The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. RESULTS: Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. CONCLUSIONS: This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.

Ethylene glycol and glycolic acid production by wild-type Escherichia coli.

Ethylene glycol and glycolic acid are bulk chemicals with a broad range of applications. The ethylene glycol and glycolic acid biosynthesis pathways have been produced by microorganisms and used as a biological route for their production. Unlike the methods that use xylose or glucose as carbon sources, xylonic acid was used as a carbon source to produce ethylene glycol and glycolic acid in this study. Amounts of 4.2 g/L of ethylene glycol and 0.7 g/L of glycolic acid were produced by a wild-type Escherichia coli W3110 within 10 H of cultivation with a substrate conversion ratio of 0.5 mol/mol. Furthermore, E. coli strains that produce solely ethylene glycol or glycolic acid were constructed. 10.3 g/L of glycolic acid was produced by E. coli ΔyqhD+aldA, and the achieved conversion ratio was 0.56 mol/mol. Similarly, the E. coli ΔaldA+yqhD produced 8.0 g/L of ethylene glycol with a conversion ratio of 0.71 mol/mol. Ethylene glycol and glycolic acid production by E. coli on xylonic acid as a carbon source provides new information on the biosynthesis pathway of these products and opens a novel way of biomass utilization.

Excellent thermoelectric properties of monolayer RbAgM (M = Se and Te): first-principles calculations.

Based on the atomic substitution method, the RbAgM monolayers (M = Se and Te), a class of derivative compounds of KAgSe, have been successfully predicted, which exhibit ultra-high mobility and poor heat transport ability, indicating their broad application potential in thermoelectric (TE) technology. Using density functional theory (DFT) and the Boltzmann transport equation (BTE), we carry out systematic studies on their electronic band structures, heat transport abilities and TE properties. Our calculated results show that the RbAgTe monolayer possesses ultra-low lattice thermal conductivity (0.90 W m-1 K-1) at room temperature and a high Seebeck coefficient (2320 µV K-1). Additionally, we also focus on the analysis of phonon velocity and Grüneisen parameter to further explain their ultra-low thermal conductivity. By combining these calculated parameters, the predicted maximum ZT values of RbAgSe and RbAgTe are as high as 2.2 and 4.1 at 700 K with optimum n-type doping, respectively, which are comparable to that of the famous TE material SnSe (ZT = 2.6 at 923 K). Our research results provide a strong theoretical basis for the experimental exploration of the TE properties of RbAgM, and help to promote further experimental verification.

Production of 2,3-dihydroxyisovalerate by Enterobacter cloacae.

2,3-Dihydroxyisovalerate is an intermediate of the valine synthesis pathway. However, neither natural microorganisms nor valine producing engineered strains have been reported yet to produce this chemical. Based on the 2,3-butanediol synthesis pathway, a biological route of 2,3-dihydroxyisovalerate production was developed using a budA and ilvD disrupted Klebsiella pneumoniae strain in our previous research. We hypothesised, that other 2,3-butanediol producing bacteria could be used for 2,3-dihydroxyisovalerate production. Here a budA disrupted Enterobacter cloacae was constructed, and this strain exhibited a high 2,3-dihydroxyisovalerate producing ability. Disruption of ilvD in E. cloacae ΔbudA further increased 2,3-dihydroxyisovalerate level. The disruption of budA, encoding an acetolactate decarboxylase, resulted in the acetolactate synthesized in the 2,3-butanediol synthesis pathway to flow into the valine synthesis pathway. The additional disruption of ilvD, encoding a dihydroxy acid dehydratase, prevented the 2,3-dihydroxyisovalerate to be further metabolized in the valine synthesis pathway. Thus, the disruption of both budA and ilvD in 2,3-butanediol producing strains might be an universal strategy for 2,3-dihydroxyisovalerate accumulation. After optimization of the medium components and culture parameters 31.2 g/L of 2,3-dihydroxyisovalerate was obtained with a productivity of 0.41 g/L h and a substrate conversion ratio of 0.56 mol/mol glucose in a fed-batch fermentation. This approach provides an economic way for 2,3-dihydroxyisovalerate production.

2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae.

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process. Key Points • A biological route of 2,3-dihydroxyisovalerate production was setup. • Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae. • Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell. • 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation.

Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae.

BACKGROUND: Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally. RESULTS: Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid. CONCLUSIONS: A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.

Phonon transport and thermal conductivity of diamond superlattice nanowires: a comparative study with SiGe superlattice nanowires.

Due to the coupling of a superlattice's longitudinal periodicity to a nanowire's radial confinement, the phonon transport properties of superlattice nanowires (SLNWs) are expected to be radically different from those of pristine nanowires. In this work, we present the comparative investigation of phonon transport and thermal conductivity between diamond SLNWs and SiGe SLNWs by using molecular dynamics simulations. In the case of period length ∼ 25 Å, the thermal conductivities of diamond SLNWs and SiGe SLNWs both increase linearly with increasing the period number, which implies the wave-like coherent phonons dominate the heat transport of SLNWs. In the case of period length ∼ 103 Å, the thermal conductivity of SiGe SLNWs is length-independent with increasing the period number, indicating that the particle-like incoherent phonons in SiGe SLNWs control the heat transport, because the phonon-phonon scattering causes phonons to not retain their phases and the coherence is destroyed before the reflection at interfaces. However in diamond SLNWs the coherent phonons still dominate heat conduction and the thermal conductivity is length-dependent, because the mean free path of phonon-phonon scattering in diamond SLNWs is much longer. The spatial distribution of phonon localized modes further supports these opinions. These results are helpful not only to understand the coherent and incoherent phonon transport, but also to modulate the thermal conductivity of SLNWs.

