Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered <i>Escherichia coli</i>

Liu, Wei; Zhang, Rubing; Tian, Ning; Xu, Xin; Cao, Yuqi; Xian, Ming; Liu, Huizhou

doi:10.1080/21655979.2015.1062188

Cited by 24 publications

(16 citation statements)

References 20 publications

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“…More recently, additional commercial applications for geraniol have been proposed, including use as a pharmaceutical agent for anti-cancer, anti-inflammatory, and pain relief purposes; and as a promising gasoline alternative [4–8]. Currently, the predominant strategies for obtaining terpenes such as geraniol are extraction from plant material and chemical synthesis; both of which are costly and inefficient [9, 10]. As such, there has been growing interest in developing more sustainable and economical biotechnological methods for terpene production [11].…”

Section: Introductionmentioning

confidence: 99%

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

et al. 2019

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Background Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds—which are often utilised for anti-microbial and anti-fungal functions in their host plant. Results In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed. Conclusion In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production. Electronic supplementary material The online version of this article (10.1186/s12934-019-1130-0) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

et al. 2019

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“…Error bars represent the standard deviation of 3–6 biological replicates. Geraniol and derivatives were omitted from the comparison as they are mainly produced by endogenous E. coli activity . A full breakdown of the product profiles can be found in Table S3.…”

Section: Resultsmentioning

confidence: 99%

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

et al. 2019

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Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α‐terpinyl cation and subsequent cyclisation and deprotonation of the α‐terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α‐terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.

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“…Exogenous geraniol synthase from Ocimum basilicum has been expressed for geraniol production in E. coli [ 26 – 28 ]. Bifunctional diacylglycerol diphosphate phosphatase (DPP1) and lipid phosphate phosphatase (LPP1) from Saccharomyces cerevisiae have been evaluated for geraniol biosynthesis [ 29 ]. The endogenous E. coli ADP-ribose pyrophosphatase (NudF) and alkaline phosphatase (PhoA) were also tested for geraniol production; however, only a tiny amount (5.3 mg/L) of geraniol was produced by PhoA in a flask culture [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…Bifunctional diacylglycerol diphosphate phosphatase (DPP1) and lipid phosphate phosphatase (LPP1) from Saccharomyces cerevisiae have been evaluated for geraniol biosynthesis [ 29 ]. The endogenous E. coli ADP-ribose pyrophosphatase (NudF) and alkaline phosphatase (PhoA) were also tested for geraniol production; however, only a tiny amount (5.3 mg/L) of geraniol was produced by PhoA in a flask culture [ 29 ]. Farnesyl phosphatases from Homo sapiens and Drosophila melanogaster can hydrolyze FPP to farnesol [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives

Zada

Wang

Park

et al. 2018

Biotechnol Biofuels

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BackgroundCurrent petroleum-derived fuels such as gasoline (C5–C12) and diesel (C15–C22) are complex mixtures of hydrocarbons with different chain lengths and chemical structures. Isoprenoids are hydrocarbon-based compounds with different carbon chain lengths and diverse chemical structures, similar to petroleum. Thus, isoprenoid alcohols such as isopentenol (C5), geraniol (C10), and farnesol (C15) have been considered to be ideal biofuel candidates. NudB, a native phosphatase of Escherichia coli, is reported to dephosphorylate isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into isopentenol. However, no attention has been paid to its promiscuous activity toward longer chain length (C10–C15) prenyl diphosphates.ResultsIn this study, the promiscuous activity of NudB toward geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) was applied for the production of isoprenoid alcohol mixtures, including isopentenol, geraniol, and farnesol, and their derivatives. E. coli was engineered to produce a mixture of C5 and C15 alcohols by overexpressing NudB (dihydroneopterin triphosphate diphosphohydrolase) and IspA (FPP synthase) along with a heterologous MVA pathway, which resulted in a total of up to 1652 mg/L mixture of C5 and C15 alcohols and their derivatives. The production was further increased to 2027 mg/L by overexpression of another endogenous phosphatase, AphA, in addition to NudB. Production of DMAPP- and FPP-derived alcohols and their derivatives was significantly increased with an increase in the gene dosage of idi, encoding IPP isomerase (IDI), indicating a potential modulation of the composition of the alcohols mixture according to the expression level of IDI. When IspA was replaced with its mutant IspA*, generating GPP in the production strain, a total of 1418 mg/L of the isoprenoid mixture was obtained containing C10 alcohols as a main component.ConclusionsThe promiscuous activity of NudB was newly identified and successfully used for production of isoprenoid-based alcohol mixtures, which are suitable as next-generation biofuels or commodity chemicals. This is the first successful report on high-titer production of an isoprenoid alcohol-based mixture. The engineering approaches can provide a valuable platform for production of other isoprenoid mixtures via a proportional modulation of IPP, DMAPP, GPP, and FPP syntheses.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1210-0) contains supplementary material, which is available to authorized users.

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Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli

Cited by 24 publications

References 20 publications

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives

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