Artificial Biosynthetic Pathway Research Articles

Recent Advances and Challenges in the Production of Hydroxylated Natural Products Using Microorganisms

Hydroxylation reaction is a significant source of structural diversity in natural products (NPs), playing a crucial role in improving the bioactivity, solubility, and stability of natural product molecules. This review summarizes the latest research progress in the field of natural product hydroxylation, focusing on several key hydroxylases involved in the biosynthesis of NPs, including cytochrome P450 monooxygenases, α-ketoglutarate-dependent hydroxylases, and flavin-dependent monooxygenases. These enzymes achieve selective hydroxylation modification of various NPs, such as terpenoids, flavonoids, and steroids, through different catalytic mechanisms. This review systematically summarizes the recent advances on the hydroxylation of NPs, such as amino acids, steroids, terpenoids, lipids, and phenylpropanoids, demonstrating the potential of synthetic biology strategies in constructing artificial biosynthetic pathways and producing hydroxylated natural product derivatives. Through metabolic engineering, enzyme engineering, genetic engineering, and synthetic biology combined with artificial intelligence-assisted technologies, a series of engineered strains have been successfully constructed for the efficient production of hydroxylated NPs and their derivatives, achieving efficient synthesis of hydroxylated NPs. This has provided new avenues for drug development, functional food, and biomaterial production and has also offered new ideas for the industrial production of these compounds. In the future, integrating artificial synthetic pathway design, enzyme directed evolution, dynamic regulation, and artificial intelligence technology is expected to further expand the application of enzyme-catalyzed hydroxylation reactions in the green synthesis of complex NPs, promoting research on natural product hydroxylation to new heights.

Microbial production of 1, 3-propanediol: a transition of feedstocks from C6 to C3 and C1 carbon sources

1, 3-propanediol (1, 3-PDO) is an important diol with wide applications in the pharmaceutical, food, and cosmetics industries. In addition, 1, 3-PDO serves as a crucial monomer in the synthesis of polytrimethylene terephthalate, an important synthetic fiber material. Microbial conversion of renewable resources such as glucose into 1, 3-PDO has been industrialized due to its environmentally friendly, energy-efficient, safe, and sustainable characteristics. It serves as a successful case in the design and application of microbial cell factories for biochemicals. However, concerns such as food scarcity and climate change are driving the exploration of non-food, low-cost, and sustainable alternatives as biomanufacturing feedstocks. The biosynthesis of 1, 3-PDO from the C3 feedstock glycerol by microorganisms has been well studied. In recent years, increasing attention has been paid to the synthesis of 1, 3-PDO from C1 feedstocks such as methanol, which has higher energy density than glucose and glycerol. Several new artificial biosynthetic pathways have been proposed and validated, laying a foundation for the sustainable bioproduction of 1, 3-PDO. This article reviews the feedstock transition from C6 to C3 and C1 carbon sources for the microbial synthesis of 1, 3-PDO and discusses the strategies for reprogramming metabolic pathway to enhance 1, 3-PDO biosynthesis from different feedstocks. Finally, the development prospects of 1, 3-PDO bioproduction from C1 feedstocks are forecasted.

Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum

Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpDA162D mutation that exhibits feedback-resistant against l-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpDA162D in C. glutamicum TP851 strain with two copies of trpDA162D in the genome led to the production of 3.1 g/L l-tryptophan in flask culture. Subsequent step for l-tryptophan chlorination into 7-chloro-l-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-l-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-l-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the l-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.

Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

BackgroundMicrobial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum.ResultsSix different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum.ConclusionsIdentification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

Cell-specific bioorthogonal tagging of glycoproteins

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

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

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

Recent advances in microbial co-culture for production of value-added compounds.

