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Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H2 and CO2 compared to organotrophic growth with fructose.

ABSTRACT: Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO), or with H2 and CO2. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H2/CO2 was compared by RNA-Seq. Upregulated genes with both syngas and H2/CO2 (compared to fructose) point to the urea cycle, uptake and degradation of peptides and amino acids, response to sulfur starvation, potentially NADPH-producing pathways involving (S)-malate and ornithine, quorum sensing, sporulation, and cell wall remodeling, suggesting a global and multicellular response to lithotrophic conditions. With syngas, the upregulated (R)-lactate dehydrogenase gene represents a route of electron transfer from ferredoxin to NAD. With H2/CO2, flavodoxin and histidine biosynthesis genes were upregulated. Downregulated genes corresponded to an intracytoplasmic microcompartment for disposal of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several cytoplasmic and membrane-associated redox-active protein genes were differentially regulated. The transcriptomic profiles of C. ljungdahlii in lithotrophic and organotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide biofuel and commodity chemical production with this species.

SUBMITTER: Aklujkar M

PROVIDER: S-EPMC5640608 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H2 and CO2 compared to organotrophic growth with fructose.

Aklujkar Muktak M Leang Ching C Shrestha Pravin M PM Shrestha Minita M Lovley Derek R DR

Scientific reports 20171013 1

Clostridium ljungdahlii derives energy by lithotrophic and organotrophic acetogenesis. C. ljungdahlii was grown organotrophically with fructose and also lithotrophically, either with syngas - a gas mixture containing hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO), or with H2 and CO2. Gene expression was compared quantitatively by microarrays using RNA extracted from all three conditions. Gene expression with fructose and with H2</su ...[more]

PMID: 29030620

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Project description:Clostridium ljungdahlii derives energy by acetogenesis as a lithotrophic pathway and as part of various organotrophic pathways. Its recently sequenced genome has made it possible to discover changes in gene expression that occur in different growth modes, from which strategies for biofuel production may be developed. C. ljungdahlii was grown with fructose as an organoheterotrophic substrate and also lithoautotrophically, either on syngas or with H2 as the electron donor and CO2 as the electron acceptor. RNA extracted from all three conditions was analyzed by hybridization to a microarray, and gene expression was compared quantitatively by RNA-Seq of C. ljungdahlii grown with fructose and with H2 and CO2. Results. Strongly upregulated (> 10-fold, p < 0.05) genes with both syngas and H2/CO2 encode enzymes that degrade aspartate and arginine through the urea cycle to control the release of ammonia from amino acids. Numerous genes for uptake and degradation of peptides and amino acids, response to sulfur starvation, and molybdopterin-dependent pathways were also significantly (> 2-fold, p < 0.05) upregulated, along with three potentially NADPH-producing pathways for which the key metabolites are (S)-malate, ornithine and 6-phospho-D-gluconate. Upregulation of genes implicated in quorum sensing, sporulation and cell wall remodeling suggests a global and multicellular response to lithoautotrophic conditions. With syngas, (R)-lactate dehydrogenase and associated electron transfer flavoproteins were upregulated, representing a route of electron transfer from ferredoxin to NAD that is independent of the proton-translocating Rnf complex. With H2/CO2, a flavodoxin and histidine biosynthesis enzymes were upregulated. Genes for degradation of purine bases entering the cell by facilitated diffusion were upregulated in lithotrophic cells, whereas genes for degradation of adenine or adenosine derivatives entering by active transport were significantly (2-fold, p < 0.05) downregulated. The most downregulated genes, after those specific to fructose metabolism, encode enzymes of pyrimidine and purine biosynthesis, arginine fermentation to ornithine, threonine biosynthesis and phosphate uptake. Genes for biogenesis of an intracytoplasmic microcompartment were downregulated, within which (S)-1,2-propanediol dehydratase and other enzymes may dispose of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several redox-active proteins, both cytoplasmic and membrane-associated, and predicted cell surface proteins were identified as differentially regulated. Conclusion. The transcriptomic profiles of C. ljungdahlii in lithoautotrophic and organoheterotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide the production of biofuels and commodity chemicals with this species.

Project description:There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C5H8), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C10 and C15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii, used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation.IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii, an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway.

Project description:BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Dataset Information

Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H2 and CO2 compared to organotrophic growth with fructose.

Publications

Transcriptomic profiles of Clostridium ljungdahlii during lithotrophic growth with syngas or H<sub>2</sub> and CO<sub>2</sub> compared to organotrophic growth with fructose.

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