Heat-shock proteins: DnaK, GroESL, and Hsp21
The so-called heat-shock proteins, including DnaK and GroEL (two highly conserved molecular chaperones), were induced by a temperature upshift (from 30 to 42°C) . The dnaK operon includes four genes: orfA (a heat shock gene with an unknown function), grpE, dnaK, and dnaJ. The groE operon includes groES and groEL. A conserved inverted repeat was found upstream of dnaK and groEL and may be implicated as a cis-acting regulatory site. Those heat-shock genes were expressed at the beginning of solvent formation and after heat stress. The mRNA levels of heat-shock genes and solvent-producing genes reached a maximum at the same time during the metabolic shift . In addition, over-expression of the heat shock proteins (GroES and GroEL) allowed an increase in the final solvent concentration, presumably by stabilizing solventogenic enzymes . Therefore, the heat-shock response in C. acetobutylicum might be part of a global regulatory network including different stress responses, such as heat shock, metabolic switching and sporulation .
The small heat shock protein Hsp21 of C. acetobutylicum was identified as a rubrerythrin-like protein. The transcription of its encoding rbr3AB operon was induced by various environmental stress conditions: heat, oxidative stress, pH of the growth medium, addition of NaCl or butanol, and incubation temperature .
The phoPR gene locus of C. acetobutylicum comprises two genes, phoP and phoR. These PhoPR proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system . It was shown that a significant increase in the mRNA transcript levels of both phoP and phoR was observed when external phosphate concentrations dropped below 0.3 mM. The autophosphorylation of PhoR and phosphorylation of PhoP were shown in vitro. An electromobility shift assay proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon (comprising five genes, pstS, pstC, pstA, pstB, and phoU) of C. acetobutylicum .
SinR and AbrB are known to be transcriptional regulators involved in sporulation initiation in Bacillus subtilis.The role of SinR and AbrB in C. acetobutylicum was studied by Scotcher et al . There were three highly similar homologs to B. subtilis abrB found in C. acetobutylicum: abrB310, abrB1941 and abrB3647. The promoters of abrB1941 and abrB3647 were inactive, whereas the abrB310 promoter was strongly active. The expression of abrB310 was elevated at the beginning of solventogenesis. Moreover, the promoter of abrB310 contains a putative 0A box. When abrB310 expression was repressed by asRNA, the acids (acetate and butyrate) accumulated to approximately twice their normal concentration. Acetone and butanol production were delayed and decreased. It was also found that sporulation in this mutant was delayed, but the morphology of the sporulating cells and spores was normal. Therefore, it was proposed that AbrB310 may act as a regulator of the transition between the acidogenic and solventogenic states. Regarding SinR, a single homolog to B. subtilis SinR was found. When the C. acetobutylicum strain was transformed with plasmids to increase or decrease SinR expression, no significant differences were observed either in growth or in acid or solvent production compared to the control strain .
Per and Fur
Peroxide repressor (PerR)-homologous protein was identified as a key repressor that plays an important role in oxidative stress defense [128,129]. In vivo, PerR from C. acetobutylicum acts as a functional peroxide sensor. Transcriptomic data showed that genes involved in the detoxification system were regulated in PerR mutant, including reverse rubrerythrins (rbr3A-rbr3B), desulfoferrodoxin (dfx), rubredoxin (rd), NADH-dependent rubredoxin oxidoreductase (NROR) and the oxygen-reducing flavodiiron proteins (FDPs, encoded by fprA1 and fprA2). Only a few targets of direct PerR regulation were identified. They include two highly expressed genes, gapN and CODH, that are putatively involved in the central energy metabolism. Under exposure to O2, C. acetobutylicum also activates the repair and biogenesis of DNA and Fe-S clusters. The genes that were downregulated when exposed to O2 were the ones involved in butyrate formation .
In the same family as PerR, a ferric uptake regulator (Fur) that helps C. acetobutylicum sense and respond to the availability of iron was identified and characterized . The Fur mutant was constructed and showed a slow-growing phenotype and an enhanced sensitivity to oxidative stress, but no dramatic change in the fermentation pattern was observed. However, the mutant overproduced riboflavin. The operon ribDBAH responsible for riboflavin synthesis 51 was significantly upregulated. Iron limitation and inactivation of Fur also affected the expression of several genes involved in energy metabolism. Two genes, encoding a lactate dehydrogenase (ldh) and a flavodoxin (fld), were highly induced .