Ultralow lattice thermal conductivity and high thermoelectric performance of monolayer KCuTe: a first principles study.

Monolayer KCuTe is a new-type of two-dimensional (2D) semiconductor material with high carrier mobility and large power energy conversion efficiencies, suggesting its potential application in thermoelectric (TE) and photoelectric fields. Based on the density functional theory (DFT) and semiclassical Boltzmann transport equation, the electronic and phonon transport properties of monolayer KCuTe are systematically studied. Our results show that it possesses an ultralow lattice thermal conductivity value of nearly ∼0.13 W m-1 K-1 at 300 K, mainly attributed to its small phonon group velocity, large Grüneisen parameters, and strong phonon-phonon scattering. Furthermore, the intralayer opposite phonon vibrations greatly restrict the heat transport. Monolayer KCuTe shows an ideal direct band gap of ∼1.21 eV, and a high twofold degeneracy appearing at the Γ point gives a high Seebeck coefficient of ∼2070 µV K-1, leading to high TE performance. Using the transport coefficients together with constant electron relaxation time, the figure of merit (ZT) can reach 2.71 at 700 K for the p-type doping, which is comparable to the well-known TE material SnSe (2.6 ± 0.3 at 935 K). Our theoretical studies may provide perspectives to TE applications of monolayer KCuTe and stimulate further experimental synthesis.

Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway.

Isobutanol is a valuable chemical and is considered a new generation biofuel. Construction of isobutanol synthesis pathways in bacteria is a hot topic in isobutanol production. Here, we show that an isobutanol synthesis pathway exists naturally in Klebsiella pneumoniae; however, this pathway is dormant in the wild-type bacterium. K. pneumoniae is a 2,3-butanediol producer, and the synthesis pathways of 2,3-butanediol, valine and isobutanol all start from condensation of two pyruvate molecules to yield α-acetolactate. Inactivation of α-acetolactate decarboxylase (encoded by budA) resulted in α-acetolactate flowing into the valine pathway, which led to synthesis of isobutanol and 2-ketoisovalerate (a precursor of isobutanol). ldhA (lactate dehydrogenase) deletion further increased the isobutanol and 2-ketoisovalerate production. In the first step of this pathway, BudB (α-acetolactate synthase) was identified as responsible for most of the α-acetolactate synthesis. Complementation of ilvBN or ilvIH (isoenzymes of budB) both resulted in a remarkable increase in 2-ketoisovalerate production. Thus, α-acetolactate formation is the rate-limiting step of 2-ketoisovalerate production. ilvC (acetohydroxy acid isomeroreductase) and ilvD (dihydroxy acid dehydratase) were identified responsible for 2-ketoisovalerate synthesis from α-acetolactate. ipdC, which encodes an indole-3-pyruvate decarboxylase, was identified responsible for most of the isobutyraldehyde formation from 2-ketoisovalerate, and isobutanol production was increased 15.7 fold in the ipdC complementation strain, with a final titer of 2.45g/L. Isobutanol dehydrogenase activity is distributed across multiple alcohol dehydrogenase enzymes expressed by K. pneumoniae. BudC, DhaT, DhaD and YqhD all had isobutanol dehydrogenase activity in vitro. YqhD uses NADPH as the coenzyme, while the other dehydrogenases use NADH. However, inactivating one or two of these dehydrogenases had no effect on isobutanol production in vivo with isobutyraldehyde as the substrate. These results reveal a novel method for biological production of isobutanol and 2-ketoisovalerate.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock.

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

The Role of the Pyruvate Acetyl-CoA Switch in the Production of 1,3-Propanediol by Klebsiella pneumoniae.

Pyruvate dehydrogenase-complex (AcoABCD) and pyruvate formate-lyase (PFL) are two pathways responsible for synthesis of acetyl-CoA from pyruvate (pyruvate acetyl-CoA switch). The two pathways were individually deleted in Klebsiella pneumoniae, and the role of the pyruvate acetyl-CoA switch in 1,3-propanediol production was investigated. Fermentation results showed that the two pathways were both active in the wild-type strain. Acetyl-CoA formation between the two pathways was equal in the wild-type strain. The pflB mutant produced high level of lactic acid, and deletion of ldhA eliminated lactic acid synthesis. The conversion ratio of glycerol to 1,3-propanediol in the pflB-ldhA mutant reached 0.541 g/g, which was 9.4 % higher than that of the wild-type strain. However, the productivity of 1,3-propanediol was decreased in the pflB-ldhA mutant. In contrast, the productivity of 1,3-propanediol was increased by 19 % in the acoABCD mutant, with the disadvantage of lower substrate conversion ratio. Regulating the pyruvate acetyl-CoA switch presents a novel way to improve the conversion ratio or productivity of 1,3-propanediol produced by K. pneumoniae.