Short chain diols (propanediols, butanediols, pentanediols) have been widely used in bulk and fine chemical industries as fuels, solvents, polymer monomers and pharmaceutical precursors. The chemical production of short chain diols from fossil resources has been developed and optimized for decades. Consideration of the exhausting fossil resources and the increasing environment issues, the bio-based process to produce short chain diols is attracting interests. Currently, a variety of biotechnologies have been developed for the microbial production of the short chain diols from renewable feed-stocks. In order to efficiently produce bio-diols, the techniques like metabolically engineering the production strains, optimization of the fermentation processes, and integration of a reasonable downstream recovery processes have been thoroughly investigated. In this review, we summarized the recent development in the whole process of bio-diols production including substrate, microorganism, metabolic pathway, fermentation process and downstream process.
Essential genes are the genes that are indispensable to cell viability. It is very important to ascertain these genes for understanding the biological phenomena, as well as drug and therapy design. In the present paper, we understand the essential and nonessential genes from the perspective of robustness. Other than traditional homology study based methods, this article use structural analysis of functional subsystems of genome-scale metabolic networks. We give the structural analysis of the citric acid cycle as a case study, the result suggests that this systems-oriented method would help to ascertain gene essentiality.
Monounsaturated fatty acids (MUFAs) are the best components for biodiesel when considering the low temperature fluidity and oxidative stability. However, biodiesel derived from vegetable oils or microbial lipids always consists of significant amounts of polyunsaturated and saturated fatty acids (SFAs) alkyl esters, which hampers its practical applications. Therefore, the fatty acid composition should be modified to increase MUFA contents as well as enhancing oil and lipid production.The model microorganism Escherichia coli was engineered to produce free MUFAs. The fatty acyl-ACP thioesterase (AtFatA) and fatty acid desaturase (SSI2) from Arabidopsis thaliana were heterologously expressed in E. coli BL21 star(DE3) to specifically release free unsaturated fatty acids (UFAs) and convert SFAs to UFAs. In addition, the endogenous fadD gene (encoding acyl-CoA synthetase) was disrupted to block fatty acid catabolism while the native acetyl-CoA carboxylase (ACCase) was overexpressed to increase the malonyl coenzyme A (malonyl-CoA) pool and boost fatty acid biosynthesis. The finally engineered strain BL21ΔfadD/pE-AtFatAssi2&pA-acc produced 82.6 mg/L free fatty acids (FFAs) under shake-flask conditions and FFAs yield on glucose reached about 3.3% of the theoretical yield. Two types of MUFAs, palmitoleate (16:1Δ9) and cis-vaccenate (18:1Δ11) made up more than 75% of the FFA profiles. Fed-batch fermentation of this strain further enhanced FFAs production to a titer of 1.27 g/L without affecting fatty acid compositions.This study demonstrated the possibility to regulate fatty acid composition by using metabolic engineering approaches. FFAs produced by the recombinant E. coli strain consisted of high-level MUFAs and biodiesel manufactured from these fatty acids would be more suitable for current diesel engines.
