Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

Heider S, Peters-Wendisch P, Wendisch VF. Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum. BMC Microbiology. 2012;12(1): 198.Background Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. Conclusion C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production

Design, construction and application of E. coli - C. glutamicum synthetic consortia

In the biorefinery concept renewable feedstocks are converted to a multitude of value-added compounds irrespective of seasonal or other variations of the complex biomass substrates. On the one hand, this can be realized by specialized single microbial strains. Alternatively, consortia of several microorganisms or strains can be used. The latter approach allows for modularity, e.g. as various strains for substrate conversion can be combined with various strains for product formation. Please click Additional Files below to see the full abstract

Systems metabolic engineering of Corynebacterium glutamicum and Bacillus methanolicus for production of new products from alternative carbon sources

Amino acid production amounts to about 2 million tons of L-lysine and 3 million tons of L-glutamate per year [1]. Corynebacterium glutamicum is widely used in industry for amino acid production from sugars, while the methylotrophic Bacillus methanolicus produces L-lysine and L-glutamate from methanol with titers of about 50 g/L [2]. Both microbial hosts have been developed for production of specialty amino acids and amines. Specifically, I will present new tools for metabolic engineering of B. methanolicus and C. glutamicum including CRISPRi/dCas9, sigma factor engineering, genome reduction, induction withphotocaged IPTG, theta-type replicating vectors and biosensors [3-10]. On the other hand, I will describe examples of metabolic engineering of these hosts for the production of specialty amino acids and diamines. C. glutamicum strains for the production of L-pipecolic acid have been constructed [11] and these were improved a titer of 14.4 g L-1, a volumetric productivity of 0.21 g L-1 h-1 and an overall yield of 0.20 g g-1 [12]. Moreover, access to production of L-pipecolic acid from glucose, glycerol, xylose, glucosamine, and starch has been enabled. Methanol-based production of the non-proteinogenic amino acid -amino butyric acid (GABA), that finds application also as precursor for bioplastics, has been achieved with B. methanolicus [13]. Efficient production of GABA from hexose and pentose sugars has been realized and optimized in recombinant C. glutamicum strains as well [14,15]. Both for C. glutamicum and B. methanolicus characterization and engineering of product export have been instrumental [16,17]. In conclusion, the microbial cell factories B. methanolicus and C. glutamicum have been programmed for efficient and sustainable production of specialty amino acids and amines from diverse carbon feedstocks to foster their biotechnological applications. [1] WENDISCH, Adv Biochem Eng Biotechnol, 10.1007/10_2016_34, 2017. [2] PFEIFENSCHNEIDER et al., Biofuel Bioprod Bioref 11: 719-731, 2017. [3] CLETO et al., ACS Synth Biol, 5, 375-385, 2016. [4] UNTHAN et al., Biotechnol J 10: 290-301, 2015. [5] BAUMGART et al. ACS Synth Biol in press, 2017. [6] BINDER et al., Appl Environ Microbiol 82: 6141-6149, 2016. [7] TANIGUCHI et al., Front Microbiol 6: 740, 2015. [8] TANIGUCHI et al., Metab Eng Comm 4: 1-11, 2017. [9] TANIGUCHI et al., BMC Microbiol 17:158. [10] IRLA et al., Front Microbiol, 7, 1481, 2016. [11] PÉREZ-GARCÍA et al., Appl Microbiol Biotechnol, 100, 8075-8090, 2016. [12] PÉREZ-GARCÍA et al., Biotechnol J, 10.1002/biot.201600646, 2017. [13] IRLA et al., Ind Crops Prod, 10.1016/j.indcrop.2016.11.050, 2017 [14] JORGE et al., Amino Acids, 48, 2519-2531, 2016. [15] JORGE et al., Biotechnol Bioeng, 10.1002/bit.26211, 2017. [16] LUBTIZ et al., Appl Microbiol Biotechnol, 100, 8465-8474, 2016. [17] NÆRDAL et al., J Biotechnol 244, 25-33, 2017

Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production

Komati Reddy G, Wendisch VF. Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production. BMC Microbiology. 2014;14(1): 54.Background Corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation. Results C. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGKHis was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg2+ for its activity. PGKHis is thermo labile and it has shown maximal activity at 50–65°C. The maximal activity of PGKHis was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively. Conclusion Unlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains

Carotenoid Production by Corynebacterium: The Workhorse of Industrial Amino Acid Production as Host for Production of a Broad Spectrum of C40 and C50 Carotenoids

Corynebacterium glutamicum is used as a workhorse of industrial biotechnology for more than 60 years since its discovery as a natural glutamate producer in the 1950s. Nowadays, L-glutamate and L-lysine are being produced with this GRAS organism in the million-ton scale every year for the food and feed markets, respectively. Sequencing of the genome and establishment of a genetic toolbox boosted metabolic engineering of this host for a broad range of industrially relevant compounds ranging from bulk chemicals to high-value products. Carotenoids, the colourful representatives of terpenoids, are high-value compounds whose bio-based production is on the rise. Since C. glutamicum is a natural producer of the rare C50 carotenoid decaprenoxanthin, this organism is well suited to establish terpenoid-overproducing platform strains with the help of metabolic engineering strategies. In this work, the carotenogenic background of C. glutamicum and the metabolic engineering strategies for the generation of carotenoid-overproducing strains are depicted

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

Markert B, Stolzenberger J, Brautaset T, Wendisch VF. Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. BMC Microbiology. 2014;14(1): 7.Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tktC) and one located on the natural occurring plasmid pBM19 (tktP). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tktP, but not of tktC during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed

Optimal Detection for Diffusion-Based Molecular Timing Channels

This work studies optimal detection for communication over diffusion-based molecular timing (DBMT) channels. The transmitter simultaneously releases multiple information particles, where the information is encoded in the time of release. The receiver decodes the transmitted information based on the random time of arrival of the information particles, which is modeled as an additive noise channel. For a DBMT channel without flow, this noise follows the L\'evy distribution. Under this channel model, the maximum-likelihood (ML) detector is derived and shown to have high computational complexity. It is also shown that under ML detection, releasing multiple particles improves performance, while for any additive channel with $\alpha$-stable noise where $\alpha<1$ (such as the DBMT channel), under linear processing at the receiver, releasing multiple particles degrades performance relative to releasing a single particle. Hence, a new low-complexity detector, which is based on the first arrival (FA) among all the transmitted particles, is proposed. It is shown that for a small number of released particles, the performance of the FA detector is very close to that of the ML detector. On the other hand, error exponent analysis shows that the performance of the two detectors differ when the number of released particles is large.Comment: 16 pages, 9 figures. Submitted for publicatio

Corynebacterium glutamicum as a platform strain for the production of a broad variety of terpenoids

Corynebacterium glutamicum is a natural carotenoid producing bacterium used in the million-ton-scale amino acid biotechnology that has been engineered for isoprenoid production1. The native membrane-bound carotenoid decaprenoxanthin is a rare C50 carotenoid. Volatile terpenoids such as valencene2 and patchoulol3 could be produced upon deletion of the first step of the specific carotenoid pathway and heterologous expression of the FPP synthase gene ispA from E. coli and terpene synthases from plant origin. However, these strains produced a yet unidentified carotenoid and only when all carotenoid biosynthetic genes were deleted, a colorless strain resulted. Expressing a codon optimized ADS from Artemisia annua in the white strain, amorphadiene, the volatile precursor for artemisinin was produced. For production of volatile terpenoids a dodecane overlay was used, a condition in which C. glutamicum benefits from its robust myco-membrane. Recently, we showed production of membrane-bound carotenoids with different length and/or cyclization status: bicyclic C50 sarcinaxanthin4, bicyclic C40 astaxanthin5, the linear lycopene6 and the linear C50 bisanhydrobacterioruberin7. This indicated that the C. glutamicum myco-membrane accepts these linear and bicyclic carotenoids. Please click Additional Files below to see the full abstract

Engineering of Corynebacterium glutamicum for growth and production of L-ornithine, L-lysine, and lycopene from hexuronic acids

Hadiati A, Krahn I, Lindner S, Wendisch VF. Engineering of Corynebacterium glutamicum for growth and production of L-ornithine, L-lysine, and lycopene from hexuronic acids. Bioresources and Bioprocessing. 2014;1(1): 25

Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum

Eberhardt D, Wendisch VF. Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum. BMC Microbiology. 2016;16(1): 235.Background Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope. Results Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin. Conclusions Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions