Taek Soon Lee

Lawrence Berkeley National Laboratory

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Jay D. Keasling

Lawrence Berkeley National Laboratory

Aindrila Mukhopadhyay

Lawrence Berkeley National Laboratory

Blake A. Simmons

Joint BioEnergy Institute

Paul D. Adams

Lawrence Berkeley National Laboratory

Christopher J. Petzold

Joint BioEnergy Institute

Edward E. K. Baidoo

Joint BioEnergy Institute

Aram Kang

Korea Institute of Radiological and Medical Sciences

Jorge Alonso-Gutiérrez

Joint BioEnergy Institute

Tanveer S. Batth

University of Copenhagen

Kevin W. George

Amyris (United States)

Cooperative Institutions

Joint BioEnergy Institute

175

Lawrence Berkeley National Laboratory

160

University of California, Berkeley

Sandia National Laboratories California

Sandia National Laboratories

Emery Oleochemicals (Malaysia)

QB3

Berkeley College

Lawrence Livermore National Security

University of California, San Diego

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CCDC 111357: Experimental Crystal Structure Determination

The Cambridge Structural Database (1999)

Hye Jin Choi Taek Soon Lee Myunghyun Paik Suh

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

10.5517/cc3qw5v

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Converting Sugars to Biofuels: Ethanol and Beyond

Bioengineering (2015)

Aram Kang Taek Soon Lee

To date, the most significant sources of biofuels are starch- or sugarcane-based ethanol, which have been industrially produced in large quantities in the USA and Brazil, respectively. However, the ultimate goal of biofuel production is to produce fuels from lignocellulosic biomass-derived sugars with optimal fuel properties and compatibility with the existing fuel distribution infrastructure. To achieve this goal, metabolic pathways have been constructed to produce various fuel molecules that are categorized into fermentative alcohols (butanol and isobutanol), non-fermentative alcohols from 2-keto acid pathways, fatty acids-derived fuels and isoprenoid-derived fuels. This review will focus on current metabolic engineering efforts to improve the productivity and the yield of several key biofuel molecules. Strategies used in these metabolic engineering efforts can be summarized as follows: (1) identification of better enzymes; (2) flux control of intermediates and precursors; (3) elimination of competing pathways; (4) redox balance and cofactor regeneration; and (5) bypassing regulatory mechanisms. In addition to metabolic engineering approaches, host strains are optimized by improving sugar uptake and utilization, and increasing tolerance to toxic hydrolysates, metabolic intermediates and/or biofuel products.

Metabolic Engineering

Isobutanol

Metabolic pathway

Lignocellulosic Biomass

Cellulosic ethanol

Bioproducts

10.3390/bioengineering2040184

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Citations (75)

Parallel Integration and Chromosomal Expansion of Metabolic Pathways

ACS Synthetic Biology (2018)

Garima Goyal Zak Costello Jorge Alonso-Gutiérrez Aram Kang Taek Soon Lee

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

Bioproducts

Metabolic pathway

Metabolic Engineering

10.1021/acssynbio.8b00243

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Citations (6)

Low-temperature combustion chemistry of biofuels: pathways in the initial low-temperature (550 K–750 K) oxidation chemistry of isopentanol

Physical Chemistry Chemical Physics (2012)

Oliver Welz Judit Zádor John D. Savee Martin Y. Ng Giovanni Meloni

The branched C5 alcohol isopentanol (3-methylbutan-1-ol) has shown promise as a potential biofuel both because of new advanced biochemical routes for its production and because of its combustion characteristics, in particular as a fuel for homogeneous-charge compression ignition (HCCI) or related strategies. In the present work, the fundamental autoignition chemistry of isopentanol is investigated by using the technique of pulsed-photolytic Cl-initiated oxidation and by analyzing the reacting mixture by time-resolved tunable synchrotron photoionization mass spectrometry in low-pressure (8 Torr) experiments in the 550–750 K temperature range. The mass-spectrometric experiments reveal a rich chemistry for the initial steps of isopentanol oxidation and give new insight into the low-temperature oxidation mechanism of medium-chain alcohols. Formation of isopentanal (3-methylbutanal) and unsaturated alcohols (including enols) associated with HO2 production was observed. Cyclic ether channels are not observed, although such channels dominate OH formation in alkane oxidation. Rather, products are observed that correspond to formation of OHvia β-C–C bond fission pathways of QOOH species derived from β- and γ-hydroxyisopentylperoxy (RO2) radicals. In these pathways, internal hydrogen abstraction in the RO2 ⇄ QOOH isomerization reaction takes place from either the –OH group or the C–H bond in α-position to the –OH group. These pathways should be broadly characteristic for longer-chain alcohol oxidation. Isomer-resolved branching ratios are deduced, showing evolution of the main products from 550 to 750 K, which can be qualitatively explained by the dominance of RO2 chemistry at lower temperature and hydroxyisopentyl decomposition at higher temperature.

