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The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains ofZygnema circumcarinatumand one strain ofZ. cylindricum) and generated chromosome-scale assemblies for all strains of the emerging model systemZ. circumcarinatum. Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.HIGHLIGHTSGenomes of four filamentous algae (Zygnema) sisters to land plantsZygnemaare rich in genes for multicellular growth and environmental acclimation: signaling, lipid modification, and transportCell wall innovations: diversification of hexameric rosette cellulose synthase in ZygnematophyceaeCo-expression networks reveal conserved modules for balancing growth and acclimation

The complete chloroplast and mitochondrial genomes of Charophyta have shed new light on the land plant terrestrialization. Here, we reported the organellar genomes of the Zygnema circumcarinatum strain UTEX 1559, and a comparative genomics investigation of 33 plastomes and 18 mitogenomes of Chlorophyta, Charophyta (including UTEX 1559 and its conspecific relative SAG 698-1a), and Embryophyta. Gene presence/absence was determined across these plastomes and mitogenomes. A comparison between the plastomes of UTEX 1559 (157,548 bp) and SAG 698-1a (165,372 bp) revealed very similar gene contents, but substantial genome rearrangements. Surprisingly, the two plastomes share only 85.69% nucleotide sequence identity. The UTEX 1559 mitogenome size is 215,954 bp, the largest among all sequenced Charophyta. Interestingly, this large mitogenome contains a 50 kb region without homology to any other organellar genomes, which is flanked by two 86 bp direct repeats and contains 15 open reading frames (ORFs). These ORFs have significant homology to proteins from bacteria and plants with functions such as primase, RNA polymerase and DNA polymerase. We conclude that (i) the previously published SAG 698-1a plastome is likely from a different Zygnema species, and (ii) the 50 kb region in the UTEX 1559 mitogenome might be recently acquired as a mobile element. © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

An endogenous homoethanol pathway (glucose/1.2 xylose => 2 pyruvate => 2 ethanol) was previously engineered in Escherichia coli SZ410 via eliminating acid-producing pathways and anaerobic expression of the pyruvate dehydrogenase complex (aceEF-lpd operon). This ethanologenic derivative was subsequently engineered through adaptive evolution and partial deletion of the RNase G, resulting in an improved strain of E. coli RM10 for ethanol production using C6 and C5 sugars. Nevertheless, compared to the ethanol tolerance and/or ethanol titer achieved by industrial yeast, further incremental improvement of RM10 was needed for ethanol production using cellulosic biomass derived C6 and C5 sugars. In this study, the role of aldB gene (encoding for acetaldehyde dehydrogenase, AldB, which oxidizes acetaldehyde to acetic acid) was evaluated for ethanol/acetaldehyde tolerance and xylose fermentation by RM10. Deletion of aldB gene decreased ethanol tolerance, fermentative cell growth and ethanol production from xylose; while overexpression of aldB gene improved fermentative cell growth, and increased ethanol production from xylose. The improvement is likely attributed to preventing acetaldehyde accumulation (a toxic intermediate of homoethanol pathway) via AldB catalyzed oxidation.

The use of microalgae as feedstocks for biodiesel is potentially limited by climatic conditions with low light and temperature levels. Monoraphidium sp. Dek19 was identified by 18S rRNA gene sequencing. This is a species indigenous to the upper Midwestern USA which grows to high densities in wastewater with bioremediation resulting in a reduction of nitrate and phosphate levels. Mesocosm pool cultures (171L) were used to characterize growth of this species in larger volumes than typical of previous lab-based studies. The alga was shown to be cold-tolerant and to grow to workable density within 15 days at relatively low light intensities in sterilized treated effluent. It was harvested by FeCl3 flocculation and filtration, and lipid content was measured at 26% of dry weight. Pool cultures yielded enough biomass to extract lipids and transesterify them to biodiesel for testing in a stationary engine. GC analysis showed FAMEs produced from Monoraphidium sp. to have a similar fatty acid profile to soybean oil. Engine testing of this algal biodiesel in blends with petrodiesel showed a significant reduction in NOx emissions. The results of this study indicate in general that searching for species of algae adapted to local environments is a good strategy for developing biodiesel feedstocks, and specifically that Monoraphidium sp. Dek19 represents a species isolate that could be used to produce this fuel economically using wastewater in Northern locations with cool climates.