Carbon storage regulator (CsrA, encoded by CAC2209) largely involved in regulating multiple pathways, including flagella assembly, oligopeptide transport, phosphotransferase transport systems, stage III sporulation and the central carbon metabolism . The disruption of CsrA resulted in a decrease in solvent production. Genes involved in iron uptake and riboflavin synthesis were also reported to be regulated by CsrA in this study.
Catabolite control protein (CcpA) mediates the utilization of hexose and pentose sugar in C. acetobutylicum. When CcpA was mutated at V302N, the utilization of xylose was greatly improved . When this mutant was coupled with the over-expression of sol genes (ctfA, ctfB and adhE1), 30% more xylose was consumed compared to the wild-type when fermented in a mixture of glucose and xylose. EMSA experiments confirmed His-CcpA binding to the promoters of xylAB, sol, and CAC1353 (PTS system transporter subunit IIC). The mutated CcpA-V302N exhibited lower affinities to those promoters than the wild type .
The RegA protein of C. acetobutylicum, a homolog of the catabolite control protein CcpA in B. subtilis, has been found to inhibit the degradation of starch by the C. acetobutylicum staA gene product in E. coli .
A redox-sensing transcriptional repressor (Rex) was recently found to modulate its DNA-binding activity in response to the NADH/NAD+ ratio and to participate in the solventogenic shift of C. acetobutylicum [110,134]. High NADH/NAD+ prevents the binding of Rex to DNA . Rex binding sites were found in the promoters of different operons: adhE2, ldh, crt, thlA, asrT, ptb, and nadA [110,134], and those operons were upregulated in the Rex mutant. Among them, ldh1 was the most strongly induced gene, followed by adhE2. Genes encoding proteins involved in detoxification, including the reverse rubrerythrins (rbr3A-rbr3B), desulfoferrodoxin (dfx), rubredoxin (rd), NADH-dependent rubredoxin oxidoreductase (nror) and the oxygen reducing flavoproteins (fprA1 and fprA2), were down regulated by 2 to 18-fold compared to the wild type. The Rex mutant produced high amounts of ethanol and butanol and a significantly lower amount of acetone and hydrogen than the wild type [110,134].
The L-arabinose utilization pathway has been studied extensively in bacteria, including Gram-positive B. subtilis. The permease encoded by araE transports L-arabinose into the cells. Subsequent genes involved in the conversion of L-arabinose belong to the operon araABDLMNPQ-abfA. The AraR gene encodes the regulatory protein for the L-arabinose metabolism in B. subtilis [135,136], which negatively controls the expression of the ara regulon. AraR exhibits a DNA binding domain at the N-terminal region and an effector binding domain at the C-terminal . In the absence of arabinose, AraR represses the expression of the ara regulons. In the AraR mutant, there was a dramatic increase in the expression of the genes involved in arabinose utilization (two araEs, araD, two araAs and araK), the genes in the pentose phosphate pathway (ptk, tal and tkt), and genes involved in arabinoside degradation (epi and arb43). EMSA experiments confirmed the affinity of AraR for the promoter of araK, araE, araD, ptk and araR itself. In the presence of L-arabinose, AraR was released from the AraR-DNA complex .