Isoprene

Bond cleavage

Hydrogen atom abstraction

10.1039/c2cp23248k

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Citations (95)

Application of targeted proteomics and biological parts assembly in E. coli to optimize the biosynthesis of an anti-malarial drug precursor, amorpha-4,11-diene

Chemical Engineering Science (2013)

Han Min Woo Gregory Murray Tanveer S. Batth Nilu Prasad Paul D. Adams

Mevalonate pathway

Heterologous

Heterologous expression

Metabolic Engineering

Metabolic pathway

Mevalonic acid

10.1016/j.ces.2013.04.033

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Citations (15)

An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

Biotechnology for Biofuels (2020)

Brett M. Garabedian Corey W. Meadows Florence Mingardon Joel M. Guenther Tristan de Rond

Abstract Background Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR) . Results Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC–MS. Conclusions With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

High-Throughput Screening

Saturated mutagenesis

10.1186/s13068-020-01821-w

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Citations (2)

폐굴껍질을 이용한 침적식 생물막법에 의한 폐수처리

한국물환경학회지 (1997)

문병현 이택순 서정윤 서희정 Taek Soon Lee

This study was conducted to use waste oyster shell as biofilm media for biological wastewater treatment. The comparison between the activated sludge and the submerged biofilm process with oyster shell media (5% of reactor volume) for domestic sewage treatment was made at various hydraulic retention times. The effluent quality of the submerged oyster shell reactor was more stable than that of activated sludge reactor. The BOD removal efficiencies of the submerged process was above 80% at the various organic loading rates of 0.12∼0.55㎏BOD/㎥/day. The COD and SS removal efficiencies of the submerged process were higher than that of the activated sludge process. Also, TKN and T-P removal efficiencies of submerged process were higher than that of the activated sludge process. The comparison between the submerged biofilm process with oyster shell media of 10% and 18% was made at various loading rates. The hydraulic retention time was fixed to 6hr. The BOD, COD_(Cr) removal efficiencies of 10% and 18% were above 80% at organic loading rates of 1.0㎏ BOD/㎥/day and 1.5㎏COD_(Cr)/㎥/day but the removal efficiencies of 18% reactor decreased as the organic loading rates were increased. The TKN removal efficiencies of 10% and 18% were above 70% at organic loading rates of 0.2㎏ TKN/㎥/day. However, the removal efficiencies of 18% reactor decreased at the higher loading rates. The phosphorus removal efficiencies of both reactors showed similar removal efficiencies.

Kjeldahl method

Hydraulic retention time

Source

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An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

Research Square (Research Square) (2020)

Brett M. Garabedian Corey W. Meadows Florence Mingardon Joel M. Guenther Tristan de Rond

Abstract Background : Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR) . Results : Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions : With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

High-Throughput Screening

Saturated mutagenesis

10.21203/rs.3.rs-52267/v1

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Citations (0)

Journal of inclusion phenomena and molecular recognition in chemistry (2001)

Hye Jin Choi Taek Soon Lee Myunghyun Paik Suh

Ethylene diamine

10.1023/a:1014436406651

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Citations (36)

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida

Biotechnology for Biofuels and Bioproducts (2022)

Xi Wang Edward E. K. Baidoo Ramu Kakumanu Silvia Xie Aindrila Mukhopadhyay

Abstract With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C 5 ) and epi-isozizaene (C 15 ) for the first time in P. putida . We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing l -glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p -coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass. Graphical Abstract

Pseudomonas putida

10.1186/s13068-022-02235-6

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Citations (12)