The D-lactic acid producing strain, Escherichia coli HBUT-D, was reengineered for L(+)-lactic acid fermentation by replacing the D-lactate dehydrogenase gene (ldhA) with an L(+)-lactate dehydrogenase gene (ldhL) from Pedicoccus acidilactici, followed by adaptive evolution in sucrose. The resulting strain, WYZ-L, has enhanced expression of the sucrose operon (cscA and cscKB). In 100 g L(-1) of sucrose fermentation using mineral salt medium, WYZ-L produced 97 g L(-1) of l(+)-lactic acid, with a yield of 90%, a maximum productivity of 3.17 g L(-1)h(-1) and an optical purity of greater than 99%. In fermentations using sugarcane molasses and corn steep liquor without additional nutrients, WYZ-L produced 75 g L(-1) of l(+)-lactic acid, with a yield of 85%, a maximum productivity of 1.18 g L(-1)h(-1), and greater than 99% optical purity. These results demonstrated that WYZ-L has the potential to use waste molasses and corn steep liquor as a resource for L(+)-lactic acid fermentation.

To establish metabolic context for radiation sensitivity by measuring autophagic flux in two different glioblastoma (GBM) cell lines. Clonogenic survival curve analysis of U87 or U251 cells exposed to γ radiation, fast neutrons, a mixed energy neutron beam (METNB) or Auger electrons from a gadolinium neutron capture (GdNC) reaction suggested other factors, beyond a defective DNA damage response, contribute to cell death of U251 cells. Altered tumor metabolism (autophagy) was hypothesized as a factor in U251 cells' clonogenic response. Each of the four different radiation modalities induced an increase in the number of autophagosomes in both U87 and U251 cells. Changes in the number of autophagosomes can be explained by either induction of autophagy or alterations in autophagic flux so autophagic flux was assayed by p62 immunoblotting or in engineered GBM cells that stably express an autophagy marker protein, LC3B-eGFP-mCherry. Perturbations in later stages of autophagy in U251 cells corresponded with radiation sensitivity of U251 cells irradiated with 10 Gy γ rays. Establishment of altered autophagic flux is a useful biomarker for metabolic stress and provided metabolic context for radiation sensitization to 10 Gy γ rays. These results provide strong evidence for the usefulness of managing tumor cell metabolism as a tool for the enhancement of radiation therapy.

The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA , which has been found to regulate secondary metabolism gene clusters.

The mitochondrial genome of the peronosporomycete water mold Saprolegnia ferax has been characterized as a 46 930 bp circle containing an 8618 bp large inverted repeat (LIR). Eighteen reading frames encode identified subunits of respiratory complexes I, III, IV and V; 16 encode polypeptides of small and large mitoribosome subunits; and one encodes a subunit of the sec-independent protein translocation pathway. Of four additional putative reading frames three are homologues of those found in the related Phytophthora infestans genome. Protein encoding loci in the tightly compacted genome typically are arranged in operon-like clusters including three abutting and two overlapping pairs of reading frames. Translational RNAs include the mitochondrial small and large subunit rRNAs and 25 tRNA species. No tRNAs are encoded to enable translation of any threonine or the arginine CGR codons. The LIR separates the molecule into 19 274 bp large and 10 420 bp small single copy regions, and it encodes intact duplicate copies of four reading frames encoding known proteins, both rRNAs, and five tRNAs. Partial 3' sequences of three additional reading frames are duplicated at single copy sequence junctions. Active recombination between LIR elements generates two distinctive gene orders and uses the duplicated 3' sequences to maintain intact copies of the partially duplicated loci.

A two‐tiered, nested molecular phylogenetic study of panicoid grasses to explore character state transitions between the C3 and C4 adaptive syndromes is presented. A broad survey of 92 panicoid species was sampled for the grass‐specific insert sequence in the chloroplast RNA polymerase locus (rpoC2), combining published and unpublished sequences. This portion of the study also included an intensive phylogenetic investigation of one clade of seven species that included Steinchisma hians, which is notable for exhibiting intermediacy between the C3 and C4 photosynthetic types. Both rpoC2 data and previously published sequences of the F subunit of an NADH‐dependent dehydrogenase were analyzed together for this small group. A rigorous phylogenetic investigation of S. hians and 13 other species of Panicoideae included in the broad survey was then performed with sequences of both rpoC2 and the externally transcribed spacer region of the nuclear ribosomal repeat. These 14 species were selected to maximize represe...

