Thermoanaerobacterium Aotearoense Research Articles

Adaptive laboratory evolution of Thermoanaerobacterium aotearoense for enhanced ethanol production from raw cassava starch

Thermophiles have the potential to utilize starch for bioethanol production in consolidated bioprocessing procedure, but they are usually limited by the intolerance of high ethanol titer. In this study, we performed adaptative laboratory evolution (ALE) on Thermoanaerobacterium aotearoense strain PRH-B3 (ΔldhΔrexΔhfsB::BCD), which produced ethanol as the only product. After hundreds of generations of domestication with the increasing ethanol concentration, two lineages of ethanol-tolerant mutants were gained and the effects of alcohol dehydrogenase (ADH) domain mutations in aldehyde/alcohol dehydrogenase AdhE were analyzed. The assay of ADH activities in vivo showed that the mutants exhibited reduced NADH-dependent ADH activities and increased NADPH-dependent ADH activities, compared to that from PRH-B3. AdhA was subsequently demonstrated to contribute to the increased NADPH-linked ADH activities in the mutants. This was accompanied by an increased expression level of NADH-dependent ferredoxin:NADP+ oxidoreductase and improved contents of NADP(H), indicating that the ethanol-tolerant strains preferred to use NADPH for ethanol synthesis with a potential regulatory mechanism. The maximum ethanol production (∼39 g/L) of the mutants had increased by 26 % after ALE from untreated cassava starch. The enhanced comprehension of the mechanisms underlying ethanol tolerance elucidated herein paves the way for the development of other ethanologenic strains with elevated ethanol titers.

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd–EtfAB (BCD) complex was overexpressed, the resulting strain PRH–B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45–0.51 g/L/h and 0.46–0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin–NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses

BackgroundSucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium.ResultsThe sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses.ConclusionsOur work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.

Engineering Thermoanaerobacterium aotearoense SCUT27 with the deficiency of a hypothetic protein regulated by ArgR1864 for enhanced ethanol production from lignocellulosic hydrolysates

Arginine repressor (ArgR1864) is a multifunctional regulator in Thermoanaerobacterium aotearoense SCUT27 (SCUT27). For better understanding its function in SCUT27, the transcriptome of SCUT27/ΔargR1864 under xylose was further investigated. 2014 (V518_2014) denoted as a hypothetic protein gene was mined, which played important roles on xylose metabolism and carbon metabolic flux distribution in SCUT27. In 2014 deletion mutant, no lactic acid could be produced along with enhanced ethanol formation. Same changes were also observed in SCUT27/ΔargR1864. ArgR1864 is the direct regulator of 2014 identified by electrophoretic mobility shift assays (EMSA). When 2014 was complemented in SCUT27/ΔargR1864, the fermentation traits of SCUT27/ΔargR1864/2014 were returned to wild-type levels, suggesting that the improved xylose utilization and ethanol formation of SCUT27/ΔargR1864 were attributed to the low expression of 2014. When cultured under sorghum stalk, corn cob and wheat straw hydrolysates, SCUT27/Δ2014 also showed the excellent capability for sugar utilization and ethanol production with the consumption rate of xylose and glucose increased by 20%–37.93 and 64.29%–190.91% separately, the ethanol production improved by 326.61%–547.89% and ethanol yield enhanced by 216.67%–337.50%. Thus, 2014, as a hypothetic protein gene, could be used as the new genetic reference to predict functional genes to engineer the microorganism for biofuel production from lignocellulosic hydrolysates.

Engineering Thermoanaerobacterium aotearoense SCUT27/Δldh with pyruvate formate lyase-activating protein (PflA) knockout for enhanced ethanol tolerance and production

Thermophilic anaerobic bacteria have received increasing interest as a promising bioprocessing platform for bioethanol production due to their broad substrate range and higher operating temperature. However, ethanol concentration is still lower than the commercial requirements on account of the high by-product formation and low ethanol tolerance. Our previous transcriptome analysis showed the gene expression level of pyruvate formate lyase-activating protein (PflA) was significantly down-regulated when Thermoanaerobacterium aotearoense SCUT27/Δldh was stimulated by various concentrations of ethanol. Based on this, we constructed the mutant strain T. aotearoense SCUT27/Δldh/ΔpflA, which not only possesses higher ethanol tolerance and production but also has enhanced ethanol/acetate ratio that is important for economical ethanol production. Compared to T. aotearoense SCUT27/Δldh, the mutant strain could resist to 5% (v/v) exogenous ethanol and produce 49 % more ethanol (38.82 g/L vs. 26.05 g/L) in 5-L bioreactor. In addition, the final concentration of ethanol reached 45.01 g/L by immobilized cell fermentation, which is the highest value obtained in T. aotearoense SCUT27. Simultaneously, the ethanol/acetate ratio of the mutant strain achieved 6.82, which was 2.16-fold higher than that of the parental strain.

Enhanced ethanol production from lignocellulosic hydrolysates by Thermoanaerobacterium aotearoense SCUT27/ΔargR1864 with improved lignocellulose-derived inhibitors tolerance

The transcriptome data of Thermoanaerobacterium aotearoense SCUT27 (SCUT27) and Thermoanaerobacterium aotearoense SCUT27/ΔargR1864 (SCUT27/ΔargR1864) under xylose showed that ArgR1864 was the negative regulator for chaperonin synthesis. With argR1864 knockout, the mutant showed up-regulated expression of DnaK-DnaJ-GrpE system and GroEL-GroES chaperonin, which not only endowed the strain with the ability to scavenge the reactive oxygen species (ROS), but also gave the mutant the capability to maintain better cell growth, xylose consumption, acetic acid and ethanol production under the stress of various lignocellulose-derived inhibitors. Diluted acid pretreated soybean straw, sorghum stalk and wheat straw hydrolysates were used to evaluate the ethanol production of SCUT27/ΔargR1864 and results showed that the mutant definitely had advantages over SCUT27 for ethanol production under lignocellulosic hydrolysates, with the xylose consumption rate increased by 25.00%–44.83%, the ethanol production improved by 113.86%–366.36% and ethanol yield enhanced by 88.89%–208.33%.

Metabolic engineering of Thermoanaerobacterium aotearoense SCUT27 for glucose and cellobiose co-utilization by identification and overexpression of the endogenous cellobiose operon

Cellobiose is abundant in partial hydrolysis of lignocellulosic hydrolysates for the weak activity of β-glucosidase, the rate-limiting step in the enzymatic hydrolysis. Thermoanaerobacterium aotearoense SCUT27 termed SCUT27 has broad substrate spectrums and can co-utilize glucose and xylose, xylose and cellobiose. However, SCUT27 can’t co-utilize glucose and cellobiose due to carbon catabolite repression (CCR), which couldn’t be eliminated after ccpA knockout. The ability of co-utilizing glucose and cellobiose is a desirable feature of SCUT27 for industrial application. Four operons related to cellobiose utilization had been explored in SCUT27, named as Cel1, Cel2, Cel3 and Cel4. RT-PCR results showed that Cel2 operon is the only intact operon including integral cellobiose PTS transport elements and glycoside hydrolase, which is very important for cellobiose utilization. The transcription of Cel2 was regulated by σ54-dependent transcription activator CelRcel2 (V518_2450). With sigma factor 54 (σ54) deletion, the transcription strength of Cel2 decreased significantly. By replacing the σ54-dependent promoter with the strong promoter of gene adhE (V518_0444), gene cluster bglpcel2EIIABCcel2 (V518_2444−2449) could be easily overexpressed and SCUT27/ΔcelRcel2/padhE obtained the ability to co-utilize glucose and cellobiose, which was a potential candidate for ethanol or lactic acid fermentation from partial hydrolysis of lignocellulosic hydrolysates.

