Entner-Doudoroff Research Articles

Phosphoketolase and KDPG aldolase metabolisms modulate photosynthetic carbon yield in cyanobacteria.

Cyanobacteria contribute to roughly a quarter of global net carbon fixation. During diel light/dark growth, dark respiration substantially lowers the overall photosynthetic carbon yield in cyanobacteria and other phototrophs. How respiratory pathways participate in carbon resource allocation at night to optimize dark survival and support daytime photosynthesis remains unclear. Here, using the cyanobacterium Synechococcus elongatus PCC 7942, we show that phosphoketolase integrates into a respiratory network in the dark to best allocate carbon resources for amino acid biosynthesis and to prepare for photosynthesis reinitiation upon photoinduction. Moreover, we show that the respiratory Entner-Doudoroff (ED) pathway in S. elongatus is incomplete, with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase exhibiting alternative oxaloacetate decarboxylation activity that modulates daytime photosynthesis. This activity allows for the bypassing of the tricarboxylic acid (TCA) cycle when ATP and NADPH consumption for biosynthesis is excessive and imbalanced relative to their production by the light reactions, thereby preventing relative NADPH accumulation and ensuring optimal photosynthetic carbon yield. Optimizing these metabolic processes offers opportunities to enhance photosynthetic carbon yield in cyanobacteria and other photosynthetic organisms under diel light/dark cycles.

The Functional Characterization of the 6-Phosphogluconate Dehydratase Operon in 2-Ketogluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01

Genus Pseudomonas bacteria mainly consume glucose through the Entner–Doudoroff (ED) route due to a lack of a functional Embden–Meyerhof–Parnas (EMP) pathway. In the present study, a 6-phosphogluconate dehydratase (edd) operon in the ED route was well investigated to find its structural characteristics and roles in the regulation of glucose consumption and 2-ketogluconic acid (2KGA) metabolism in the industrial 2KGA-producer P. plecoglossicida JUIM01. The edd operon contained four structural genes of edd, glk, gltR, and gtrS, encoding 6-PG dehydratase Edd, glucokinase Glk, response regulatory factor GltR, and histidine kinase GtrS, respectively. A promoter region was observed in the 5′-upstream of the edd gene, with a transcriptional start site located 129 bp upstream of the edd gene and in a pseudo-palindromic sequence of 5′-TTGTN7ACAA-3′ specifically binding to the transcription factor HexR. The knockout of the edd gene showed a remarkably negative effect on cell growth and re-growth using 2KGA as a substrate, beneficial to 2KGA production, with an increase of 8%. The deletion of glk had no significant effect on the cell growth or glucose metabolism, while showing an adverse impact on the 2KGA production, with a decrease of 5%. The outputs of the present study would provide a theoretical basis for 2KGA-producer improvement with metabolic engineering strategies and the development and optimization of P. plecoglossicida as the chassis cells.

The primary carbon metabolism in cyanobacteria and its regulation.

Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis. Many cyanobacterial strains can live in different trophic modes, ranging from photoautotrophic and heterotrophic to mixotrophic growth. However, the regulatory mechanisms allowing a flexible switch between these lifestyles are poorly understood. As anabolic fixation of CO2 in the Calvin-Benson-Bassham (CBB) cycle and catabolic sugar-degradation pathways share intermediates and enzymatic capacity, a tight regulatory network is required to enable simultaneous opposed metabolic fluxes. The Entner-Doudoroff (ED) pathway was recently predicted as one glycolytic route, which cooperates with other pathways in glycogen breakdown. Despite low carbon flux through the ED pathway, metabolite analyses of mutants deficient in the ED pathway revealed a distinct phenotype pointing at a strong regulatory impact of this route. The small Cp12 protein downregulates the CBB cycle in darkness by inhibiting phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase. New results of metabolomic and redox level analyses on strains with Cp12 variants extend the known role of Cp12 regulation towards the acclimation to external glucose supply under diurnal conditions as well as to fluctuations in CO2 levels in the light. Moreover, carbon and nitrogen metabolism are closely linked to maintain an essential C/N homeostasis. The small protein PirC was shown to be an important regulator of phosphoglycerate mutase, which identified this enzyme as central branching point for carbon allocation from CBB cycle towards lower glycolysis. Altered metabolite levels in the mutant ΔpirC during nitrogen starvation experiments confirm this regulatory mechanism. The elucidation of novel mechanisms regulating carbon allocation at crucial metabolic branching points could identify ways for targeted redirection of carbon flow towards desired compounds, and thus help to further establish cyanobacteria as green cell factories for biotechnological applications with concurrent utilization of sunlight and CO2.