Rapid climate change and intensified human activities have resulted in water table lowering (WTL) and enhanced nitrogen (N) deposition in Tibetan alpine wetlands. These changes may alter the magnitude and direction of greenhouse gas (GHG) emissions, affecting the climate impact of these fragile ecosystems. We conducted a mesocosm experiment combined with a metagenomics approach (GeoChip 5.0) to elucidate the effects of WTL (-20 cm relative to control) and N deposition (30 kg N ha-1 yr-1 ) on carbon dioxide (CO2 ), methane (CH4 ) and nitrous oxide (N2 O) fluxes as well as the underlying mechanisms. Our results showed that WTL reduced CH4 emissions by 57.4% averaged over three growing seasons compared with no-WTL plots, but had no significant effect on net CO2 uptake or N2 O flux. N deposition increased net CO2 uptake by 25.2% in comparison with no-N deposition plots and turned the mesocosms from N2 O sinks to N2 O sources, but had little influence on CH4 emissions. The interactions between WTL and N deposition were not detected in all GHG emissions. As a result, WTL and N deposition both reduced the global warming potential (GWP) of growing season GHG budgets on a 100-year time horizon, but via different mechanisms. WTL reduced GWP from 337.3 to -480.1 g CO2 -eq m-2 mostly because of decreased CH4 emissions, while N deposition reduced GWP from 21.0 to -163.8 g CO2 -eq m-2 , mainly owing to increased net CO2 uptake. GeoChip analysis revealed that decreased CH4 production potential, rather than increased CH4 oxidation potential, may lead to the reduction in net CH4 emissions, and decreased nitrification potential and increased denitrification potential affected N2 O fluxes under WTL conditions. Our study highlights the importance of microbial mechanisms in regulating ecosystem-scale GHG responses to environmental changes.
Abstract BackgroundFluorinases play a unique role in producing fluorinated organic molecules through a biological method. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.ResultsA gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused protein was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and Immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase-containing surface display system with improved whole-cell catalytic efficiency and stability showed low growth pressure on the protein expressing host. The conversion rate of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) achieved 55%.ConclusionsHere, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of Escherichia coli and maintained its catalytic activity. The surface display offers a new solution for the industrial application of biological fluorination.
Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l−1 for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.
Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l−1 for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.
ABSTRACT Heavy ion beam (HIB) irradiation is widely utilized in studies of cosmic rays-induced cellular effects and microbial breeding. Establishing an accurate dose-survival relationship is crucial for selecting the optimal irradiation dose. Typically, after irradiating logarithmic-phase cell suspensions with HIB, the survival fraction (SF) is determined by the ratio of clonal-forming units in irradiated versus control groups. However, our findings indicated that SF measurements were time sensitive. For the Saccharomyces cerevisiae model, the observed SF initially declined and subsequently increased in a eutrophic state; conversely, in an oligotrophic state, it remained relatively stable within 120 minutes. This time effect of SF observations in the eutrophic state can be ascribed to HIB-exposed cells experiencing cell cycle arrest, whereas the control proliferated rapidly, resulting in an over-time disproportionate change in viable cell count. Therefore, an alternative involves irradiating oligotrophic cells, determining SF thereafter, and transferring cells to the eutrophic state to facilitate DNA repair-mutation. Transcriptomic comparisons under these two trophic states yield valuable insights into the DNA damage response. Although DNA repair was postponed in an oligotrophic state, cells proactively mobilized specific repair pathways to advance this process. Effective nutritional supplementation should occur within 120 minutes, beyond this window, a decline in SF indicates an irreversible loss of repair capability. Upon transition to the eutrophic state, S. cerevisiae swiftly adapted and completed the repair. This study helps to minimize time-dependent variability in SF observations and to ensure effective damage repair and mutation in microbial breeding using HIB or other mutagens. It also promotes the understanding of microbial responses to complex environments. IMPORTANCE Mutation breeding is a vital means of developing excellent microbial resources. Consequently, understanding the mechanisms through which microorganisms respond to complex environments characterized by mutagens and specific physiological-biochemical states holds significant theoretical and practical values. This study utilized Saccharomyces cerevisiae as a microbial model and highly efficient heavy ion beam (HIB) radiation as a mutagen, it revealed the time dependence of observations of survival fractions (SF) in response to HIB radiation and proposed an alternative to avoid the indeterminacy that this variable brings. Meanwhile, by incorporating an oligotrophic state into the alternative, this study constructed a dynamic map of gene expression during the fast-repair and slow-repair stages. It also highlighted the influence of trophic states on DNA repair. The findings apply to the survival-damage repair-mutation effects of single-celled microorganisms in response to various mutagens and contribute to elucidating the biological mechanisms underlying microbial survival in complex environments.
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