From methane to value-added bioproducts: microbial metabolism, enzymes, and metabolic engineering.

Methane is abundant in nature, and excessive emissions will cause the greenhouse effect. Methane is also an ideal carbon and energy feedstock for biosynthesis. In the review, the microorganisms, metabolism, and enzymes for methane utilization, and the advances of conversion to value-added bioproducts were summarized. First, the physiological characteristics, classification, and methane oxidation process of methanotrophs were introduced. The metabolic pathways for methane utilization and key intermediate metabolites of native and synthetic methanotrophs were summarized. Second, the enzymatic properties, crystal structures, and catalytic mechanisms of methane-oxidizing and metabolizing enzymes in methanotrophs were described. Third, challenges and prospects in metabolic pathways and enzymatic catalysis for methane utilization and conversion to value-added bioproducts were discussed. Finally, metabolic engineering of microorganisms for methane biooxidation and bioproducts synthesis based on different pathways were summarized. Understanding the metabolism and challenges of microbial methane utilization will provide insights into possible strategies for efficient methane-based synthesis.

Co-conversion of lignocellulose-derived glucose, xylose, and aromatics to polyhydroxybutyrate by metabolically engineered Cupriavidus necator.

Utilization of all major components of lignocellulose is essential for biomass biorefining. Glucose, xylose, and lignin-derived aromatics can be generated from cellulose, hemicellulose, and lignin of lignocellulose degradation through pretreatment and hydrolysis. In present work, Cupriavidus necator H16 was engineered to utilize glucose, xylose, p-coumaric acid, and ferulic acid simultaneously by multi-step genetic engineering. Firstly, genetic modification and adaptive laboratory evolution were performed to promote glucose transmembrane transport and metabolism. Xylose metabolism was then engineered by integrating genes xylAB (xylose isomerase and xylulokinase) and xylE (proton-coupled symporter) in the locus of ldh (lactate dehydrogenase) and ackA (acetate kinase) on the genome, respectively. Thirdly, p-coumaric acid and ferulic acid metabolism was achieved by constructing an exogenous CoA-dependent non-ß-oxidation pathway. Using corn stover hydrolysates as carbon sources, the resulting engineered strain Reh06 simultaneously converted all components of glucose, xylose, p-coumaric acid, and ferulic acid to produce 11.51 g/L polyhydroxybutyrate.

Biosynthesis of value-added bioproducts from hemicellulose of biomass through microbial metabolic engineering.

Hemicellulose is the second most abundant carbohydrate in lignocellulosic biomass and has extensive applications. In conventional biomass refinery, hemicellulose is easily converted to unwanted by-products in pretreatment and therefore can't be fully utilized. The present study aims to summarize the most recent development of lignocellulosic polysaccharide degradation and fully convert it to value-added bioproducts through microbial and enzymatic catalysis. Firstly, bioprocess and microbial metabolic engineering for enhanced utilization of lignocellulosic carbohydrates were discussed. The bioprocess for degradation and conversion of natural lignocellulose to monosaccharides and organic acids using anaerobic thermophilic bacteria and thermostable glycoside hydrolases were summarized. Xylose transmembrane transporting systems in natural microorganisms and the latest strategies for promoting the transporting capacity by metabolic engineering were summarized. The carbon catabolite repression effect restricting xylose utilization in microorganisms, and metabolic engineering strategies developed for co-utilization of glucose and xylose were discussed. Secondly, the metabolic pathways of xylose catabolism in microorganisms were comparatively analyzed. Microbial metabolic engineering for converting xylose to value-added bioproducts based on redox pathways, non-redox pathways, pentose phosphate pathway, and improving inhibitors resistance were summarized. Thirdly, strategies for degrading lignocellulosic polysaccharides and fully converting hemicellulose to value-added bioproducts through microbial metabolic engineering were proposed.

Microbial redox coenzyme engineering and applications in biosynthesis.

