Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

From waste to health-supporting molecules: biosynthesis of natural products from lignin-, plastic- and seaweed-based monomers using metabolically engineered Streptomyces lividans.

BACKGROUND: Transforming waste and nonfood materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry to optimize the use of resources while reducing environmental footprint. However, despite these advancements, the production of high-value natural products often continues to depend on the use of first-generation substrates, underscoring the intricate processes and specific requirements of their biosyntheses. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce a wide array of natural products, which is attributed to its genetic versatility and potent secondary metabolic activity. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and nonfood substrates. RESULTS: We metabolically engineered S. lividans to heterologously produce the ribosomally synthesized and posttranslationally modified peptide bottromycin, as well as the polyketide pamamycin. The modified strains successfully produced these compounds using waste and nonfood model substrates such as protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. In terms of production efficiency, S. lividans showed remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, which led to enhanced and highly selective bottromycin production. Additionally, the strain generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract with no additional nutrients. CONCLUSION: Our study showcases the successful production of high-value natural products based on the use of varied waste and nonfood raw materials, circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience when grown on these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could be focused on enhancing S. lividans substrate utilization pathways to process the intricate mixtures commonly found in waste and nonfood sources more efficiently.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production.

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

ECHDC1 knockout mice accumulate ethyl-branched lipids and excrete abnormal intermediates of branched-chain fatty acid metabolism.

The cytosolic enzyme ethylmalonyl-CoA decarboxylase (ECHDC1) decarboxylates ethyl- or methyl-malonyl-CoA, two side products of acetyl-CoA carboxylase. These CoA derivatives can be used to synthesize a subset of branched-chain fatty acids (FAs). We previously found that ECHDC1 limits the synthesis of these abnormal FAs in cell lines, but its effects in vivo are unknown. To further evaluate the effects of ECHDC1 deficiency, we generated knockout mice. These mice were viable, fertile, showed normal postnatal growth, and lacked obvious macroscopic and histologic changes. Surprisingly, tissues from wild-type mice already contained methyl-branched FAs due to methylmalonyl-CoA incorporation, but these FAs were only increased in the intraorbital glands of ECHDC1 knockout mice. In contrast, ECHDC1 knockout mice accumulated 16-20-carbon FAs carrying ethyl-branches in all tissues, which were undetectable in wild-type mice. Ethyl-branched FAs were incorporated into different lipids, including acylcarnitines, phosphatidylcholines, plasmanylcholines, and triglycerides. Interestingly, we found a variety of unusual glycine-conjugates in the urine of knockout mice, which included adducts of ethyl-branched compounds in different stages of oxidation. This suggests that the excretion of potentially toxic intermediates of branched-chain FA metabolism might prevent a more dramatic phenotype in these mice. Curiously, ECHDC1 knockout mice also accumulated 2,2-dimethylmalonyl-CoA. This indicates that the broad specificity of ECHDC1 might help eliminate a variety of potentially dangerous branched-chain dicarboxylyl-CoAs. We conclude that ECHDC1 prevents the formation of ethyl-branched FAs and that urinary excretion of glycine-conjugates allows mice to eliminate potentially deleterious intermediates of branched-chain FA metabolism.

Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(ß-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(ß-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake.

The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.

Engineering the precursor pool to modulate the production of pamamycins in the heterologous host S. albus J1074.

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger, induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus.

Variants in the ethylmalonyl-CoA decarboxylase (ECHDC1) gene: a novel player in ethylmalonic aciduria?

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short-chain acyl-CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl-CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss-of-function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2.

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions.

BACKGROUND: The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. RESULTS: In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. CONCLUSION: Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

The synthesis of branched-chain fatty acids is limited by enzymatic decarboxylation of ethyl- and methylmalonyl-CoA.

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function.

Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway.

