Search

Metabolic engineering (ME) and synthetic biology (SynBio) are two intersecting fields with different focal points. While SynBio focuses more on genomic aspects to build novel cell devices, ME emphasizes the phenotypic outputs (e.g., production). SynBio has the potential to revolutionize the bio-productions; however, the introduction of synthetic devices/pathways often consumes significant cellular resources and incurs fitness costs. Currently, SynBio applications still lack guidelines in re-allocating cellular carbon and energy fluxes. To resolve this, ME principles may help the SynBio community. First, 13C MFA (metabolic flux analysis) can characterize the burdens of genetic infrastructures and reveal optimal strategies for distributing cellular resources. Second, novel microbial chassis should be explored to employ their unique metabolic features for product synthesis. Third, standardization and classification of bio-production papers will not only improve the communication between ME and SynBio, but also facilitate text mining and machine learning to harness information for rational strain design. Ultimately, the data-driven modeling and 13C MFA will be integral components of the SynBio design-build-test-learn cycle for generating novel microbial cell factories.

Green sulfur bacteria, which live in extremely low-light environments, use chlorosomes to harvest light. A chlorosome is the most efficient, and arguably the simplest, light-harvesting antenna complex, which contains hundreds of thousands of densely packed bacteriochlorophylls (BChls). To harvest light efficiently, BChls in a chlorosome form supramolecular aggregates; thus, it is of great interest to determine the organization of the BChls in a chlorosome. In this study, we conducted a (13)C solid-state nuclear magnetic resonance and Mg K-edge X-ray absorption analysis of chlorosomes from wild-type Chlorobaculum tepidum. The X-ray absorption results indicated that the coordination number of the Mg in the chlorosome must be >4, providing evidence that electrostatic interactions formed between the Mg of a BChl and the carbonyl group or the hydroxyl group of the neighboring BChl molecule. According to the intermolecular distance constraints obtained on the basis of (13)C homonuclear dipolar correlation spectroscopy, we determined that the molecular assembly of BChls is dimer-based and that the hydrogen bonds among the BChls are less extensive than commonly presumed because of the twist in the orientation of the BChl dimers. This paper also reports the first (13)C homonuclear correlation spectrum acquired for carotenoids and lipids-which are minor, but crucial, components of chlorosomes-extracted from wild-type Cba. tepidum.

Chlorobaculum [Cba.] tepidum is known to grow optimally at 48-52 °C and can also be cultured at ambient temperatures. In this paper, we prepared constant temperature, temperature shift, and temperature shift followed by backshift cultures and investigated the intrinsic properties and spectral features of chlorosomes from those cultures using various approaches, including temperature-dependent measurements on circular dichroism (CD), UV-visible, and dynamic light scattering. Our studies indicate that (1) chlorosomes from constant temperature cultures at 50 and 30 °C exhibited more resistance to heat relative to temperature shift cultures; (2) as temperature increases bacteriochlorophyll c (BChl c) in chlorosomes is prone to demetalation, which forms bacteriopheophytin c, and degradation under aerobic conditions. Some BChl c aggregates inside reduced chlorosomes prepared in low-oxygen environments can reform after heat treatments; (3) temperature shift cultures synthesize and incorporate more BChl c homologs with a smaller substituent at C-8 on the chlorin ring and less BChl c homologs with a larger long-chain alcohol at C-17(3) versus constant-temperature cultures. We hypothesize that the long-chain alcohol at C-17(3) (and perhaps together with the substituent at C-8) may account for thermal stability of chlorosomes and the substituent at C-8 may assist self-assembling BChls; and (4) while almost identical absorption spectra are detected, chlorosomes from different growth conditions exhibited differences in the rotational length of the CD signal, and aerobic and reduced chlorosomes also display different Qy CD intensities. Further, chlorosomes exhibited changes of CD features in response to temperature increases. Additionally, we compare temperature-dependent studies for the Cba. tepidum chlorosomes and previous studies for the Chloroflexus aurantiacus chlorosomes. Together, our work provides useful and novel insights on the properties and organization of chlorosomes.

Chloroflexus aurantiacus is an anoxygenic phototrophic bacterium. Its unique CO2 fixation pathway and primitive light-harvesting antenna complexes have attracted extensive research attentions. In this work, we investigated the photoheterotrophic growth of C. aurantiacus J-10-fl using acetate [at 55°C and without H2(g)]. The results indicate that glycine can promote anaerobic biomass production in a minimal medium by threefold to fivefold. Via (13)C-metabolite analysis, we observed that glycine was involved in serine synthesis. Instead of being used as a major carbon source, glycine was degraded to produce C1 units and NAD(P)H. Tracer experiments also suggest that photoheterotrophic cultures growing with a exogenous glycine source exhibited capabilities of assimilating CO2 via multiple routes (including the 3-hydroxypropionate pathway). Finally, glycylglycine, a commonly used culture buffer, also significantly enhanced photoheterotrophic growth of C. aurantiacus, probably due to its thermal or enzymatic breakdown to glycine.