Many biosynthetic processes, either in vivo or in vitro, involve redox reactions catalyzed by oxidoreductases - which depend on coenzymes as electron carriers. Redox balance is regulated mainly by coenzymes NAD(P)+ and NAD(P)H and is essential for biosynthesis. New techniques for the regulation and regeneration of coenzymes have recently advanced our understanding of, and demonstrated promising applications in, synthetic biology.

The Overexpression of Phasin and Regulator Genes Promoting the Synthesis of Polyhydroxybutyrate in Cupriavidus necator H16 under Nonstress Conditions.

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO2. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. IMPORTANCE Microbial fixation of CO2 is an effective way to reduce greenhouse gases. Some microbes, such as C. necator H16, usually accumulate PHB when they grow under stress. Low-oxygen stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO2, H2, and O2, while under stress, growth is restricted, and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO2 into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes upregulated under nonstress conditions were identified through comparative transcriptome analysis and overexpression of phasin, and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.

Wide-bound salt tolerance of the inocula from marine sediment and their specific microbial community.

The microorganisms in marine sediment are promising candidates for the treatment of the saline wastes due to their property of salt tolerance. However, the knowledge about the microbial community and property of the marine sediments is still limited. In the present study, the salt tolerance of the microorganisms in the marine sediment that was collected from a marine fish farm was investigated by being used as inoculum for anaerobic digestion. The microbial communities were analyzed by high-throughput sequencing. The inoculum from the wastewater plant (IWTP) was taken as a control. The inoculum from the marine sediment (IMS) showed excellent capacity for anaerobic digestion at salinities of 0.3%-6%. Even at a salinity of 9%, the methane yield remained 60% of the highest yield. IMS provides promising microbial resources for the treatment of both fresh-water and saliferous organic wastes. While the IWTP was sensitive to salt, the methane yield decreased to 56% of the highest yield at the salinity of 3%. The bacterial taxonomic richness of IMS was about half of that in IWTP. Eighty-one genera were identified only in IWTP but not in IMS. The IMS possessed fewer bacterial members related to the nitrogen cycle than IWTP, but more members related to the sulfur cycle. The members of animal parasites or symbionts in IMS were significantly fewer than those in IWTP. The archaeal compositions of IMS and IWTP were different. The relative abundance of the unidentified archaea in IMS was much higher than that in IWTP with 12.52% vs 0.06% at phylum level. The findings of this work expand our understanding of the microorganisms in marine sediments and will promote the application of them in waste treatment.

Depolymerization and conversion of lignin to value-added bioproducts by microbial and enzymatic catalysis.

Lignin, the most abundant renewable aromatic compound in nature, is an excellent feedstock for value-added bioproducts manufacturing; while the intrinsic heterogeneity and recalcitrance of which hindered the efficient lignin biorefinery and utilization. Compared with chemical processing, bioprocessing with microbial and enzymatic catalysis is a clean and efficient method for lignin depolymerization and conversion. Generally, lignin bioprocessing involves lignin decomposition to lignin-based aromatics via extracellular microbial enzymes and further converted to value-added bioproducts through microbial metabolism. In the review, the most recent advances in degradation and conversion of lignin to value-added bioproducts catalyzed by microbes and enzymes were summarized. The lignin-degrading microorganisms of white-rot fungi, brown-rot fungi, soft-rot fungi, and bacteria under aerobic and anaerobic conditions were comparatively analyzed. The catalytic metabolism of the microbial lignin-degrading enzymes of laccase, lignin peroxidase, manganese peroxidase, biphenyl bond cleavage enzyme, versatile peroxidase, and ß-etherize was discussed. The microbial metabolic process of H-lignin, G-lignin, S-lignin based derivatives, protocatechuic acid, and catechol was reviewed. Lignin was depolymerized to lignin-derived aromatic compounds by the secreted enzymes of fungi and bacteria, and the aromatics were converted to value-added compounds through microbial catalysis and metabolic engineering. The review also proposes new insights for future work to overcome the recalcitrance of lignin and convert it to value-added bioproducts by microbial and enzymatic catalysis.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions.

