Yan Chen

Yan Chen
Lawrence Berkeley National Laboratory | LBL · Biological Systems & Engineering

PhD

About

86
Publications
8,856
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1,142
Citations
Citations since 2017
76 Research Items
1038 Citations
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2017201820192020202120222023050100150200250300
2017201820192020202120222023050100150200250300

Publications

Publications (86)
Article
The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars deriv...
Preprint
Full-text available
The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars deriv...
Article
Full-text available
X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alterna...
Article
Cyclopropane-functionalized hydrocarbons are excellent fuels due their high energy density. However, the organic synthesis of these molecules is challenging. In this work, we produced polycyclopropanated fatty acids in bacteria. These molecules can be converted into renewable fuels for energy-demanding applications such as shipping, long-haul trans...
Article
Full-text available
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteom...
Article
Full-text available
Rhamnolipids (RL) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low-cost...
Preprint
This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample prepa...
Preprint
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negati...
Preprint
This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". Th...
Preprint
This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best...
Article
Full-text available
In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer struct...
Preprint
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negati...
Article
Full-text available
Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrome...
Preprint
This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample prepa...
Preprint
This protocol details steps in discovery proteomic data-dependent acquisition with a standard-flow UHPLC-Obitrap system and a subsequent Mascot database search. It was adapted from González Fernández-Niño, S. M., et al. "Standard flow liquid chromatography for shotgun proteomics in bioenergy research."Frontiers in bioengineering and biotechnology,...
Preprint
This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best...
Article
Full-text available
Harnessing the microbiota for beneficial outcomes is limited by our poor understanding of the constituent bacteria, as the functions of most of their genes are unknown. Here, we measure the growth of a barcoded transposon mutant library of the gut commensal Bacteroides thetaiotaomicron on 48 carbon sources, in the presence of 56 stress-inducing com...
Article
Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to cr...
Preprint
This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". Th...
Article
Full-text available
High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets fo...
Article
Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY·FliMN with and...
Preprint
Full-text available
We adapted a high-throughput sample preparation workflow for Gram-negative bacteria to work with yeast. It consists of a zymolyase cell wall digestion step, cell lysis, protein precipitation, protein resuspension, protein quantification, and normalization of protein concentration followed by standard bottom-up proteomic procedures of reducing and b...
Article
Full-text available
Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secon...
Article
Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and spe-cialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated β-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a f...
Preprint
Full-text available
This protocol details steps in discovery proteomic data-dependent acquisition with a standard-flow UHPLC-QTOF system and a subsequent Mascot database search. It was adapted from González Fernández-Niño, S. M., et al. "Standard flow liquid chromatography for shotgun proteomics in bioenergy research."Frontiers in bioengineering and biotechnology, 3 (...
Preprint
Full-text available
This protocol details steps in targeted proteomic data acquisition with a standard-flow UHPLC-QQQ system. It was adapted from Chen, Y. et al. “A rapid methods development workflow for high-throughput quantitative proteomic applications.” PloS ONE 14,2 e0211582. 14 Feb. 2019, doi:10.1371/journal.pone.0211582.
Preprint
Full-text available
Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significan...
Article
While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of...
Article
Full-text available
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Article
There is strong interest in valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin‐derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble aromatic‐rich fractions which we...
Preprint
Full-text available
Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to...
Article
The genomic revolution ushered in an era of discovery and characterization of enzymes from novel organisms that fueled engineering of microbes to produce commodity and high-value compounds. Over the past decade advances in synthetic biology tools in recent years contributed to significant progress in metabolic engineering efforts to produce both bi...
Article
Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural ana...
Article
The method of X-ray Footprinting and Mass Spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary...
Article
Full-text available