Microbial central carbon metabolism in a tidal freshwater marsh and an upland mixed conifer soil under oxic and anoxic conditions.

The central carbon (C) metabolic network is responsible for most of the production of energy and biosynthesis in microorganisms and is therefore key to a mechanistic understanding of microbial life in soil communities. Many upland soil communities have shown a relatively high C flux through the pentose phosphate (PP) or the Entner-Doudoroff (ED) pathway, thought to be related to oxidative damage control. We tested the hypothesis that the metabolic organization of the central C metabolic network differed between two ecosystems, an anoxic marsh soil and oxic upland soil, and would be affected by altering oxygen concentrations. We expected there to be high PP/ED pathway activity under high oxygen concentrations and in oxic soils and low PP/ED activity in reduced oxygen concentrations and in marsh soil. Although we found high PP/ED activity in the upland soil and low activity in the marsh soil, lowering the oxygen concentration for the upland soil did not reduce the relative PP/ED pathway activity as hypothesized, nor did increasing the oxygen concentration in the marsh soil increase the PP/ED pathway activity. We speculate that the high PP/ED activity in the upland soil, even when exposed to low oxygen concentrations, was related to a high demand for NADPH for biosynthesis, thus reflecting higher microbial growth rates in C-rich soils than in C-poor sediments. Further studies are needed to explain the observed metabolic diversity among soil ecosystems and determine whether it is related to microbial growth rates.IMPORTANCEWe observed that the organization of the central carbon (C) metabolic processes differed between oxic and anoxic soil. However, we also found that the pentose phosphate pathway/Entner-Doudoroff (PP/ED) pathway activity remained high after reducing the oxygen concentration for the upland soil and did not increase in response to an increase in oxygen concentration in the marsh soil. These observations contradicted the hypothesis that oxidative stress is a main driver for high PP/ED activity in soil communities. We suggest that the high PP/ED activity and NADPH production reflect higher anabolic activities and growth rates in the upland soil compared to the anaerobic marsh soil. A greater understanding of the molecular and biochemical processes in soil communities is needed to develop a mechanistic perspective on microbial activities and their relationship to soil C and nutrient cycling. Such an increased mechanistic perspective is ecologically relevant, given that the central carbon metabolic network is intimately tied to the energy metabolism of microbes, the efficiency of new microbial biomass production, and soil organic matter formation.

Engineering Pseudomonas putida KT2440 for Dipicolinate Production via the Entner-Doudoroff Pathway.

Dipicolinic acid (DPA), a cyclic diacid, has garnered significant interest due to its potential applications in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. However, its natural bioproduction is limited since DPA is only accumulated in Bacillus and Clostridium species during sporulation. Thus, heterologous production by engineered strains is of paramount importance for developing a sustainable biological route for DPA production. Pseudomonas putida KT2440 has emerged as a promising host for the production of various chemicals thanks to its robustness, metabolic versatility, and genetic tractability. The dominant Entner-Doudoroff (ED) pathway for glucose metabolism in this strain offers an ideal route for DPA production due to the advantage of NADPH generation and the naturally balanced flux between glyceraldehyde-3-phosphate and pyruvate, which are both precursors for DPA synthesis. In this study, DPA production via the ED pathway was in silico designed in P. putida KT2440. The systematically engineered strain produced dipicolinate with a titer of 11.72 g/L from glucose in a 5 L fermentor. This approach not only provides a sustainable green route for DPA production but also expands our understanding of the metabolic potential of the ED pathway in P. putida KT2440.