Rhodobacter sphaeroides is able to use 3-hydroxypropionate as the sole carbon source through the reductive conversion of 3-hydroxypropionate to propionyl coenzyme A (propionyl-CoA). The ethylmalonyl-CoA pathway is not required in this process because a crotonyl-CoA carboxylase/reductase (Ccr)-negative mutant still grew with 3-hydroxypropionate. Much to our surprise, a mutant defective for another specific enzyme of the ethylmalonyl-CoA pathway, mesaconyl-CoA hydratase (Mch), lost its ability for 3-hydroxypropionate-dependent growth. Interestingly, the Mch-deficient mutant was rescued either by introducing an additional ccr in-frame deletion that resulted in the blockage of an earlier step in the pathway or by heterologously expressing a gene encoding a thioesterase (YciA) that can act on several CoA intermediates of the ethylmalonyl-CoA pathway. The mch mutant expressing yciA metabolized only less than half of the 3-hydroxypropionate supplied, and over 50% of that carbon was recovered in the spent medium as free acids of the key intermediates mesaconyl-CoA and methylsuccinyl-CoA. A gradual increase in growth inhibition due to the blockage of consecutive steps of the ethylmalonyl-CoA pathway by gene deletions suggests that the growth defects were due to the titration of free CoA and depletion of the CoA pool in the cell rather than to detrimental effects arising from the accumulation of a specific metabolite. Recovery of carbon in mesaconate for the wild-type strain expressing yciA demonstrated that carbon flux through the ethylmalonyl-CoA pathway occurs during 3-hydroxypropionate-dependent growth. A possible role of the ethylmalonyl-CoA pathway is proposed that functions outside its known role in providing tricarboxylic acid intermediates during acetyl-CoA assimilation.IMPORTANCE Mutant analysis is an important tool utilized in metabolic studies to understand which role a particular pathway might have under certain growth conditions for a given organism. The importance of the enzyme and of the pathway in which it participates is discretely linked to the resulting phenotype observed after mutation of the corresponding gene. This work highlights the possibility of incorrectly interpreting mutant growth results that are based on studying a single unit (gene and encoded enzyme) of a metabolic pathway rather than the pathway in its entirety. This work also hints at the possibility of using an enzyme as a drug target although the enzyme may participate in a nonessential pathway and still be detrimental to the cell when inhibited.

Structural basis for substrate specificity of methylsuccinyl-CoA dehydrogenase, an unusual member of the acyl-CoA dehydrogenase family.

(2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD) and is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation. It catalyzes the oxidation of (2S)-methylsuccinyl-CoA to α,ß-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. Here we report the crystal structure of MCD at a resolution of 1.37 Å. The enzyme forms a homodimer of two 60-kDa subunits. Compared with other ACDs, MCD contains an ∼170-residue-long N-terminal extension that structurally mimics a dimer-dimer interface of these enzymes that are canonically organized as tetramers. MCD catalyzes the unprecedented oxidation of an α-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. MCD apparently evolved toward preventing the nonspecific oxidation of succinyl-CoA, which is a close structural homolog of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism. For different metabolic engineering and biotechnological applications, however, an enzyme that can oxidize succinyl-CoA to fumaryl-CoA is sought after. Based on the MCD structure, we were able to shift substrate specificity of MCD toward succinyl-CoA through active-site mutagenesis.

Introduction of Glyoxylate Bypass Increases Hydrogen Gas Yield from Acetate and l-Glutamate in Rhodobacter sphaeroides.

Rhodobacter sphaeroides produces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as l-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2 yield from acetate in the presence of l-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H2 production from acetate and l-glutamate but not for growth and acetate consumption in the presence of l-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2 production, although its H2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H2 production from acetate and l-glutamate when both acetate-assimilating pathways are present.IMPORTANCE As an alternative to fossil fuel, H2 is a promising renewable energy source. Although photofermentative H2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2 yields from acetate and l-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H2 yields from acetate and l-glutamate. Therefore, this study provides a novel strategy for improving H2 yields from acetate in the presence of l-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H2 production.

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.

BACKGROUND: Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production. RESULTS: The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L). CONCLUSIONS: The successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species.

Understanding the physiological roles of polyhydroxybutyrate (PHB) in Rhodospirillum rubrum S1 under aerobic chemoheterotrophic conditions.

Polyhydroxybutyrate (PHB) is an important biopolymer accumulated by bacteria and associated with cell survival and stress response. Here, we make two surprising findings in the PHB-accumulating species Rhodospirillum rubrum S1. We first show that the presence of PHB promotes the increased assimilation of acetate preferentially into biomass rather than PHB. When R. rubrum is supplied with (13)C-acetate as a PHB precursor, 83.5 % of the carbon in PHB comes from acetate. However, only 15 % of the acetate ends up in PHB with the remainder assimilated as bacterial biomass. The PHB-negative mutant of R. rubrum assimilates 2-fold less acetate into biomass compared to the wild-type strain. Acetate assimilation proceeds via the ethylmalonyl-CoA pathway with (R)-3-hydroxybutyrate as a common intermediate with the PHB pathway. Secondly, we show that R. rubrum cells accumulating PHB have reduced ribulose 1,5-bisphosphate carboxylase (RuBisCO) activity. RuBisCO activity reduces 5-fold over a 36-h period after the onset of PHB. In contrast, a PHB-negative mutant maintains the same level of RuBisCO activity over the growth period. Since RuBisCO controls the redox potential in R. rubrum, PHB likely replaces RuBisCO in this role. R. rubrum is the first bacterium found to express RuBisCO under aerobic chemoheterotrophic conditions.