Corynebacterium glutamicum ATCC 13032 is an established and industrially-relevant microbial host that has been utilized for the expression of many desirable bioproducts. Tetra-methylpyrazine (TMP) is a naturally occurring alkylpyrazine with broad applications spanning fragrances to resins. We identified an engineered strain of C. glutamicum which p...
Article
The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and...
Preprint
Full-text available
Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliM N ). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliM N with a...
Preprint
Full-text available
There is strong interest in the valorization of lignin derived from plant biomass to produce valuable products; however, the structural complexity of this biopolymer has been a major bottleneck to conversion. Chemical pretreatment liberates soluble fractions of lignin that may be upgraded by biological conversion. Here, ionic liquid pretreatment wa...
Article
Mass spectrometry–based quantitative proteomic analysis has proven valuable for clinical and biotechnology related research and development. Driving this value have been improvements in the sensitivity, resolution, and robustness of mass analyzers. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lea...
Preprint
Full-text available
Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural ana...
Article
Full-text available
Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that...
Preprint
Full-text available
Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that...
Article
The Design–Build–Test–Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and bio-based products. Here, we report on implementation of...
Preprint
Obligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the esse...
Article
Plants are an attractive source of renewable carbon for conversion to biofuels and bio‐based chemicals. Conversion strategies often use a fraction of the biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as aromatics and amino acids may improve the efficiency of biomass conversion. Pseudomonas p...
Article
Significance Despite the importance of immune receptors in the biology of plants and animals, the precise biochemical mechanisms leading to the biosynthesis and secretion of the peptide activators of these immune receptors remain unexplored. Here, we describe the biosynthesis, processing, and secretion of a peptide activator that binds a rice immun...
Preprint
Full-text available
The genomic catalogue of the human microbiota has expanded dramatically in recent years, and insights derived from human microbiota genomics has vast potential to generate treatments for human diseases. However, predictably harnessing the microbiota for beneficial outcomes is currently limited by our lack of understanding of the physiology of the c...
Article
Full-text available
Background Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass,...
Article
Full-text available
Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven significant progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance change...
Data
The relative abundances of Glycolysis, Pentose phosphate pathway, TCA cycle, and Ribosome proteins in P. putida grown on 10 mM of glucose, p-coumarate, and 5-aminovalerate carbon sources in MOPS media. The error bar shows the standard deviation of measured peak area of three biological replicates. Statistical significance of p-coumarate and 5-amino...
Data
Peptide retention time prediction of industrial interested pathway proteins from various in house built spectra libraries. (XLSX)
Data
Peptide retention time prediction based on spectra library built with in house MS data. (XLSX)
Data
Peptide retention time prediction based on spectra library built with public depository MS data. (XLSX)
Article
Full-text available
Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtil...
Preprint
Plant biomass is an attractive source of renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the total biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as plant-derived aromatics and amino acids have the potential to improve the eff...
Article
How membrane viscosity affects respiration In bacteria, energy production by the electron transport chain occurs at cell membranes and can be influenced by the lipid composition of the membrane. Budin et al. used genetic engineering to influence the concentration of unsaturated branched-chain fatty acids and thus control membrane viscosity (see the...
Article
Full-text available
Background Aviation fuels are an important target of biofuels research due to their high market demand and competitive price. Isoprenoids have been demonstrated as good feedstocks for advanced renewable jet fuels with high energy density, high heat of combustion, and excellent cold-weather performance. In particular, sesquiterpene compounds (C15),...
Preprint
The rice immune receptor XA21 is activated by the sulfated microbial peptide RaxX (required for activation of XA21-mediated immunity X) produced by Xanthomonas oryzae pv. oryzae ( Xoo ). Mutational studies and targeted proteomics revealed that RaxX is processed and secreted by the protease/transporter RaxB, whose function can be partially fulfilled...
Article
Full-text available
Background: Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated...
Article
Full-text available
We previously engineered E. coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid-derived pathways face the systematic meta...
Article
Full-text available
Streptomyces genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a Streptomyces-derived, well-characterized modular p...
Article
Full-text available
The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary bod...