Chloroflexus Research Articles

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

BackgroundIn the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs.ResultsHere we describe the metabolic engineering of baker’s yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella entericaSEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L−1 3HP with a yield of 13 % C-mol C-mol−1 glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by 13C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed.ConclusionsIn this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0451-5) contains supplementary material, which is available to authorized users.

Digalactosyldiacylglycerol is essential in Synechococcus elongatus PCC 7942, but its function does not depend on its biosynthetic pathway

Digalactosyldiacylglycerol (DGDG) is a major component of thylakoid membranes, occupying approximately 20% of the membrane system. This lipid composition is conserved from cyanobacteria to the chloroplasts of terrestrial plants, suggesting that DGDG is important for the function of photosynthetic membranes. Here we isolated the gene for DGDG synthase in the cyanobacterium Synechococcus elongatus PCC 7942 (7942dgdA) and found that this gene is essential for this species. 7942dgdA could be knocked out only when genes for cyanobacterial or plant DGDG synthases were expressed, indicating that the important factor was not the specific synthetic pathway but the lipid product. Lack of DGDG could not be compensated by the other membrane lipids in S. elongatus PCC 7942 or by glucosylgalactosyldiacylglycerol synthesized by the β-GlcT gene of Chloroflexus aurantiacus. These results reveal that DGDG has an indispensable role in S. elongatus PCC 7942 and that the second galactose molecule is key. Conservation and distribution of the galactolipid synthetic pathway among oxygenic phototrophs is discussed. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Editorial: Enzymes from Extreme Environments.

Editorial: Enzymes from Extreme Environments.

Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions.

The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

Glycine Cleavage Powers Photoheterotrophic Growth of Chloroflexus aurantiacus in the Absence of H 2.

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 13C-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.

Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli

In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h.

Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin.

Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis, and is required by all three domains of life. Here, we report the purification and biochemical and structural characterization of enolase from Chloroflexus aurantiacus, a thermophilic anoxygenic phototroph affiliated with the green non-sulfur bacteria. The protein was purified as a homodimer with a subunit molecular weight of 46 kDa. The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25 and 80°C were 147 and 300 U mg−1 of protein, respectively. Km values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03 mM, respectively. The Km values for Mg2+ binding at these temperatures were 2.5 and 1.9 mM, respectively. When compared to enolase from mesophiles, the biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted. These data are consistent with the results of our phylogenetic analysis of enolase, which reveal that enolase has a thermophilic origin.

Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species.

Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.

Genomic distribution of B-vitamin auxotrophy and uptake transporters in environmental bacteria from the Chloroflexi phylum.

Bacteria from the Chloroflexi phylum are dominant members of phototrophic microbial mat communities in terrestrial thermal environments. Vitamins of B group are key intermediates (precursors) in the biosynthesis of indispensable enzyme cofactors driving numerous metabolic processes in all forms of life. A genomics-based reconstruction and comparative analysis of respective biosynthetic and salvage pathways and riboswitch regulons in over 20 representative Chloroflexi genomes revealed a widespread auxotrophy for some of the vitamins. The most prominent predicted phenotypic signature, auxotrophy for vitamins B1 and B7 was experimentally confirmed for the best studied model organism Chloroflexus aurantiacus. These observations along with identified candidate genes for the respective uptake transporters pointed to B vitamin cross-feeding as an important aspect of syntrophic metabolism in microbial communities. Inferred specificities of homologous substrate-binding components of ABC transporters for vitamins B1 (ThiY) and B2 (RibY) were verified by thermofluorescent shift approach. A functional activity of the thiamine-specific transporter ThiXYZ from C. aurantiacus was experimentally verified by genetic complementation in E. coli. Expanding the integrative approach, which was applied here for a comprehensive analysis of B-vitamin metabolism in Chloroflexi would allow reconstruction of metabolic interdependencies in microbial communities.

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area, Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54 degrees C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected irn the samples from both the thermophilic and mesophilic mats. Cultures ofnonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolatd from the mats developing at high (50.6-49.4 degrees C) and low temperatures (45-20 degrees C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealedin low-temperature mats. Truly thermophilic purple and gree sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfuret communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophylla-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20 degrees C) mat is of interest.

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus.

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.

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus.

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of~18nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.

Evaluating the annotation of protein-coding genes in bacterial genomes: Chloroflexus aurantiacus strain J-10-fl and Natrinema sp J7-2 as case studies.

Gene annotation plays a key role in subsequent biochemical and molecular biological studies of various organisms. There are some errors in the original annotation of sequenced genomes because of the lack of sufficient data, and these errors may propagate into other genomes. Therefore, genome annotation must be checked from time to time to evaluate newly accumulated data. In this study, we evaluated the gene density of 2606 bacteria or archaea, and identified 2 with extreme values, the minimum value (Chloroflexus aurantiacus strain J-10-fl) and maximum value (Natrinema sp J7-2), to conduct genome re-annotation. In the genome of C. aurantiacus strain J-10-fl, we identified 17 new genes with definite functions and eliminated 34 non-coding open-reading frames; in the genome of Natrinema sp J7-2, we eliminated 118 non-coding open reading frames. Our re-annotation procedure may provide a reference for improving the annotation of other bacterial genomes.

