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Biotechnology and Bioengineering

Katia Tarasava, Rongming Liu, Andrew Garst, Ryan T Gill
Optimization of metabolic flux is a difficult and time-consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial-and-error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I-E CRISPR-Cas system and an iterative cloning strategy for construction of guide RNA arrays...
March 14, 2018: Biotechnology and Bioengineering
Oz Habiby, Omri Nahor, Alvaro Israel, Alexander Liberzon, Alexander Golberg
Marine macroalgae are a potential feedstock for biorefineries that can reduce dependence on fossil fuels and contribute to bioeconomy. New knowledge and technologies for efficient conversion of solar energy into macroalgae biomass are needed to increase biomass yields and energy conversion efficiency. In this work, we show that the green macroalgae from Ulva sp. can grow under the pulsed light in a photobioreactor with higher exergy conversion efficiency in comparison to cultivation under constant light with the same intensity...
March 14, 2018: Biotechnology and Bioengineering
Wei Xiong, Luis H Reyes, William E Michener, Pin-Ching Maness, Katherine J Chou
Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates...
March 14, 2018: Biotechnology and Bioengineering
Gert Peters, Brecht De Paepe, Lien De Wannemaeker, Dries Duchi, Jo Maertens, Jeroen Lammertyn, Marjan De Mey
Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high-throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences with- out the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental...
March 13, 2018: Biotechnology and Bioengineering
Stanley Chung, Jun Tian, Zhijun Tan, Jie Chen, Jongchan Lee, Michael Borys, Zheng Jian Li
Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts towards developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants...
March 13, 2018: Biotechnology and Bioengineering
Javier Viña-Gonzalez, Katarina Elbl, Xavier Ponte, Francisco Valero, Miguel Alcalde
Aryl-alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H2 O2 . Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure-guided evolution...
March 13, 2018: Biotechnology and Bioengineering
Fayin Zhu, Yuanshan Wang, Ka-Yiu San, George N Bennett
It is of great economic interest to produce succinate from low-grade carbon sources, which can enhance the competitiveness of the biological route. In this study, succinate producer E. coli CT550/pHL413KF1 was further engineered to efficiently use the mixed sugars from non-food based soybean hydrolysate to produce succinate under anaerobic conditions. Since many common E. coli strains fail to use galactose anaerobically even if they can use it aerobically, the glucose and galactose related sugar transporters were deactivated individually and evaluated...
March 6, 2018: Biotechnology and Bioengineering
Zhengxiong Zhou, Qing Li, Hao Huang, Hao Wang, Yang Wang, Guocheng Du, Jian Chen, Zhen Kang
Chondroitin sulfate has been widely used in both medical and clinical applications. Commercial chondroitin sulfate has been mainly acquired from animal tissue extraction. Here we report a new two-step biological strategy for producing chondroitin sulfate A and chondroitin sulfate C. First, the chondroitin biosynthesis pathway in a recombinant Bacillus subtilis strain using sucrose as carbon source was systematically optimized and the titer of chondroitin was significantly enhanced to 7.15 g/L. Then, specific sulfation transformation systems were successfully constructed and optimized by combining the purified aryl sulfotransferase IV (ASST IV), chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST)...
February 27, 2018: Biotechnology and Bioengineering
Samarthya Bhagia, Jorge F S Ferreira, Ninad Kothari, Angelica Nunez, Xuan Liu, Nildo da Silva Dias, Donald L Suarez, Rajeev Kumar, Charles E Wyman
Currently, major biofuel crops are also food crops that demand fertile soils and good-quality water. Jerusalem artichoke (Helianthus tuberosus, Asteraceae) produces high tonnage of tubers that are rich in sugars, mainly in the form of inulin. In this study, plants of the cultivar 'White Fuseau' grown under five salinity levels were evaluated for tuber yield. Results indicated that this cultivar is moderately salt-tolerant if the goal is tuber production. Hydraulic pressings of the tubers produced juice that contained 15% (wet weight) or 55% (dry weight) free sugars, with 70% of these in the form of inulin and the rest as fructose, sucrose, and glucose...
February 24, 2018: Biotechnology and Bioengineering
Wen-Fang Wang, Han Xiao, Jian-Jiang Zhong
Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of 72 candidates of cytochrome P450 monooxygenase (CYP450) genes from G...
