Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor.

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L-1 could be achieved. When the isopropanol concentration has reached about 8 g. L-1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L-1 of IPA compared to the reference strain (8 g. L-1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L-1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L- 1.

Thermodynamic and Kinetic Modeling Directs Pathway Optimization for Isopropanol Production in a Gas-Fermenting Bacterium.

Rational engineering of gas-fermenting bacteria for high yields of bioproducts is vital for a sustainable bioeconomy. It will allow the microbial chassis to renewably valorize natural resources from carbon oxides, hydrogen, and/or lignocellulosic feedstocks more efficiently. To date, rational design of gas-fermenting bacteria such as changing the expression levels of individual enzymes to obtain the desired pathway flux is challenging, because pathway design must follow a verifiable metabolic blueprint indicating where interventions should be executed. Based on recent advances in constraint-based thermodynamic and kinetic models, we identify key enzymes in the gas-fermenting acetogen Clostridium ljungdahlii that correlate with the production of isopropanol. To this extent, we integrated a metabolic model in comparison with proteomics measurements and quantified the uncertainty for a variety of pathway targets needed to improve the bioproduction of isopropanol. Based on in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, we identified the top two significant flux control sites, i.e., acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC), overexpression of which could lead to increased isopropanol production. Our predictions directed iterative pathway construction, which enabled a 2.8-fold increase in isopropanol production compared to the initial version. The engineered strain was further tested under gas-fermenting mixotrophic conditions, where more than 4 g/L isopropanol was produced when CO, CO2, and fructose were provided as the substrates. In a bioreactor environment sparging with CO, CO2, and H2 only, the strain produced 2.4 g/L isopropanol. Our work highlighted that the gas-fermenting chasses can be fine-tuned for high-yield bioproduction by directed and elaborative pathway engineering. IMPORTANCE Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering. Here, we provide a case study by engineering isopropanol production in gas-fermenting Clostridium ljungdahlii. We demonstrate that a modeling approach based on the thermodynamic and kinetic analysis at the pathway level can provide actionable insights into strain engineering for optimal bioproduction. This approach may pave the way for iterative microbe redesign for the conversion of renewable gaseous feedstocks.

Co-expression of an isopropanol synthetic operon and eGFP to monitor the robustness of Cupriavidus necator during isopropanol production.

Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol/acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighted the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered.

An acetate-independent pathway for isopropanol production via HMG-CoA in Escherichia coli.

Isopropanol has a good potential as a new fuel substitution. In the model biosynthesis pathway of isopropanol synthesis, acetoacetyl-CoA is converted to acetoacetate by acetoacetyl-CoA transferases, which requires an acetate molecule as a substrate. Herein, a novel isopropanol synthesis pathway based on mammalian ketone metabolic pathway was developed. In this pathway, acetoacetyl-CoA is condensed with acetyl-CoA to generate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, and then catalyzed by HMG-CoA lyase to generate acetoacetate. This process is acetate-independent. Under the same experimental system using glycerol as carbon source, the E. coli strain MG::ISOP1 containing the novel pathway produced 11.7 times more isopropanol than the strain MG::ISOP0 containing the model pathway. The pta-ackA knockout mutant strain MG∆pta-ackA::ISOP1, which reduced the conversion of acetyl-CoA to acetate, further increased the production from 76 mg/L to 360 mg/L. In another strategy, knocking out atoDA to block the acetoacetate degradation pathway in strain MG∆atoDA::ISOP1 increased the production to 680 mg/L. By knocking out both of pta-ackA and atoDA, strain MGΔpta-ackAΔatoDA::ISOP1 produced 964 mg/L of isopropanol, which was 12.7 times that of MG::ISOP1. This study indicated that the novel pathway is competent for isopropanol synthesis, and provides a new perspective for biosynthesis of isopropanol.

Isopropanol biosynthesis from crude glycerol using fatty acid precursors via engineered oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Isopropanol is widely used as a biofuel and a disinfectant. Chemical preparation of isopropanol destroys the environment, which makes biological preparation of isopropanol necessary. Previous studies focused on the use of expensive glucose as raw material. Therefore, the microbial cell factory that ferments isopropanol with cheap raw materials will provide a greener way to produce isopropanol. RESULTS: This study converted crude glycerol into isopropanol using Y. lipolytica. As a microbial factory, the active natural lipid and fatty acid synthesis pathway endows Y. lipolytica with high malonyl-CoA production capacity. Acetoacetyl-CoA synthase (nphT7) and isopropanol synthesis genes are integrated into the Y. lipolytica genome. The nphT7 gene uses the accumulated malonyl-CoA to synthesize acetoacetyl-CoA, which increases isopropanol production. After medium optimization, the best glycerol medium was found and resulted in a 4.47-fold increase in isopropanol production. Fermenter cultivation with pure glycerol medium resulted in a maximum isopropanol production of 1.94 g/L. In a crude glycerol fermenter, 1.60 g/L isopropanol was obtained, 82.53% of that achieved with pure glycerol. The engineered Y. lipolytica in this study has the highest isopropanol titer reported. CONCLUSIONS: The engineered Y. lipolytica successfully produced isopropanol by using crude glycerol as a cheap carbon source. This is the first study demonstrating the use of Y. lipolytica as a cell factory to produce isopropanol. In addition, this is also a new attempt to accumulate lipid synthesis precursors to synthesize other useful chemicals by integrating exogenous genes in Y. lipolytica.

