From microbial upcycling to biology-oriented synthesis: combining whole-cell production and chemo-enzymatic functionalization for sustainable taxanoid delivery

Hirte, Max; Mischko, Wolfgang; Kemper, Katarina; Rohrer, Stefanie; Huber, Claudia; Fuchs, Monika; Eisenreich, Wolfgang; Minceva, Mirjana; Brück, Thomas

doi:10.1039/c8gc03126f

Cited by 13 publications

(10 citation statements)

References 71 publications

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“…In the same study, another functionalized diterpenoid was produced in a similar way by combining miltiradiene-producing E. coli with S. cerevisiae expressing a CYP450 required for subsequent conversion to ferruginol at 18 mg L −1 . Very interesting with regard to cheap feedstock utilization was the recent engineering of E. coli for the production of 364 mg L −1 of taxadiene utilizing corn steep liquor and glycerol as carbon source (Hirte et al 2018). On the other hand, S. cerevisiae has been engineered to produce about 800 mg/L of jolkinol C and a record > 1 g L −1 of oxidized casbanes that are potential intermediates for the synthesis of various pharmaceuticals (Wong et al 2018).…”

Section: Microbial Production Of Different Terpenoid Classesmentioning

confidence: 99%

Identifying and engineering the ideal microbial terpenoid production host

Moser

Pichler

2019

Appl Microbiol Biotechnol

120

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More than 70,000 different terpenoid structures are known so far; many of them offer highly interesting applications as pharmaceuticals, flavors and fragrances, or biofuels. Extraction of these compounds from their natural sources or chemical synthesis is-in many cases-technically challenging with low or moderate yields while wasting valuable resources. Microbial production of terpenoids offers a sustainable and environment-friendly alternative starting from simple carbon sources and, frequently, safeguards high product specificity. Here, we provide an overview on employing recombinant bacteria and yeasts for heterologous de novo production of terpenoids. Currently, Escherichia coli and Saccharomyces cerevisiae are the two best-established production hosts for terpenoids. An increasing number of studies have been successful in engineering alternative microorganisms for terpenoid biosynthesis, which we intend to highlight in this review. Moreover, we discuss the specific engineering challenges as well as recent advances for microbial production of different classes of terpenoids. Rationalizing the current stages of development for different terpenoid production hosts as well as future prospects shall provide a valuable decision basis for the selection and engineering of the cell factory(ies) for industrial production of terpenoid target molecules.

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Section: Microbial Production Of Different Terpenoid Classesmentioning

confidence: 99%

Identifying and engineering the ideal microbial terpenoid production host

Moser

Pichler

2019

Appl Microbiol Biotechnol

120

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“…The employed expression system, also includes the native E. coli non-mevalonate pathway (MEP) bottleneck enzymes 1-deoxy- d -xylulose-5-phosphate synthase (DXS; WP_099145004.1) and isopentenyl-pyrophosphate isomerase (IDI; AAC32208.1) to enhance sesquiterpene production as previously described [ 19 , 20 ]. In addition, the ORFs of the TPS candidates were also cloned into a two-plasmid diterpene production system, which includes a geranylgeranyl diphosphate synthase (crtE from Pantoea ananatis ; ADD79325.1) [ 21 ], to test for possible catalytic activity towards diterpene production. In preliminary experiments Copu6, Copu7, Copu10 and Copu11 did not show any catalytic activity when tested for sesquiterpene production using the single operon expression vector nor for diterpene production applying the two-plasmid system.…”

Section: Resultsmentioning

confidence: 99%

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

et al. 2022

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Background Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. Results In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l−1 and 395 mg l−1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. Conclusion The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis. Graphical Abstract

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“…Furthermore, decreasing the metabolic burden of the plasmid construct on the native host metabolism [85] can be achieved by using polycistronic operons to reduce the amount of plasmid in a cell. Additionally, computer aided fine-tuning [86] of transcription rates by promotor [24] and RBS [87] variations will further enhance the production rate [25]. Alternatively, permanent integration of the heterologous genes into the host genome is an alternative strategy to circumvent metabolic stress by antibiotics, which are required to maintain a plasmid in the production host [28].…”

Section: Reviewmentioning

confidence: 99%

“…Using host microorganisms, such as bacteria or baker’s yeast for the heterologous synthesis of terpenes increases the sustainability of bioactive terpene production by saving resources, as the production host can be fed with residual organic waste streams, such as milling or forestry waste. Additionally, the heterologous terpene production minimizes waste generation as the targeted production of a single compound reduces extraction and purification steps [24–25]. Additionally, heterologous production enables protein engineering to optimize product ratios or to alter the native product portfolio of the enzyme [9,26–27].…”

Section: Introductionmentioning

confidence: 99%

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

Driller¹,

Garbe²,

Mehlmer³

et al. 2019

Beilstein J. Org. Chem.

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CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

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From microbial upcycling to biology-oriented synthesis: combining whole-cell production and chemo-enzymatic functionalization for sustainable taxanoid delivery

Abstract: Conversion of low-value, by-product streams into taxadiene in conjunction with new purification and processing options expanding the taxanoids’ chemical space.

Cited by 13 publications

References 71 publications

Identifying and engineering the ideal microbial terpenoid production host

Identifying and engineering the ideal microbial terpenoid production host

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

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