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Heteroexpression of Osa-miR319b improved switchgrass biomass yield and feedstock quality by repression of PvPCF5.

BACKGROUND: Switchgrass (Panicum virgatum L.), a C4 perennial grass, has been recognized as one of the most potentially important lignocellulose biofuel crops. MicroRNA319 (miR319) plays a key role in plant development, abiotic resistance, and cell wall biosynthesis by repressing expression of its target TCP genes. We hypothesized miR319-TCP pathway could play important roles in switchgrass feedstock characteristics for biofuel production, and produced switchgrass transgenic plants overexpressing miR319 (by ectopic expressing Osa-MIR319b gene), blocking miR319 (by overexpressing a target mimicry of miR319/MIM319) and repression of miR319 target gene PvPCF5. Plant phenotype, biomass yield, and feedstock quality of transgenic plants were analyzed. RESULTS: Overexpression of miR319 in switchgrass promoted leaf elongation and expansion of transgenic plants, increased plant height, stem diameter, and resulted in a significant increase in plant biomass yield. Transgenic plants overexpressing of miR319 reduced lignin content, showed significantly higher enzymatic hydrolysis efficiency compared to the wild type plant. However, opposite results were observed in the MIM319 plants. Furthermore, suppression of miR319 target gene PvPCF5 activity also reduced lignin content, increased lignin monomer S/G ratio and the proportion of ß-O-4 linkages, while significantly improving the sugar production per plant. Quantitative real-time (qRT-PCR) analysis indicated that expression of PvMYB58/63B and PvHCT with predicted TCP binding sites in their promoter regions was negatively regulated by miR319-PvPCF5 module. CONCLUSIONS: MiR319-PvPCF5 module plays positive roles in regulating biomass yield and quality of switchgrass. It can be utilized as a candidate molecular tool in regulating biomass yield and feedstock quality. The finding could also be transferred to other grasses for forage quality improvement through genetic manipulation.

Nitrogen-doped porous carbon from biomass with superior catalytic performance for acetylene hydrochlorination.

Acetylene hydrochlorination is an important aspect of the industrial synthesis of polyvinyl chloride, but it requires a toxic mercury chloride catalyst. Here we report a green, highly efficient and low cost nitrogen-doped soybean meal carbon (SBMC) catalyst obtained from the simple carbonization of biomass soybean meal (SBM) in the presence of zinc chloride. This material exhibits excellent catalytic performance during acetylene hydrochlorination, with an initial acetylene conversion greater than 99% and 98% selectivity for vinyl chloride at 200 °C over 110 h. Analyses by X-ray photoelectron spectroscopy and temperature programmed desorption as well as catalytic activity evaluations show that pyridinic species are the active sites for hydrogen chloride, while pyrrolic N species are the main active sites for acetylene. An analysis of charge calculations based on model catalysts further indicates that the activity of pyrrolic N species essentially determines the performance of the SBMC catalyst. This investigation of the mechanism of acetylene hydrochlorination over SBMC confirms that such nitrogen-doped catalysts have two different active sites for the adsorption and activation of hydrogen chloride and acetylene molecules. This mechanism is different from that associated with metal chloride catalysts such as HgCl2. This SBMC catalyst is a potential alternative to HgCl2@AC catalysts for vinyl chloride synthesis and suggests a new means of designing carbon catalysts with basic surfaces for acetylene hydrochlorination.

Corrosion behavior and mechanism of carbon steel influenced by interior deposit microflora of an in-service pipeline.

