway. To enhance the efficiency of the last methylation step, two strategies were used, and strengthening S-adenosylmethionine (SAM) regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect. Then, the optimized pathway was assembled in a single cell. However, the biosynthetic efficiency was still low, and was not significantly improved by modular optimization of pathway genes. Thus, coculturing engineering strategy was adopted. At the optimal inoculation ratio, the titer reached 328.9 mg/L. Further, gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain. As a result, the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation. These are the highest reported titers of vanillyl alcohol so far. This work provides an effective strategy for sustainable production of vanillyl alcohol.

Engineering a Prokaryotic Non-P450 Hydroxylase for 3'-Hydroxylation of Flavonoids.

Plant-derived cytochrome P450-dependent flavonoid 3'-hydroxylases are the rate-limiting enzymes in flavonoid biosynthesis. In this study, the large component (HpaB) of a prokaryotic 4-hydroxyphenylacetate (4-HPA) 3-hydroxylase was engineered for efficient 3'-hydroxylation of naringenin. First, we selected four HpaBs through database search and phylogenetic analysis and compared their catalytic activities toward 4-HPA and naringenin. HpaB from Rhodococcus opacus B-4 (RoHpaB) showed better preference toward naringenin. To elucidate the underlying mechanism, we analyzed the structural differences of HpaBs through homologous modeling, molecular docking, and molecular dynamics simulation, and the substrate preference of RoHpaB was mainly attributed to the shorter chain length of loop 212-222 and the larger substrate binding pocket. RoHpaB was further engineered by alanine scanning and structural replacement, and the RoHpaBY215A variant was obtained, whose catalytic efficiency (kcat/Km) toward naringenin is 25.3 times higher than that of RoHpaB. In addition, RoHpaBY215A also showed significantly improved activity toward flavonoids apigenin and kaempferol. This work opens the possibility of using engineered HpaB as a versatile hydroxylase to produce functionalized flavonoids.

Biosynthesis of plant hemostatic dencichine in Escherichia coli.

Dencichine is a plant-derived nature product that has found various pharmacological applications. Currently, its natural biosynthetic pathway is still elusive, posing challenge to its heterologous biosynthesis. In this work, we design artificial pathways through retro-biosynthesis approaches and achieve de novo production of dencichine. First, biosynthesis of the two direct precursors L-2, 3-diaminopropionate and oxalyl-CoA is achieved by screening and integrating microbial enzymes. Second, the solubility of dencichine synthase, which is the last and only plant-derived pathway enzyme, is significantly improved by introducing 28 synonymous rare codons into the codon-optimized gene to slow down its translation rate. Last, the metabolic network is systematically engineered to direct the carbon flux to dencichine production, and the final titer reaches 1.29 g L-1 with a yield of 0.28 g g-1 glycerol. This work lays the foundation for sustainable production of dencichine and represents an example of how synthetic biology can be harnessed to generate unnatural pathways to produce a desired molecule.

Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid.

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S-adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH2 for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH2-dependent hydroxylation.

Biosynthesis of allantoin in Escherichia coli via screening a highly effective urate oxidase.

Allantoin is an important fine chemical that can be widely used in pharmaceutical, cosmetic and agricultural industries. Currently, allantoin is mainly produced by plant extraction or chemical synthesis. Due to the cost and environmental concerns, biosynthesis of allantoin from renewable feedstock is much more desirable. However, microbial production of allantoin from simple carbon sources has not yet been achieved so far. In this study, de novo biosynthesis of allantoin was achieved by constructing an artificial biosynthetic pathway. First, screening of efficient urate oxidases and xanthine dehydrogenases enabled allantoin production from hypoxanthine, a natural intermediate in purine metabolic pathway in Escherichia coli. Then, assemble of the entire pathway resulted in 13.9 mg/L allantoin from glucose in shake flask experiments. The titer was further improved to 639.8 mg/L by enhancing the supply of the precursor, redistribution of carbon flux, and reduction of acetate. Finally, scale-up production of allantoin was conducted in a 1-L fermentor under fed-batch culture conditions, which enabled the synthesis of 2360 mg/L allantoin, representing a 170-fold increase compared with the initial strain. This study not only demonstrates the potential for industrial production of allantoin, but also provides a bacterial platform for synthesis of other purines-derived high-value chemicals.