Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering

Abstract

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Recent advances in the metabolic pathways and microbial production of coenzyme Q

“…As a consequence, the highest 4-HBA product titer up to about 37 g/L was achieved in a two-stage bioprocess (Kitade et al 2018 ). Thus, to provide aromatic precursor for CoQ 10 biosynthesis, feedback-resistant CPL and AroG from E. coli were introduced into a mutant C. glutamicum , in which pobA , pcaHG , and qsuABD encoding 4-HBA hydroxylase, PCA dioxygenase, and QSU pathway enzymes (putative shikimate importer, 3-dehydroshikimate dehydratase, and quinate/shikimate dehydrogenase), respectively, were deleted (Burgardt et al 2021 ). Meanwhile, P. taiwanensis was tailored to produce 4-HBA via l -tyrosine (Lenzen et al 2019 ).…”

“…A bacterium natively lacking CoQ biosynthesis has recently been engineered for CoQ 10 production (Table 1 ) (Burgardt et al 2021 ). Previously, C. glutamicum was engineered for high-level production of the aromatic CoQ 10 precursor 4-HBA (Kitade et al 2018 ; Purwanto et al 2018 ).…”

“…Two steps were required to enable CoQ 10 production by the 4-HBA producing C. glutamicum strain. First, overproduction of the prenyl precursor of CoQ 10 , decaprenyl diphosphate (DPP), was achieved by heterologous expression of DPP synthase gene ddsA from Paracoccus denitrificans (Burgardt et al 2021 ). Second, genes for the whole ubiquinone pathway from E. coli were expressed and the resulting strain produced 0.43 mg/L (Burgardt et al 2021 ).…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…It is an interesting compound. C. glutamicum was the first microbe not natively synthesising CoQ10 that was engineered for CoQ10 production [113] (Table 3). A carotenoid-deficient strain with increased supply of the precursor FPP was constructed to synthesise decaprenyl diphosphate (DPP) and was the first CoQ10 precursor to express the DPP synthase gene ddsA isolated from Paracoccus denitrificans.…”

“…Metabolic engineering of the shikimate pathway provided para-hydroxybenzoate (pHBA) as the second CoQ10 precursor. Using ubi genes from E. coli allowed the prenylation of pHBA with DPP followed by decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 [113].…”