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Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. Key points: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli. [ABSTRACT FROM AUTHOR]

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10β-hydroxylase (T10βH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10β-ol (T10OH), was investigated in Escherichiacoli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10βH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. Key points: • The T10βH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10βH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L. [ABSTRACT FROM AUTHOR]

Sakuranetin is a plant-natural product, which has increasingly been utilized in cosmetic and pharmaceutical industries for its extensive anti-inflammatory, anti-tumor, and immunomodulatory effects. Sakuranetin was mostly produced by extraction technology from plants, which is limited to natural conditions and biomass supply. In this study, a de novo biosynthesis pathway of sakuranetin by engineered S. cerevisiae was constructed. After a series of heterogenous gene integration, a biosynthetic pathway of sakuranetin from glucose was successfully constructed in S. cerevisiae whose sakuranetin yield reached only 4.28 mg/L. Then, a multi-module metabolic engineering strategy was applied for improving sakuranetin yield in S. cerevisiae: (1) adjusting the copy number of sakuranetin synthesis genes, (2) removing the rate-limiting factor of aromatic amino acid pathway and optimizing the synthetic pathway of aromatic amino acids to enhance the supply of carbon flux for sakuranetin, and (3) introducing acetyl-CoA carboxylase mutants ACC1S659A,S1157A and knocking out YPL062W to strengthen the supply of malonyl-CoA which is another synthetic precursor of sakuranetin. The resultant mutant S. cerevisiae exhibited a more than tenfold increase of sakuranetin titer (50.62 mg/L) in shaking flasks. Furthermore, the sakuranetin titer increased to 158.65 mg/L in a 1-L bioreactor. To our knowledge, it is the first report on the sakuranetin de novo synthesis from glucose in S. cerevisiae. Key points: • De novo biosynthesis of sakuranetin was constructed by engineered S. cerevisiae. • Sakuranetin production was enhanced by multi-module metabolic engineering strategy. • It is the first report on the sakuranetin de novo synthesis in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Several de novo amino acid, nucleotide, and vitamin biosynthesis pathways are essential for the survival and virulence of pathogenic bacteria in the host. There is a high potential for narrow-spectrum therapeutics with nutrient stress targets specific to the pathogen or site of infection. Innovative high-throughput screening platforms have been developed to identify inhibitors of nutrient stress that can be combined with rapid and effective target identification techniques. Differences between the human and mouse nutrient environment have led to vitamin biosynthesis being overlooked as an antibiotic target. Idiosyncratic connections between antibiotic resistance and nutrient biosynthesis pathways hold promise for unique combination approaches against multidrug-resistant pathogens. Novel approaches are required to address the looming threat of pan-resistant Gram-negative pathogens and forestall the rise of untreatable infections. Unconventional targets that are uniquely important during infection and tractable to high-throughput drug discovery methods hold high potential for innovation in antibiotic discovery programs. In this context, inhibitors of bacterial nutrient stress are particularly exciting candidates for future antibiotic development. Amino acid, nucleotide, and vitamin biosynthesis pathways are critical for bacterial growth in nutrient-limiting conditions in the laboratory and the host. Although historically dismissed as dispensable for pathogens, a wealth of transposon mutagenesis and single-mutant studies have emerged which demonstrate that several such pathways are critical for infection. Indeed, high-throughput screens of diverse synthetic compounds and natural products have uncovered inhibitors of nutrient biosynthesis. Herein, we review bacterial nutrient biosynthesis and its role during host infection. Further, we explore screening platforms developed to search for inhibitors of these targets and highlight successes among these. Finally, we feature important and sometimes surprising connections between bacterial nutrient biosynthesis, antibiotic activity, and antibiotic resistance. [ABSTRACT FROM AUTHOR]

Poly(3-hydroxybutyrate-co-3-hydroxypropionate) [P(3HB-co-3HP)] is a biodegradable and biocompatible polyester with improved and expanded material properties compared with poly(3-hydroxybutyrate) (PHB). This study engineered a robust malonyl-CoA pathway in Cupriavidus necator for the efficient supply of a 3HP monomer, and could achieve the production of [P(3HB-co-3HP)] from variable oil substrates. Flask level experiments followed by product purification and characterization found the optimal fermentation condition (soybean oil as carbon source, 0.5 g/L arabinose as induction level) in general consideration of the PHA content, PHA titer and 3HP molar fraction. A 5 L fed-batch fermentation (72 h) further increased the dry cell weight (DCW) to 6.08 g/L, the titer of [P(3HB-co-3HP)] to 3.11 g/L and the 3HP molar fraction to 32.25%. Further improving the 3HP molar fraction by increasing arabinose induction failed as the engineered malonyl-CoA pathway was not properly expressed under the high-level induction condition. With several promising advantages (broader range of economic oil substrates, no need for expensive supplementations such as alanine and VB12), this study indicated a candidate route for the industrial level production of [P(3HB-co-3HP)]. For future prospects, further studies are needed to further improve the strain and the fermentation process and expand the range of relative products. [ABSTRACT FROM AUTHOR]

