scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Vitamin B ◽  
E Coli ◽  
De Novo Biosynthesis ◽  
Optimization Of Fermentation ◽  
Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

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

2021 ◽  
Vol 12 ◽  
Author(s):  
Kyung Taek Heo ◽  
Byeongsan Lee ◽  
Jae-Hyuk Jang ◽  
Jung-Oh Ahn ◽  
Young-Soo Hong
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Chain Elongation ◽  
Piper Nigrum ◽  
Glucose Medium ◽  
E Coli ◽  
Acid Catalyzed ◽  
Simple Sugar

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.

scholarly journals Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Huan Fang ◽  
Dong Li ◽  
Jie Kang ◽  
Pingtao Jiang ◽  
Jibin Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Vitamin B12 ◽  
De Novo ◽  
De Novo Biosynthesis

scholarly journals Transport engineering for improving production and secretion of valuable alkaloids in Escherichia coli

2021 ◽  
Author(s):  
Yasuyuki Yamada ◽  
Miya Urui ◽  
Hidehiro Oki ◽  
Kai Inoue ◽  
Haruyuki Matsui ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Plasmid Stability ◽  
Plant Metabolites ◽  
E Coli ◽  
Specialized Metabolites ◽  
Important Intermediate ◽  
High Production ◽  
Suitable Expression

AbstractMetabolic engineering of microorganisms to produce specialized plant metabolites has been established. However, these methods are limited by low productivity and the intracellular accumulation of metabolites. Here, we aimed to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. We established a reticuline-producing Escherichia coli strain and introduced a multidrug and toxic compound extrusion transporter, Arabidopsis AtDTX1, into it. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 significantly enhanced reticuline production by 11-fold; produced reticuline was secreted into the medium. AtDTX1 expression conferred high plasmid stability, and up- or downregulated genes associated with biological processes including metabolic pathways for reticuline biosynthesis, leading to a high production and secretion of reticuline. The successful application of a transporter for alkaloid production suggests that the transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

scholarly journals Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Quentin M Dudley ◽  
Connor J Nash ◽  
Michael C Jewett
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Enzymatic Synthesis ◽  
Biosynthetic Pathway ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Design Criteria ◽  
Acetyl Coa ◽  
E Coli ◽  
Enzymatic Pathways

Abstract Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l−1) limonene over 24 h, a productivity of 3.8 mg l−1 h−1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

scholarly journals Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

2016 ◽  
Vol 113 (12) ◽  
pp. 3209-3214 ◽  
Author(s):  
Bradley Walters Biggs ◽  
Chin Giaw Lim ◽  
Kristen Sagliani ◽  
Smriti Shankar ◽  
Gregory Stephanopoulos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Complex Molecules ◽  
E Coli ◽  
Precursor Synthesis ◽  
P450 Expression ◽  
High Level

Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

scholarly journals Flavin Adenine Dinucleotide-Dependent 4-Phospho-d-Erythronate Dehydrogenase Is Responsible for the 4-Phosphohydroxy-l-Threonine Pathway in Vitamin B6 Biosynthesis in Sinorhizobium meliloti

2006 ◽  
Vol 188 (13) ◽  
pp. 4635-4645 ◽  
Author(s):  
Masaaki Tazoe ◽  
Keiko Ichikawa ◽  
Tatsuo Hoshino
Keyword(s):  
Escherichia Coli ◽  
Vitamin B6 ◽  
Sinorhizobium Meliloti ◽  
Biosynthetic Pathway ◽  
Flavin Adenine Dinucleotide ◽  
Resonance Spectroscopy ◽  
Vitamin B ◽  
Major Pathway ◽  
E Coli ◽  
K 12

ABSTRACT The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-d-xylulose 5-phosphate and 4-phosphohydroxy-l-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-d-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-α-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to d-erythrulose, d-erythrulose 4-phosphate, and d-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and d-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.

scholarly journals Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Xueying Wang ◽  
Yongjin J. Zhou ◽  
Lei Wang ◽  
Wujun Liu ◽  
Yuxue Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Nicotinic Acid ◽  
De Novo ◽  
Colorimetric Assay ◽  
Reduced Form ◽  
Content Type ◽  
E Coli ◽  
Nad Biosynthesis ◽  
Nicotinamide Mononucleotide

ABSTRACT NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.

scholarly journals Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate l-pipecolic acid in Escherichia coli

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Hanxiao Ying ◽  
Sha Tao ◽  
Jing Wang ◽  
Weichao Ma ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Pathway ◽  
De Novo ◽  
Pipecolic Acid ◽  
De Novo Biosynthesis

scholarly journals Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5391-5397 ◽  
Author(s):  
Si Jae Park ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Biosynthetic Pathway ◽  
Pha Synthase ◽  
Enzymatic Analysis ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Enoyl Coa Hydratase

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

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