Investigation of carbon steel corrosion influenced by in-situ microbial communities can provide reliable information about microbiologically influenced corrosion (MIC) in the oil and gas field. Here, we investigated the 90-day corrosion behavior of Q235 carbon steel influenced by interior deposit microflora of an in-service pipeline using open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS). Linear sweep voltammetry (LSV), 16S rRNA gene sequencing, and surface analysis were used to comprehensively analyze the corrosion mechanisms. The results indicated that OCP was decreased while the charge transfer resistance (Rct) was increased, and that steel corrosion was inhibited during the first 45 days. Subsequently, OCP was significantly increased while Rct was rapidly decreased, and steel corrosion was enhanced. After 90-day immersion, severe pitting corrosion with a maximum pit depth of 89.6 µm occurred on the steel surface. Viable microbes in the final biofilm significantly increased the cathodic current. Iron carbonate, chukanovite and cementite were identified as the main corrosion products on the steel surface. Methanobacterium dominated the final biofilm community. These observations indicate that the corrosion mechanism of the final biofilm can be explained by extracellular electron transfer MIC in which microbes corrode steel by direct electron uptake.

The extracellular endo-ß-1,4-xylanase with multidomain from the extreme thermophile Caldicellulosiruptor lactoaceticus is specific for insoluble xylan degradation.

BACKGROUND: The extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus can degrade and metabolize untreated lignocellulosic biomass containing xylan. The mechanism of the bacterium for degradation of insoluble xylan in untreated biomass has not been revealed. RESULTS: In the present study, the only annotated extracellular endo-ß-1,4-xylanase (Xyn10B) with multidomain structures in C. lactoaceticus genome was biochemically characterized. Xyn10B contains three N-terminal consecutive family 22 carbohydrate-binding modules (CBMs), one GH10 catalytic domain (CD), two family 9 CBMs and two S-layer homology (SLH) modules in the C-terminal. CBM22a shares 27.1% and 27.2% sequence homology with CBM22b and CBM22c, respectively. The sequence homology between two CBM9 s and two SLHs is 26.8% and 25.6%, respectively. To elucidate the effect of multiple domains on the enzymatic properties of Xyn10B, the truncated variants of which (Xyn10B-TM1: CBM22a-CBM22b-CBM22c-CD10; Xyn10B-TM2: CBM22c-CD10; Xyn10B-TM3: CBM22c-CD10-CBM9a; and Xyn10B-TM4: CD10-CBM9a) were separately reconstructed, recombinantly expressed and biochemically characterized. Enzymatic properties studies showed that the optimal temperature for all four Xyn10B truncations was 65 °C. Compared to Xyn10B-TM3 and Xyn10B-TM4, Xyn10B-TM1 and Xyn10B-TM2 had higher hydrolytic activity, thermostability and affinity on insoluble substrates. It is noteworthy that Xyn10B-TM1 and Xyn10B-TM2 have higher enzymatic activity on insoluble xylan than the soluble counterparts, whereas Xyn10B-TM3 and Xyn10B-TM4 showed opposite characteristics. The kinetic parameters analysis of Xyn10B-TM1 on xylan showed V max was 5740, 1300, 1033, and 3925 U/µmol on insoluble oat spelt xylan (OSX), soluble beechwood xylan (BWX), soluble sugar cane xylan (SCX), and soluble corncob xylan (CCX), respectively. The results indicated that CBM22s especially CBM22c promoted the hydrolytic activity, thermostability and affinity on insoluble substrates of the Xyn10B truncations. The functions of CBM22, CBM9, CD and SLH are different, while contribute synergetically to the thermostability, protein structure integrity, substrate binding, and high hydrolytic activity on insoluble xylan of untreated lignocellulosic biomass. The domains of CBM22, CBM9, CD and SLH have different characteristics, which synergistically promote the thermostability, protein structure integrity, affinity on insoluble substrates and enzymatic activity properties of Xyn10B. CONCLUSIONS: The extracellular endo-ß-1,4-xylanase with multidomain structures of CBM, CD and SLH promote the biodegradation of insoluble xylan in untreated lignocellulosic biomass by thermophilic C. lactoaceticus.

The mutual influence between corrosion and the surrounding soil microbial communities of buried petroleum pipelines.