Cordycepin is one of the nucleoside compounds with various nutraceutical and health-promoting functions, which makes it widely used in nutraceutical and pharmaceutical fields. Traditionally, cordycepin is mainly extracted from the natural fruiting bodies of Cordyceps spp.. However, the collection of natural Cordyceps will lead to the depletion of wild resources and the decline of biodiversity, as well as damage to the local fragile ecological environment. This review provides the microbial resources of cordycepin and introduces the relationship between key metabolic pathways, transcription factors, and cordycepin biosynthesis in artificial cultured cordycepin-producing strains. In addition, the methods of strain improvement and genetic engineering to increase cordycepin production were reviewed, and the possibility of using synthetic biology to construct cell factories to increase cordycepin production was discussed. The analysis of the cordycepin biosynthesis pathway in fungi is of great guiding significance for the improvement of cordycepin production capacity. The construction of cell factories using synthetic biology and systematic metabolic engineering can be used as a green and energy-saving alternative method for the industrial production of cordycepin. • Cordycepin is a nucleoside compound with multiple biological activities. • Microbial sources and de novo biosynthesis of cordycepin are summarized. • Strain improvement is beneficial to enhance the production of cordycepin. • Synthetic biology makes it possible to industrial produce cordycepin. [ABSTRACT FROM AUTHOR]

Isobavachalcone (IBC) is a prenylated chalcone mainly distributed in some Fabaceae and Moraceae species. IBC exhibits a wide range of pharmacological properties, including anti-bacterial, anti-viral, anti-inflammatory, and anticancer activities. In this study, we attempted to construct the heterologous biosynthesis pathway of IBC in tobacco (Nicotiana tabacum). Four previously reported prenyltransferases, including GuILDT from Glycyrrhiza uralensis, HlPT1 from Humulus lupulus, and SfILDT and SfFPT from Sophora flavescens, were subjected to an in planta screening to verify their activities for the biosynthesis of IBC, by using tobacco transient expression with exogenous isoliquiritigenin as the substrate. Only SfFPT and HlPT1 could convert isoliquiritigenin to IBC, and the activity of SfFPT was higher than that of HlPT1. By co-expression of GmCHS8 and GmCHR5 from Glycine max, endogenous isoliquiritigenin was generated in tobacco leaves (21.0 mg/g dry weight). After transformation with a multigene vector carrying GmCHS8, GmCHR5, and SfFPT, de novo biosynthesis of IBC was achieved in transgenic tobacco T0 lines, in which the highest amount of IBC was 0.56 mg/g dry weight. The yield of IBC in transgenic plants was nearly equal to that in SfFPT transient expression experiments, in which substrate supplement was sufficient, indicating that low IBC yield was not attributed to the substrate supplement. Our research provided a prospect to produce valuable prenylflavonoids using plant-based metabolic engineering. [ABSTRACT FROM AUTHOR]

Abstract Steroidal compounds are of great interest in the pharmaceutical field, with steroidal drugs as the second largest category of medicine in the world. Advances in synthetic biology and metabolic engineering have enabled de novo biosynthesis of sterols and steroids in yeast, which is a green and safe production route for these valuable steroidal compounds. In this review, we summarize the metabolic engineering strategies developed and employed for improving the de novo biosynthesis of sterols and steroids in yeast based on the regulation mechanisms, and introduce the recent progresses in de novo synthesis of some typical sterols and steroids in yeast. The remaining challenges and future perspectives are also discussed.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae , converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo . Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

Anthocyanins are natural pigments found in various plants. As multifunctional natural compounds, anthocyanins are widely used in food, pharmaceuticals, health products, and cosmetics. At present, the anthocyanins are heterologously biosynthesized in prokaryotes from flavan-3-ols, which is rather expensive. This study aimed to metabolically engineer Saccharomyces cerevisiae for anthocyanin production. Anthocyanin production has been extensively studied to understand the metabolic pathway enzymes in their natural hosts, including CHS (chalcone synthase); FLS (flavonol synthase); CHI (chalcone isomerase); F3H (flavanone 3-hydroxylase); F3′H (flavonoid 3′-hydroxylase); F3′5′H (flavonoid 3′,5′-hydroxylase); DFR (dihydroflavonol 4-reductase); ANS (anthocyanidin synthase); LAR (leucoanthocyanidin reductase); and UFGT (flavonoid 3-O-glucosyltransferase). The anthocyanin transporter MdGSTF6 was first introduced and proven to be indispensable for the biosynthesis of anthocyanins. By expressing MdGSTF6, FaDFR, PhANS0, and Dc3GT and disrupting EXG1 (the main anthocyanin-degrading enzyme), the BA-22 strain produced 261.6 mg/L (254.5 mg/L cyanidin-3-O-glucoside and 7.1 mg/L delphinidin-3-O-glucoside) anthocyanins from 2.0 g/L dihydroflavonols, which was known to be the highest titer in eukaryotes. Finally, 15.1 mg/L anthocyanins was obtained from glucose by expressing the de novo biosynthesis pathway in S. cerevisiae, which is known to be the highest de novo production. It is the first study to show that through the introduction of a plant anthocyanin transporter and knockout of a yeast endogenous anthocyanin degrading enzyme, the anthocyanin titer has been increased by more than 100 times.

Poly(3-hydroxybutyrate-co-3-hydroxypropionate) [P(3HB-co-3HP)] is a biodegradable and biocompatible polyester with improved and expanded material properties compared with poly(3-hydroxybutyrate) (PHB). This study engineered a robust malonyl-CoA pathway in Cupriavidus necator for the efficient supply of a 3HP monomer, and could achieve the production of [P(3HB-co-3HP)] from variable oil substrates. Flask level experiments followed by product purification and characterization found the optimal fermentation condition (soybean oil as carbon source, 0.5 g/L arabinose as induction level) in general consideration of the PHA content, PHA titer and 3HP molar fraction. A 5 L fed-batch fermentation (72 h) further increased the dry cell weight (DCW) to 6.08 g/L, the titer of [P(3HB-co-3HP)] to 3.11 g/L and the 3HP molar fraction to 32.25%. Further improving the 3HP molar fraction by increasing arabinose induction failed as the engineered malonyl-CoA pathway was not properly expressed under the high-level induction condition. With several promising advantages (broader range of economic oil substrates, no need for expensive supplementations such as alanine and VB12), this study indicated a candidate route for the industrial level production of [P(3HB-co-3HP)]. For future prospects, further studies are needed to further improve the strain and the fermentation process and expand the range of relative products.

Abstract Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

[Display omitted] • Integrated systematic bio-engineering with a temperature-sensitive switch to decouple cell growth from DPA production. • Enhanced pyruvate conservation by controlling glucose uptake, EMP, and TCA pathways. • Redirected carbon flux from the TCA cycle by lowering cultivation temperature from 37 °C to 30 °C. • Achieved DPA titer (97.2 g/L) and yield (0.64 g/g glucose) in 5-liter fed-batch fermentation, much higher than those in previous reports. For industrial strain construction, rational allocation of carbon flux is of paramount importance especially for decoupling cell growth and chemical productions to get maximum titer, rate, yield (TRY), which become Gordian Knot. Here, a temperature-sensitive switch and genetic circuits was used for effectively decoupling cell growth from D-pantothenic acid (DPA) production, along with systematically metabolic engineering including blocking redundant pathways of pyruvate and enhancing DPA driving force. Afterwards, rapid biomass accumulation only happened during growth stage, and subsequent high-efficient DPA production was initiated with reducing fermentation temperature. Finally, 97.20 g/L DPA and 0.64 g/g glucose conversion rate were achieved in 5-liter fed-batch fermentation. These undisputedly represent a milestone for the biosynthesis of DPA. With using strategies for decoupling cell growth from chemical productions, it would serve as "Alexander's sword" to cut Gordian Knot to get industrial chassis cells with excellent TRY for de novo biosynthesis of valuable chemicals. [ABSTRACT FROM AUTHOR]

