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Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

ABSTRACT: Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.

SUBMITTER: Srinivasan P

PROVIDER: S-EPMC6690885 | biostudies-other | 2019 Aug

REPOSITORIES: biostudies-other

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Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

Srinivasan Prashanth P Smolke Christina D CD

Nature communications 20190812 1

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast ...[more]

PMID: 31406117

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Project description:BackgroundAnthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.ResultsSaccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.ConclusionThe results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

Project description:BACKGROUND:(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. RESULTS:As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. CONCLUSION:We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

Dataset Information

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

Publications

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

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