High efficiency production of 5-hydroxyectoine Zusing metabolically engineered Escherichia coli.

The 5-hydroxyectoine is a natural protective agent with long-lasting moisturising and radiation resistance properties. It can be naturally synthesized by some extremophiles using the "bacterial milking" process, but this can corrode bioreactors and downstream purification may cause environmental pollution. In this study, an engineered Escherichia coli (E. coli) strain was constructed for the 5-hydroxyectoine production. First, three ectoine hydroxylases were characterised and the enzyme from Halomonas elongata was the most effective. The L-2,4-diaminobutyrate transaminase mutant was introduced into the engineered strain, which could accumulate 2.8 g/L 5-hydroxyectoine in shake flasks. By activating the glyoxylate cycle and balancing the α-ketoglutarate distribution, the 5-hydroxyectoine titer was further increased to 3.4 g/L. Finally, the optimized strain synthesized 58 g/L 5-hydroxyectoine via a semi-continuous feeding process in a NaCl-free medium. Overall, this study reported the highest titer of 5-hydroxyectoine synthesized by E. coli and established a low-salt fermentation process through the aforementioned efforts.

[Production of neohesperidin from hesperetin by an engineered strain of Escherichia coli].

Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana, rhamnose synthase VvRHM-NRS from Vitis vinifera, and rhamnose transferase Cm1,2RhaT from Citrus maxima. After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced 4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

De Novo Biosynthesis of Dihydroquercetin in Saccharomyces cerevisiae.

Dihydroquercetin is a vital flavonoid compound with a wide range of physiological activities. However, factors, such as metabolic regulation, limit the heterologous synthesis of dihydroquercetin in microorganisms. In this study, flavanone 3-hydroxylase (F3H) and flavanone 3'-hydroxylase (F3'H) were screened from different plants, and their co-expression in Saccharomyces cerevisiae was optimized. Promoter engineering and redox partner engineering were used to optimize the corresponding expression of genes involved in the dihydroquercetin synthesis pathway. Dihydroquercetin production was further improved through multicopy integration pathway genes and systems metabolic engineering. By increasing NADPH and α-ketoglutarate supply, the catalytic efficiency of F3'H and F3H was improved, thereby effectively increasing dihydroquercetin production (235.1 mg/L). Finally, 873.1 mg/L dihydroquercetin titer was obtained by fed-batch fermentation in a 5-L bioreactor, which is the highest dihydroquercetin production achieved through de novo microbial synthesis. These results established a pivotal groundwork for flavonoids synthesis.

Machine learning-assisted substrate binding pocket engineering based on structural information.

Engineering enzyme-substrate binding pockets is the most efficient approach for modifying catalytic activity, but is limited if the substrate binding sites are indistinct. Here, we developed a 3D convolutional neural network for predicting protein-ligand binding sites. The network was integrated by DenseNet, UNet, and self-attention for extracting features and recovering sample size. We attempted to enlarge the dataset by data augmentation, and the model achieved success rates of 48.4%, 35.5%, and 43.6% at a precision of ≥50% and 52%, 47.6%, and 58.1%. The distance of predicted and real center is ≤4 Å, which is based on SC6K, COACH420, and BU48 validation datasets. The substrate binding sites of Klebsiella variicola acid phosphatase (KvAP) and Bacillus anthracis proline 4-hydroxylase (BaP4H) were predicted using DUnet, showing high competitive performance of 53.8% and 56% of the predicted binding sites that critically affected the catalysis of KvAP and BaP4H. Virtual saturation mutagenesis was applied based on the predicted binding sites of KvAP, and the top-ranked 10 single mutations contributed to stronger enzyme-substrate binding varied while the predicted sites were different. The advantage of DUnet for predicting key residues responsible for enzyme activity further promoted the success rate of virtual mutagenesis. This study highlighted the significance of correctly predicting key binding sites for enzyme engineering.

Engineering Gluconbacter oxydans with efficient co-utilization of glucose and sorbitol for one-step biosynthesis of 2-keto-L-gulonic.

As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004.

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Enhancing (2S)-naringenin production in Saccharomyces cerevisiae by high-throughput screening method based on ARTP mutagenesis.

(2S)-Naringenin, a dihydro-flavonoid, serves as a crucial precursor for flavonoid synthesis due to its extensive medicinal values and physiological functions. A pathway for the synthesis of (2S)-naringenin from glucose has previously been constructed in Saccharomyces cerevisiae through metabolic engineering. However, this synthetic pathway of (2S)-naringenin is lengthy, and the genes involved in the competitive pathway remain unknown, posing challenges in significantly enhancing (2S)-naringenin production through metabolic modification. To address this issue, a novel high-throughput screening (HTS) method based on color reaction combined with a random mutagenesis method called atmospheric room temperature plasma (ARTP), was established in this study. Through this approach, a mutant (B7-D9) with a higher titer of (2S)-naringenin was obtained from 9600 mutants. Notably, the titer was enhanced by 52.3% and 19.8% in shake flask and 5 L bioreactor respectively. This study demonstrates the successful establishment of an efficient HTS method that can be applied to screen for high-titer producers of (2S)-naringenin, thereby greatly improving screening efficiency and providing new insights and solutions for similar product screenings.

Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae.

Campesterol is a kind of important functional food additive. Therefore, stable and efficient campesterol biosynthesis is significant. Herein, we first knocked out the sterol 22-desaturase gene in Saccharomyces cerevisiae and expressed sterol Δ7-reductase from Pangasianodon hypophthalmus, obtaining a strain that produced 6.6 mg/L campesterol. Then, the modular expression of campesterol synthesis enzymes was performed, and a campesterol titer of 88.3 mg/L was achieved. Because campesterol is a lipid-soluble macromolecule, we promoted lipid droplet formation by exploring regulatory factors, and campesterol production was improved to 169.20 mg/L. Next, triacylglycerol lipase was used to achieve compartment campesterol synthesis. After enhancing the expression of sterol Δ7-reductase and screening cations, the campesterol titer reached 438.28 mg/L in a shake flask and 1.44 g/L in a 5 L bioreactor, which represents the highest campesterol titer reported to date. Metabolic regulation combined with lipid droplet engineering may be useful for the synthesis of other steroids as well.

Efficient synthesis of squalene by cytoplasmic-peroxisomal engineering and regulating lipid metabolism in Yarrowia lipolytica.

Squalene, a high-value acyclic triterpenoid compound, is broadly used in the food and medical industries. Although the large acetyl-CoA pool and hydrophobic space of Yarrowia lipolytica are suitable for the accumulation of squalene, the current production level in Y. lipolytica is still not sufficient for industrial production. In this study, two rounds of multicopy integration of genes encoding key enzymes were performed to enhance squalene anabolic flux in the cytoplasm. Furthermore, the mevalonate pathway was imported into peroxisomes through the compartmentalization strategy, and the production of squalene was significantly increased. By augmenting the acetyl-CoA supply in peroxisomes and the cytoplasm, the squalene was boosted to 2549.1 mg/L. Finally, the squalene production reached 51.2 g/L by fed-batch fermentation in a 5-L bioreactor. This is the highest squalene production reported to date for microbial production, and this study lays the foundation for the synthesis of steroids and squalene derivatives.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the ß-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

Chromatin regulator Eaf3p regulates nitrogen metabolism in Saccharomyces cerevisiae as a trans-acting factor.

IMPORTANCE: In this study, the mechanism of chromatin regulator Eaf3p regulating nitrogen metabolism in S. cerevisiae was investigated. It provides theoretical support for epigenetic modifications of cells to alter the level of histone modifications, coordinate the expression of multiple genes, and make it more conducive to the co-metabolism of multiple nitrogen sources. Moreover, it provides new ideas for industrial brewing yeast strains to achieve nitrogen source metabolism balance, reduce the accumulation of harmful nitrogen metabolites, and improve fermentation efficiency. This study provides a reference for changing the performance of microbial strains and improving the quality of traditional fermentation products and provides a theoretical basis for studying epigenetic modification and nitrogen metabolism regulation. It has an important theoretical explanation and practical application value. In addition, this study also provides useful clues for the study.

Enhancing Glycosylation of Flavonoids by Engineering the Uridine Diphosphate Glucose Supply in Escherichia coli.

Glycosylation can enhance the solubility and stability of flavonoids. The main limitation of the glycosylation process is low intracellular uridine diphosphate glucose (UDPG) availability. This study aimed to create a glycosylation platform strain in Escherichia coli BL21(DE3) by multiple metabolic engineering of the UDPG supply. Glycosyltransferase TcCGT1 was introduced to synthesize vitexin and orientin from apigenin and luteolin, respectively. To further expand this glycosylation platform strain, not only were UDP rhamnose and UDP galactose synthesis pathways constructed, but rhamnosyltransferase (GtfC) and galactosyltransferase (PhUGT) were also introduced, respectively. In a 5 L bioreactor with apigenin, luteolin, kaempferol, and quercetin as glycosyl acceptors, vitexin, orientin, afzelin, quercitrin, hyperoside, and trifolin glycosylation products reached 17.2, 36.5, 5.2, 14.1, 6.4, and 11.4 g/L, respectively, the highest titers reported to date for all. The platform strain has great potential for large-scale production of glycosylated flavonoids.

Engineering Saccharomyces cerevisiae for synthesis of ß-myrcene and (E)-ß-ocimene.