Micro-organisms have often been used to produce bioactive compounds as antibiotics, antifungals, and anti-tumors, etc. due to their easy and applicable culture, genetic manipulation, and extraction, etc. Mainly, microbial mono-cultures have been applied to produce value-added compounds and gotten numerous valuable results. However, mono-culture also has several complicated problems, such as metabolic burdens affecting the growth and development of the host, leading to a decrease in titer of the target compound. To circumvent those limitations, microbial co-culture has been technically developed and gained much interest compared to mono-culture. For example, co-culture simplifies the design of artificial biosynthetic pathways and restricts the recombinant host's metabolic burden, causing increased titer of desired compounds. This paper summarizes the recent advanced progress in applying microbial platform co-culture to produce natural products, such as flavonoid, terpenoid, alkaloid, etc. Furthermore, importantly different strategies for enhancing production, overcoming the metabolic burdens, building autonomous modulation of cell growth rate and culture composition in response to a quorum-sensing signal, etc., were also described in detail.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Development of Artificial Synthetic Pathway of Endophenazines in Pseudomonas chlororaphis P3.

Simple SummaryTerpenoid phenazines generally produced in Streptomyces exhibit potential antitumor and antibacterial activities. In this study, we designed and constructed an artificial biosynthetic pathway for the synthesis of terpenoid phenazines in Pseudomonas chlororaphis P3. We successfully synthesized endophenazine A and endophenazine A1 for the first time in Pseudomonas by introducing the prenyltransferase PpzP. Moreover, we revealed the biosynthetic pathway of endophenazine A1 in P. chlororaphis P3. This study enriches the diversity of phenazines in P. chlororaphis P3 and provides a reference for the heterologous synthesis of terpenoid phenazines.Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in Streptomyces, limiting its large-scale industrial development. Pseudomonas chlororaphis P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P. chlororaphis P3. Primarily, the prenyltransferase PpzP from Streptomyces anulatus 9663 was introduced into P. chlororaphis P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P. chlororaphis P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.

Metabolic Division in an Escherichia coli Coculture System for Efficient Production of Kaempferide.

Kaempferide, a plant-derived natural flavonoid, exhibits excellent pharmacological activities with nutraceutical and medicinal applications in human healthcare. Efficient microbial production of complex flavonoids suffers from metabolic crosstalk and burden, which is a big challenge for synthetic biology. Herein, we identified 4'-O-methyltransferases and divided the artificial biosynthetic pathway of kaempferide into upstream, midstream, and downstream modules. By combining heterologous genes from different sources and fine-tuning the expression, we optimized each module for the production of kaempferide. Furthermore, we designed and evaluated four division patterns of synthetic labor in coculture systems by plug-and-play modularity. The linear division of three modules in a three-strain coculture showed higher productivity of kaempferide than that in two-strain cocultures. The U-shaped division by co-distributing the upstream and downstream modules in one strain led to the best performance of the coculture system, which produced 116.0 ± 3.9 mg/L kaempferide, which was 510, 140, and 50% higher than that produced by the monoculture, two-strain coculture, and three-strain coculture with the linear division, respectively. This is the first report of efficient de novo production of kaempferide in a robust Escherichia coli coculture. The strategy of U-shaped pathway division in the coculture provides a promising way for improving the productivity of valuable and complex natural products.

Combined active pocket and hinge region engineering to develop an NADPH-dependent phenylglycine dehydrogenase

NADPH-dependent amino acid dehydrogenases (AADHs) are favorable enzymes to construct artificial biosynthetic pathways in whole-cell for high-value noncanonical amino acids (NcAAs) production. Glutamate dehydrogenases (GluDHs) represent attractive candidates for the development of novel NADPH-dependent AADHs. Here, we report the development of a novel NADPH-dependent phenylglycine dehydrogenase by combining active pocket engineering and hinge region engineering of a GluDH from Pseudomonas putida (PpGluDH). The active pocket of PpGluDH was firstly tailored to optimize its binding mode with bulky substrate α-oxobenzeneacetic acid (α-OA), and then, the hinge region was further engineered to tune the protein conformational dynamics, which finally resulted in a mutant M3 (T196A/T121I/L123D) with a 103-fold increase of catalytic efficiency (kcat/Km) toward α-OA. The M3 mutant exhibited high catalytic performance in both in vitro biocatalysis preparation and in vivo biosynthesis of l-phenylglycine, indicating its promising practical applications. Our results demonstrated that co-engineering of the active pocket and hinge region is an effective strategy for developing novel NADPH-dependent AADHs from GluDHs for NcAAs production.