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k cat/K m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.

Heliobacteria are a group of anoxygenic phototrophic bacteria that use a unique pigment, bacteriochlorophyll g, for photosynthetic energy conversion within a type I homodimeric reaction center. Like their nonphotosynthetic relatives the clostridia, heliobacteria have a gram-positive cell structure and can form heat-resistant endospores. Heliobacteria are also unusual in that they are the only anaerobic anoxygenic phototrophs that lack a mechanism for autotrophic growth. Growth of heliobacteria is therefore dependent upon the presence of usable organic carbon sources and occurs either photoheterotrophically or chemotrophically (via pyruvate fermentation). While knowledge of heliobacterial photosynthesis and physiology has steadily increased since the relatively recent discovery of these phototrophs in the 1980s, high-resolution structural data pertaining to features of the heliobacterial photosynthetic apparatus are not yet available. This chapter summarizes our current understanding of energy conservation in heliobacteria as it relates to central carbon metabolism (in both light and dark conditions), electron transport, and light harvesting and photochemistry within the reaction center.

After feeding microbes with a defined 13 C substrate, unique isotopic patterns (isotopic fingerprints) can be formed in their metabolic products. Such labelling information not only can provide novel insights into functional pathways but also can determine absolute carbon fluxes through the metabolic network via metabolic modelling approaches. This technique has been used for finding pathways that may have been mis-annotated in the past, elucidating new enzyme functions, and investigating cell metabolisms in microbial communities. In this review paper, we summarize the applications of 13 C approaches to analyse novel cell metabolisms for the past 3 years. The isotopic fingerprints (defined as unique isotopomers useful for pathway identifications) have revealed the operations of the Entner–Doudoroff pathway, the reverse tricarboxylic acid cycle, new enzymes for biosynthesis of central metabolites, diverse respiration routes in phototrophic metabolism, co-metabolism of carbon nutrients and novel CO 2 fixation pathways. This review also discusses new isotopic methods to map carbon fluxes in global metabolisms, as well as potential factors influencing the metabolic flux quantification (e.g. metabolite channelling, the isotopic purity of 13 C substrates and the isotopic effect). Although 13 C labelling is not applicable to all biological systems (e.g. microbial communities), recent studies have shown that this method has a significant value in functional characterization of poorly understood micro-organisms, including species relevant for biotechnology and human health.

The chlorosome is a highly specialized supramolecular light-harvesting antenna complex found in green photosynthetic bacteria and is composed of self-assembled bacteriochlorophyll (BChl) pigments entrapped in a lipid vesicle. These organelles are of interest for development of synthetic devices for solar harvesting and conversion because the organization and packing of BChls in the chlorosome provides a highly efficient light collection and energy funneling mechanism with properties that are superior to similar artificial systems based on self-assembled BChl pigment analogues. In this study, we investigated sol–gel chemistry as an approach to entrap and stabilize chlorosomes isolated from Chloroflexus aurantiacus. Two distinct synthesis approaches that differed in the H2O/Si ratio in the gels were investigated. Spectrophotometric analysis showed that the chlorosomes were intact when encapsulated in sol–gels and did not suffer any deleterious effects during the entrapment process. In addition, the integrity of the chlorosomes was unaffected by methanol levels that can result during the formation of sol–gels. Using small-angle neutron scattering it was not only possible to characterize the properties of the sol–gel matrix but also the size, shape and aggregation state of the entrapped chlorosomes. The sol–gels formed at a higher H2O/Si ratio (FH gels) resulted in a more branched gel structure with a larger pore size compared to the gels formed at lower H2O/Si ratio (PH gels). The chlorosomes entrapped in FH gels had dimensions of � 16.0 � 51.1 � 180.1 nm which agrees well with the size of chlorosomes previously determined using cryo-transmission electron microscopy, while the chlorosomes in the PH gels appear to be aggregated. The approach described here offers new possibilities for the development of artificial solar-harvesting and energy conversion devices based on naturally occurring photosynthetic systems.

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome - the light absorbing antenna complex - in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of Cba. tepidum grows slower and incorporates less BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays: (a) smaller cross-sectional radius and overall size; (b) simplified BChl c homologues with smaller side chains; (c) blue-shifted Qy absorption maxima and (d) a sigmoid-shaped circular dichroism (CD) spectra. Using a theoretical model we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis., 32 pages, 14 figures