De Novo Biosynthesis of 4-Vinylanisole in Engineered Escherichia coli.

4-Vinylanisole is an aggregation pheromone of the locust. Both gregarious and solitary locusts exhibit a strong attraction toward 4-vinylanisole, irrespective of gender or age. Therefore, 4-vinylanisole can be used for trapping and monitoring locusts. In this study, the construction of a de novo 4-vinylanisole pathway in Escherichia coli has been demonstrated for the first time. Subsequently, by increasing the supply of precursor substrates, we further improved the biosynthesis of 4-vinylanisole. Finally, a two-phase organic overlay culture was used to increase the titer to 206 mg/L. It presents a sustainable and ecofriendly alternative for the synthesis of 4-vinylanisole.

De Novo Biosynthesis of Anisyl Alcohol and Anisyl Acetate in Engineered Escherichia coli.

Anisyl alcohol and its ester anisyl acetate are both important fragrance compounds and have a wide range of applications in the cosmetics, perfumery, and food industries. The currently commercially available anisyl alcohol and anisyl acetate are based on chemical synthesis. However, consumers increasingly prefer natural fragrance compounds. Therefore, it is of great significance to construct microbial cell factories to produce anisyl alcohol and anisyl acetate. In this study, we first established a biosynthetic pathway in engineered Escherichia coli MG1655 for the production of anisyl alcohol from simple carbon sources. We further increased the anisyl alcohol production to 355 mg/L by the increasing availability of erythrose-4-phosphate and phosphoenolpyruvate. Finally, we further demonstrated the production of anisyl acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent acetylation of anisyl alcohol to produce anisyl acetate. To our knowledge, this is the first report on the biosynthesis of anisyl alcohol and anisyl acetate directly from a renewable carbon source.

De novo biosynthesis of N-acetyltyramine in engineered Escherichia coli.

N-acetyltyramine as a tyramine alkaloid has drawn great attention because of its excellent anti-free radical, antithrombotic, and antitumour activity. Therefore, it is an attractive compound. In this study, we reported for the first time the construction a synthetic pathway of N-acetyltyramine in engineered Escherichia coli. First, the tyrosine decarboxylase tdc gene and arylalkylamine N-acyltransferase aanat gene were introduced into E. coli to generate a recombinant N-acetyltyramine producer with L-tyrosine as substrate. Subsequently, overexpressing aroGfbr and TyrAfbr enhance the availability of L-tyrosine to achieve de novo biosynthesis of N-acetyltyramine from glucose. Finally, overexpressing the transketolase I tktA and phosphoenolpyruvate synthase ppsA genes improved the N-acetyltyramine production to 854 mg/L.

De Novo Biosynthesis of Methyl Cinnamate in Engineered Escherichia coli.

Methyl cinnamate with a fruity balsamic odor is an important fragrance ingredient in perfumes and cosmetics. Chemical processes are currently the only means of producing methyl cinnamate. But consumers prefer natural flavors. Therefore, it is necessary to design and develop microbial cell factories for the production of methyl cinnamate. In this study, we established for the first time a biosynthetic pathway in engineered Escherichia coli for production of methyl cinnamate from glucose. We further increased the methyl cinnamate production to 302 mg/L by increasing the availability of the metabolic precursors. Finally, the titer was increased to 458 mg/L in a two-phase culture system.

De novo biosynthesis of τ-cadinol in engineered Escherichia coli.

τ-Cadinol is a sesquiterpene that is widely used in perfume, fine chemicals and medicines industry. In this study, we established a biosynthetic pathway for the first time in engineered Escherichia coli for production of τ-cadinol from simple carbon sources. Subsequently, we further improved the τ-cadinol production to 35.9 ± 4.3 mg/L by optimizing biosynthetic pathway and overproduction of rate-limiting enzyme IdI. Finally, the titer was increased to 133.5 ± 11.2 mg/L with a two-phase organic overlay-culture medium system. This study shows an efficient method for the biosynthesis of τ-cadinol in E. coli with the heterologous hybrid MVA pathway.

Biosynthesis of actarit using engineered Escherichia coli.

Actarit is widely regarded as a safe and effective drug for the treatment of rheumatoid arthritis. There is no report on the bioproductin of actarit so far. In this study, we demonstrated for the first time the development of an artificial actarit biosynthetic pathway in Escherichia coli. First, 4-aminophenylacetic acid is selected as precursor substrates for the production of actarit. Second, an N-acetyltransferase that can efficiently catalyse the esterification of acetyl-CoA and 4-aminophenylacetic acid to form actarit was discovered. Subsequently, an engineered E. coli that allows production of actarit from simple carbon sources was established. Finally, we further increased the production of actarit to 206 ± 16.9 mg/L by overexpression of shikimate dehydrogenase ydiB and shikimate kinase aroK.