Crocins are bioactive natural products that rarely exist in plants. High costs and resource shortage severely limit its development and application. Synthetic biology studies on crocins are of considerable global interest. However, the lack of high-efficiency genetic tools and complex cascade biocatalytic systems have substantially hindered progress in crocin biosynthesis-related research. Based on mutagenesis, a high-efficiency GjCCD4a mutant (N212m) was constructed with a catalytic efficiency that was 25.08-fold higher than that of the wild-type. Solubilized GjCCD4a was expressed via fusion with an MBP tag. Moreover, N212m and ten other genes were introduced into Escherichia coli for the de novo biosynthesis of five crocins. The engineered E57 strain produced crocins III and V with a total yield of 11.50 mg/L, and the E579 strain produced crocins I-V with a total output of 8.43 mg/L at shake-flask level. This study identified a marvelous genetic element (N212m) for crocin biosynthesis and achieved its de novo biosynthesis in E. coli using glucose. This study provides a reference for the large-scale production of five crocins using E. coli cell factories. [ABSTRACT FROM AUTHOR]

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio-alkane has gained attention as an ideal drop-in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof-of-principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α-dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C12-18 free fatty acids to C11-17 aldehydes. Co-expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole-cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232-237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Steroidal compounds are of great interest in the pharmaceutical field, with steroidal drugs as the second largest category of medicine in the world. Advances in synthetic biology and metabolic engineering have enabled de novo biosynthesis of sterols and steroids in yeast, which is a green and safe production route for these valuable steroidal compounds. In this review, we summarize the metabolic engineering strategies developed and employed for improving the de novo biosynthesis of sterols and steroids in yeast based on the regulation mechanisms, and introduce the recent progresses in de novo synthesis of some typical sterols and steroids in yeast. The remaining challenges and future perspectives are also discussed. [ABSTRACT FROM AUTHOR]

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p -coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures. [ABSTRACT FROM AUTHOR]

Lactones are volatile organic compounds widely present in foods. These chemicals are applied as flavors and fragrances in the food, cosmetics and pharmaceutical industries. Recently, the potential of lactones as green solvents and fuel precursors reinforced their role as platform compounds of future bio-based economies. However, their current mode of production needs to change. Lactones are mainly obtained through chemical synthesis or microbial biotransformation of hydroxy fatty acids. The latter approach is preferred but still needs to use more sustainable substrates. Hydroxy fatty acids are non-abundant and non-sustainable substrates from environmental, health and economic points of view. Therefore, it is urgent to identify and engineer microorganisms with the rare ability to biosynthesize lactones from carbohydrates or renewable lipids. Here, we firstly address the variety and importance of lactones. Then, the current understanding of the biosynthetic pathways involved in lactone biosynthesis is presented, making use of the knowledge acquired in microorganisms and fruits. From there, we present and make the distinction between biotransformation processes and de novo biosynthesis of lactones. Finally, the opportunities and challenges towards more sustainable production in addition to the relevance of two well-known industrial microbes, the filamentous fungus Ashbya gossypii and the yeast Yarrowia lipolytica, are discussed. [ABSTRACT FROM AUTHOR]

The industrially important meta-cresol (m-cresol, 3-methylphenol) is mainly produced from fossil resources by chemical methods. The microbial production of m-cresol was rarely investigated. Herein, we constructed a platform for the overproduction of m-cresol in a modified fungus Aspergillus nidulans FGSC no. A1145∆ST∆EM, which gave a gram-level titer using starch as carbon resource. For the biosynthesis of m-cresol, the 6-methyl salicylic acid synthase (MSAS)-encoding gene patK and 6-methyl salicylic acid decarboxylase-encoding gene patG from A. clavatus were co-expressed in the host A. nidulans. Multiple strategies, including promotor engineering, gene multiplication, and fed-batch fermentation, were applied to raise the production of m-cresol, which resulted in the titers of 1.29 g/L in shaking flasks and 2.03 g/L in fed-batch culture. The chassis cell A. nidulans A1145∆ST∆EM was proved to possess better tolerance to m-cresol than yeast, as it could grow in the liquid medium containing up to 2.5 g/L of m-cresol. These results showed that A. nidulans has great potential to be further engineered for industrial production of m-cresol. Key points • m-Cresol was de novo biosynthesized by a fungal chassis cell Aspergillus nidulans. • Promoter engineering and gene multiplication implemented the fine-tuned genes expression. • The titer of m-cresol reached 2.03 g/L via fed-batch culture. [ABSTRACT FROM AUTHOR]

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal , sam5 , com , and 4cl2nt , along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds. [ABSTRACT FROM AUTHOR]

