Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

Sonntag, Frank; Kroner, Cora; Lubuta, Patrice; Peyraud, Rémi; Horst, Andréa; Buchhaupt, Markus; Schrader, Jens

doi:10.1016/j.ymben.2015.09.004

Cited by 95 publications

(59 citation statements)

References 70 publications

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“…Accordingly, engineering pathways de novo for a given substrate–product pair is a better approach to metabolic engineering than depending on pathways that consume glucose for any and all biochemical products. The diversification of feedstocks away from glucose, syngas, methane, methanol and glycerol, supports our idea3031323334. Our framework applies principles of orthogonality to design metabolic processes that are tailored for the conversion of a specific substrate to a product in the most efficient way possible.…”

Section: Discussionsupporting

confidence: 63%

Redesigning metabolism based on orthogonality principles

2017

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Modifications made during metabolic engineering for overproduction of chemicals have network-wide effects on cellular function due to ubiquitous metabolic interactions. These interactions, that make metabolic network structures robust and optimized for cell growth, act to constrain the capability of the cell factory. To overcome these challenges, we explore the idea of an orthogonal network structure that is designed to operate with minimal interaction between chemical production pathways and the components of the network that produce biomass. We show that this orthogonal pathway design approach has significant advantages over contemporary growth-coupled approaches using a case study on succinate production. We find that natural pathways, fundamentally linked to biomass synthesis, are less orthogonal in comparison to synthetic pathways. We suggest that the use of such orthogonal pathways can be highly amenable for dynamic control of metabolism and have other implications for metabolic engineering.

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Section: Discussionsupporting

confidence: 63%

Redesigning metabolism based on orthogonality principles

2017

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“…In all of these expression systems, the α‐humulene synthase (ZSSI) from the shampoo ginger plant Zingiber zerumbet was used. The co‐expression of a synthetic mevalonate (MVA) pathway in addition to the native methylerythritol 4‐phosphate (MEP) pathway increased the final product concentrations ,. In engineered E. coli JM109 heterologously expressing the mevalonate pathway, an IPP isomerase, and ZSSI, final concentrations of 263 mg L −1 and 1039 mg L −1 were produced in a complex medium supplied with 0.5 g L −1 lithium acetoacetate and 1 g L −1 d ‐mevalonolactone, respectively, two days after induction with isopropyl β‐ d ‐1‐thiogalactopyranoside (IPTG) .…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, heterologous α‐humulene production on glucose in defined medium was established in another engineered E. coli strain, achieving a concentration of 60 mg L −1 α‐humulene within 24 h. Sonntag et al. described the production of α‐humulene by M. extorquens CM502 (pFS62b) on methanol . Upon additional expression of a heterologous MVA pathway from M. xanthus , the α‐humulene concentration could be increased three‐fold compared to a strain lacking the MVA pathway and generating terpenes exclusively through the native MEP pathway.…”

Section: Methodsmentioning

confidence: 99%

“…Upon additional expression of a heterologous MVA pathway from M. xanthus , the α‐humulene concentration could be increased three‐fold compared to a strain lacking the MVA pathway and generating terpenes exclusively through the native MEP pathway. During a methanol‐limited fed‐batch fermentation, up to 1.65 g L −1 α‐humulene were produced after approximately 100 h with a space time yield of 15 mg L −1 h −1 …”

Section: Methodsmentioning

confidence: 99%

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CO₂ to Terpenes: Autotrophic and Electroautotrophic α‐Humulene Production with Cupriavidus necator

et al. 2018

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We show that CO can be converted by an engineered "Knallgas" bacterium (Cupriavidus necator) into the terpene α-humulene. Heterologous expression of the mevalonate pathway and α-humulene synthase resulted in the production of approximately 10 mg α-humulene per gram cell dry mass (CDW) under heterotrophic conditions. This first example of chemolithoautotrophic production of a terpene from carbon dioxide, hydrogen, and oxygen is a promising starting point for the production of different high-value terpene compounds from abundant and simple raw materials. Furthermore, the production system was used to produce 17 mg α-humulene per gram CDW from CO and electrical energy in microbial electrosynthesis (MES) mode. Given that the system can convert CO by using electrical energy from solar energy, it opens a new route to artificial photosynthetic systems.

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“…This was likely due to differences in intracellular MvaE and MvaS activities, because the only genotypic differences among the strains were in the promoter region of the mvaES operon. Previous studies showed that coordinated and well‐balanced enhancement of the expression of the isoprene biosynthesis pathway genes is important for increasing isoprenoid production (Sonntag et al ; Liu et al ). Therefore, it would be so important to maintain a balance in metabolic flux when integrating additional gene expression cassettes to increase yield and productivity in our future study.…”

Section: Discussionmentioning

confidence: 99%

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis

et al. 2019

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Aims Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. Methods and Results We constructed isoprene‐producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual‐phase isoprene production, where the initial cell growth phase under Pi‐saturating conditions was uncoupled from the subsequent isoprene production phase under Pi‐limiting conditions. In fed‐batch fermentation using our best strain (SWITCH‐PphoC/pIspSM) in a 1‐l bioreactor, isoprene concentration in the off‐gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH‐PphoC/pIspSM produced totally 2·5 g l−1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. Conclusions This proof‐of‐concept study demonstrated that our Pi‐dependent dual‐phase production system using a P. ananatis strain as a producer has potential for industrial‐scale isoprene fermentation. Significance and Impact of the Study This Pi‐dependent dual‐phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

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Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

Cited by 95 publications

References 70 publications

Redesigning metabolism based on orthogonality principles

Redesigning metabolism based on orthogonality principles

CO₂ to Terpenes: Autotrophic and Electroautotrophic α‐Humulene Production with Cupriavidus necator

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis

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Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

Cited by 95 publications

References 70 publications

Redesigning metabolism based on orthogonality principles

Redesigning metabolism based on orthogonality principles

CO2 to Terpenes: Autotrophic and Electroautotrophic α‐Humulene Production with Cupriavidus necator

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis

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CO₂ to Terpenes: Autotrophic and Electroautotrophic α‐Humulene Production with Cupriavidus necator