Secondary metabolism and development are linked in Aspergillus through the conserved regulatory velvet complex composed of VeA, VelB, and LaeA. The founding member of the velvet complex, VeA, shuttles between the cytoplasm and nucleus in response to alterations in light. Here we describe a new interaction partner of VeA identified through a reverse genetics screen looking for LaeA-like methyltransferases in Aspergillus nidulans. One of the putative LaeA-like methyltransferases identified, LlmF, is a negative regulator of sterigmatocystin production and sexual development. LlmF interacts directly with VeA and the repressive function of LlmF is mediated by influencing the localization of VeA, as over-expression of llmF decreases the nuclear to cytoplasmic ratio of VeA while deletion of llmF results in an increased nuclear accumulation of VeA. We show that the methyltransferase domain of LlmF is required for function; however, LlmF does not directly methylate VeA in vitro. This study identifies a new interaction partner for VeA and highlights the importance of cellular compartmentalization of VeA for regulation of development and secondary metabolism., Author Summary In recent years there has been increased interest in bioactive small molecules produced by filamentous fungi. Members of the genus Aspergillus are prolific producers of natural products such as penicillin, the cholesterol lowering drug lovastatin, in addition to several toxins, the most famous being aflatoxin. The genetic regulation of fungal natural products is coupled with developmental differentiation through a conserved protein complex termed the velvet complex. The founding member of the complex, velvet (VeA), is a light-regulated protein that shuttles between the cytoplasm and nucleus in response to illumination. Once in the nucleus, VeA interacts with the putative methyltransferase LaeA to positively regulate production of secondary metabolites and with VelB to induce sexual development. We have identified a new interaction partner of VeA that has sequence homology to LaeA. The putative LaeA-like methyltransferase LlmF controls the subcellular localization of VeA in response to light, thereby regulating the downstream outputs of secondary metabolism and development. While the mechanism of the velvet complex remains an enigma, our data suggest that manipulation of protein subcellular localization is an approach that can be used to control production of secondary metabolites.

Previously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobic expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). The resulting ethanol pathway involves glycolysis, PDH, and alcohol dehydrogenase (AdhE). The E. coli B-derived ethanologenic strain SZ420 was then further improved for ethanol tolerance (up to 40 g l−1 ethanol) through adaptive evolution. However, the resulting ethanol tolerant mutant, SZ470, was still unable to complete fermentation of 75 g l−1 xylose, even though the theoretical maximum ethanol titer would have been less than 40 g l−1 should the fermentation have reached completion. In this study, the cra (encoding for a catabolite repressor activator) and the HSR2 region of rng (encoding for RNase G) were deleted from SZ470 in order to improve xylose fermentation. Deletion of the HSR2 domain resulted in significantly increased mRNA levels (47-fold to 409-fold) of multiple glycolytic genes (pgi, tpiA, gapA, eno), as well as the engineered ethanol pathway genes (aceEF-lpd, adhE) and the transcriptional regulator Fnr (fnr). The higher adhE mRNA level resulted in increased AdhE activity (>twofold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. The increased enzymes would then enable the resulting strain, RM10, to achieve increased cell growth and complete fermentation of 75 g l−1 xylose with an 84% improved ethanol titer (35 g l−1), compared to that (19 g l−1) obtained by the parent, SZ470. However, deletion of cra resulted in a negative impact on cell growth and xylose fermentation, suggesting that Cra is important for long-term fermentative cell growth.

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B 12 , penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B 12 through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.