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

Polychaetes can be successfully employed to recover essential fatty acids (EFA) from wasted uneaten aquafeeds present in aquaculture effluents. The optimization of the timeframe required to produce premium ragworms (Hediste diversicolor) biomass rich in EFA is paramount to make available to the aquafeeds industry another alternative ingredient to fish meal and fish oil. The present study aimed to evaluate the potential enrichment of ragworms fatty acid (FA) profile when fed a commercial aquafeed during 10, 20, and 40 days (D10, D20, and D40) under different combinations of water temperature (20 and 25°C) and salinity (15, 20, and 25). Total FA incremented progressively overtime, with D40 polychaetes exhibiting average values ranging between 70 and 90 μg mg–1 DW. The average values of n-6 FA ranged between 13 and 17 μg mg–1 DW, while that of n-3 FA varied between 17 and 19 μg mg–1 DW at D40. No significant differences were found in the FA profile of H. diversicolor cultured under different combinations of temperature and salinity. The FA profile of cultured polychaetes exhibited between 28 and 31% dissimilarity from that of wild conspecifics and displayed a higher content of two essential n-3 FA: eicosapentaenoic (20:5 n-3, EPA) and docosahexaenoic acids (22:6 n-3, DHA) (values ranging between 9.6–11.2% and 4.3–5.0% of total FA, respectively). A higher similarity in FA profile was recorded between D40 polychaetes and aquafeed than with initially stocked or wild specimens. Palmitic (16:0), oleic (18:1 n-9), linoleic (18:2 n-6), eicosadienoic (20:2 n-6), EPA (20:5 n-3), and DHA (22:6 n-3) were the FA whose concentration exhibited the highest increment. Evidence of de novo FA biosynthesis was observed through the formation of some FA that were neither present in the initially stocked biomass, nor in the aquafeed supplied, such as 5,11-eicosadienoate (Δ5,1120:2), 7,13,16-docosatrienoate (Δ7,13,1622:3), dihomo-gamma-linolenic (20:3 n-6), eicosatrienoic (20:3 n-3) and eicosatetraenoic (20:4 n-3) acids. A plateau of total FA, n-6, and n-3 FA was not reached over the study period. Overall, the present study highlights the potential of H. diversicolor as an extractive species for integrated multi-trophic aquaculture (IMTA) applications.

Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories. [ABSTRACT FROM AUTHOR]

Abstract Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.

Biofuels and biochemicals derived from renewable resources are sconsidered as potential solutions for the energy crisis and associated environmental problems that human beings are facing today. However, so far the available types of bioderived chemicals are rather limited, and production efficiency is generally low. Expanding the realm of bioderived chemicals and relevant derivatives can help motivate the development of bioenergy and the general bioeconomy. Aldehydes, possessing unique reactivity, hold great promise as platform chemicals for producing a large portfolio of bioproducts. In this review, we focus on production of aldehydes from renewable bioresources and derivatization of aldehydes through chemocatalysis, biocatalysis, or de novo biosynthesis. Perspectives on combining protein engineering and cascade reactions for advanced aldehyde derivatization are also provided. • Aldehydes, possessing unique reactivity, hold great promise as platform chemicals for producing various bioproducts. • Bioaldehydes can be produced from renewable bioresources including agriculture residuals and municipal wastes. • De novo biosynthesis and chemocatalysis or biocatalysis for aldehyde and derivative production were presented. • Advanced aldehyde derivatization can be achieved by combining protein engineering and cascade reactions. [ABSTRACT FROM AUTHOR]

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R-lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S-lipoic acid or precursors with undesired chirality. Toward sustainable production of R-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R-lipoic acid biosynthesis in S. cerevisiae. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R-lipoic acid production.