Engineering Thermoanaerobacterium aotearoense SCUT27 with argR knockout for enhanced ethanol production from lignocellulosic hydrolysates

Although Thermoanaerobacterium aotearoense SCUT27 (SCUT27) could co-utilize glucose and xylose, the presence of glucose still repressed xylose catabolism. Arginine repressors (ArgRs) were involved in several key metabolic pathways and might be the global regulator. In SCUT27, three genes (V518_0585; V518_1870; V518_1864) were annotated as argR and only the deficiency of argR1864 could greatly improve the co-utilization of glucose and xylose, due to the enhanced activity of xylose isomerase, xylulokinase and the higher energy level. The metabolic flux of SCUT27/ΔargR1864 indicated that new carbon distribution had been re-established and the ethanol yield had increased by 82.95%, strains growth and acetate yield improved by ~35.91% without detectable lactate for the poor activity of lactate dehydrogenase. The improved concentration of ATP and NAD(H) in SCUT27/ΔargR1864 provided more energy to respond the stress, which enabled the mutant the better cell viability to utilize lignocellulosic hydrolysates for enhanced ethanol formation.

The redox-sensing transcriptional repressor Rex is important for regulating the products distribution in Thermoanaerobacterium aotearoense SCUT27.

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD+ concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol production.

A Recombinant β-Mannanase from Thermoanaerobacterium aotearoense SCUT27: Biochemical Characterization and Its Thermostability Improvement.

β-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 β-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for β-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 °C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 °C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.

Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen\xa0production from lignocellulose hydrolysates

BackgroundAs a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P)+, which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated.ResultsCompared with the parental strain, the NADH/NAD+ ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% (p < 0.01) and (13.24 ± 1.12)% (p < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% (p < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/∆nfnAB mutant produced 190.63–209.31 mmol/L hydrogen, with a yield of 1.66–1.77 mol/mol and productivity of 12.71–13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid.ConclusionsThe T. aotearoense SCUT27/∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates.

Enhanced ethanol production from lignocellulosic hydrolysates by inhibiting the hydrogen synthesis in Thermoanaerobacterium aotearoense SCUT27(Δldh)

AbstractBACKGROUNDThermophilic anaerobic bacteria have gained increased interest as a desirable biological catalyst for bioethanol production from lignocellulosic biomass. However, by‐products such as lactic acid, acetic acid and hydrogen (H2) not only lead to low ethanol yield but also increase the difficulty of ethanol purification. Considering that ethanol production is dependent on the supply of NADH, blocking or inhibiting lactic acid (by knocking out ldh) and H2 formation should be a reliable strategy to further increase the ethanol production.RESULTSComparative genome analysis indicated that three hydrogenase gene clusters (hyd, ech and hfs) were identified in Thermoanaerobacterium aotearoense SCUT27, and they were proved to be related to H2 production by gene knockout. Deletion of hyd or ech in SCUT27(Δldh) showed a decrease in substrate consumption and ethanol production, while hfsB gene knockout resulted in significant improvement in overall fermentation performance. Compared with SCUT27(Δldh), the ethanol concentration, yield and productivity of SCUT27(Δldh/ΔhfsB) were increased from 25.15 ± 0.45 g L−1, 0.33 ± 0.01 g g−1 and 0.30 ± 0.01 g L−1 h−1 to 39.64 ± 0.16 g L−1, 0.36 ± 0.00 g g−1 and 0.41 ± 0.01 g L−1 h−1 respectively using glucose in fed‐batch fermentation. The enhanced ethanol production was due to the increased NADH supply and alcohol dehydrogenase activity. Finally, the feasibility of ethanol production by SCUT27(Δldh/ΔhfsB) from hydrolysates of soybean hull, wheat straw, corn cob and corn straw was demonstrated.CONCLUSIONKnocking out the ldh and hfsB in SCUT27 is an efficient strategy for diverting carbon flux and NADH towards ethanol production, and the resulting strain SCUT27(Δldh/ΔhfsB) has great potential in the production of ethanol from lignocellulosic hydrolysates. © 2019 Society of Chemical Industry

Enhanced Purification Efficiency and Thermal Tolerance of Thermoanaerobacterium aotearoense β-Xylosidase through Aggregation Triggered by Short Peptides.

To simplify purification and improve heat tolerance of a thermostable β-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total β-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( Kcat/ Km) was increased from 21.31 mM-1 s-1 for ThXylC to 32.19 mM-1 s-1 for ThXylC-ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.

