COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Can Genome Sequencing Coupled to Flux Balance Analyses Offer Precision Guidance for Industrial Strain Development? The Lessons from Carbon Trafficking in Corynebacterium glutamicum ATCC 21573.

Systems biology tools offer new prospects for industrial strain selection. For bacteria that are significant for industrial applications, whole-genome sequencing coupled to flux balance analysis (FBA) can help unpack the complex relationships between genome mutations and carbon trafficking. This work investigates the l-tyrosine (l-Tyr) overproducing model system Corynebacterium glutamicum ATCC 21573 with an eye to more rational and precision strain development. Using genome-wide mutational analysis of C. glutamicum, we identified 27,611 single nucleotide polymorphisms and 479 insertion/deletion mutations. Mutations in the carbon uptake machinery have led to phosphotransferase system-independent routes as corroborated with FBA. Mutations within the central carbon metabolism of C. glutamicum impaired the carbon flux, as evidenced by the lower growth rate. The entry to and flow through the tricarboxylic acid cycle was affected by mutations in pyruvate and α-ketoglutarate dehydrogenase complexes, citrate synthase, and isocitrate dehydrogenase. FBA indicated that the estimated flux through the shikimate pathway became larger as the l-Tyr production rate increased. In addition, protocatechuate export was probabilistically impossible, which could have contributed to the l-Tyr accumulation. Interestingly, aroG and cg0975, which have received previous attention for aromatic amino acid overproduction, were not mutated. From the branch point molecule, prephenate, the change in the promoter region of pheA could be an influential contributor. In summary, we suggest that genome sequencing coupled with FBA is well poised to offer rational guidance for industrial strain development, as evidenced by these findings on carbon trafficking in C. glutamicum ATCC 21573.

Metabolic engineering of Corynebacterium glutamicum for l-tyrosine production from glucose and xylose.

Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.

Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production.

Coenzyme Q10 (CoQ10) is a lipid-soluble compound with important physiological functions and is sought after in the food and cosmetic industries owing to its antioxidant properties. In our previous proof of concept, we engineered for CoQ10 biosynthesis the industrially relevant Corynebacterium glutamicum, which does not naturally synthesize any CoQ. Here, liquid chromatography-mass spectrometry (LC-MS) analysis identified two metabolic bottlenecks in the CoQ10 production, i.e., low conversion of the intermediate 10-prenylphenol (10P-Ph) to CoQ10 and the accumulation of isoprenologs with prenyl chain lengths of not only 10, but also 8 to 11 isopentenyl units. To overcome these limitations, the strain was engineered for expression of the Ubi complex accessory factors UbiJ and UbiK from Escherichia coli to increase flux towards CoQ10, and by replacement of the native polyprenyl diphosphate synthase IspB with a decaprenyl diphosphate synthase (DdsA) to select for prenyl chains with 10 isopentenyl units. The best strain UBI6-Rs showed a seven-fold increased CoQ10 content and eight-fold increased CoQ10 titer compared to the initial strain UBI4-Pd, while the abundance of CoQ8, CoQ9, and CoQ11 was significantly reduced. This study demonstrates the application of the recent insight into CoQ biosynthesis to improve metabolic engineering of a heterologous CoQ10 production strain.

Fermentative Production of Halogenated Tryptophan Derivatives with Corynebacterium glutamicum Overexpressing Tryptophanase or Decarboxylase Genes.

The aromatic amino acid l-tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in Corynebacterium glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L-1 7-Cl-indole and 23 mg L-1 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L-1 upon expression of AADC from B. atrophaeus. AADC enzymes were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L-1 7-Br-tryptamine with a volumetric productivity of 8.3 mg L-1 h-1 in a fed-batch fermentation.

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

Coenzyme Q (CoQ) 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. Here, we review discovery of the pathway with a particular focus on its superstructuration and regulation, and we summarize the metabolic engineering strategies for overproduction of CoQ by microorganisms. Studies in model microorganisms elucidated the details of CoQ biosynthesis and revealed the existence of multiprotein complexes composed of several enzymes that catalyze consecutive reactions in the CoQ pathways of Saccharomyces cerevisiae and Escherichia coli. Recent findings indicate that the identity and the total number of proteins involved in CoQ biosynthesis vary between species, which raises interesting questions about the evolution of the pathway and could provide opportunities for easier engineering of CoQ production. For the biotechnological production, so far only microorganisms have been used that naturally synthesize CoQ10 or a related CoQ species. CoQ biosynthesis requires the aromatic precursor 4-hydroxybenzoic acid and the prenyl side chain that defines the CoQ species. Up to now, metabolic engineering strategies concentrated on the overproduction of the prenyl side chain as well as fine-tuning the expression of ubi genes from the ubiquinone modification pathway, resulting in high CoQ yields. With expanding knowledge about CoQ biosynthesis and exploration of new strategies for strain engineering, microbial CoQ production is expected to improve.

Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum.

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine-derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylA Xc B Cg (xylA from Xanthomonas campestris) and araBAD Ec from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcC Ec from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDA Ec from E. coli or with putrescine oxidase gene puo Rq from Rhodococcus qingshengii and patD Ec . Deletion of the l-glutamate dehydrogenase-encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcC Ec -patDA Ec ) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

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

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.

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum.

The N-functionalized amino acid N-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid O-isopropyl N-methylanthranilate, the flavor compound O-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to N-alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium Corynebacterium glutamicum are operated industrially at the million tons per annum scale. Fermentative processes using C. glutamicum for N-alkylated amino acids based on an imine reductase have been developed, while N-alkylation of the aromatic amino acid anthranilate with S-adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene anmt encoding anthranilate N-methyltransferase from Ruta graveolens resulted in production of N-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene sahH improved N-methylanthranilate production. In a test bioreactor culture, the metabolically engineered C. glutamicum C1* strain produced NMA to a final titer of 0.5 g·L-1 with a volumetric productivity of 0.01 g·L-1·h-1 and a yield of 4.8 mg·g-1 glucose.

Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis.

l-2-hydroxyglutarate (l-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural l-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of l-2HG. Since l-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined l-lysine overproducing strain was required to enable the conversion of l-lysine to l-2HG in a six-step synthetic pathway. To this end, l-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported l-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of l-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved l-2HG production. The availability of Fe2+ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to l-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a l-2HG titer of 3.5 g L-1 was obtained at the higher of two tested aeration levels. Production of l-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of l-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability.