YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica.

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

Development of a growth coupled and multi-layered dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin biosynthesis in Escherichia coli.

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution.

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

Systems Metabolic Engineering of Saccharomyces cerevisiae for the High-Level Production of (2S)-Eriodictyol.

(2S)-eriodictyol (ERD) is a flavonoid widely found in citrus fruits, vegetables, and important medicinal plants with neuroprotective, cardioprotective, antidiabetic, and anti-obesity effects. However, the microbial synthesis of ERD is limited by complex metabolic pathways and often results in a low production performance. Here, we engineered Saccharomyces cerevisiae by fine-tuning the metabolism of the ERD synthesis pathway. The results showed that the ERD titer was effectively increased, and the intermediate metabolites levels were reduced. First, we successfully reconstructed the de novo synthesis pathway of p-coumaric acid in S. cerevisiae and fine-tuned the metabolic pathway using promoter engineering and terminator engineering for the high-level production of (2S)-naringenin. Subsequently, the synthesis of ERD was achieved by introducing the ThF3'H gene from Tricyrtis hirta. Finally, by multiplying the copy number of the ThF3'H gene, the production of ERD was further increased, reaching 132.08 mg L-1. Our work emphasizes the importance of regulating the metabolic balance to produce natural products in microbial cell factories.

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.

Design and Assembly of a Biofactory for (2S)-Naringenin Production in Escherichia coli: Effects of Oxygen Transfer on Yield and Gene Expression.

The molecule (2S)-naringenin is a scaffold molecule with several nutraceutical properties. Currently, (2S)-naringenin is obtained through chemical synthesis and plant isolation. However, these methods have several drawbacks. Thus, heterologous biosynthesis has emerged as a viable alternative to its production. Recently, (2S)-naringenin production studies in Escherichia coli have used different tools to increase its yield up to 588 mg/L. In this study, we designed and assembled a bio-factory for (2S)-naringenin production. Firstly, we used several parametrized algorithms to identify the shortest pathway for producing (2S)-naringenin in E. coli, selecting the genes phenylalanine ammonia lipase (pal), 4-coumarate: CoA ligase (4cl), chalcone synthase (chs), and chalcone isomerase (chi) for the biosynthetic pathway. Then, we evaluated the effect of oxygen transfer on the production of (2S)-naringenin at flask (50 mL) and bench (4 L culture) scales. At the flask scale, the agitation rate varied between 50 rpm and 250 rpm. At the bench scale, the dissolved oxygen was kept constant at 5% DO (dissolved oxygen) and 40% DO, obtaining the highest (2S)-naringenin titer (3.11 ± 0.14 g/L). Using genome-scale modeling, gene expression analysis (RT-qPCR) of oxygen-sensitive genes was obtained.

Engineering a Novel Metabolic Pathway for Improving Cellular Malonyl-CoA Levels in Escherichia coli.

Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media.

Glycosylation Modification Enhances (2S)-Naringenin Production in Saccharomyces cerevisiae.

(2S)-Naringenin is an important flavonoid precursor, with multiple nutritional and pharmacological activities. Both (2S)-naringenin and other flavonoid production are hindered by poor water solubility and inhibited cell growth. To address this, we increased solubility and improved cell growth by partially glycosylating (2S)-naringenin to naringenin-7-O-glucoside, which facilitated increased extracellular secretion, by knocking out endogenous glycosyl hydrolase genes, EXG1 and SPR1, and expressing the glycosyltransferase gene (UGT733C6). Naringenin-7-O-glucoside synthesis was further improved by optimizing UDP-glucose and shikimate pathways. Then, hydrochloric acid was used to hydrolyze naringenin-7-O-glucoside to (2S)-naringenin outside the cell. Thus, our optimized Saccharomyces cerevisiae strain E32T19 produced 1184.1 mg/L (2S)-naringenin, a 7.9-fold increase on the starting strain. Therefore. we propose that glycosylation modification is a useful strategy for the efficient heterologous biosynthesis of (2S)-naringenin in S. cerevisiae.

Engineering caveolin-mediated endocytosis in Saccharomyces cerevisiae.

As a potential substitute for fatty acids, common low-cost oils could be used to produce acetyl-CoA derivatives, which meet the needs of low-cost industrial production. However, oils are hydrophobic macromolecules and cannot be directly transported into cells. In this study, caveolin was expressed in Saccharomyces cerevisiae to absorb exogenous oils. The expression of caveolin fused with green fluorescent protein showed that caveolin mediated the formation of microvesicles in S. cerevisiae and the addition of 5,6-carboxyfluorescein showed that caveolae had the ability to transport exogenous substances into cells. The intracellular and extracellular triacylglycerol levels were then detected after the addition of soybean oil pre-stained with Nile Red, which proved that caveolae had the ability to absorb the exogenous oils. Lastly, caveolin for oils absorption and lipase from Bacillus pumilus for oil hydrolysis were co-expressed in the naringenin-producing Saccharomyces cerevisiae strain, resulting in naringenin production increasing from 222 mg/g DCW (dry cell weight) (231 mg/L) to 269 mg/g DCW (241 mg/L). These results suggested that the caveolin-mediated transporter independent oil transport system would provide a promising strategy for the transport of hydrophobic substrates.

Improving (2S)-naringenin production by exploring native precursor pathways and screening higher-active chalcone synthases from plants rich in flavonoids.

Flavonoids are a group of valuable compounds with a variety of health benefits. (2 S)-Naringenin is an important flavonoid skeleton, which can be tailored into almost all flavonoids. In this study, the Saccharomyces cerevisiae native precursor pathways were explored and higher-active CHSs from plants rich in flavonoids were screened. The results indicated that overexpressing the native precursor pathways is not an efficient approach to improving (2 S)-naringenin production in our chassis strain. On the other hand, by screening from plants rich in flavonoids, we obtained four CHSs with higher activities than the commonly used PhCHS. Among these CHSs, SjCHS1 increased the (2 S)-naringenin titer by 48.38% in shaking flasks. Finally, we combined the native precursor pathways optimization with the higher-active CHS that screened, and further increased the (2 S)-naringenin titer to 203.49 mg/L from glucose in shaking flasks. The results achieved in this study indicated that plants rich in flavonoids are good sources for higher-active CHS screening, and that the heterologous pathway and chassis precursor flux should be synergistically engineered to achieve higher production.

Systematically Engineered Fatty Acid Catabolite Pathway for the Production of (2S)-Naringenin in Saccharomyces cerevisiae.

The (2S)-naringenin is an important natural flavonoid with several bioactive effects on human health. It is also a key precursor in the biosynthesis of other high value compounds. The production of (2S)-naringenin is significantly influenced by the acetyl-CoA available in the cytosol. In this study, we increased the acetyl-CoA supply via the ß-oxidation of fatty acids in the peroxisomes of Saccharomyces cerevisiae. Several lipases from different sources and PEX11, FOX1, FOX2, and FOX3, the key genes of the fatty acid ß-oxidation pathway, were overexpressed during the production of (2S)-naringenin in yeast. The level of acetyl-CoA was 0.205 nmol higher than that in the original strain and the production of (2S)-naringenin increased to 286.62 mg/g dry cell weight when PEX11 was overexpressed in S. cerevisiae strain L07. Remarkable (2S)-naringenin production (1129.44 mg/L) was achieved with fed-batch fermentation, with the highest titer reported in any microorganism. Our results demonstrated the use of fatty acid ß-oxidation to increase the level of cytoplasmic acetyl-CoA and the production of its derivatives.

Promoter-Library-Based Pathway Optimization for Efficient (2S)-Naringenin Production from p-Coumaric Acid in Saccharomyces cerevisiae.

Pathway optimization plays an important role in fine-tuning metabolic pathways. In most conditions, more than three genes are involved in the biosynthesis pathway of a specific target product. To improve the titer of products, rational regulation of a group of genes by a series of promoters with different strengths is essential. On the basis of a series of RNA-Seq data, a set of 66 native promoters was chosen to fine-tune gene expression in Saccharomyces cerevisiae. Promoter strength was characterized by measuring the fluorescence strength of the enhanced green fluorescent protein through fluorescence-activated cell sorting. The expressions of PTDH1, PPGK1, PINO1, PSED1, and PCCW12 were stronger than that of PTDH3, whereas those of another 15 promoters were stronger than that of PTEF1. Then, 30 promoters were chosen to optimize the biosynthesis pathway of (2S)-naringenin from p-coumaric acid. With a high-throughput screening method, the highest titer of (2S)-naringenin in a 5 L bioreactor reached 1.21 g/L from p-coumaric acid, which is the highest titer according to the currently available reports.

Efficient Biosynthesis of (2S)-Naringenin from p-Coumaric Acid in Saccharomyces cerevisiae.

(2S)-Naringenin, a (2S)-flavanone, is widely used in the food, chemical, and pharmaceutical industries because of its diverse physiological activities. The production of (2S)-naringenin in microorganisms provides an ideal source that reduces the cost of the flavonoid. To achieve efficient production of (2S)-naringenin in Saccharomyces cerevisiae (S. cerevisiae), we constructed a biosynthetic pathway from p-coumaric acid, a cost-effective and more efficient precursor. The (2S)-naringenin synthesis pathway genes were integrated into the yeast genome to obtain a (2S)-naringenin production strain. After gene dosage experiments, the genes negatively regulating the shikimate pathway and inefficient chalcone synthase activity were verified as factors limiting (2S)-naringenin biosynthesis. With fed-batch process optimization of the engineered strain, the titer of (2S)-naringenin reached 648.63 mg/L from 2.5 g/L p-coumaric acid. Our results indicate that the constitutive production of (2S)-naringenin from p-coumaric acid in S. cerevisiae is highly promising.

