Biological Engineering Faculty Publications Copyright (c) 2013 Utah State University All rights reserved. http://digitalcommons.usu.edu/bioeng_facpub Recent documents in Biological Engineering Faculty Publications en-us Wed, 22 May 2013 01:43:26 PDT 3600 Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases http://digitalcommons.usu.edu/bioeng_facpub/71 http://digitalcommons.usu.edu/bioeng_facpub/71 Mon, 20 May 2013 12:10:58 PDT BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins.

]]> Dayu Yu et al. Engineered Production of Fungal Anticancer Cyclooligomer Depsipeptides in Saccharo-Myces Cerevisiae http://digitalcommons.usu.edu/bioeng_facpub/70 http://digitalcommons.usu.edu/bioeng_facpub/70 Tue, 23 Apr 2013 14:50:42 PDT Two fungal cyclooligomer depsipeptide synthetases (CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC 7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.82±1.41 mg/l) and bassianolide (21.74±0.08 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding D-hydroxyisovaleric acid (D-Hiv) and the corresponding L-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of D-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins. The total titer of beauvericin and its congeners (beauvericins A, B and C) was increased to 61.73±2.96 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC 7159. Supplement of L-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.76±2.12 mg/l. Using this yeast system, we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.

]]> Dayu Yu et al. Rational Reprogramming of Fungal Polyketide First Ring Cyclization http://digitalcommons.usu.edu/bioeng_facpub/69 http://digitalcommons.usu.edu/bioeng_facpub/69 Thu, 28 Feb 2013 14:31:19 PST Resorcylic acid lactones (RAL) and dihydroxyphenylacetic acid lactones (DAL) represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS (hrPKS) whose product is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates towards defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the DAL dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal versus the bacterial folding mode difference is portable upon creating hybrid enzymes, and structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations, and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first ring cyclizations will facilitate efforts towards the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

]]> Yuquan Xu et al. Flexible graphene bio-nanosensor for lactate http://digitalcommons.usu.edu/bioeng_facpub/68 http://digitalcommons.usu.edu/bioeng_facpub/68 Thu, 07 Feb 2013 11:25:29 PST The development of a flexible nanosensor for detecting lactate could expand opportunities for using graphene, both in fundamental studies for a variety of device platforms and in practical applications. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with ultrasensitive sensing capabilities. Lactic acid is important for clinical analysis, sports medicine, and the food industry. Recently, wearable and flexible bioelectronics on plastics have attracted great interest for healthcare, sports and defense applications due to their advantages of being light-weight, bendable, or stretchable. Here, we demonstrate for the first time the development of a flexible graphene-based bio-nanosensor to detect lactate. Our results show that flexible lactate biosensors can be fabricated on a variety of plastic substrates. The sensor can detect lactate sensitively from 0.08 μM to 20 μM with a fast steady-state measuring time of 2 s. The sensor can also detect lactate under different mechanical bending conditions, the sensor response decreased as the bending angle and number of bending repetitions increased. We anticipate that these results could open exciting opportunities for fundamental studies of flexible graphene bioelectronics by using other bioreceptors, as well as a variety of wearable, implantable, real-time, or on-site applications in fields ranging from clinical analysis to defense.

]]> P. Labroo et al. An Indigoidine Biosynthetic Gene Cluster from Streptomyces Chromofuscus ATCC 49982 Contains an Unusual IndB Homologue http://digitalcommons.usu.edu/bioeng_facpub/67 http://digitalcommons.usu.edu/bioeng_facpub/67 Fri, 09 Nov 2012 09:07:06 PST A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.