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments.

BACKGROUND: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. RESULTS: We present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. CONCLUSIONS: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol.

Rational design and modification of autotrophic bacteria to efficiently produce high-value chemicals and biofuels are crucial for establishing a sustainable and economically viable process for one-carbon (C1) source utilization, which, however, remains a challenge in metabolic engineering. In this study, autotrophic Clostridium ljungdahlii was metabolically engineered to efficiently co-produce three important bulk chemicals, isopropanol, 3-hydroxybutyrate (3-HB), and ethanol (together, IHE), using syngas (CO2/CO). An artificial isopropanol-producing pathway was first constructed and optimized in C. ljungdahlii to achieve an efficient production of isopropanol and an unexpected product, 3-HB. Based on this finding, an endogenous active dehydrogenase capable of converting acetoacetate to 3-HB was identified in C. ljungdahlii, thereby revealing an efficient 3-HB-producing pathway. The engineered strain was further optimized to reassimilate acetic acid and synthesize 3-HB by introducing heterologous functional genes. Finally, the best-performing strain was able to produce 13.4, 3.0, and 28.4 g/L of isopropanol, 3-HB, and ethanol, respectively, in continuous gas fermentation. Therefore, this work represents remarkable progress in microbial production of bulk chemicals using C1 gases.

A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations.

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.

Activation of short-chain ketones and isopropanol in sulfate-reducing bacteria.

BACKGROUND: Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA. RESULTS: Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B12-dependent mutase, and a NAD+-dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B12-dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B12-dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone. CONCLUSIONS: According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410.

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

Isopropanol production from carbon dioxide in Cupriavidus necator in a pressurized bioreactor.

A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L-1 level from CO2 for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO2, H2 and air inputs and the ability to operate at high pressures. The bioreactor configuration and cultivation strategy enabled growth of Cupriavidus necator strains for long periods, to reach over 3 g L-1 dry cell weight. No negative impact of the high pressure was observed on viability of the strains up to more than 4 bar overpressure. The cultivation was then carried out using an engineered isopropanol producing strain; in this case, 3.5 g L-1 isopropanol was obtained from CO2 as the sole carbon source. This is the first reported demonstration of a successful production from engineered bacteria of product in the g L-1 range on CO2, raising the prospect of future development of CO2-based bioprocesses.

The Role of Saccharomyces cerevisiae Yeast and Lactic Acid Bacteria in the Formation of 2-Propanol from Acetone during Fermentation of Rye Mashes Obtained Using Thermal-Pressure Method of Starch Liberation.

This study set out to assess the acetone content in rye sweet mashes prepared using the thermal-pressure method of starch liberation, and to investigate the formation of 2-propanol during the fermentation process. In the first set of experiments, we evaluated the correlation between the color and the content of acetone and furfural in industrially produced sweet mashes (n = 37). The L * value was negatively correlated with the content of both acetone and furfural, while chromatic parameters a * and b * and the yellowness index (YI) had strong positive correlations with acetone (r > 0.9) and furfural (r > 0.8 for a * and r > 0.9 for b * and YI). In the second set of experiments, we assessed the concentration of acetone and 2-propanol in distillery rye mashes, fermented by S. cerevisiae yeast and lactic acid bacteria. The influence of fermentation temperature on the formation of 2-propanol was also evaluated. The presence of 2-propanol in the post-fermentation media was confirmed, while a decrease in acetone content was observed. Fermentation temperature (27 °C or 35 °C) was found to have a significant effect on the concentration of 2-propanol in trials inoculated with lactic bacteria. The content of 2-propanol was more than 11 times higher in trials fermented at the higher temperature. In the case of yeast-fermented mashes, the temperature did not affect 2-propanol content. The acetone in the sweet mash was assumed to be a precursor of 2-propanol, which was found in the fermented mashes.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.

A molecular simulation and spectroscopic approach to the binding affinity between trypsin and 2-propanol and protein conformation.