Plastid ancestors lacked a complete Entner-Doudoroff pathway, limiting plants to glycolysis and the pentose phosphate pathway

The Entner–Doudoroff (ED) pathway provides an alternative to glycolysis. It converts 6-phosphogluconate (6-PG) to glyceraldehyde-3-phosphate and pyruvate in two steps consisting of a dehydratase (EDD) and an aldolase (EDA). Here, we investigate its distribution and significance in higher plants and determine the ED pathway is restricted to prokaryotes due to the absence of EDD genes in eukaryotes. EDDs share a common origin with dihydroxy-acid dehydratases (DHADs) of the branched chain amino acid pathway (BCAA). Each dehydratase features strict substrate specificity. E. coli EDD dehydrates 6-PG to 2-keto-3-deoxy-6-phosphogluconate, while DHAD only dehydrates substrates from the BCAA pathway. Structural modeling identifies two divergent domains which account for their non-overlapping substrate affinities. Coupled enzyme assays confirm only EDD participates in the ED pathway. Plastid ancestors lacked EDD but transferred metabolically promiscuous EDA, which explains the absence of the ED pathway from the Viridiplantae and sporadic persistence of EDA genes across the plant kingdom.

Optimizing Hexose Utilization Pathways of Cupriavidus necator for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions.

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner-Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD600 of the phaC1AB1-knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD600 of phaC1AB1-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator, an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator.

Multicolor optogenetics for regulating flux ratio of three glycolytic pathways using EL222 and CcaSR in Escherichia coli.

Optogenetics is an attractive synthetic biology tool for controlling the metabolic flux distribution. Here, we demonstrated optogenetic flux ratio control of glycolytic pathways consisting of the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways by illuminating multicolor lights using blue light-responsive EL222 and green/red light-responsive CcaSR in Escherichia coli. EL222 forms a dimer and binds to a particular DNA sequence under blue light; therefore, target gene expression can be reduced or induced by inserting a recognition sequence into its promoter regions. First, a flux ratio between the PP and ED pathways was controlled by blue light using EL222. After blocking the EMP pathway, the EL222-recognition sequence was inserted between the -35 and -10 regions of gnd to repress the PP flux and was also inserted upstream of the -35 region of edd to induce ED flux. After adjusting light intensity, the PP:ED flux ratios were 60:39% and 29:70% under dark and blue light conditions, respectively. Finally, a CcaSR-based pgi expression system was implemented to control the flux ratio between the EMP and PP + ED pathways by illuminating green/red light. The EMP:PP:ED flux ratios were 80:9:11%, 14:35:51%, and 33:5:62% under green, red, and red and blue light, respectively.

Characterization of the Entner-Doudoroff pathway in Pseudomonas aeruginosa catheter-associated urinary tract infections.

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen responsible for a subset of catheter-associated urinary tract infections (CAUTI). In a murine model of P. aeruginosa CAUTI, we previously demonstrated that urea within urine suppresses quorum sensing and induces the Entner-Doudoroff (E-D) pathway. The E-D pathway consists of the genes zwf, pgl, edd, and eda. Zwf and Pgl convert glucose-6-phosphate into 6-phosphogluconate. Edd hydrolyzes 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Finally, Eda cleaves KDPG to glyceraldehyde-3-phosphate and pyruvate, which enters the citric acid cycle. Here, we generated in-frame E-D mutants in the strain PA14 and assessed their growth phenotypes on chemically defined and complex media. These E-D mutants have a growth defect when grown on glucose or gluconate as the sole carbon source, which is similar to results previously reported for PAO1 mutants lacking E-D genes. RNA-sequencing following short exposure to urine revealed minimal gene regulation differences compared to the wild type. In a murine CAUTI model, virulence testing of E-D mutants revealed that two mutants lacking zwf and pgl showed minor fitness defects. Infection with the ∆pgl strain exhibited a 20% increase in host survival, and the ∆zwf strain displayed decreased colonization of the catheter and kidneys. Consequently, our findings suggest that the E-D pathway in P. aeruginosa is dispensable in this model of CAUTI. IMPORTANCE Prior studies have shown that the Entner-Doudoroff pathway is up-regulated when Pseudomonas aeruginosa is grown in urine. Pseudomonads use the Entner-Doudoroff (E-D) pathway to metabolize glucose instead of glycolysis, which led us to ask whether this pathway is required for urinary tract infection. Here, single-deletion mutants of each gene in the pathway were tested for growth on chemically defined media with single-carbon sources as well as complex media. The effect of each mutant on global gene expression in laboratory media and urine was characterized. The virulence of these mutants in a murine model of catheter-associated urinary tract infection revealed that these mutants had similar levels of colonization indicating that glucose is not the primary carbon source utilized in the urinary tract.

