Roman Netzer

Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis

In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.

Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum.

ABSTRACT Despite its key position in central metabolism, l-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, l-serine is consumed at rates up to 19.4 ± 4.0 nmol min−1 (mg [dry weight])−1, resulting in the complete consumption of 100 mM l-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled l-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of l-serine is mainly converted to pyruvate-derived metabolites such as l-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional l-serine dehydratase (l-SerDH) activity, and therefore conversion of l-serine to pyruvate, and (ii) growth of the recombinant strain on l-serine as the single substrate. In contrast, deletion of sdaA decreased the l-serine cometabolism rate with glucose by 47% but still resulted in degradation of l-serine to pyruvate. Cystathionine β-lyase was additionally found to convert l-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal l-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular l-serine conversion to pyruvate might occur, although probably the weak affinity of l-SerDH (apparent Km, 11 mM) prevents substantial l-serine degradation.

Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Microbial communities related to biodegradation of dispersed Macondo oil at low seawater temperature with Norwegian coastal seawater.

The Deepwater Horizon (DWH) accident in 2010 created a deepwater plume of small oil droplets from a deepwater well in the Mississippi Canyon lease block 252 (‘Macondo oil’). A novel laboratory system was used in the current study to investigate biodegradation of Macondo oil dispersions (10 μm or 30 μm median droplet sizes) at low oil concentrations (2 mg l−1) in coastal Norwegian seawater at a temperature of 4–5°C. Whole metagenome analyses showed that oil biodegradation was associated with the successive increased abundances of Gammaproteobacteria, while Alphaproteobacteria (Pelagibacter) became dominant at the end of the experiment. Colwellia and Oceanospirillales were related to n‐alkane biodegradation, while particularly Cycloclasticus and Marinobacter were associated with degradation of aromatic hydrocarbons (HCs). The larger oil droplet dispersions resulted in delayed sequential changes of Oceanospirillales and Cycloclasticus, related with slower degradation of alkanes and aromatic HCs. The bacterial successions associated with oil biodegradation showed both similarities and differences when compared with the results from DWH field samples and laboratory studies performed with deepwater from the Gulf of Mexico.

Continuous Control of the Flow in Biochemical Pathways through 5 Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm†

ABSTRACT The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (Pantitet ), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50 carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus

ABSTRACT We investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for l-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased l-lysine production levels.

Concentrations of viable oil-degrading microorganisms are increased in feces from Calanus finmarchicus feeding in petroleum oil dispersions.

Zooplankton are suggested to be biotic contributors to the transport and weathering of oil in marine environments due to their ingestion of oil. In the present experiment, feeding activity and microbial communities in feces from Calanus finmarchicus feeding in oil dispersions were characterized. Feeding activity was significantly reduced in oil dispersions. The microbial communities in clean and oil-containing copepod feces were dominated by Rhodobacteraceae family bacteria (Lesingera, Phaeobacter, Rugeria, and Sulfitobacter), which were suggested to be indigenous to copepod feces. The results also indicated that these bacteria were metabolizing oil compounds, as a significant increase in the concentrations of viable oil degrading microorganisms was observed in oil-containing feces. This study shows that bacteria in feces from copepods feeding in dilute oil dispersions have capacity for degradation of oil. Zooplankton may therefore contribute to weathering of oil by excreting feces with microbial communities already adapted to degradation of oil.

Biodegradation of dispersed oil in seawater is not inhibited by a commercial oil spill dispersant

Chemical dispersants are well-established as oil spill response tools. Several studies have emphasized their positive effects on oil biodegradation, but recent studies have claimed that dispersants may actually inhibit the oil biodegradation process. In this study, biodegradation of oil dispersions in natural seawater at low temperature (5°C) was compared, using oil without dispersant, and oil premixed with different concentrations of Slickgone NS, a widely used oil spill dispersant in Europe. Saturates (nC10-nC36 alkanes), naphthalenes and 2- to 5-ring polycyclic aromatic hydrocarbons (PAH) were biotransformed at comparable rates in all dispersions, both with and without dispersant. Microbial communities differed primarily between samples with or without oil, and they were not significantly affected by increasing dispersant concentrations. Our data therefore showed that a common oil spill dispersant did not inhibit biodegradation of oil at dispersant concentrations relevant for response operations.

Biotransformation of petroleum hydrocarbons and microbial communities in seawater with oil dispersions and copepod feces

To determine biotransformation of components in crude oil dispersions in the presence of feces from marine copepods, dispersed oil was incubated alone, with the addition of clean or oil-containing feces. We hypothesized that the feces would contribute with nutrients to bacteria, and higher concentrations of oil-degrading bacteria, respectively. Presence of clean feces resulted in higher degradation of aromatic oil compounds, but lower degradation of n-alkanes. Presence of oil-containing feces resulted in higher degradation of n-alkanes. The effect of clean feces on aromatic compounds are suggested to be due to higher concentrations of nutrients in the seawater where aromatic degradation takes place, while the lower degradation of n-alkanes are suggested to be due to a preference by bacteria for feces over these compounds. Large aggregates were observed in oil dispersions with clean feces, which may cause sedimentation of un-weathered lipophilic oil compounds towards the seafloor if formed during oil spills.

Biodegradation of Petroleum Oil in Cold Marine Environments

The cold regions of the Earth are exposed to petroleum oil exploration, production, and transport, with risk of oil spills. Biodegradation is an essential petroleum weathering process and may remove discharged petroleum compounds completely by mineralization processes. These processes are most apparent for soluble compounds and with dispersed oil. Surface and subsurface spills will generate different situations, and in addition freezing of oil in marine ice may transport the oil over large distances. A variety of marine psychrophilic or psychrotolerant bacteria from both shallow and deepwater environments have been reported to degrade hydrocarbons in seawater or marine sediments, most of these affiliated within the phyla Proteobacteria and Bacteroidetes. Several of these may also act on hydrocarbons in sea ice, and active bacterial respiration in sea ice has been shown down to temperatures of −20 °C. The cold environments require several microbial survival and catabolism strategies, including productions of exopolysaccharides, cold-active enzymes, cold-shock, cold-acclimation and anti-freeze proteins, as well as adjusting their membrane lipid composition. Oil biodegradation in cold environments is well documented by laboratory and field studies, and even oil frozen in marine ice will stimulate bacterial metabolism. Flocculation processes have also been associated with oil biodegradation, raising discussions on the fate of the oil, especially after the Deepwater Horizon blowout. Bioremediation in cold marine environments has been investigated as a labor-effective technology which generates no harmful by-products, mainly by adding fertilizers to stimulate the oil biodegradation by the indigenous bacteria (biostimulation), but also inoculation of exogenic hydrocarbonoclastic cultures (bioaugmentation) has been suggested.