Chemical composition of Acacia farnesiana (L) wild fruits and its activity against Mycobacterium tuberculosis and dysentery bacteria.

ETHNOPHARMACOLOGICAL RELEVANCE: In Mexico, plants are an important element of traditional medicine, and many are considered part of Mexican cultural heritage from prehispanic and colonial times. Nevertheless, relatively few systematic scientific studies have been conducted to fully characterize the chemical composition and pharmacological activities of Mexican medicinal plants. Acacia farnesiana is used in Mexican traditional medicine to treat dysentery and tuberculosis and therefore could have bioactive compounds that may explain its traditional use. AIMS OF THE STUDY: i) To isolate and characterize the compounds from the hexanic, chloroformic and methanolic extracts; ii) to identify the volatile compounds from methylated hexanic and chloroformic extracts using GC-FID and GC-MS methods; iii) to identify the compounds from methanolic and aqueous extracts using HPLC-Q-TOF-MS; iv) to test the activity of extracts and isolated compounds against Mycobacterium tuberculosis and dysentery bacteria. MATERIAL AND METHODS: A. farnesiana fruits were collected in Acatlán de Osorio, Puebla, Mexico. Hexanic, chloroformic, methanolic and aqueous extracts were prepared and analyzed by different chromatographic techniques including column chromatography, flash chromatography, GC-FID, GC-MS and HPLC-Q-TOF-MS. Structural elucidation was carried out by NMR spectroscopic analysis. The activity of extracts, phytochemicals and semi-synthetic derivatives against Mycobacterium tuberculosis H37Rv and G122 as well as dysentery bacteria (Campylobacter jejuni, Shigella flexneri, Salmonella enteritidis, Yersinia enterocolitica and enterohemorrhagic Escherichia coli) was determined by the broth microdilution method and reported as minimal inhibitory concentration (MIC µg/mL). RESULTS: From both hexane and chloroform extracts, tetracosanoic acid (2S)-2,3-dihydroxypropyl ester (1) and (3ß,22E)-estigmasta-5,22-dien-3-yl ß-D-glucopyranoside (2) were isolated and characterized. From the methanolic extract, methyl gallate (3), gallic acid (4), (3ß,22E)-estigmasta-5,22-dien-3-yl ß-D-glucopyranoside (2), (2S) naringenin 7-O-ß-glucopyranoside (prunin, 5), pinitol (6) and sucrose (7) were isolated and characterized. Furthermore, hexanic and chloroformic extracts were analyzed by GC-FID and GC-MS and 18 methylated fatty acids were identified for each extract in addition to three sterols. The methanolic and aqueous extracts were analyzed separately by HPLC-Q-TOF-MS, and 15 compounds were identified in each extract. The compounds 1, 2, and 7, in addition to 13 fatty acids and eight phenolic compounds, were identified for the first time in A. farnesiana. The extracts showed antitubercular (MIC 100-200 µg/mL) and antidysentery activity (MIC 100-200 µg/mL). Methyl gallate and its acetylated derivative showed activity against the sensible strain M. tuberculosis H37Rv with MIC values of 50-25 µg/mL, respectively. The flavanone prunin showed activity against multidrug resistant M. tuberculosis G122 (MIC 50 µg/mL). Methyl gallate, gallic acid and prunin showed activity against C. jejuni (MIC 50 µg/mL). CONCLUSIONS: The activity of tested extracts and isolated compounds against M. tuberculosis and dysentery bacteria justifies the ethnomedical use of A. farnesiana fruits for the treatment of tuberculosis and dysentery.

Studies of flavonoids from Glycyrrhizae Radix et Rhizoma / 中草药

Objective: To study the chemical constituents of flavonoids from Glycyrrhizae Radix et Rhizoma. Methods: The compounds were isolated and purified by column chromatography over HP-20 macroporous resin, silica gel, Sephadex LH-20, and preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. Results: Ten flavonoids were isolated and identified as 4’,6,7-trihydroxy-2’-methoxyl-chalcone (1), 3’,4’,5,7-tetrahydroxy-8-(3-hydroxy-3- methylbutyl)-isoflavone (2), isoliquiritigenin (3), isoliquiritin (4), echinatin (5), orobol (6), ononin (7), 2(S)-3’,5’,7-trihydroxy- flavanone (8), 2(S)-naringenin-4’-O-β-D-glucopyranoside (9), and 4’,7-dihydroxyflavone (10). Conclusion: Compounds 1 and 2 are new compounds named isolicochalcone B and licoisoflavone G, while compound 9 is isolated from the genus for the first time.