Monoterpenes are among the important natural plant terpenes. Monoterpenes usually have the characteristics of volatility and strong aroma. ß-Myrcene and its isomer (E)-ß-ocimene are typical acyclic monoterpenes. They are high-value monoterpenes that have been widely applied in foods, cosmetics, and medicines. However, large-scale commercial production of ß-myrcene and (E)-ß-ocimene is restricted by their production method that mainly involves extraction from plant essential oils. Currently, an alternative synthetic route utilizing an engineered microbial platform was proposed for effective production. This study used a Saccharomyces cerevisiae strain previously constructed for squalene production as the starting strain. Farnesyl diphosphate synthase (Erg20) expression was weakened by promoter replacement and screened for optimal myrcene synthase (MS) and ocimene synthase (OS) activities. In the resulting S. cerevisiae engineered for ß-myrcene and (E)-ß-ocimene synthesis, titers of ß-myrcene and (E)-ß-ocimene were enhanced by a fusion expressing a mutant Erg20* with the obtained monoterpene synthase and optimizing the added solvent in a two-phase fermentation system. Finally, by scaling up in a 5-L fermenter, 8.12 mg/L of ß-myrcene was obtained, which was first reported in yeast, and 34.56 mg/L of (E)-ß-ocimene was obtained, which is the highest reported to date. This study provides a new synthesis route for ß-myrcene and (E)-ß-ocimene. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03818-2.

Metabolic engineering combined with enzyme engineering for overproduction of ectoine in Escherichia coli.

Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysCT311I from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.

Efficient production of 2-keto-L-gulonic acid from D-glucose in Gluconobacter oxydans ATCC9937 by mining key enzyme and transporter.

Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.

Chromatin regulator Ahc1p co-regulates nitrogen metabolism via interactions with multiple transcription factors in Saccharomyces cerevisiae.

Chromatin regulation is an important gene expression/regulation system, but little is known about how it affects nitrogen metabolism in Saccharomyces cerevisiae. A previous study demonstrated the regulatory role of the chromatin regulator Ahc1p on multiple key genes of nitrogen metabolism in S. cerevisiae, but the regulatory mechanism remains unknown. In this study, multiple key nitrogen metabolism genes directly regulated by Ahc1p were identified, and the transcription factors interacting with Ahc1p were analyzed. It was ultimately found that Ahc1p may regulate some key nitrogen metabolism genes in two ways. First, Ahc1p acts as a co-factor and is recruited with transcription factors such as Rtg3p or Gcr1p to facilitate transcription complex binding to target gene core promoters and promote transcription initiation. Second, Ahc1p binds at enhancers to promote the transcription of target genes in concert with transcription factors. This study furthers the understanding of the regulatory network of nitrogen metabolism in S. cerevisiae from an epigenetic perspective.

Regulation of Ethanol Assimilation for Efficient Accumulation of Squalene in Saccharomyces cerevisiae.

Squalene is a triterpene that can be obtained from fish and plant oils. It is important in cosmetics and vaccines and is a precursor for many high-value terpenes and steroids. In order to increase squalene accumulation, the mevalonate pathway was systematically enhanced. Accumulation of squalene tended to increase when ethanol was added as a carbon source during fermentation, but a high concentration of ethanol affected both the strain growth and accumulation of products. By overexpressing the key trehalose synthesis gene TPS1 and the heat shock protein gene HSP104, the content of trehalose by Saccharomyces cerevisiae (S. cerevisiae) was enhanced, and stress caused by ethanol was relieved. The OD600 value of the modified S. cerevisiae strain was increased by 80.2%, its ethanol tolerance was increased to 30 g/L, and it retained excellent activity with 50 g/L ethanol. After optimizing the fermentation conditions, the squalene titer in a 5 L bioreactor reached 27.3 g/L and the squalene content was 650 mg/g dry cell weight, the highest squalene production parameters reported to date for a microorganism.

De novo biosynthesis of carminic acid in Saccharomyces cerevisiae.

Carminic acid is a natural red dye extracted from the insect Dactylopius coccus. Due to its ideal dying effect and high safety, it is widely used in food and cosmetics industries. Previous study showed that introduction of polyketide synthase (OKS) from Aloe arborescens, cyclase (ZhuI) and aromatase (ZhuJ) from Streptomyces sp. R1128, and C-glucosyltransferase (UGT2) from D. coccus into Aspergillus nidulans could achieve trace amounts of de novo production. These four genes were introduced into Saccharomyces cerevisiae, but carminic acid was not detected. Analysis of the genome of A. nidulans revealed that 4'-phosphopantetheinyl transferase (NpgA) and monooxygenase (AptC) are essential for de novo biosynthesis of carminic acid in S. cerevisiae. Additionally, endogenous hydroxylase (Cat5) from S. cerevisiae was found to be responsible for hydroxylation of flavokermesic acid to kermesic acid. Therefore, all enzymes and their functions in the biosynthesis of carminic acid were explored and reconstructed in S. cerevisiae. Through systematic pathway engineering, including regulating enzyme expression, enhancing precursor supply, and modifying the ß-oxidation pathway, the carminic acid titer in a 5 L bioreactor reached 7580.9 µg/L, the highest yet reported for a microorganism. Heterologous reconstruction of the carminic acid biosynthetic pathway in S. cerevisiae has great potential for de novo biosynthesis of anthraquinone dye.