De novo biosynthesis of tyrosol acetate and hydroxytyrosol acetate from glucose in engineered Escherichia coli.

Tyrosol and hydroxytyrosol derived from virgin olive oil and olives extract, have wide applications both as functional food components and as nutraceuticals. However, they have low bioavailability due to their low absorption and high metabolism in human liver and small intestine. Acetylation of tyrosol and hydroxytyrosol can effectively improve their bioavailability and thus increase their potential use in the food and cosmeceutical industries. There is no report on the bioproductin of tyrosol acetate and hydroxytyrosol acetate so far. Thus, it is of great significance to develop microbial cell factories for achieving tyrosol acetate or hydroxytyrosol acetate biosynthesis. In this study, a de novo biosynthetic pathway for the production of tyrosol acetate and hydroxytyrosol acetate was constructed in Escherichia coli. First, an engineered E. coli that allows production of tyrosol from simple carbon sources was established. Four aldehyde reductases were compared, and it was found that yeaE is the best aldehyde reductase for tyrosol accumulation. Subsequently, the pathway was extended for tyrosol acetate production by further overexpression of alcohol acetyltransferase ATF1 for the conversion of tyrosol to tyrosol acetate. Finally, the pathway was further extended for hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase HpaBC.

Development of an artificial biosynthetic pathway for biosynthesis of (S)-reticuline based on HpaBC in engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionally these compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)-Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)-reticuline biosynthetic pathway based on 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) in Escherichia coli. Then, we further improved the (S)-reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4-dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.

De novo biosynthesis of linalool from glucose in engineered Escherichia coli.

Linalool is an important terpenoids of floral scents and has wide applications. In the past, several groups reported on a strategy to establish biosynthesis of linalool in yeast based on co-expression of Saccharomyces cerevisiae farnesyl diphosphate synthase ERG20 and Actinidia arguta linalool synthase LIS. However, ERG20 has both geranyl diphosphate synthase and farnesyl diphosphate synthase activities, which can lead to metabolic flow to farnesyl diphosphate. In this study, a heterologous linalool biosynthesis pathway was constructed in Escherichia coli and showed that using Abies grandis geranyl diphosphate synthase GPPS2 instead of ERG20 can effectively improve linalool biosynthesis. Subsequently, we further improved the biosynthesis of linalool by overexpression of isopentenyl diphosphate isomerase Idi.

De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris.

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odor, which is widely used in cosmetics and food industries. Natural 2-PE is costly and cannot meet the market demand due to the relative low content of 2-PE in the plants. Thus, there is an increasing interest in the search for alternative routes for 2-PE production. Here we demonstrate the engineering of Pichia pastoris to produce 2-PE directly from simple sugars for the first time. We first demonstrated that improving downstream pathway from phenylpyruvate to 2-PE by overexpressing ARO10 and ADH6 could increase the biosynthesis of 2-PE. Then several genetic engineering strategies were developed to increase phenylpyruvate availability to improve 2-PE production. 1169 mg/L of 2-PE was accumulated in the final engineered strain. This study showed the potential of P. pastoris as a host strain to produce industrially interested 2-PE by metabolic engineering.

De Novo Biosynthesis of Indole-3-acetic Acid in Engineered Escherichia coli.

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.

Biosynthesis of nerol from glucose in the metabolic engineered Escherichia coli.

In this study, nerol was biosynthesized in the metabolic engineered Escherichia coli from glucose for the first time. Firstly, the truncated neryl diphosphate synthase gene tNDPS1 was expressed that catalyzes isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form neryl diphosphate (NPP), and then the nerol synthase gene GmNES was co-expressed to synthesize the final product nerol from NPP. The engineered strain LZ001 accumulated 0.053 ±â€¯0.015 mg/L of nerol. Secondly, the IDI1, MVD1, ERG8, ERG12, tHMG1 and ERG13 were co-expressed to increase the supply of IPP and DMAPP. Finally, the heterologous ERG10 gene was overexpressed, and the recombinant strain LZ005 produced 1.564 ±â€¯0.102 mg/L of nerol in shaking-flask culture, which represents a 29.51-fold increase over LZ001 strain. This study shows the novel method for the biosynthesis of nerol and provides new metabolic engineering strategy for the production of terpenoids.

Biosynthesis of advanced biofuel farnesyl acetate using engineered Escherichia coli.