Small regulatory RNA
Small noncoding RNAs are widespread in all kingdoms of life, where they serve to regulate and tune gene expression, subsequently influencing a wide range of cellular processes that include environmental stress responses and virulence processes in pathogens . Many sRNAs have been described in Gram-negative bacteria, but our understanding of their role in Gram-positive bacteria has been slower . Hundreds of sRNAs have recently been identified in Clostridia. Chen et al  have developed a method to identify potential sRNAs in the Clostridium genus. By genomics comparison and the in-silico prediction of rho-independent terminators and promoters, they have predicted sRNAs in the following 21 Clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, eight C. botulinum strains, C. cellulolyticum, C. difficile, two C. kluyveri strains, C. novyi, three C. perfringens strains, C. phytofermentans, C. tetani, and C. thermocellum. Most of the predicted sRNAs were found in noncoding regions. Seven of these sRNAs have Shine-Dalgarno (SD) sequences, and some possess a start codon downstream of the SD sequences. Thirty sRNAs (of 113 predicted) of C. acetobutylicum and 21 sRNAs of C. botulinum were validated by northern blot analysis and quantitative reverse transcription PCR (qRT-PCR). A conserved, novel sRNA was found to be responsive to the antibiotic clindamycin . Lately, Venkataramanan et al.  have used RNA deep sequencing to examine the sRNome of C. acetobutylicum that is responsive to butanol and butyrate and have identified 46 additional sRNAs. Under metabolite stress, these 159 sRNAs display divergent expression patterns. The expression of sRNAs affecting transcriptional (6S, S-box, and solB) and translational (tmRNA and SRP-RNA) processes and RNA chaperone Hfq were found to be stress-related .
This PhD thesis has two main parts, as follows: the metabolic engineering of C. acetobutylicum for the production of fuels and chemicals and understanding the regulatory role of an uncharacterized Cap0037 protein.
The metabolic engineering part has three objectives:
• Detail the homologous recombination technique by replicative plasmid that has been used successfully to construct C. acetobutylicum mutants.
• Construct a C. acetobutylicum mutant that produces n-butanol with high selectivity and yield on glucose. By the homologous recombination method, the metabolic pathways leading to by-products including lactate, butyrate and acetone were eliminated. The carbon fluxes were guided to n-butanol formation by replacing hbd with hbd1 and thla with atoB genes. The final strain CAB1060 also contains inactivated Rex protein which globally regulates metabolism in response to the intracellular NADH/NAD+ concentration. This CAB1060 mutant was then evaluated for its ability to produce butanol at high yield and selectivity in a continuous bioreactor coupling alcohol extraction by vacuum distillation to a cell recycling in order to improve productivities.
• Construct a C. acetobutylicum platform strain unable to produce hydrogen and useful for further engineering to produce chemicals and fuels. In this part, a new homologous recombination system using non-replicative plasmid was developed.
In the second part, the Cap0037protein of unknown function was primarily studied. To do so, a Cap0037 inactivated mutant was constructed using group II intron. A thorough comparison of the Cap0037::int mutant and the wild-type strain was analyzed from the transcriptomics, fluxomics, and proteomics aspects. In addition, DNA binding activity of Cap0037 was also studied. The potential Cap0037 regulon was predicted by bio-informatics tools.
Table of contents :
1 Chapter 1 Background
1.1 The history of acetone-butanol-ethanol (ABE) fermentation
1.2 Clostridium acetobutylicum strain
1.3 The central metabolism of Clostridium acetobutylicum
1.3.2 Enzymes involved in electron fluxes
1.3.3 Central pathway from acetyl-CoA to butyryl-CoA
1.3.4 Acetate formation
1.3.5 Butyrate formation
1.3.6 Lactate and acetoin formation
1.3.7 Acetone formation
1.3.8 Ethanol and butanol formation
1.4 Metabolic engineering tools in Clostridium acetobutylicum
1.4.1 Shuttle vectors for gene over-expression
1.4.2 Inducible promoter/repressor systems