Metabolic engineering of Escherichia coli for enhanced production of 1,3-butanediol from glucose

1,3-Butanediol (1,3-BDO) finds versatile applications in the cosmetic, chemical, and food industries. This study focuses on the metabolic engineering of Escherichia coli K12 to achieve efficient production of 1,3-BDO from glucose via acetoacetyl-CoA, 3-hydroxybutyryl-CoA, and 3-hydroxybutyraldehyde. The accumulation of an intermediary metabolite (pyruvate) and a byproduct (3-hydroxybutyric acid) was reduced by disruption of the negative transcription factor (PdhR) for pyruvate dehydrogenase complex (PDHc) and employing an efficient alcohol dehydrogenase (YjgB), respectively. Additionally, to improve NADPH availability, carbon flux was redirected from the Embden-Meyerhof-Parnas (EMP) pathway to the Entner–Doudoroff (ED) pathway. One resulting strain achieved a record-high titer of 790 mM (∼71.1 g/L) with a yield of 0.65 mol/mol for optically pure (R)-1,3-BDO, with an enantiomeric excess (e.e.) value of 98.5 %. These findings are useful in the commercial production of 1,3-BDO and provide valuable insights into the development of an efficient cell factory for other acetyl-CoA derivatives.

A parallel glycolysis provides a selective advantage through rapid growth acceleration.

Glycolysis is a universal metabolic process that breaks down glucose to produce adenosine triphosphate (ATP) and biomass precursors. The Entner-Doudoroff (ED) pathway is a glycolytic pathway that parallels textbook glycolysis but yields half as much ATP. Accordingly, in organisms that possess both glycolytic pathways (for example, Escherichia coli), its raison d'être remains a mystery. In this study, we found that the ED pathway provides a selective advantage during growth acceleration. Upon carbon and nitrogen upshifts, E. coli accelerates growth faster with than without the ED pathway. Concurrent isotope tracing reveals that the ED pathway flux increases faster than that of textbook glycolysis. We attribute the fast response time of the ED pathway to its strong thermodynamic driving force and streamlining of glucose import. Intermittent nutrient supply manifests the evolutionary advantage of the parallel glycolysis; thus, the dynamic nature of an ostensibly redundant pathway's role in promoting rapid adaptation constitutes a metabolic design principle.

Effect of long-term influx of tertiary treated wastewater on native bacterial communities in a dry valley topsoil: 16S rRNA gene-based metagenomic analysis of composition and functional profile.

Although dumping treated wastewater into soil might provide nutrients and organic matter, it can also expose the ecosystem to biological and chemical risks. A vital indication of soil health and quality is the soil microbial community. The current work used next-generation 16S rRNA gene amplicon sequencing to evaluate the effects of the long-term influx of tertiary treated wastewater (TWW) into Wadi Uranah, a dry valley in Makkah city, Saudi Arabia, on native topsoil bacterial community composition and predicted functions. The findings demonstrated that neither the compositions of microbial communities nor their predicted functions using PICRUSt2 differed significantly (p>0.05) between polluted valley soil (PolVS) and unpolluted valley soil (UPVS). Alpha and beta diversity, however, showed that the PolVS samples had a considerably higher level of diversity and variability. Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the most prevalent phyla in both groups. Noticeable relative variations existed in some metabolic pathways such as cofactor, prosthetic group, electron carrier degradation, aldehyde degradation, and Entner-Doudoroff (ED) pathways. Overall, our findings suggest that because both groups have very similar core microbiomes and functions, the long-term disposal of tertiary TWW into Wadi Uranah may have little to no influence on the composition and function of soil bacterial communities. In addition, the long-term discharge of tertiary TWW after partially treated wastewater's initial disposal may have helped the native soil microbial community recover.

