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Enquist-Newman M.,Bio Architecture Laboratory Inc. | Faust A.M.E.,Bio Architecture Laboratory Inc. | Bravo D.D.,Bio Architecture Laboratory Inc. | Santos C.N.S.,Bio Architecture Laboratory Inc. | And 35 more authors.
Nature | Year: 2014

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-l-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l-1) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks. © 2014 Macmillan Publishers Limited. Source

Santos C.N.S.,Manus Biosynthesis, Inc. | Regitsky D.D.,Calysta Energy Inc. | Yoshikuni Y.,Bio Architecture Laboratory Inc.
Nature Communications | Year: 2013

Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable instalment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a ∼40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (∼330%) and productivity (∼1,200%) after 50 generations. Source

Au-Yeung P.H.,Dow Chemical Company | Resnick S.M.,Dow Chemical Company | Resnick S.M.,Calysta Energy Inc. | Witt P.M.,Dow Chemical Company | And 3 more authors.
AIChE Journal | Year: 2013

A novel horizontal reactive distillation apparatus and a new overall process scheme are proposed for continuous multicomponent chiral resolution via reversible enantioselective acylation of a chiral (racemic) substrate by a chiral (racemic) acyl donor. The process enables simultaneous production of up to four enantiomers with enhanced chiral purity. Kinetic studies, miniplant experiments, and process simulation results are described for a model lipase-catalyzed reaction: (R)-enantioselective transesterification of (R,S)-1-n-butoxy-2-propanol with (R,S)-1-methoxy-2-acetoxypropane to produce (R)-1-n-butoxy-2-acetoxypropane, (R)-1-methoxy-2-propanol, and the two unreacted (S)-enantiomers of the (R,S)-reagents. A horizontal, compartmentalized reactive distillation vessel is specified instead of a conventional reactive distillation column to provide longer liquid-phase residence time needed for adequate conversion. Low vapor-traffic pressure drop allows operation under vacuum at reduced temperatures for good enzyme stability and enantioselectivity. The general technology has potential as a means to producing a wide range of chiral synthons used in asymmetric syntheses of chiral pharmaceuticals and other biologically active products. © 2013 American Institute of Chemical Engineers. Source

Silverman J.,Calysta Energy Inc.
Fuels and Petrochemicals Division 2014 - Core Programming Area at the 2014 AIChE Spring Meeting and 10th Global Congress on Process Safety | Year: 2014

Methane, from a variety of sources including natural gas, represents an abundant domestic resource. Chemical approaches developing gas-to-liquids (GTL) technology to improve the use of methane as a fuel have met with only limited success to date despite significant investment. In contrast, little effort has been expended to deploy modern bioengineering approaches towards GTL process development. Several limitations, most notably the cost of sugar feedstocks, have prevented the economical production of biofuels using microbial systems. Exploiting inexpensive, domestically abundant carbon feedstocks such as methane represents an attractive strategy towards economically sustainable biofuel production Important progress has recently been made toward engineering a number of phototrophic and fermentative microorganisms for the production of fuels and chemicals. Several limitations, most notably the ever-increasing cost of sugar feedstocks, currently prevent the economical production of fuels from microbial systems. Exploiting methane, an inexpensive, domestically abundant carbon feedstock, represents an attractive strategy towards economically sustainable production of next generation transportation fuels. Calysta Energy has developed a genetic engineering platform for host organisms (methanotrophs) capable of metabolizing methane to a variety of biofuels and biochemicals. The genetic tools, together with innovative fermentation and bioprocess approaches, enable the rapid implementation of well-characterized pathways to utilize natural gas as a biological feedstock instead of sugar. Source

Seo J.-S.,University of Utah | Lee S.,University of Utah | Lee S.,Calysta Energy Inc. | Poulter C.D.,University of Utah
Journal of the American Chemical Society | Year: 2013

Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies. © 2013 American Chemical Society. Source

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