An experimental point of view on hydration/solvation in halophilic proteins

Protein-solvent interactions govern the behaviors of proteins isolated from extreme halophiles. In this work, we compared the solvent envelopes of two orthologous tetrameric malate dehydrogenases (MalDHs) from halophilic and non-halophilic bacteria. The crystal structure of the MalDH from the non-halophilic bacterium Chloroflexus aurantiacus (Ca MalDH) solved, de novo, at 1.7 Å resolution exhibits numerous water molecules in its solvation shell. We observed that a large number of these water molecules are arranged in pentagonal polygons in the first hydration shell of Ca MalDH. Some of them are clustered in large networks, which cover non-polar amino acid surface. The crystal structure of MalDH from the extreme halophilic bacterium Salinibacter ruber (Sr) solved at 1.55 Å resolution shows that its surface is strongly enriched in acidic amino acids. The structural comparison of these two models is the first direct observation of the relative impact of acidic surface enrichment on the water structure organization between a halophilic protein and its non-adapted counterpart. The data show that surface acidic amino acids disrupt pentagonal water networks in the hydration shell. These crystallographic observations are discussed with respect to halophilic protein behaviors in solution

Acetate formation in the photoheterotrophic bacteriumChloroflexus aurantiacusinvolves an archaeal type ADP-forming acetyl-CoA synthetase isoenzyme I

The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate during photohetero trophic growth on glucose and in resting cell suspensions. Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells, the activities of phosphotransacetylase and acetate kinase, usually involved in acetate formation in Bacteria, could not be detected; instead, the cells contained an acetyl-CoA synthetase (ADP-forming) (ACD) (acetyl-CoA + ADP + Pi → acetate + ATP + CoA), an enzyme so far reported in prokaryotes to be specific for acetate-forming Archaea. ACD, which was induced 10-fold during growth on glucose, was purified and the encoding gene was identified as Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl-CoA/acetate and other acyl-CoA esters/acids were determined, showing similarity of the C. aurantiacus ACD to archaeal ACD I isoenzymes, which are involved in acetate formation from sugars. This is the first report of a functional ACD involved in acetate formation in the domain of Bacteria.

Metalloproteins Diversified: The Auracyanins Are a Family of Cupredoxins That Stretch the Spectral and Redox Limits of Blue Copper Proteins

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display the entire known spectral and redox variations known in the type 1 copper site. C. aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon.

Reconstruction of rod self-aggregates of natural bacteriochlorophylls-c from Chloroflexus aurantiacus

Bacteriochlorophyll(BChl)s-c were extracted, isolated and purified from cultured cells of a green photosynthetic bacterium, Chloroflexus (Cfl.) aurantiacus. Their self-aggregates were prepared from a hydrophobic hexane-based solution and the obtained self-aggregate solids were examined by electronic absorption and circular dichroism (CD) spectroscopy as well as atomic force microscopy (AFM). Visible/near-infrared absorption and CD spectra of the BChl-c self-aggregates were very similar to those in cells of Cfl. aurantiacus. AFM analysis indicated that some self-aggregates had rods with a 5-nm diameter and a 3.5-μm length at longest. The rod diameter was identical to that reported for natural chlorosomal rods of Cfl. aurantiacus. Rod self-aggregates of naturally occurring BChls-c with a 5-nm diameter were first reconstructed here in vitro.

Functional analysis and expression of the mono-heme containing cytochrome c subunit of alternative complex III in Chloroflexus aurantiacus

The filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus possesses an unusual electron transfer complex called Alternative Complex III instead of the cytochrome bc or bf type complex found in nearly all other known groups of phototrophs. Earlier work has confirmed that Alternative Complex III behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. In this work, we focus on elucidating the contribution of individual subunits to the overall function of Alternative Complex III. The monoheme subunit ActE has been expressed and characterized in Escherichia coli. A partially dissociated Alternative Complex III missing subunit ActE and subunit ActG was obtained by treatment with the chaotropic agent KSCN, and was then reconstituted with the expressed ActE. Enzymatic activity of the partially dissociated Alternative Complex III was greatly reduced and was largely restored in the reconstituted complex. The redox potential of the heme in the recombinant ActE was +385mV vs. NHE, similar to the highest potential heme in the intact complex. The results strongly suggest that the monoheme subunit, ActE, is the terminal electron carrier for Alternative Complex III.

Temperature shift effect on the Chlorobaculum tepidum chlorosomes

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.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD− double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC−(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.