Dynamic cell responses in Thermoanaerobacterium sp. under hyperosmotic stress

As a nongenetic engineering technique, adaptive evolution is an effective and easy-to-operate approach to strain improvement. In this work, a commercial Thermoanaerobacterium aotearoense SCUT27/Δldh-G58 was successfully isolated via sequential batch fermentation with step-increased carbon concentrations. Mutants were isolated under selective high osmotic pressures for 58 passages. The evolved isolate rapidly catabolized sugars at high concentrations and subsequently produced ethanol with good yield. A 1.6-fold improvement of ethanol production was achieved in a medium containing 120 g/L of carbon substrate using the evolved strain, compared to the start strain. The analysis of transcriptome and intracellular solute pools suggested that the adaptive evolution altered the synthesis of some compatible solutes and activated the DNA repair system in the two Thermoanaerobacterium sp. evolved strains. Overall, the results indicated the potential of adaptive evolution as a simple and effective tool for the modification and optimization of industrial microorganisms.

Enhanced Biodegradation of Sugar Cane Bagasse by Coculture of Clostridium thermocellum and Thermoanaerobacterium aotearoense Supplemented with CaCO3

The pretreated sugarcane bagasse (SCB) could be directly degraded by a designed co-culture system with Clostridium thermocellum and Thermoanaerobacterium aotearoense for biological hydrogen and ethanol production, and the production was remarkably improved by CaCO3 supplementation. Here, the effects of CaCO3 concentration (10~100 mM) on the production of hydrogen and ethanol were investigated. Under the optimal CaCO3 concentration of 40 mM, the hydrogen production reached 87.56±4.08 mmol/L from 2% pretreated SCB with a yield of 4.38 mmol H2/g SCB, an 88.62% increase over the culture without added CaCO3 (46.42±1.22mmol/L, 2.32 mmol H2/g SCB). Additionally, the maximum ethanol concentration reached 10.60±0.81 mM, a 192.82% increase over the control (3.62±0.14 mM). The stimulatory effect of CaCO3 on biodegradation of SCB was primarily ascribed to the buffering capacity of CO32-.This study developed a novel strategy to improve SCB biodegradation for biofuel production.

Biohydrogen Production from Waste Biomass

Biohydrogen production from waste biomass has recently gained more attention. At the South China University of Technology, our research group focuses on biohydrogen production from waste biomass that is based on a consolidated bioprocessing strategy. Cassava pulp, paper sludge, sugarcane bagasse, and spent mushroom compost have all been demonstrated to be feasible feedstocks for hydrogen production with Clostridium thermocellum . Furthermore, co-culture of C. thermocellum and Thermoanaerobacterium aotearoense can enhance the hydrogen production [1]. Besides, our studies showed that surfactant PEG8000 and CaCO 3 both have positive effects on the hydrogen production by C. thermocellum [2-4]. How does biohydrogen production from waste biomass catch our eyes and attract us to devote our efforts on it? With the rapid development of economy, all kinds of waste biomass produced every year are increasing. Recycle of waste biomass can avoid pollution following unsatisfactory disposal such as discard, landfill, and incineration in the field directly. Moreover, waste biomass as raw materials have no conflict with food supply. Thus, there is no doubt that biohydrogen from waste biomass is a promising environmentally friendly energy carrier for its high energy content, and its burning feature of producing only water as the by-product. Biohydrogen from waste biomass is renewable and has no contribution to environmental pollution and climate change, which are valuable for the current situation of increasingly serious energy and environmental issues in China and all over the world. Bioprocesses for hydrogen production from a variety of biomass materials even waste biomass are very promising in the near future. It is wonderful to produce biohydrogen from waste biomass, however, there are some barriers blocking us out of industrial production. Though the utilization of abundant waste biomass can lower the feedstock cost, the cost of biohydrogen production is still less competitive over traditional fuel oil. Furthermore, an inexpensive and simple pretreatment of waste biomass is needed for biohydrogen production. Technological barriers like hydrogen storage, compressor and distribution networks, lack of durable fuel cell technologies, and integration with the existing infrastructure remain to be overcome [5]. The significant problem of low hydrogen yield could be solved by metabolic engineering or co-culture of microorganisms with different advantages. Ideal biohydrogen production should meet the demand of high hydrogen yield and productivity simultaneously. Appropriate bioreactor design, hydrogen production in non-sterile conditions, and feasible techniques for separation or purification of hydrogen are beneficial to practical application. It is important to take the application requirement into consideration, when we are busy in studying biological technologies to improve the hydrogen yield. Advances in optimized processes and technologies would bring us closer to industrially viable biohydrogen production from waste biomass. A comprehensive consideration on industrial feasibility and laboratory investigation would accelerate the large scale production of biohydrogen. Citation: Zhu, M.-J., and Li, H.-N. (2016). Biohydrogen Production from Waste Biomass. Trends in Renewable Energy, 2(2), 54-55. DOI: 10.17737/tre.2016.2.2.0021

Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense.

The strictly anaerobic, Gram-positive bacterium, Thermoanaerobacterium aotearoense SCUT27, is capable of producing ethanol, hydrogen and lactic acid by directly fermenting glucan, xylan and various lignocellulosically derived sugars. By using non-metabolizable and metabolizable sugars as substrates, we found that cellobiose, galactose, arabinose and starch utilization was strongly inhibited by the existence of 2-deoxyglucose (2-DG). However, the xylose and mannose consumptions were not markedly affected by 2-DG at the concentration of one-tenth of the metabolizable sugar. Accordingly, T. aotearoense SCUT27 could consume xylose and mannose in the presence of glucose. The carbon catabolite repression (CCR) related genes, ccpA, ptsH and hprK were confirmed to exist in T. aotearoense SCUT27 through gene cloning and protein characterization. The highly purified Histidine-containing Protein (HPr) could be specifically phosphorylated at Serine 46 by HPr kinase/phosphatase (HPrK/P) with no need to add fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc-6-P) in the reaction mixture. The specific protein-interaction of catabolite control protein A (CcpA) and phosphorylated HPr was proved via affinity chromatography in the absence of formaldehyde. The equilibrium binding constant (K D) of CcpA and HPrSerP was determined as 2.22 ± 0.36 nM by surface plasmon resonance (SPR) analysis, indicating the high affinity between these two proteins.

Valorisation of mixed bakery waste in non-sterilized fermentation for l-lactic acid production by an evolved Thermoanaerobacterium sp. strain

In this study, an advanced biorefinery technology that uses mixed bakery waste has been developed to produce l-lactic acid using an adaptively evolved Thermoanaerobacterium aotearoense LA1002-G40 in a non-sterilized system. Under these conditions, mixed bakery waste was directly hydrolysed by Aspergillus awamori and Aspergillus oryzae, resulting in a nutrient-rich hydrolysate containing 83.6g/L glucose, 9.5g/L fructose and 612mg/L free amino nitrogen. T. aotearoense LA1002-G40 was evaluated and then adaptively evolved to grow in this nutrient-rich hydrolysate. Using a 5-L fermenter, the overall lactic acid production from mixed bakery waste was 0.18g/g with a titer, productivity and yield of 78.5g/L, 1.63g/L/h and 0.85g/g, respectively. This is an innovative procedure involving a complete bioconversion process for l-lactic acid produced from mixed bakery waste under non-sterilized conditions. The proposed process could be potentially applied to turn food waste into l-lactic acid in an economically feasible way.

Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Draft Genome Sequence of an Anaerobic, Thermophilic Bacterium, Thermoanaerobacterium aotearoense SCUT27, Isolated from a Hot Spring in China

Thermoanaerobacterium aotearoense SCUT27, isolated from a hot spring in China, is a strictly anaerobic, thermophilic bacterium capable of degrading xylan and converting both pentose and hexose to ethanol with high yields. Here, we report the draft genome sequence of SCUT27, which reveals insights into the mechanisms of carbon source coutilization and xylan degradation in this thermophilic microorganism.