Increasing the stability and activity of enzymes is one of the most popular ideas in biochemistry studies. The current study focused on the interactions between 2-propanol as an osmolyte and trypsin to increase the enzyme thermal stability by the modification of the solvent environment. To determine the binding mechanism of 2-propanol with trypsin, fluorescence emission quenching was observed as a static mode of quenching upon the binding of 2-propanol to trypsin. With the formation of hydrogen bonds and lower hydrophobicity levels after the addition of 2-propanol, Tm of complexes were increased. Also, the α-helix content of trypsin was increased as obtained by far-UV CD. CD results analysis showed that there was no significant perturbation in the structure of trypsin upon an increase in the concentration of 2-propanol. Molecular docking results also indicated that 2-propanol could bind to trypsin and hydrophobic interactions and hydrogen bond contributions played the major role in this binding. Consequently, the results of the molecular dynamics simulation showed that the stability of trypsin-2 propanol was obtained to be about 2.5 nm in the equilibrium state, indicating the stability and rigidity of the trypsin-2 propanol complex. Upon 2-propanol conjugation, the residual activity of the enzyme was increased. 2-propanol, therefore, acted as a stabilizer and activator for trypsin.

Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423.

BACKGROUND: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. RESULTS: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. CONCLUSIONS: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.

Sustainable approach in phlorotannin recovery from macroalgae.

In this present study, alcohol/salt liquid biphasic system was used to extract phlorotannin from brown macroalgae. Liquid biphasic system is a new green technology that integrated with various processes into one-step, by concentrating, separating and purifying the bioproduct in a unit operation. The solvent used is non-toxic and there is potential for solvent recovery which is beneficial to the environment. Phlorotannin is a bioactive compound that has gained much attention due to its health beneficial effect. Therefore, the isolation of phlorotannin is lucrative as it contains various biological activities that are capable to be utilised into food and pharmaceutical application. By using 2-propanol/ammonium sulphate system, the highest recovery of phlorotannin was 76.1% and 91.67% with purification factor of 2.49 and 1.59 from Padina australis and Sargassum binderi, respectively. A recycling study was performed and the salt phase of system was recycled where maximum salt recovery of 41.04% and 72.39% could be obtained from systems containing P. australis and S. binderi, respectively. Similar recovery of phlorotannin was observed after performing two cycles of the system, this concludes that the system has good recyclability and eco-friendly.

The Draft Genome Sequence of Clostridium beijerinckii NJP7, a Unique Bacterium Capable of Producing Isopropanol-Butanol from Hemicellulose Through Consolidated Bioprocessing.

A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76 Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

Biochemical Gas Sensors (Biosniffers) Using Forward and Reverse Reactions of Secondary Alcohol Dehydrogenase for Breath Isopropanol and Acetone as Potential Volatile Biomarkers of Diabetes Mellitus.

This study describes two biosniffers to determine breath acetone and isopropanol (IPA) levels and applies them for breath measurement in healthy subjects and diabetic patients. Secondary alcohol dehydrogenase (S-ADH) can reduce acetone and oxidize nicotinamide adenine dinucleotide (NADH to NAD+) in a weak acid environment. NADH can be excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. S-ADH can also oxidize IPA and reduce NAD+ to NADH when it is in an alkaline environment. Thus, IPA can be detected by the increase of fluorescence. The developed biosniffers show rapid response, high sensitivity and high selectivity. The breath acetone and IPA analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb and 15.4 ± 11.3 ppb. Both acetone and IPA did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (p < 1 × 10-6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (p < 0.01). The breath IPA also had a higher concentration in diabetic patients (23.1 ± 20.1 ppb, p < 0.01), but only T2D patients presented a statistical difference (23.9 ± 21.3 ppb, p < 0.01). These findings are worthwhile in the study of breath biomarkers for diabetes mellitus diagnosis. Additionally, the developed biosniffers provide a new technique for volatolomics research.

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production.

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4gL-1 isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15gL-1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8gL-1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16gL-1 after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9-18% increase in the isopropanol yield on fructose.

Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitrate-mediated microbially enhanced oil recovery.

Amendment of reservoir fluid with injected substrates can enhance the growth and activity of microbes. The present study used isopropyl alcohol (IPA) or acetone to enhance the indigenous anaerobic nitrate-reducing bacterium Thauera sp. TK001. The strain was able to grow on IPA or acetone and nitrate. To monitor effects of strain TK001 on oil recovery, sand-packed columns containing heavy oil were flooded with minimal medium at atmospheric or high (400psi) pressure. Bioreactors were then inoculated with 0.5 pore volume (PV) of minimal medium containing Thauera sp. TK001 with 25mM of acetone or 22.2mM of IPA with or without 80mM nitrate. Incubation without flow for two weeks and subsequent injection with minimal medium gave an additional 17.0±6.7% of residual oil in place (ROIP) from low-pressure bioreactors and an additional 18.3% of ROIP from the high-pressure bioreactors. These results indicate that acetone or IPA, which are commonly used organic solvents, are good substrates for nitrate-mediated microbial enhanced oil recovery (MEOR), comparable to glucose, acetate or molasses, tested previously. This technology may be used for coupling biodegradation of IPA and/or acetone in waste streams to MEOR where these waste streams are generated in close proximity to an oil field.