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Microbes can be engineered to produce next-generation biofuels; however, the accumulation of toxic biofuels can limit yields. Previous studies have shown that efflux pumps can increase biofuel tolerance and improve production. Here, we asked whether expressing multiple pumps in combination could further increase biofuel tolerance. Pump overexpression inhibits cell growth, suggesting a trade-off between biofuel and pump toxicity. With multiple pumps, it is unclear how the fitness landscape is impacted. To address this, we measured tolerance of Escherichia coli to the biojet fuel precursor α-pinene in one-pump and two-pump strains. To support our experiments, we developed a mathematical model describing toxicity due to biofuel and overexpression of pumps. We found that data from one-pump strains can accurately predict the performance of two-pump strains. This result suggests that it may be possible to dramatically reduce the number of experiments required for characterizing the effects of combined biofuel tolerance mechanisms.

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The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

The identification of nicotinamide-N1-oxide as a metabolite in the urine of a schizophrenic patient prompted a study of the relative metabolism of nicotinic acid and nicotinamide in mental patients and healthy volunteers. Metabolites quantified included N1-methyl-2-pyridone-5-carboxamide, N1-methyl-4-pyridone-3-carboxamide, N1-methylnicotinamide, nicotinuric acid, and nicotinamide-N1-oxide. More of most of these metabolites evidently was excreted after nicotinamide ingestion than after nicotinic acid. At the highest doses (3000 mg/day), the relative proportions of these metabolites in the urine were changed. There were only slight difference between healthy individuals and mental patients in the quantities of metabolites excreted, and no statistically significant trends were noted.

Despite the absence of phenotypic manifestations in alternating generations characteristic of X-linked disorders, a thesis is presented that a major type of Kinsey grades 5 and 6 male homosexuality is determined by a gene in the Xq28 region. A total of 133 families in 78 kinshps of male and female homosexual probands, in addition to 116 families (including those of 40 famous homosexuals) from the literature, revealed an unbalanced secondary sex ratio in the maternal generation of male, but not of female, homosexuals. On the maternal side, in this study, the ratio of all uncles to all aunts of 90 males homosexuals was 132/209, chi 2 = 8.52, p = 0.004. On the maternal side for the total of all sources, the ratio of uncles to aunts of male homosexuals was 241/367, chi 2 = 13.20; p < 0.0001. The male/female ratio of the total number of maternal sibships bearing homosexuals (310/628: 0.491) was a measure of fetal wastage of the mothers' male sibs; 49%. This ratio was very close to that of the total number of children born to fathers affected with any one of nine Xq28-linked male semilethal conditions (255/508: ratio 0.556); for the difference between the two populations chi 2 = 0.859, p = 0.354. The male/female ratio of the total number of children born to female carriers of any one of these same conditions (1,232/1,062: ratio 1.16), chi 2 = 13.8 p < or = 0.0001, is close to that of the total number of children in homosexual sibships: 511/413, chi 2 = 10.4, p = 0.005. Between the number of children born to Xq28 mothers and to those born of mothers of homosexuals chi 2 = 0.581, p = 0.446. One may readily surmise that the maternal influence so often related to homosexuality may lie in the mother being a genetic carrier, with traits thereto associated. In this study, 65% of the mothers of homosexuals had no or only one live-born brother. Additional support for a genetic hypothesis is found in the occurrence of multiple instances--almost exclusively among maternal relatives--of infertility, spontaneous abortions, miscarriages, stillbirths, remaining single past age 30, and suicide. Of 109 male and 43 female homosexual index cases in the present series there were 6 instances of brother/sister homosexual sibships.(ABSTRACT TRUNCATED AT 400 WORDS)