1.4.3 Reporter systems
1.4.4 Antisense RNA (asRNA)
1.4.5 Group II intron
1.4.6 Genomic mutagenesis by homologous recombination system
1.4.7 Random mutagenesis by mariner transposon system
1.5 Clostridium acetobutylicum as an n-butanol producer-a challenging task
1.6 Genome scale metabolic (GSM) models for Clostridium acetobutylicum
1.7 Regulatory network in Clostridium acetobutylicum
1.7.1 Spo0A and Sigma factors
1.7.2 The controversial SolR
1.7.3 Agr quorum sensing
1.7.4 Heat-shock proteins: DnaK, GroESL, and Hsp21
1.7.7 Per and Fur
1.7.12 Small regulatory RNA
2 Chapter 2 Thesis Objectives
3 Chapter 3 Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum
3.3 Results and Discussion
3.3.1 MGCΔcac1502 strain, a C. acetobutylicum strain that is transformable without previous in vivo plasmid methylation
3.3.2 Construction of the MGCΔcac1502Δupp strain: the first marker-free C. acetobutylicum strain with two deleted genes
3.3.3 Deletion of the CA_C3535 gene in the MGCΔcac1502Δupp strain using the upp/5-FU system as a counter-selectable marker for the loss of plasmid
3.3.4 Determination of the recognition sequence of Cac824II encoded by CA_C3535
3.3.5 Deletion of the ctfAB genes in the MGCΔcac1502ΔuppΔcac3535 to create a strain no longer producing acetone
3.3.6 Deletion of the ldhA gene in the MGCΔcac1502ΔuppΔcac3535 to create a strain no longer producing lactate
3.6 Experimental Procedure
3.6.1 Bacterial strain, plasmids and oligonucleotides
3.6.2 Culture and growth conditions
3.6.3 DNA manipulation techniques
3.6.4 Construction of pUC18-FRT-MLS2
3.6.5 Construction of pCons2.1
3.6.6 Construction of pCIP2-1
3.6.7 Construction of pREPcac15
3.6.8 Construction of pCIPcac15
3.6.9 Construction of pREPupp
3.6.10 Construction of pCLF1
3.6.11 Construction of pCons::upp
3.6.12 Construction of pREPcac35::upp
3.6.13 Construction of pREPctfAB::upp
3.6.14 Construction of pREPldhA::upp
3.6.15 Construction of pCLF::upp
4 Chapter 4 The Weizmann process revisited for the continuous production of n-butanol
4.2 Main text
4.3 Supplementary Materials and Methods
4.3.1 Bacterial strains, plasmids and primers
4.3.2 Chemostat culture of recombinant strains
4.3.3 Continuous extractive high density cell recycle fermentation
4.3.4 Measurement of fermentation parameters
4.3.5 General protocol for gene deletion by homologous recombination system using replicative plasmid pSOS95-MLSr [167,174]
4.3.6 Construction of pSOS95-MLSr-upp-Drex-catP and CAB1058 strain
4.3.7 Construction of pEryupp-atoB plasmid and CAB1059 strain
4.3.8 Construction of pSOS95-upp-hbd1-catP-oriRepA and CAB1060 strain
4.3.9 Enzyme activity measurements
5 Chapter 5 Construction of a hydrogenase minus mutant of Clostridium acetobutylicum : a platform strain for the continuous production of fuels and chemicals
5.1 Main text
5.3 Material and Methods
5.3.1 Knockout of hydA and thlA using LltrB intron
5.3.2 Construction and integration by single crossing over of the pEryUppΔthlA plasmid in the chromosome of C. acetobutylicumuppcac1502
5.3.3 Construction and integration by single crossing over of the pCatUppHydA270 plasmid in the chromosome of C. acetobutylicumuppcac1502
5.3.4 Simultaneous inactivation of thlA and hydA in C. acetobutylicumuppcac1502 116
6 Chapter 6 Cap0037, a novel global regulator of Clostridium acetobutylicum metabolism
6.3 Results and Discussion
6.3.1 The phylogenetic tree and bioinformatics analysis of Cap0037/Cap0036
6.3.2 Disruption of CA_P0037 by pCUI-cap37 (189s)
6.3.3 Carbon and electron fluxes of the Cap37::intron mutant under different physiological conditions
6.3.4 Determination of the CA_P0037 DNA binding site (BS)
6.3.5 Determination of putative Cap0037 regulon
6.3.6 Global transcription changes in the Cap37::int mutant
6.5 Experimental Procedure
6.5.1 Bacterial strains, plasmids and culture media
6.5.2 Plasmid construction
6.5.3 Continuous culture
6.5.4 Isolation of total mRNA and microarray
6.5.5 Analytical methods
6.5.6 Southern blot analysis
6.5.7 Expression and purification of His-tagged protein
6.5.8 Electromobility shift assays (EMSAs)
6.5.9 DNase I protection assays (DNA Footprinting)
6.5.10 Bioinformatic tools
6.5.11 Microarray data accession number
7 Chapter 7 Conclusion and future perspectives