Tools of pathway reconstruction and production of economically relevant plant secondary metabolites in recombinant microorganisms

Dziggel, Clarissa; Schäfer, Holger; Wink, Michael

doi:10.1002/biot.201600145

Cited by 37 publications

(24 citation statements)

References 121 publications

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“…6.6% of lovastatin residue isn't a small number for the following separation and purification at the industrial scale. Several strategies can be applied to improve the in vivo hydrolysis efficiency to minimize lovastatin residues, such as regulation of PcEST expression through codon and promoter optimization, increase of PcEST catalytic efficiency through rational design and directed evolution of the enzyme, and adjustment of fermentation process. Here we adopted a directed evolution strategy.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

et al. 2018

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Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 °C increased T over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale.

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Section: Discussionmentioning

confidence: 99%

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

et al. 2018

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“…Bioengineering the genes of plants and microbes is becoming a very competitive strategy in synthesis to produce pharmacologically relevant plant natural products and their unnatural analogues. During the last 20 years, several plant genes have been identified, cloned, and expressed successfully, and this has unlocked de novo production of plant metabolites such as morphine, strictosidine, artemisinin, Taxol, and resveratrol . Future development for accelerating the discovery of gene candidates relies on innovative computational methods combined with transcriptomics and metabolomics analysis to make plant genome mining more feasible .…”

Section: Methodsmentioning

confidence: 99%

“…Strictosidine is an emblematic natural product, as it is the common precursor of the vast majority of monoterpene indole alkaloids (MIAs) found in the plant families of Apocynaceae, Loganiaceae, and Rubiaceae. Although its biosynthesis pathway has been elucidated completely and reconstructed successfully in tobacco and in yeast, the downstream pathways that generate the tremendous diversity of MIAs (>3000 structures) from strictosidine remain unknown with almost none of the biosynthetic genes identified. Thus, it is not surprising that the production of the MIA anticancer drugs vinblastine and vincristine, as well as cantharantine, is still inaccessible today by protein engineering.…”

Section: Methodsmentioning

confidence: 99%

“…

DedicatedtoMichel Rohmer on the occasion of his 69th birthdayBioengineering the genes of plants and microbes is becoming av ery competitive strategy in synthesis to produce pharmacologicallyr elevant plant natural products and their unnatural analogues.D uring the last 20 years, severalp lant genes have been identified, cloned,a nd expressed successfully,a nd this has unlocked de novo production of plant metabolites such as morphine, strictosidine, artemisinin, Taxol, and resveratrol. [1] Future development for accelerating the discoveryo fg ene candidates relies on innovative computational methods combined with transcriptomics and metabolomics analysis to make plant genome mining more feasible. [2] Nevertheless, in many cases the proteins of interest are produced in low amounts, which prevents in vivo extraction, or they are inactiveorg ive a differentp roduct after heterologous expression in am odel system.O ur understanding of the biocatalytic cascade or multistep reactions made by enzymes in different cellular compartment remains limited, [3] and yet deciphering new biocatalytic systemsi nn ature for future bioinspired synthetic strategies represents as timulating interdisciplinary field for chemists and biologists.

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confidence: 99%

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Tandem Biocatalysis Unlocks the Challenging de Novo Production of Plant Natural Products

Duplais

Estevez

2017

ChemBioChem

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Intimate partnership: Knowledge of the biocatalytic cascades in different cellular compartments is limited, but deciphering these systems in nature can be used to inspire synthetic strategies. Two studies report new insights into the biosynthesis of alkaloids and sesterterpenoids in plants. This highlight presents these novel biotransformations to illustrate how tandem biocatalysis can impact the future of natural product production.

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“…Consistent with the wide array of triterpene bioactivities, there is considerable effort in developing strategies for pathway identification and characterization of the corresponding enzymatic activities [ 18 ]. However, heterologous biosynthesis of cyclic plant-type triterpenes in microbial cells was thus far largely restricted to yeast systems, most notably Saccharomyces cerevisiae [ 17 ], and could only recently also be demonstrated for the triterpene dammarenediol-II in Escherichia coli [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis

et al. 2017

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Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.

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Tools of pathway reconstruction and production of economically relevant plant secondary metabolites in recombinant microorganisms

Cited by 37 publications

References 121 publications

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

Tandem Biocatalysis Unlocks the Challenging de Novo Production of Plant Natural Products

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis

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