Flavonoids are a large family of plant and fungal natural products, among which many have been found to possess outstanding biological activities. Utilization of engineered microbes as surrogate hosts for heterologous biosynthesis of flavonoids has been investigated extensively. However, current microbial biosynthesis strategies mostly rely on using one microbial strain to accommodate the long and complicated flavonoid pathways, which presents a major challenge for production optimization. Here, we adapt the emerging modular co-culture engineering approach to rationally design, establish and optimize an Escherichia coli co-culture for de novo biosynthesis of flavonoid sakuranetin from simple carbon substrate glucose. Specifically, two E. coli strains were employed to accommodate the sakuranetin biosynthesis pathway. The upstream strain was engineered for pathway intermediate p-coumaric acid production, whereas the downstream strain converted p-coumaric acid to sakuranetin. Through step-wise optimization of the co-culture system, we were able to produce 29.7 mg/L sakuranetin from 5 g/L glucose within 48 h, which is significantly higher than the production by the conventional monoculture-based approach. The co-culture biosynthesis was successfully scaled up in a fed-batch bioreactor, resulting in the production of 79.0 mg/L sakuranetin. To our knowledge, this is the highest bioproduction concentration reported so far for de novo sakuranetin biosynthesis using the heterologous host E. coli. The findings of this work expand the applicability of modular co-culture engineering for addressing the challenges associated with heterologous biosynthesis of complex natural products. Key points: • De novo biosynthesis of sakuranetin was achieved using E. coli-E. coli co-cultures. • Sakuranetin production by co-cultures was significantly higher than the mono-culture controls. • The co-culture system was optimized by multiple metabolic engineering strategies. • The co-culture biosynthesis was scaled up in fed-batch bioreactor. [ABSTRACT FROM AUTHOR]

Warming occurs in the Arctic twice as fast as the global average, which in turn leads to a large enhancement in terpenoid emissions from vegetation. Volatile terpenoids are the main class of biogenic volatile organic compounds (VOCs) that play crucial roles in atmospheric chemistry and climate. However, the biochemical mechanisms behind the temperature‐dependent increase in VOC emissions from subarctic ecosystems are largely unexplored. Using 13CO2‐labeling, we studied the origin of VOCs and the carbon (C) allocation under global warming in the soil–plant–atmosphere system of contrasting subarctic heath tundra vegetation communities characterized by dwarf shrubs of the genera Salix or Betula. The projected temperature rise of the subarctic summer by 5°C was realistically simulated in sophisticated climate chambers. VOC emissions strongly depended on the plant species composition of the heath tundra. Warming caused increased VOC emissions and significant changes in the pattern of volatiles toward more reactive hydrocarbons. The 13C was incorporated to varying degrees in different monoterpene and sesquiterpene isomers. We found that de novo monoterpene biosynthesis contributed to 40%–44% (Salix) and 60%–68% (Betula) of total monoterpene emissions under the current climate, and that warming increased the contribution to 50%–58% (Salix) and 87%–95% (Betula). Analyses of above‐ and belowground 12/13C showed shifts of C allocation in the plant–soil systems and negative effects of warming on C sequestration by lowering net ecosystem exchange of CO2 and increasing C loss as VOCs. This comprehensive analysis provides the scientific basis for mechanistically understanding the processes controlling terpenoid emissions, required for modeling VOC emissions from terrestrial ecosystems and predicting the future chemistry of the arctic atmosphere. By changing the chemical composition and loads of VOCs into the atmosphere, the current data indicate that global warming in the Arctic may have implications for regional and global climate and for the delicate tundra ecosystems. [ABSTRACT FROM AUTHOR]

The aromatic amino acid biosynthesis pathway, together with its downstream branches, represents one of the most commercially valuable biosynthetic pathways, producing a diverse range of complex molecules with many useful bioactive properties. Aromatic compounds are crucial components for major commercial segments, from polymers to foods, nutraceuticals, and pharmaceuticals, and the demand for such products has been projected to continue to increase at national and global levels. Compared to direct plant extraction and chemical synthesis, microbial production holds promise not only for much shorter cultivation periods and robustly higher yields, but also for enabling further derivatization to improve compound efficacy by tailoring new enzymatic steps. This review summarizes the biosynthetic pathways for a large repertoire of commercially valuable products that are derived from the aromatic amino acid biosynthesis pathway, and it highlights both generic strategies and specific solutions to overcome certain unique problems to enhance the productivities of microbial hosts. • The aromatic amino acid biosynthesis pathway yields commercially valuable products. • The broad scope of products from aromatic amino acid biosynthesis is described. • Biosynthetic pathways and interconnections among the sub-branches are presented. • High-level organization of enzymes of the upstream module is illustrated. • The exemplary sophisticated regulations in the upstream pathway at various levels are highlighted. [ABSTRACT FROM AUTHOR]

The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p -hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species. • PYK1 D147N expression supplies an improved PEP flux towards 2PE synthesis. • TYR1 expression driven by YEN1pr results in >90% reduction of pOH2PE. • Expression of a Bb Xfpk and Ck pta shunt did not result in improve 2PE production. • 2PE production is limited by product inhibition. • The best performing strain produced 13mM of 2PE with a 0.113 mol mol-1 [ABSTRACT FROM AUTHOR]