Diminishing petroleum reserves and the rapid accumulation of greenhouse gases lead to increasing interest in microbial biofuels. In this study, a heterologous farnesyl acetate biosynthesis pathway was constructed in Escherichia coli for the first time. Firstly, the AtoB, ERG13, tHMG1, ERG12, ERG8, MVD1, Idi, IspA and PgpB were expressed to accumulate farnesol in the E. coli cells. Then the alcohol acetyltransferase (ATF1) was heterologous overexpressed for the subsequent esterification farnesol to farnesyl acetate. The engineered strain DG 106 accumulated 128 ±â€¯10.5 mg/L of farnesyl acetate. Finally, the isopentenyl-diphosphate isomerase was further overexpressed, and the recombinant strain DG107 produced 201 ±â€¯11.7 mg/L of farnesyl acetate. This study shows the novel method for the biosynthesis of the advanced biofuel farnesyl acetate directly from glucose and highlight the enormous designing strategies for metabolic engineering of bioproducts.

Metabolic Engineering of Escherichia coli for Production of 2-Phenylethanol and 2-Phenylethyl Acetate from Glucose.

Rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester 2-phenylethyl acetate (2-PEAc) are two important aromatic compounds and have wide applications. In the past, 2-PE and 2-PEAc were mainly produced from l-phenylalanine. In this study, Escherichia coli was engineered to de novo biosynthesis of 2-PE and 2-PEAc from glucose: first, overexpression of deregulated 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase aroG fbr and chorismate mutase/prephenate dehydratase pheA fbr for increasing phenylpyruvate production in E. coli, subsequently, heterologous expression of decarboxylase kdc and overexpression of reductase yjgB for the conversion of phenylpyruvate to 2-PE, with the engineered strain DG01 producing 578 mg/L 2-PE, and, finally, heterologous expression of an aminotransferase aro8 to redirect the metabolic flux to phenylpyruvate. 2-PE (1016 mg/L) was accumulated in the engineered strain DG02. Alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae can esterify a wide variety of alcohols, including 2-PE. We have further demonstrated the biosynthesis of 2-PEAc from glucose by overexpressing atf1 for the subsequent conversion of 2-PE to 2-PEAc. The engineered strain DG03 produced 687 mg/L 2-PEAc.

Biosynthesis of the fatty acid isopropyl esters by engineered Escherichia coli.

The fatty acid methyl esters and fatty acid ethyl esters are known as biodiesels which are considered to be renewable, nontoxic and biodegradable biofuels. However, the conventional biodiesels show a high crystallization temperature which is one of the most critical obstacles against the widespread biodiesel usage. The high crystallization temperature of biodiesel can be reduced by replacing the methyl or ethyl ester with an isopropyl moiety. Here we report on a strategy to establish biosynthesis of the fatty acid isopropyl esters(FAIPEs) from the simple substrate glucose in Escherichia coli with heterologous coexpression of atoB encoded acetyl-CoA acetyltransferase and atoAD encode acetoacetyl-CoA transferase from E. coli, ADC encode acetoacetate decarboxylase from Clostridium acetobutylicum, ADH encoded NADP-dependent alcohol dehydrogenase from Clostridium beijerinckii, 'TesA encoded a truncated fatty acyl-ACP thioesterase and FadD encoded fatty acyl-CoA synthetase from E. coli, and the WS/DGAT encoded acyltransferase from Acinetobacter baylyi strain ADP1. It was found that the yield of FAIPEs was up to 203.4mg/L and accounted for around 6.4% (wt/wt) of the dry cell weight. Our results indicates that it is a feasible strategy to improve the yield of FAIPEs by increasing fatty acyl-CoA availability in biosynthetic pathway and exhibit a promising method for production of biodiesels with good low-temperature flow properties.

Metabolic engineering of Escherichia coli to high efficient synthesis phenylacetic acid from phenylalanine.

Phenylacetic acid (PAA) is a fine chemical with a high industrial demand for its widespread uses. Whereas, microorganic synthesis of PAA is impeded by the formation of by-product phenethyl alcohol due to quick, endogenous, and superfluous conversion of aldehydes to their corresponding alcohols, which resulted in less conversation of PAA from aldehydes. In this study, an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that does duty for a platform for aromatic aldehyde biosynthesis was used to prompt more PAA biosynthesis. We establish a microbial biosynthetic pathway for PAA production from the simple substrate phenylalanine in E. coli with heterologous coexpression of aminotransferase (ARO8), keto acid decarboxylase (KDC) and aldehyde dehydrogenase H (AldH) gene. It was found that PAA transformation yield was up to ~94% from phenylalanine in E. coli and there was no by-product phenethyl alcohol was detected. Our results reveal the high efficiency of the RARE strain for production of PAA and indicate the potential industrial applicability of this microbial platform for PAA biosynthesis.

Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine.

