Coproduction of Phase-Separated Carotenoids and β-Farnesene as a Yeast Biomass Valorization Strategy.

Valorization, the process whereby waste materials are converted into more valuable products, is rarely practiced in industrial fermentation. We developed a model valorization system whereby that had previously been engineered to produce high concentrations (>100 g/L) of extracellular β-farnesene was further engineered to simultaneously produce intracellular carotenoids, both products being isoprenoids. Thus, a single fermentation generates two valuable products, namely, β-farnesene in the liquid phase and carotenoids in the solid biomass phase.

Do physical activity levels differ by number of children at home in women aged 25-44 in the general population?

Objectives: While physical activity is important for health, many women do not meet recommended levels, particularly mothers. The purpose of this study was to assess whether physical activity levels differ by number of children at home in women aged 25-44 in the general US population.

Methods: This cross-sectional analysis used 2017 Behavioral Risk Factor Surveillance System data for females aged 25-44 (N = 6266) from California, Colorado, New York, Texas, and Utah.

Rewriting yeast central carbon metabolism for industrial isoprenoid production.

A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (CH), a plant sesquiterpene with versatile industrial applications, is straightforward.

Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage).

Rapid and reliable DNA assembly via ligase cycling reaction.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs.

Comparative study on oxidative treatments of NAPL containing chlorinated ethanes and ethenes using hydrogen peroxide and persulfate in soils.

The goal of this study was to assess the oxidation of NAPL in soil, 30% of which were composed of chlorinated ethanes and ethenes, using catalyzed hydrogen peroxide (CHP), activated persulfate (AP), and H(2)O(2)-persulfate (HP) co-amendment systems. Citrate, a buffer and iron ligand, was amended to the treatment system to enhance oxidative treatment. Four activation/catalysis methods were employed: (1) oxidant only, (2) oxidant-citrate, (3) oxidant-iron(II), and (4) oxidant-citrate-iron(II).

Influence of vitamin B12 and cocultures on the growth of Dehalococcoides isolates in defined medium.

Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B(12), and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia.

Reductive dehalogenase gene expression as a biomarker for physiological activity of Dehalococcoides spp.

This study characterizes the transcriptional expression of the reductive dehalogenase (RDase)-encoding tceA and vcrA genes and evaluates their applicability as potential biological markers of Dehalococcoides activity. When Dehalococcoides ethenogenes 195 was provided with trichloroethene (TCE) as the electron acceptor, the expression of the tceA gene increased by 90-fold relative to that in cells starved of chlorinated ethenes, demonstrating that tceA gene expression is indicative of the active physiological state of this strain. In a Dehalococcoides-containing enrichment culture that contains both the tceA and vcrA genes, the tceA gene was up-regulated in response to TCE and cis-1,2-dichloroethene (cDCE) exposure, while the vcrA gene was up-regulated in response to TCE, cDCE, and vinyl chloride (VC).

Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes.

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard.

Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities.

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains.

Transcriptional expression of the tceA gene in a Dehalococcoides-containing microbial enrichment.

Dynamic changes in the transcriptional expression of the tceA gene, which encodes a trichloroethene reductive dehalogenase, were characterized in a Dehalococcoides-containing microbial enrichment culture. Expression was quantified by real-time PCR as the number of tceA transcripts per tceA gene. Expression of tceA increased 40-fold after chlorinated ethene-starved cells were exposed to trichloroethene (TCE), cis-dichloroethene (DCE), or 1,1-DCE but did not increase after exposure to tetrachloroethene or vinyl chloride.

An internal reference technique for accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive dehalogenase gene.

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (ref mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding tceA gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment.

Structure of reaction intermediates formed during Saccharomyces cerevisiae Rad51-catalyzed strand transfer.

The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a double-stranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates.

C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis.

Chromosome segregation and X-chromosome gene regulation in Caenorhabditis elegans share the component MIX-1, a mitotic protein that also represses X-linked genes during dosage compensation. MIX-1 achieves its dual roles through interactions with different protein partners. To repress gene expression, MIX-1 acts in an X-chromosome complex that resembles the mitotic condensin complex yet lacks chromosome segregation function.