]]> Dayu Yu et al. Bioassays- Procedures and Results http://digitalcommons.usu.edu/bioeng_facpub/66 http://digitalcommons.usu.edu/bioeng_facpub/66 Tue, 25 May 2010 11:22:23 PDT A. F. Maciorowski et al. Bioassays- Procedures and Results http://digitalcommons.usu.edu/bioeng_facpub/64 http://digitalcommons.usu.edu/bioeng_facpub/64 Tue, 25 May 2010 11:22:22 PDT A. F. Maciorowski et al. Fate of Polynuclear Aromatic Compounds (PNAs) in Soil-Plant Systems http://digitalcommons.usu.edu/bioeng_facpub/65 http://digitalcommons.usu.edu/bioeng_facpub/65 Tue, 25 May 2010 11:22:22 PDT Ronald C. Sims et al. Reovirus Removal and Inactivation by Slow Rate Sand Filtration http://digitalcommons.usu.edu/bioeng_facpub/62 http://digitalcommons.usu.edu/bioeng_facpub/62 Tue, 25 May 2010 11:22:21 PDT L. K. McConnell et al. Human Health Effects Assays http://digitalcommons.usu.edu/bioeng_facpub/63 http://digitalcommons.usu.edu/bioeng_facpub/63 Tue, 25 May 2010 11:22:21 PDT Ronald C. Sims et al. Mobility of Organics in Land Treatment Systems http://digitalcommons.usu.edu/bioeng_facpub/60 http://digitalcommons.usu.edu/bioeng_facpub/60 Tue, 25 May 2010 11:22:20 PDT R. J. Mahmood et al. The Application and Effectiveness of Slow Sand Filtration in the United States http://digitalcommons.usu.edu/bioeng_facpub/61 http://digitalcommons.usu.edu/bioeng_facpub/61 Tue, 25 May 2010 11:22:20 PDT L. A. Slezak et al. Human Health Effects Assays http://digitalcommons.usu.edu/bioeng_facpub/59 http://digitalcommons.usu.edu/bioeng_facpub/59 Tue, 25 May 2010 11:22:19 PDT Ronald C. Sims et al. The Effect of Temperature on Polycyclic Aromatic Hydrocarbon Persistence in an Unacclimated Agricultural Soil http://digitalcommons.usu.edu/bioeng_facpub/57 http://digitalcommons.usu.edu/bioeng_facpub/57 Tue, 25 May 2010 11:22:18 PDT M. P. Coover et al. Review and Evaluation of Current Design and Management Practices for Land Treatment Units Receiving Petroleum Wastes http://digitalcommons.usu.edu/bioeng_facpub/58 http://digitalcommons.usu.edu/bioeng_facpub/58 Tue, 25 May 2010 11:22:18 PDT J. P. Martin et al. Extraction of Polycyclic Aromatic Hydrocarbons from Spiked Soil http://digitalcommons.usu.edu/bioeng_facpub/55 http://digitalcommons.usu.edu/bioeng_facpub/55 Tue, 25 May 2010 11:22:17 PDT M. P. Coover et al. Human Health Effects Assays http://digitalcommons.usu.edu/bioeng_facpub/56 http://digitalcommons.usu.edu/bioeng_facpub/56 Tue, 25 May 2010 11:22:17 PDT Ronald C. Sims et al. Evaluation of Mobility of Pesticides in Soil Systems Using U.S. EPA Methodology http://digitalcommons.usu.edu/bioeng_facpub/53 http://digitalcommons.usu.edu/bioeng_facpub/53 Tue, 25 May 2010 11:22:16 PDT J. E. McLean et al. The Rate of Benzo[a]pyrene Apparent Loss in a Natural and Manure Amended Clay Loam Soil http://digitalcommons.usu.edu/bioeng_facpub/54 http://digitalcommons.usu.edu/bioeng_facpub/54 Tue, 25 May 2010 11:22:16 PDT M. P. Coover et al. A Mathematical Model for the Fate of Hazardous Substances in Soil: Model Description and Experimental Results http://digitalcommons.usu.edu/bioeng_facpub/52 http://digitalcommons.usu.edu/bioeng_facpub/52 Tue, 25 May 2010 11:22:15 PDT W. Greeney et al.