In order to meet the need of consumer preferences for natural flavor compounds, microbial synthesis method has become a very attractive alternative to the chemical production. The 2-phenylethanol (2-PE) and its ester 2-phenylethylacetate (2-PEAc) are two extremely important flavor compounds with a rose-like odor. In recent years, Escherichia coli and yeast have been metabolically engineered to produce 2-PE. However, a metabolic engineering approach for 2-PEAc production is rare. Here, we designed and expressed a 2-PEAc biosynthetic pathway in E. coli. This pathway comprised four steps: aminotransferase (ARO8) for transamination of L-phenylalanine to phenylpyruvate, 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde, aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-PE, alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc. Using the engineered E. coli strain for shake flasks cultivation with 1 g/L L-phenylalanine, we achieved co-production of 268 mg/L 2-PEAc and 277 mg/L 2-PE. Our results suggest that approximately 65% of L-phenylalanine was utilized toward 2-PEAc and 2-PE biosynthesis and thus demonstrate potential industrial applicability of this microbial platform.

Metabolic engineering of Escherichia coli for production of biodiesel from fatty alcohols and acetyl-CoA.

Microbial production of biodiesel from renewable feedstock has attracted intensive attention. Biodiesel is known to be produced from short-chain alcohols and fatty acyl-CoAs through the expression of wax ester synthase/fatty acyl-CoA: diacylglycerol acyltransferase that catalyzes the esterification of short-chain alcohols and fatty acyl-CoAs. Here, we engineered Escherichia coli to produce various fatty alcohol acetate esters, which depend on the expression of Saccharomyces cerevisiae alcohol acetyltransferase ATF1 that catalyzes the esterification of fatty alcohols and acetyl-CoA. The fatty acid biosynthetic pathways generate fatty acyl-ACPs, fatty acyl-CoAs, or fatty acids, which can be converted to fatty alcohols by fatty acyl-CoA reductase, fatty acyl-ACP reductase, or carboxylic acid reductase, respectively. This study showed the biosynthesis of biodiesel from three fatty acid biosynthetic pathway intermediates.

Metabolic engineering of microbes for branched-chain biodiesel production with low-temperature property.

BACKGROUND: The steadily increasing demand for diesel fuels calls for renewable energy sources. This has attracted a growing amount of research to develop advanced, alternative biodiesel worldwide. Several major disadvantages of current biodiesels are the undesirable physical properties such as high viscosity and poor low-temperature operability. Therefore, there is an urgent need to develop novel and advanced biodiesels. RESULTS: Inspired by the proven capability of wax ester synthase/acyl-coenzyme A, diacylglycerol acyltransferase (WS/DGAT) to generate fatty acid esters, de novo biosynthesis of fatty acid branched-chain esters (FABCEs) and branched fatty acid branched-chain esters (BFABCEs) was performed in engineered Escherichia coli through combination of the (branched) fatty acid biosynthetic pathway and the branched-chain amino acid biosynthetic pathway. Furthermore, by modifying the fatty acid pathway, we improved FABCE production to 273 mg/L and achieved a high proportion of FABCEs at 99.3 % of total fatty acid esters. In order to investigate the universality of this strategy, Pichia pastoris yeast was engineered and produced desirable levels of FABCEs for the first time with a good starting point of 169 mg/L. CONCLUSIONS: We propose new pathways of fatty acid ester biosynthesis and establish proof of concept through metabolic engineering of E. coli and P. pastoris yeast. We were able to produce advanced biodiesels with high proportions FABCEs and BFABCEs. Furthermore, this new strategy promises to achieve advanced biodiesels with beneficial low-temperature properties.

Expression, characterization and crystal structure of thioredoxin from Schistosoma japonicum.

Schistosoma japonicum, a human blood fluke, causes a parasitic disease affecting millions of people in Asia. Thioredoxin-glutathione system of S. japonicum plays a critical role in maintaining the redox balance in parasite, which is a potential target for development of novel antischistosomal agents. Here we cloned the gene of S. japonicum thioredoxin (SjTrx), expressed and purified the recombinant SjTrx in Escherichia coli. Functional assay shows that SjTrx catalyses the dithiothreitol (DTT) reduction of insulin disulphide bonds. The coupling assay of SjTrx with its endogenous reductase, thioredoxin glutathione reductase from S. japonicum (SjTGR), supports its biological function to maintain the redox homeostasis in the cell. Furthermore, the crystal structure of SjTrx in the oxidized state was determined at 2.0 Å resolution, revealing a typical architecture of thioredoxin fold. The structural information of SjTrx provides us important clues for understanding the maintenance function of redox homeostasis in S. japonicum and pathogenesis of this chronic disease.