Construct a synthetic Entner–Doudoroff pathway in Bacillus licheniformis for enhancing lichenysin production

Lichenysin, a cyclic lipopeptide biosurfactant produced by Bacillus licheniformis, is composed of aspartate, glutamine, valine, leucine, isoleucine, and branched chain fatty acids. The synthesis of these amino acids and fatty acids requires pyruvate and NADPH as the primary precursor and cofactor. Therefore, a sufficient supply of pyruvate and NADPH is crucial for lichenysin production. This study aimed to increase lichenysin production by constructing a synthetic ED pathway in B. licheniformis WX02 through introducing phosphogluconate dehydratase (encoded by gene edd) and 2-keto-3-deoxygluconate 6-phosphate aldolase (encoded by gene eda) from Escherichia coli. Additionally, the NADP+-dependent glucose-6-phosphate dehydrogenase (encoded by gene zwf) was overexpressed, resulting in an engineered strain WX02/pHY-edda(Ec)-zwf. Analysis of the fermentation process revealed that the concentrations of pyruvate, aspartate, glutamine, valine, leucine, branched-chain fatty acids (iC15:0, aC15:0, iC16:0, iC17:0), and NADPH in WX02/pHY-edda(Ec)-zwf were increased by 77.21%, 80.41%, 85.31%, 141.64%, 44.94%, 35.08%, 38.08%, 19.33%, 21.16%, and 425%, respectively, compared to the control strain WX02/pHY300, which resulted in a 45.43% increase of lichenysin titer. This work took advantage of the ED pathway to increase lichenysin production for the first time, and provides a promising strategy for boosting the productivity of biochemicals that require pyruvate and NADPH as precursor and cofactor.

Production of N-acetylglucosamine from carbon dioxide by engineering Cupriavidus necator H16

The conversion of CO2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner–Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO2 under normal conditions..

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner–Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer′s productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis.

Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for the commercial production of desirable bioproducts. Sugar transporters are responsible for the import of substrate sugars and the conversion of ethanol and other products. Glucose-facilitated diffusion protein Glf is responsible for facilitating the diffusion of glucose uptake in Z. mobilis. However, another sugar transporter-encoded gene, ZMO0293, is poorly characterized. We employed gene deletion and heterologous expression mediated by the CRISPR/Cas method to investigate the role of ZMO0293. The results showed that deletion of the ZMO0293 gene slowed growth and reduced ethanol production and the activities of key enzymes involved in glucose metabolism in the presence of high concentrations of glucose. Moreover, ZMO0293 deletion caused different transcriptional changes in some genes of the Entner Doudoroff (ED) pathway in the ZM4-ΔZM0293 strain but not in ZM4 cells. The integrated expression of ZMO0293 restored the growth of the glucose uptake-defective strain Escherichia coli BL21(DE3)-ΔptsG. This study reveals the function of the ZMO0293 gene in Z. mobilis in response to high concentrations of glucose and provides a new biological part for synthetic biology.

Comparative genomics and transcriptomic response to root exudates of six rice root-associated Burkholderia sensu lato species

Beyond being a reliable nutrient provider, some bacteria will perceive the plant as a potential host and undertake root colonization leading to mutualistic or parasitic interactions. Bacteria of the Burkholderia and Paraburkholderia genera are frequently found in the rhizosphere of rice. While the latter are often described as plant growth promoting species, Burkholderia are often studied for their human opportunistic traits. Here, we used root exudate stimulation on three Burkholderia and three Paraburkholderia strains isolated from rice roots to characterize their preliminary adaptation to the rice host at the transcriptomic level. Instead of the awaited genus-dependent adaptation, we observed a strongly species-specific response for all tested strains. While all bacteria originate from the rice environment, there are great disparities in their levels of adaptation following the sensing of root exudates. We further report the shared major functions that were differentially regulated in this early step of bacterial adaptation to plant colonization, including amino acids and putrescine metabolism, the Entner-Doudoroff (ED) pathway as well as cyclic diguanylate monophosphate (c-di-GMP) cycling.