Received 21 March 2011/Accepted 25 May 2011 Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi. Nitric oxide (NO) is a signaling compound of great importance in biological systems (7, 10, 21). This molecule can also cause nitrosative stress which is potentially remediated by the widely distributed flavohemoglobins (FHbs) found in both eukaryotes and prokaryotes (4, 7). Extensive research on FHb proteins from bacteria and yeast revealed their structure, function, and mechanism of action (3, 7, 17). Earlier studies have shown that FHbs in organisms such as Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar steady-state NO dioxygenation kinetics (7). Through a dioxygenase-mediated reaction, FHbs, in the presence of molecular O 2, converts NO into nontoxic nitrate ions. FHb proteins contain a hemoglobin-like domain with a noncovalently bound heme B protein and a reductase domain with binding sites for FAD and NAD(P)H. It is known that fhb genes are activated by various agents, such as nitrate, nitrite, NO, and NO-releasing agents (5, 7, 8, 11, 20, 24). In aspergilli, the conversion of NO to NO 3 by FHbs, Fhb1 and Fhb2 in Aspergillus oryzae (30) and FhbA and FhbB in the filamentous fungus model Aspergillus nidulans, has been demonstrated (24). The present work involves the study of the role of FHbs and NO in fungal development and secondary metabolism. Fungal strains and growth conditions. Aspergillus nidulans Cib08 (biA1 yA2), CibA (biA1 yA2 fhbA::argB), CibB (biA1 yA2 fhbB::argB) (described in reference 24), and FGSCA4 were used in this study. The strains were cultured on glucose minimum medium (GMM) plus the appropriate supplements for the corresponding auxotrophic markers (13). Medium was supplemented with 1.5 mM diethylenetriamine-NoNoate (Sigma), a NO-releasing compound, after sterilization when indicated. Solid medium was prepared by adding 10 g/liter agar. Strains were stored as 30% glycerol stocks at 80°C. Morphological studies. Plates containing 25 ml of solid GMM plus the appropriate supplements were top agar inoculated with 5 10 6 spores per plate of medium. The cultures were wrapped and incubated at 37°C in the dark. Cores (8-mm diameter) were collected from each spread plate and examined for cleistothecium production. Ethanol (70%) was sprayed on the core surface to facilitate the visualization of cleistothecia under the microscope. The experiments included five repli

The mitochondrial genome of the peronosporomycete water mold Saprolegnia ferax has been characterized as a 46 930 bp circle containing an 8618 bp large inverted repeat (LIR). Eighteen reading frames encode identified subunits of respiratory complexes I, III, IV and V; 16 encode polypeptides of small and large mitoribosome subunits; and one encodes a subunit of the sec-independent protein translocation pathway. Of four additional putative reading frames three are homologues of those found in the related Phytophthora infestans genome. Protein encoding loci in the tightly compacted genome typically are arranged in operon-like clusters including three abutting and two overlapping pairs of reading frames. Translational RNAs include the mitochondrial small and large subunit rRNAs and 25 tRNA species. No tRNAs are encoded to enable translation of any threonine or the arginine CGR codons. The LIR separates the molecule into 19 274 bp large and 10 420 bp small single copy regions, and it encodes intact duplicate copies of four reading frames encoding known proteins, both rRNAs, and five tRNAs. Partial 3' sequences of three additional reading frames are duplicated at single copy sequence junctions. Active recombination between LIR elements generates two distinctive gene orders and uses the duplicated 3' sequences to maintain intact copies of the partially duplicated loci.

Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pls), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pls of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies.

DNA is believed to be the molecular target for the cytotoxic activities of platinum (Pt) anticancer drugs. We report here a class of platinum(II)- and platinum(IV)-pyrophosphato complexes that exhibit cytotoxicity comparable with and, in some cases, better than cisplatin in ovarian cell lines (A2780, A2780/C30, and CHO), yet they do not show any evidence of covalent binding to DNA. Moreover, some of these compounds are quite effective in cisplatin- and carboplatin-resistant cell line A2780/C30. The lack of DNA binding was demonstrated by the absence of a detectable Pt signal by atomic absorption spectroscopy using isolated DNA from human ovarian cells treated with a platinum(II)-pyrophosphato complex, ( trans -1,2-cyclohexanediamine)(dihydrogen pyrophosphato) platinum(II), (pyrodach-2) and from NMR experiments using a variety of nucleotides including single- and double-stranded DNA. Furthermore, pyrodach-2 exhibited reduced cellular accumulations compared with cisplatin in cisplatin- and carboplatin-resistant human ovarian cells, yet the IC 50 value for the pyrophosphato complex was much less than that of cisplatin. Moreover, unlike cisplatin, pyrodach-2 treated cells overexpressed fas and fas-related transcription factors and some proapoptotic genes such as Bak and Bax. Data presented in this report collectively indicate that pyrodach-2 follows different cytotoxic mechanisms than does cisplatin. Unlike cisplatin, pyrodach-2 does not undergo aquation during 1 week and is quite soluble and stable in aqueous solutions. Results presented in this article represent a clear paradigm shift not only in expanding the molecular targets for Pt anticancer drugs but also in strategic development for more effective anticancer drugs.