Lactones are volatile organic compounds widely present in foods. These chemicals are applied as flavors and fragrances in the food, cosmetics and pharmaceutical industries. Recently, the potential of lactones as green solvents and fuel precursors reinforced their role as platform compounds of future bio-based economies. However, their current mode of production needs to change. Lactones are mainly obtained through chemical synthesis or microbial biotransformation of hydroxy fatty acids. The latter approach is preferred but still needs to use more sustainable substrates. Hydroxy fatty acids are non-abundant and non-sustainable substrates from environmental, health and economic points of view. Therefore, it is urgent to identify and engineer microorganisms with the rare ability to biosynthesize lactones from carbohydrates or renewable lipids. Here, we firstly address the variety and importance of lactones. Then, the current understanding of the biosynthetic pathways involved in lactone biosynthesis is presented, making use of the knowledge acquired in microorganisms and fruits. From there, we present and make the distinction between biotransformation processes and de novo biosynthesis of lactones. Finally, the opportunities and challenges towards more sustainable production in addition to the relevance of two well-known industrial microbes, the filamentous fungus Ashbya gossypii and the yeast Yarrowia lipolytica, are discussed.

Marmesin is essential in plant defense systems and exhibits various biological activities. In this study, we reconstituted the marmesin biosynthetic pathway in the Saccharomyces cerevisiae BY4741 chassis. We engineered the aromatic amino acid (AAA) biosynthetic pathways by introducing Escherichia coli -derived ppsA to improve the availability of the AAA precursor phosphoenolpyruvate, overexpressing the feedback inhibition resistance genes ARO4 K229L and ARO7 G141S to direct the metabolic flux toward the tyrosine branch, and deleting ARO10 , PDC5 , and PDC6 to reduce the byproducts from the Ehrlich pathway. The umbelliferone 6-dimethylallyltransferase (U6DT) and marmesin synthase (MS) involved in marmesin synthesis were optimized to increase marmesin production. Marmesin production was improved by truncating the transmembrane domains of PcU6DT , FcMS , and AtCPR1 and increasing the copy numbers of the genes encoding the truncated enzymes. Finally, a marmesin titer of 27.7 mg/L was obtained in shake flasks using the engineered yeast strain MU5. The constructed marmesin-producing strain provides the foundation for the green and large-scale production of pharmaceutically important furanocoumarins.

Obligate intracellular pathogens have coevolved with their host, leading to clever strategies to access nutrients, to combat the host’s immune response, and to establish a safe niche for intracellular replication. The host, on the other hand, has also developed ways to restrict the replication of invaders by limiting access to nutrients required for pathogen survival. In this review, we describe the recent advancements in both computational methods and high-throughput –omics techniques that have been used to study and interrogate metabolic functions in the context of intracellular parasitism. Specifically, we cover the current knowledge on the presence of amino acid biosynthesis and uptake within the Apicomplexa phylum, focusing on human-infecting pathogens: Toxoplasma gondii and Plasmodium falciparum. Given the complex multi-host lifecycle of these pathogens, we hypothesize that amino acids are made, rather than acquired, depending on the host niche. We summarize the stage specificities of enzymes revealed through transcriptomics data, the relevance of amino acids for parasite pathogenesis in vivo, and the role of their transporters. Targeting one or more of these pathways may lead to a deeper understanding of the specific contributions of biosynthesis versus acquisition of amino acids and to design better intervention strategies against the apicomplexan parasites.

• Mcl-PHA biosynthesis from different central metabolic routes was tested at low DO. • De novo mcl-PHA synthesis was not induced by oxygen limitation in P. putida LS46. • The content of monomers from β-oxidation intermediates was enriched at low DO. • The oxygen transfer rate could be used to control the content of certain monomers. Biosynthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) requires oxygen, but little is known of how mcl-PHA production is impacted in low dissolved oxygen (DO) environments, particularly when synthesized from monomers obtained via a de novo pathway. Growth and mcl-PHA synthesis characteristics under microaerophilic bioreactor conditions were observed in Pseudomonas putida LS46 when grown on carbon substrates requiring a de novo pathway, to varying extent, for mcl-PHA synthesis. Negligible PHA accumulated from glucose, acetic acid, or valeric acid under oxygen limitation (DO ≤ 0.15 mg L−1), while final mcl-PHA content reached 19.5% of cell dry mass (CDM) from hexanoic acid. The C6 content of the polymer obtained under low DO was 10% higher than in a DO excess (NH 4 -limited) control experiment, with less of the C10 monomers indicative of de novo synthesis. In all cases, the carbon-to-PHA yield was poor (< 0.1 C-mol C-mol−1). We conclude that, as opposed to utilizing β-oxidation intermediates, de novo biosynthesis of mcl-PHA using P. putida LS46 is not favorable in microaerophilic environments. In addition to scale-up implications, this work also suggests that lowering the bioreactor oxygen transfer rate can decrease the contribution of monomers incorporated using de novo synthesis for carbon feedstocks with mixed composition. [ABSTRACT FROM AUTHOR]

