Curt R. Fischer

Selection and optimization of microbial hosts for biofuels production

Currently, the predominant microbially produced biofuel is starch- or sugar-derived ethanol. However, ethanol is not an ideal fuel molecule, and lignocellulosic feedstocks are considerably more abundant than both starch and sugar. Thus, many improvements in both the feedstock and the fuel have been proposed. In this paper, we examine the prospects for bioproduction of four second-generation biofuels (n-butanol, 2-butanol, terpenoids, or higher lipids) from four feedstocks (sugars and starches, lignocellulosics, syngas, and atmospheric carbon dioxide). The principal obstacle to commercial production of these fuels is that microbial catalysts of robust yields, productivities, and titers have yet to be developed. Suitable microbial hosts for biofuel production must tolerate process stresses such as end-product toxicity and tolerance to fermentation inhibitors in order to achieve high yields and titers. We tested seven fast-growing host organisms for tolerance to production stresses, and discuss several metabolic engineering strategies for the improvement of biofuels production.

Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae

ABSTRACT The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

ABSTRACT The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h−1 and by 3 orders of magnitude at a lower dilution rate (0.327 h−1). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h−1 and persisted until the end of the experiment (∼200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations

Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux.

Identifying Functionally Important Mutations from Phenotypically Diverse Sequence Data

ABSTRACT Here we present a simple statistical method to determine the phenotypic contribution of a single mutation from libraries of mutants with diverse phenotypes in which each mutant contains a multitude of mutations. The central premise of this method is that, given M phenotypic classes, mutations that do not affect the phenotype should partition among the M classes according to a multinomial distribution. Deviations from this distribution are indicative of a link between specific mutations and phenotypes. We suggest that this method will aid the engineering of functional nucleic acids, proteins, and other biomolecules by uncovering target sites for rational mutagenesis. As a proof of the principle, we show how the method can be used to deduce the individual effects of mutations in a set of 69 PL-λ promoter variants. Each of these promoters was generated by error-prone PCR and incorporated numerous mutations. The activity of the promoters was assayed using flow cytometry to measure the fluorescence of a green fluorescent protein reporter gene. Our analysis of the sequences of these mutants revealed seven positions having a statistically significant correlation with promoter activity. Using site-directed mutagenesis, we constructed point mutations for several sites, both statistically significant and insignificant, and combinations of these sites. Our results show that the statistical method correctly elucidated the phenotypic manifestations of these mutations. We suggest that this method may be useful for expediting directed evolution experiments by allowing both desired and undesired mutations to be identified and incorporated between rounds of mutagenesis.

The intelligent design of evolution

Mol Syst Biol. 2: 2006.0020 The debate between intelligent design and evolution in education may still rage in school boards and classrooms, but intelligent design is making headway in the laboratory. In this case, though, the designer turned out to be just some clever scientist. A recent paper in Nature (Yoshikuni et al , 2006) presented the iterative evolution of highly specific catalysts from a promiscuous wild‐type enzyme via what the authors refer to as designed divergent evolution. The paper investigated whether catalytic functionality could be rationally engineered into a protein, without recourse to the high‐throughput screening techniques necessary for directed evolution. Yoshikuni et al (2006) started with a terpene synthase enzyme, γ‐humulene synthase, that is promiscuous not in its substrate specificity but in its product selectivity—it catalyzes the formation of 52 different sesquiterpene products from one single substrate, farnesyl diphosphate. (Sesquiterpenes naturally occur in a variety of plants, and their derivatives are used in applications ranging from chemical feedstocks to antifungal compounds.) The predominant product for the wild‐type enzyme is γ‐humulene, but Yoshikuni et al designed seven mutant variants with improved selectivities for eight of the products. How did they do it? Using prior knowledge of the active sites in the terpene synthase family and the crystal structure of another terpene synthase, the authors identified a set of 19 candidate ‘plasticity’ residues in γ‐humulene synthase that lie along the contour of the active site. …