The velvet gene, veA, co-ordinates asexual and sexual development in the homothallic fungal species Aspergillus nidulans. Studies in Aspergillus parasiticus and Aspergillus fumigatus demonstrated that veA also regulates morphological differentiation in these species. Whether veA has the same role in morphogenesis in other fungal genera has not been investigated. In this work, we studied the role of the veA homologue, FvVE1, in the heterothallic fungus Fusarium verticillioides. Deletion of FvVE1 suppressed aerial hyphal growth and reduced colony surface hydrophobicity on solid media. In submerged cultures, FvVE1 deletion caused alterations in hyphal polarity, marked activation of conidiation and yeast-like growth. The latter was promoted by shaking to increase aeration of cultures. In addition, FvVE1 deletion markedly increased the ratio of macroconidia to microconidia. Supplementation of osmotic stabilizers restored the wild-type phenotype to deletion mutants, suggesting phenotypic alterations caused by FvVE1 deletion are related to cell wall defects. This is consistent with the hypersensitivity of FvVE1 deletion mutants to SDS and with the significant reduction in the mannoprotein content of mutants compared with the wild-type strain. However, no dramatic cell wall alterations were observed when mutants were examined by transmission electron microscopy. Our data strongly suggest that FvVE1 is important for cell wall integrity, cell surface hydrophobicity, hyphal polarity and conidiation pattern.

There are a number of aspects of lipid metabolism for which desired changes are approachable using biotechnology. One is the alteration of fatty acid composition of seed oils, and another is the change in the formation of fatty acid-derived flavor and aroma compounds. We have identified polypeptides associated with control of linolenate content in plant tissues using mutants and a chemical modulator of lipid composition. Work is in progress involving use of an ω-9 desaturase gene to decrease saturated fatty acid content of plant lipids. We have elucidated the role of specific lipoxygenase isozymes in the formation of C 6 aldehydes in soybean seed preparations and have produced transgenic plants with altered lipoxygenases. Finally, we have developed improved somatic embryogenesis systems for soybeans, facilitating genetic transformation and the study of the control of lipid biosynthesis and peroxidative metabolism.

To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. ‘KY 14’ by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5′-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3′ to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1–0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50–529% increase in C6-aldehyde production. The impact on C6-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C6-aldehydes than that of other well-characterized LOX isozymes.

A portion of the surface of detached Graptopetalum paraquayense E. Walther leaves can be used to assay small amounts of reagents in lanolin for their ability to induce shoots only at the site of application. The cytokinins benzyladenine, kinetin, and 6-(gamma,gamma-dimethylallylamino)purine (DMAAP) were tested, and DMAAP was most effective in bud induction at concentrations below 1%. The higher the hormone concentration, the sooner the appearance of leaf primordia and the higher the ultimate yield of buds. Leaves treated with DMAAP for 2 days developed buds as rapidly as those with longer treatments.

Background Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. Results The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over that achieved by AI05 (pAI02). Conclusions Increasing reducing power (NADH equivalent) output per glucose catabolized was achieved by anaerobic expression of both the pdh operon (pyruvate dehydrogenase) and the sdhCDAB-sucABCD operon, resulting in a strain capable of generating 10 NADH equivalent per glucose under anaerobic condition. The new E. coli strain AI21 (pAI02) achieved an actual 96 % conversion of xylose to xylitol (via synthetic respiration), and 6 xylitol (from xylose) per glucose catabolized (YRPG = 6, the highest known value). This strategy can be used to engineer microbial strains for the production of other reduced products from redox neutral sugars using glucose as a source of reducing power.