The activity of RubisCO and energy demands for its biosynthesis. Comparative studies with CO2-reductases.

Most CO2 on Earth is fixed into organic matter via reactions catalysed by enzymes called carboxylases. CO2-fixation via carboxylases occurs in the Calvin-Benson-Bassham (CBB) cycle, and the crucial role in this cycle is played by RubisCO (D-ribulose 1,5-bisphosphate carboxylase/oxygenase). CO2 can also be fixed by pathways, where a reduction of CO2 to formate or carbon monoxide (CO) occurs. The latter reactions are performed by so-called CO2-reductases e.g. formate dehydrogenase (FDH), carbon-monooxide (CO) dehydrogenase (CODH), and crotonyl-CoA reductase/carboxylase (CCR). In general, a simple model of enzymatic activity based only on a turnover rate of an enzyme for an appropriate substrate (kcat) is insufficient. Based on estimated metabolic costs of each amino acid, the average energetic costs of amino acid biosynthesis (Eaa), and the total costs (ET) for selected CO2-fixing enzymes were analyzed concerning 1) kcat for CO2 (kC), and 2) specificity factor (Srel) for RubisCO. A comparison of Eaa and ET to their kC showed that CODH and FDHs do not need to be more efficient enzymes in CO2 capturing pathways than some forms of RubisCO. CCR was the only both low-cost and highly active CO2-fixing enzyme. The obtained results showed also that there exists an evolutionarily conserved trade-off between Srel of RubisCOs and the energetic demands needed for their biosynthesis. Phylogenetic analysis demonstrated that RubisCO, CODH, FDH, and CCR are enzymes formed as a result of parallel evolution. Moreover, the kinetic parameters (kC) of CO2-fixing enzymes were plausibly optimized already at the early stages of life evolution on Earth.

TCA Cycle Replenishing Pathways in Photosynthetic Purple Non-Sulfur Bacteria Growing with Acetate.

Purple non-sulfur bacteria (PNSB) are anoxygenic photosynthetic bacteria harnessing simple organic acids as electron donors. PNSB produce a-aminolevulinic acid, polyhydroxyalcanoates, bacteriochlorophylls a and b, ubiquinones, and other valuable compounds. They are highly promising producers of molecular hydrogen. PNSB can be cultivated in organic waste waters, such as wastes after fermentation. In most cases, wastes mainly contain acetic acid. Therefore, understanding the anaplerotic pathways in PNSB is crucial for their potential application as producers of biofuels. The present review addresses the recent data on presence and diversity of anaplerotic pathways in PNSB and describes different classifications of these pathways.

Silencing of ECHDC1 inhibits growth of gemcitabine-resistant bladder cancer cells.

Combined gemcitabine and cisplatin (GC) treatment is a first line chemotherapy for bladder cancer. However, acquired resistance to GC has been a major problem. To address the mechanism of gemcitabine resistance, and to identify potential biomarkers or target proteins for its therapy, we aimed to identify candidate proteins associated with gemcitabine resistance using proteomic analysis. We established gemcitabine-resistant human bladder cancer cell lines (UMUC3GR and HT1376GR) from gemcitabine-sensitive human bladder cancer cell lines (UMUC3 and HT1376). We compared the protein expression of parental and gemcitabine-resistant cell lines using isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography tandem mass spectrometry. Among the identified proteins, ethylmalonyl-CoA decarboxylase (ECHDC1) expression was significantly increased in both of the gemcitabine-resistant cell lines compared to the respective parental cell lines. Silencing of ECHDC1 reduced ECHDC1 expression and significantly inhibited the proliferation of UMUC3GR cells. Furthermore, silencing of ECHDC1 induced upregulation of p27, which is critical for cell cycle arrest in the G1 phase, and induced G1 arrest. In conclusion, ECHDC1 expression is increased in gemcitabine-resistant bladder cancer cells, and is involved in their cell growth. ECHDC1, which is a metabolite proofreading enzyme, may be a novel potential target for gemcitabine-resistant bladder cancer therapy.

Replacing the Ethylmalonyl-CoA Pathway with the Glyoxylate Shunt Provides Metabolic Flexibility in the Central Carbon Metabolism of Methylobacterium extorquens AM1.

The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium Methylobacterium extorquens AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in M. extorquens AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in M. extorquens AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future.