Tailoring Corynebacterium glutamicum towards increased malonyl-CoA availability for efficient synthesis of the plant pentaketide noreugenin

Lars Milke,Nicolai Kallscheuer,Jan Marienhagen,Jannick Kappelmann

doi:10.1186/s12934-019-1117-x

Lars Milke, Nicolai Kallscheuer + Show 2 more

Open Access

https://doi.org/10.1186/s12934-019-1117-x

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Abstract

BackgroundIn the last years, different biotechnologically relevant microorganisms have been engineered for the synthesis of plant polyphenols such as flavonoids and stilbenes. However, low intracellular availability of malonyl-CoA as essential precursor for most plant polyphenols of interest is regarded as the decisive bottleneck preventing high product titers.ResultsIn this study, Corynebacterium glutamicum, which emerged as promising cell factory for plant polyphenol production, was tailored by rational metabolic engineering towards providing significantly more malonyl-CoA for product synthesis. This was achieved by improving carbon source uptake, transcriptional deregulation of accBC and accD1 encoding the two subunits of the acetyl-CoA carboxylase (ACC), reduced flux into the tricarboxylic acid (TCA) cycle, and elimination of anaplerotic carboxylation of pyruvate. The constructed strains were used for the synthesis of the pharmacologically interesting plant pentaketide noreugenin, which is produced by plants such as Aloe arborescens from five molecules of malonyl-CoA. In this context, accumulation of the C1/C6 cyclized intermediate 1-(2,4,6-trihydroxyphenyl)butane-1,3-dione (TPBD) was observed, which could be fully cyclized to the bicyclic product noreugenin by acidification.ConclusionThe best strain C. glutamicum Nor2 C5 mufasOBCD1 PO6-iolT1 ∆pyc allowed for synthesis of 53.32 mg/L (0.278 mM) noreugenin in CGXII medium supplemented with casamino acids within 24 h.

Highlights

In the last years, different biotechnologically relevant microorganisms have been engineered for the synthesis of plant polyphenols such as flavonoids and stilbenes
Establishing a heterologous pathway for the synthesis of noreugenin For establishing noreugenin synthesis in C. glutamicum, a codon-optimized, synthetic variant of the pcs gene originating from A. arborescens was cloned into the vector pMKEx2 yielding pMKEx2-pcsAaCg, which allows for isopropyl β-d-thiogalactopyranoside (IPTG) inducible gene expression of pcsAaCg from the strong T7 promoter [24]
After transformation of C. glutamicum C7 selected as production strain due to its improved capability to supply malonyl-CoA, the resulting strain C. glutamicum C7 pMKEx2-pcsAaCg was cultivated in defined CGXII minimal medium with 4% glucose and 1 mM IPTG

Summary

Introduction

Different biotechnologically relevant microorganisms have been engineered for the synthesis of plant polyphenols such as flavonoids and stilbenes. Polyphenols constitute the third large group of plant secondary metabolites [1]. In plants, polyphenols such as flavonoids, stilbenes or lignans do not contribute directly to growth or propagation, they rather protect the plant from UV radiation, aid in the defense against pathogens or herbivores, and color petals and fruits to attract animals [2, 3]. Several stilbenes (e.g. resveratrol and its O-methylated derivatives) as well as flavonoids (e.g. naringenin, kaempferol and quercetin) could be produced with C. glutamicum from supplemented phenylpropanoid precursors or directly from glucose [10, 11].

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