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0–C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.

Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed. [ABSTRACT FROM AUTHOR]

Background: Pterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems. Results: We identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular l-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of l-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using l-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-l-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis. Conclusion: As a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a l-methionine containing medium in combination with a strain that had an engineered metabolic pathway for l-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme. [ABSTRACT FROM AUTHOR]

3-Phenylpropionic acid (3PPA) and its derivative 3-phenylpropyl acetate (3PPAAc) are important aromatic compounds with broad applications in the cosmetics and food industries. In this study, we constructed a plasmid-free 3PPA-producing Escherichia coli strain and designed a novel 3PPAAc biosynthetic pathway. A module containing tyrosine ammonia lyase and enoate reductase, evaluated under the control of different promoters, was combined with phenylalanine-overproducing strain E. coli ATCC31884, enabling the plasmid-free de novo production of 218.16 ± 43.62 mg L -1 3PPA. The feasibility of the pathway was proved by screening four heterologous alcohol acetyltransferases, which catalyzed the transformation of 3-phenylpropyl alcohol into 3PPAAc. Afterward, 94.59 ± 16.25 mg L -1 3PPAAc was achieved in the engineered E. coli strain. Overall, we have not only demonstrated the potential of de novo synthesis of 3PPAAc in microbes for the first time but also provided a platform for the future of biosynthesis of other aromatic compounds.

Resveratrol is a well-known triphenolic natural product present in red wine. For its contribution to human health, the demand for resveratrol as a food and nutrition supplement has increased significantly. In recent years, the rapid development of synthetic biology has promoted extensive work to increase the production of resveratrol in microbes. However, supplementation of expensive phenylpropanoic precursors was required in current engineered strains. Here, we first utilized the site-specific integration strategy to produce resveratrol in Escherichia coli. The genes tal, 4cl and sts were site-specific integrated into the loci of genes tyrR and trpED in the chromosome of E. coli BW25113 (DE3). The final strain was capable of producing 4.612 mg L-1 of resveratrol from glucose. [ABSTRACT FROM AUTHOR]

An even medium-chain triglyceride ( MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase ( VLCAD) deficiency ( VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids ( MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient ( VLCAD−/−) mice after 1 year of supplementation as compared with a control diet. The de novo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT- PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC- MS. Long-term application of even and odd MCFAs strongly induced de novo biosynthesis and elongation of fatty acids in both wild-type and VLCAD−/− mice, leading to an alteration of the hepatic fatty acid profiles. We detected de novo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD−/− mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing. [ABSTRACT FROM AUTHOR]

Background: Styrene and its derivatives as monomers and petroleum-based feedstocks are valuable as raw materials in industrial processes. The chemical reaction for styrene production uses harsh reaction conditions such as high temperatures or pressures, or requires base catalysis with microwave heating. On the other hand, production of styrene and its derivatives in Escherichia coli is an environmental friendly process to produce conventional petroleum-based feedstocks. Results: An artificial biosynthetic pathway was developed in E. coli that yields 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene from simple carbon sources. This artificial biosynthetic pathway has a codonoptimized phenolic acid decarboxylase (pad) gene from Bacillus and some of the phenolic acid biosynthetic genes. E. coli strains with the tal and pad genes, the tal, sam5, and pad genes, and the tal, sam5, com, and pad genes produced 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. Furthermore, these pathways were expressed in a tyrosine overproducing E. coli. The yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation. Conclusions: Our system is the first to use E. coli with artificial biosynthetic pathways for the de novo synthesis of 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in a simple glucose medium. Similar approaches using microbial synthesis from simple sugar could be useful in the synthesis of plant-based aromatic chemicals. [ABSTRACT FROM AUTHOR]