A novel alginate lyase and its domain functions for the preparation of unsaturated monosaccharides.

Brown algae are considered promising crops for the production of sustainable biofuels. However, the commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency toward polymannuronic acid (polyM), polyguluronic acid (polyG), and alginate sodium, with kcat of 39.42 ± 1.9s-1, 32.53 ± 0.88s-1, and 38.30 ± 2.12s-1, respectively. AlyPL17 showed maximum activity at 45°C and pH 9.0. The domain truncation did not change the optimal temperature and optimal pH, but greatly reduced the activity. In addition, AlyPL17 degrades alginate through the cooperative action of two structural domains in an exolytic mode. The minimal degradation substrate of AlyPL17 is a disaccharide. Furthermore, AlyPL17 and AlyPL6 can synergistically degrade alginate to prepare unsaturated monosaccharides that can be converted to 4-deoxy-L-erythron-5-hexoseuloseuronate acid (DEH). DEH is reduced to KDG by DEH reductase (Sdr), which enters the Entner-Doudoroff (ED) pathway as a common metabolite and is converted to bioethanol. KEY POINTS: • Biochemical characterization of alginate lyase from Pedobacter hainanensis NJ-02 and its truncated form. • Degradation patterns of AlyPL17 and the role of its domains in product distribution and mode of action. • Potential of synergistic degradation system for efficient preparation of unsaturated monosaccharides.

Microbial survival mechanisms within serpentinizing Mariana forearc sediments.

Marine deep subsurface sediment is often a microbial environment under energy-limited conditions. However, microbial life has been found to persist and even thrive in deep subsurface environments. The Mariana forearc represents an ideal location for determining how microbial life can withstand extreme conditions including pH 10-12.5 and depleted nutrients. The International Ocean Discovery Program Expedition 366 to the Mariana Convergent Margin sampled three serpentinizing seamounts located along the Mariana forearc chain with elevated concentrations of methane, hydrogen, and sulfide. Across all three seamount summits, the most abundant transcripts were for cellular maintenance such as cell wall and membrane repair, and the most abundant metabolic pathways were the Entner-Doudoroff pathway and tricarboxylic acid cycle. At flank samples, sulfur cycling involving taurine assimilation dominated the metatranscriptomes. The in situ activity of these pathways was supported by the detection of their metabolic intermediates. All samples had transcripts from all three domains of Bacteria, Archaea, and Eukarya, dominated by Burkholderiales, Deinococcales, and Pseudomonales, as well as the fungal group Opisthokonta. All samples contained transcripts for aerobic methane oxidation (pmoABC) and denitrification (nirKS). The Mariana forearc microbial communities show activity not only consistent with basic survival mechanisms, but also coupled metabolic reactions.

Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum

l-Homoserine is an important platform compound that is widely used to produce many valuable bio-based products, but production of l-homoserine in Corynebacterium glutamicum remains low. In this study, an efficient l-homoserine-producing strain was constructed. Native pentose phosphate pathway (PPP) was enhanced and heterologous Entner-Doudoroff (ED) pathway was carefully introduced into l-homoserine-producing strain, which increased the l-homoserine titer. Coexpression of NADH-dependent aspartate-4-semialdehyde dehydrogenase and aspartate dehydrogenase could increase the titer from 11.3 to 13.3 g/L. Next, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD) was coexpressed with that of NAD+-dependent (NAD-GPD) to construct dual-channel glycolysis for balance of intracellular cofactors, which increased the l-homoserine titer by 48.6 % to 16.8 g/L. Finally, engineered strain Cg18-1 accumulated 63.5 g/L l-homoserine after 96 h in a 5 L bioreactor, the highest titer reported to date for C. glutamicum. This dual-channel glycolysis strategy provides a reference for automatic cofactor regulation to promote efficient biosynthesis of other target products.