Buried petroleum pipeline corrosion and leaks cause inevitable changes in the microbial communities of the surrounding soils. In addition, soils with different microbial communities can make different contributions to buried pipeline corrosion. Three kinds of soil samples of buried petroleum pipelines under different corrosion and petroleum contamination conditions were collected from the Shengli Oilfield of China to investigate the mutual influence between corrosion and the microbial communities of the surrounding soil. The 16S rRNA gene high-throughput Illumina MiSeq sequencing was used to analyze the microbial communities of different surrounding soils. Electrochemical tests were performed for steel corrosion investigation. The results showed that the microbial diversity of the surrounding soils of corroded pipelines with/without petroleum contamination (O-soil and C-soil, respectively) decreased significantly as compared with that of the non-corroded and non-contaminated ones (NC-soil). The C-soil contained more abundant Balneolaceae (Balneola, KSA1), Flavobacteriaceae (Muricauda, Gramella) and Desulfuromonadaceae (Pelobacter, Geoalkalibacter). The O-soil possessed a greater abundance of Halomonas, Pseudoalteromonas, Psychrobacter and Dietzia, which were reported to have a capacity for hydrocarbon degradation. Moreover, electrochemical measurements indicated that the microcosm of the C-soil and NC-soil promoted steel corrosion, while the C-soil community showed a slightly higher corrosion rate. However, the O-soil community mitigated the steel corrosion. These observations suggested that pipeline corrosion increased proportions of microorganisms, which are likely related to fermentation, sulfur respiration, iron respiration and manganese respiration in surrounding soils and enhanced the soil corrosivity, while petroleum contamination weakened the corrosion ability and promoted the growth of hydrocarbon-degrading organisms in the microbial community.

Simultaneous biosynthesis of (R)-acetoin and ethylene glycol from D-xylose through in vitro metabolic engineering.

(R)-acetoin is a four-carbon platform compound used as the precursor for synthesizing novel optically active materials. Ethylene glycol (EG) is a large-volume two-carbon commodity chemical used as the anti-freezing agent and building-block molecule for various polymers. Currently established microbial fermentation processes for converting monosaccharides to either (R)-acetoin or EG are plagued by the formation of undesirable by-products. We show here that a cell-free bioreaction scheme can generate enantiomerically pure acetoin and EG as co-products from biomass-derived D-xylose. The seven-step, ATP-free system included in situ cofactor regeneration and recruited enzymes from Escherichia coli W3110, Bacillus subtilis shaijiu 32 and Caulobacter crescentus CB 2. Optimized in vitro biocatalytic conditions generated 3.2 mM (R)-acetoin with stereoisomeric purity of 99.5% from 10 mM D-xylose at 30 °C and pH 7.5 after 24 h, with an initial (R)-acetoin productivity of 1.0 mM/h. Concomitantly, EG was produced at 5.5 mM, with an initial productivity of 1.7 mM/h. This in vitro biocatalytic platform illustrates the potential for production of multiple value-added biomolecules from biomass-based sugars with no ATP requirement.

Sequential processing with fermentative Caldicellulosiruptor kronotskyensis and chemolithoautotrophic Cupriavidus necator for converting rice straw and CO2 to polyhydroxybutyrate.

Unpretreated rice straw was fermented by the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis, generating solubilized carbohydrates, organic acids, lignin-derived aromatics, H2 , and CO2 , which were subsequently used to produce polyhydroxybutyrate (PHB) by the chemolithoautotrophic bacterium Cupriavidus necator. The fermented liquid significantly enhanced the growth of C. necator, leading to a five-fold cell biomass yield, and a nine-fold PHB yield compared to what was obtained from conventional mineral media. This integrated process utilized all products of lignocellulose fermentation without H (electron) loss and carbon emission, while concomitantly enhancing CO2 fixation by C. necator for PHB production. The sequential coupling of C. kronotskyensis and C. necator provides not only a new biorefinery paradigm characterized by reduced pretreatment and saccharification requirements but also an efficient way for enhancing CO2 fixation.