February 24, 2018: Biotechnology and Bioengineering
S Kushwaha, A K Marcus, B E Rittmann
In situ bioreduction of soluble hexavalent uranium [U(VI)] to insoluble U(IV) (as UO2 ) has been proposed as a means of preventing U migration in the groundwater. This work focuses on the bioreduction of U(VI) and precipitation of U(IV). It uses anaerobic batch reactors with Desulfovibrio vulgaris, a well-known sulfate, iron, and U(VI) reducer, growing on lactate as the electron donor, in the absence of sulfate, and with a 30-mM bicarbonate buffering. In the absence of sulfate, D. vulgaris reduced >90% of the total soluble U(VI) (1 mM) to form U(IV) solids that were characterized by X-ray diffraction and confirmed to be nano-crystalline uraninite with crystallite size 2...
February 24, 2018: Biotechnology and Bioengineering
Lee R Moore, Daichi Mizutani, Tomoya Tanaka, Amy Buck, Mark Yazer, Maciej Zborowski, Jeffrey J Chalmers
The ability to separate RBCs from the other components of whole blood has a number of useful clinical and research applications ranging from removing RBCs from typical clinical blood draw, bone marrow transplants to transfusions of these RBCs to patients after significant blood loss. Viewed from a mechanistic/process perspective, there are three routine methodologies to remove RBCs: 1) RBCs lysis, 2) separation of the RBCs from the nucleated cells (i.e., stem cells) based on density differences typically facilitated through centrifugation or sedimentation agents, and 3) antibody based separation in which a targeted RBC is bound with an affinity ligand that facilitates its removal...
February 24, 2018: Biotechnology and Bioengineering
Xiaowei Peng, Robert M Kelly, Yejun Han
Unpretreated rice straw was fermented by the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensis, generating solubilized carbohydrates, organic acids, lignin-derived aromatics, H2 , and CO2 , which were subsequently used to produce polyhydroxybutyrate (PHB) by the chemolithoautotrophic bacterium Cupriavidus necator. The fermented liquid significantly enhanced the growth of C. necator, leading to a 5-fold cell biomass yield and a 9-fold PHB yield compared to what was obtained from conventional mineral media...
February 24, 2018: Biotechnology and Bioengineering
Liang Guo, Fan Zhang, Can Zhang, Guipeng Hu, Cong Gao, Xiulai Chen, Liming Liu
The compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency; however, prokaryotes are unicellular organisms that lack membrane-bound organelles. To mimic this natural compartmentalization, we present here the targeting of the reductive tricarboxylic acid (rTCA) pathway to the periplasm to enhance the production of malate. A multigene combination knockout strategy was used to construct a phosphoenolpyruvate (PEP) pool. Then, the genes encoding phosphoenolpyruvate carboxykinase and malate dehydrogenase were combinatorially overexpressed to construct a cytoplasmic rTCA pathway for malate biosynthesis; however, the efficiency of malate production was low...
February 24, 2018: Biotechnology and Bioengineering
Jeffrey J Marlow, Amit Kumar, Brandon Enalls, Linda M Reynard, Noreen Tuross, Gregory Stephanopoulos, Peter Girguis
Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy-rich, yet the most efficient methane-activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by-products at a comparable rate and in near-stoichiometric proportion to methane consumption...
February 20, 2018: Biotechnology and Bioengineering
Eunji Jo, June Seok Heo, Ja-Yun Lim, Bo-Ram Lee, Chul Joo Yoon, Jinkwan Kim, Jeewon Lee
The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. 24 copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv β3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle...
February 20, 2018: Biotechnology and Bioengineering
Jimmy T Trinh, Masfer H Alkahtani, Isaac Rampersaud, Arfaan Rampersaud, Marlan Scully, Ryland F Young, Philip Hemmer, Lanying Zeng
Rapid identification of specific bacterial strains within clinical, environmental, and food samples can facilitate the prevention and treatment of disease. Fluorescent nanodiamonds (FNDs) are being developed as biomarkers in biology and medicine, due to their excellent imaging properties, ability to accept surface modifications, and lack of toxicity. Bacteriophages, the viruses of bacteria, can have exquisite specificity for certain hosts. We propose to exploit the properties of FNDs and phages to develop phages conjugated with FNDs as long-lived fluorescent diagnostic reagents...
February 20, 2018: Biotechnology and Bioengineering
Jiazhang Lian, Zehua Bao, Sumeng Hu, Huimin Zhao
The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency...
February 20, 2018: Biotechnology and Bioengineering
Joshua G Gober, Amy E Rydeen, Timothy D Schwochert, Evan J Gibson-O'Grady, Eric M Brustad
Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h ...
February 20, 2018: Biotechnology and Bioengineering
Mohammad HamediRad, Jiazhang Lian, Hejun Li, Huimin Zhao
Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8...
February 20, 2018: Biotechnology and Bioengineering
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