De Novo Biosynthesis and Whole-Cell Catalytic Production of 2-Acetamidophenol in Escherichia coli.

2-Acetamidophenol (AAP) is an aromatic product with promising activities in agricultural applications and medical research. At present, AAP is synthesized by chemical methods from nonrenewable fossil fuel resources, which cause environmental pollution and the reaction conditions are harsh. In this study, we constructed the artificial biosynthetic pathway of AAP with five different expressed proteins in Escherichia coli for the first time. By introducing the hydrogen peroxide degrading enzyme catalase and improving cell tolerance to toxic intermediates or products, the yield of AAP reached 33.54 mg/L using shaking-flask culture. The best-engineered strain could produce 568.57 mg/L AAP by fed-batch fermentation from glucose and precursor (2-aminophenol, 2-AP) addition. Furthermore, a one-pot whole-cell cascade biocatalytic pathway to AAP and analogues was developed and optimized. This method can efficiently produce 1.8 g/L AAP using the methyl anthranilate hydrolysis product as the substrate. This study provides not only the de novo artificial biosynthetic pathway of AAP in E. coli but also a promising sustainable and efficient strategy to enable the synthesis of AAP on a gram scale.

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

Design and construction of an artificial pathway for biosynthesis of acetaminophen in Escherichia coli

Acetaminophen (AAP) is one of the most commonly used drug ingredients that possesses antipyretic and analgesic effects. As an unnatural chemical, AAP is commercially produced by chemical processes using petroleum-derived carbohydrates, such as phenol, as raw materials, which is unsustainable and eco-unfriendly. In this study, we report design and construction of an artificial biosynthetic pathway for de novo production of AAP from simple carbon source. By exploring and expanding the substrate repertoire of natural enzymes, we identified and characterized a novel p-aminobenzoic acid (p-ABA) monooxygenase and an p-aminophenol (p-AP) N-acetyltransferase, which enabled the bacterial production of AAP from p-ABA. Then, we constructed an p-ABA over-producer by screening of p-ABA synthases and enhancing glutamine availability, resulting in 836.43 mg/L p-ABA in shake flasks in E. coli. Subsequent assembly of the entire biosynthetic pathway permitted the de novo production of AAP from glycerol for the first time. Finally, pathway engineering by dynamically regulating the expression of pathway genes via a temperature-inducible controller enabled production enhancement of AAP with a titer of 120.03 mg/L. This work not only constructs a microbial platform for AAP production, but also demonstrates design and construction of artificial biosynthetic pathways via discovering novel bioreactions based on existing enzymes.

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, traditionallythese 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.

Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli.

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.

Modular metabolic engineering for production of phloretic acid, phloretin and phlorizin in Escherichia coli

Phloretic acid, phloretin and phlorizin (phloretin 2′-O-glucoside), plant-derived natural products, are well known for human health benefits and medical application. Here, we construct an artificial biosynthetic pathway of phlorizin by modular engineering strategy and realize the production of phloretic acid, phloretin and phlorizin in Escherichia coli. Extending from L-tyrosine biosynthetic pathway, we firstly established the phloretic acid module comprised of heterologous TAL and ER enzymes, and highest titer of phloretic acid was achieved from glucose. Then, through biochemical screening of functional microbial Aa4CL and structure-based mutagenesis of MdCHS3 in phloretin module, we successfully constructed the phloretin biosynthetic pathway. Finally, we tested several glycosyltransferases in phlorizin module, and phloretic acid was bioconverted to phlorizin. This is the first report on biosynthesis of phloretin and phlorizin in engineered E. coli, indicating the promising potential of microbial cell factories to produce natural and unnatural dihydrochalcones.