Conversion of cellulose and hemicellulose of biomass simultaneously to acetoin by thermophilic simultaneous saccharification and fermentation.

BACKGROUND: Acetoin (3-hydroxy-2-butanone), the precursor of biofuel 2,3-butanediol, is an important bio-based platform chemical with wide applications. Fermenting the low-cost and renewable plant biomass is undoubtedly a promising strategy for acetoin production. Isothermal simultaneous saccharification and fermentation (SSF) is regarded as an efficient method for bioconversion of lignocellulosic biomass, in which the temperature optima fitting for both lignocellulose-degrading enzymes and microbial strains. RESULTS: A thermotolerant (up to 52 °C) acetoin producer Bacillus subtilis IPE5-4 which simultaneously consumed glucose and xylose was isolated and identified. By compound mutagenesis, the mutant IPE5-4-UD-4 with higher acetoin productivity was selected. When fermenting at 50 °C in a 5-L bioreactor using glucose as the feedstock by strain IPE5-4-UD-4, the acetoin concentration reached 28.83 ± 0.91 g L-1 with the acetoin yield and productivity of 0.34 g g-1 glucose and 0.60 g L-1 h-1, respectively. Furthermore, an optimized and thermophilic SSF process operating at 50 °C was conducted for acetoin production from alkali-pretreated corncob (APC). An acetoin concentration of 12.55 ± 0.28 g L-1 was achieved by strain IPE5-4-UD-4 in shake flask SSF, with the acetoin yield and productivity of 0.25 g g-1 APC and 0.17 g L-1 h-1. Meanwhile, the utilization of cellulose and hemicellulose in the SSF approach reached 96.34 and 93.29%, respectively. When further fermented at 50 °C in a 5-L bioreactor, the concentration of acetoin reached the maximum of 22.76 ± 1.16 g L-1, with the acetoin yield and productivity reaching, respectively, 0.46 g g-1 APC and 0.38 g L-1 h-1. This was by far the highest acetoin yield in SSF from lignocellulosic biomass. CONCLUSIONS: This thermophilic SSF process provided an efficient and economical route for acetoin production from lignocellulosic biomass at ideal temperature for both enzymatic hydrolysis and microbial fermentation.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus.

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 ß-l-arabinopyranosidase (CpAbp27A), and two GH127 ß-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved ß-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusIMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as ß-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate.

Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65-70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h-1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

Changes in composition, cellulose degradability and biochemical methane potential of Miscanthus species during the growing season.

The composition, cellulose degradability and biochemical methane potential (BMP) of M. sinensis, M. floridulus, Miscanthus×giganteus and M. lutarioriparius were investigated concomitantly at different growth/harvest times during their growing season. For all the four species, there was only a slight change in the compositional content. Meanwhile there was a huge change in the BMP values. At the growth time of 60days the BMPs ranged from 247.1 to 266.5mlg-1VS. As growth time was prolonged, the BMPs decreased by 11-35%. For each species, the BMP was positively correlated to the cellulose degradability with the correlation coefficients (R2) ranging from 0.8055 to 0.9925. This suggests that besides the biomass yield, it is justifiable to consider cellulose degradability when selecting the suitable harvest time for biofuels production from Miscanthus, especially in tropical and subtropical regions where Miscanthus can be harvested twice or more within a year.

Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea.

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO2 in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase). The model correctly predicted previously observed features of the cycle: the 35-65% split of carbon flux through the acetyl-CoA and succinate branches, the high abundance and relative ratio of acetyl-CoA/propionyl-CoA carboxylase (ACC) and MCR, and the significance of ACC and hydroxybutyryl-CoA synthetase (HBCS) as regulated control points for the cycle. The model was then used to assess metabolic engineering strategies for incorporating CO2 into chemical intermediates and products of biotechnological importance: acetyl-CoA, succinate, and 3-hydroxypropionate.