Biology subjects

Trinh, C. T.

Publications and source records attributed to Trinh, C. T..

7 recordsLinked to original sources

bioRxiv2018

Multiobjective Strain Design: A Framework for Modular Cell Engineering

Diversity of cellular metabolism can be harnessed to produce a large space of molecules. However, development of optimal strains with high product titers, rates, and yields required for industrial production is laborious and expensive. To accelerate the strain engineering process, we have recently introduced a modular cell design concept that enables rapid generation of optimal production strains by systematically assembling a modular cell with an exchangeable production module(s) to produce target molecules efficiently. In this study, we formulated the modular cell design concept as a general multiobjective optimization problem with flexible design objectives derived from mass action. We developed algorithms and an associated software package, named ModCell2 to implement the design. We demonstrated that ModCell2 can systematically identify genetic modifications to design modular cells that can couple with a variety of production modules and exhibit a minimal tradeoff among modularity, performance, and robustness. Analysis of the modular cell designs revealed both intuitive and complex metabolic architectures enabling modular production of these molecules. We envision ModCell2 provides a powerful tool to guide modular cell engineering and sheds light on modular design principles of biological systems.

bioengineering↗

bioRxiv2018

Exceptional Solvent Tolerance in Yarrowia lipolytica Is Enhanced by Sterols

Microbial biocatalysis in organic solvents such as ionic liquids (ILs) is attractive for making fuels and chemicals from complex substrates including lignocellulosic biomass. However, low IL concentrations of 0.5-1.0 % (v/v) can drastically inhibit microbial activity. In this study, we engineered an exceptionally robust oleaginous yeast Yarrowia lipolytica, YlCW001, by adaptive laboratory evolution (ALE). The mutant YlWC001 shows robust growth in up to 18% (v/v) 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), which makes it the most IL-tolerant microorganism published to our knowledge. Remarkably, YlCW001 exhibits broad tolerance in most commonly used hydrophilic ILs beyond [EMIM][OAc]. Scanning electron microscopy revealed that ILs significantly damage cell wall and/or membrane of wildtype Y. lipolytica with observed cavities, dents, and wrinkles while YlCW001 maintains healthy morphology even in high concentrations of ILs up to 18% (v/v). By performing comprehensive metabolomics, lipidomics, and transcriptomics to elucidate this unique phenotype, we discovered that both wildtype Y. lipolytica and YlCW001 reconfigured membrane composition (e.g., glycerophospholipids and sterols) and cell wall structure (e.g., chitin) under IL-stressful environments. By probing the steroid pathway at transcriptomic, enzymatic, and metabolic levels, we validated that sterols (i.e., ergosterol) are a key component of the cell membrane that enables Y. lipolytica to resist IL-responsive membrane damage and hence tolerate high IL concentrations. This study provides a better understanding of exceptional robustness of Y. lipolytica that can be potentially harnessed as a microbial manufacturing platform for production of fuels and chemicals in organic solvents.

microbiology↗

bioRxiv2018

In Silico Processing of the Complete CRISPR-Cas Spacer Space for Identification of PAM Sequences

Despite extensive exploration of the diversity of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, biological applications have been mostly confined to Class 2 systems, specifically the Cas9 and Cas12 (formerly Cpf1) single effector proteins. A key limitation of exploring and utilizing other CRISPR-Cas systems with unique functionalities, particularly Class I types and their multi-protein effector complex, is the knowledge of the systems protospacer adjacent motif (PAM) sequence identity. In this work, we developed a systematic pipeline, named CASPERpam, that enables us to comprehensively assess the PAM sequences of all the available CRISPR-Cas systems in the NCBI database of bacterial genomes. The CASPERpam analysis revealed that within the 30,389 assemblies previously screen for CRISPR arrays, there exists 26,364 spacers that match somewhere in the viral, bacterial, and plasmid databases of NCBI, using the constraints of 95% sequence identity and 95% sequence coverage for blast hits. When grouping these results by species, we were able to identify putative PAM sequences for 1,049 among 1,493 unique species. The remaining species either have insufficient data or an undetermined result from the analysis. Finally, we were able to infer certain design principles that are relevant for understanding PAM diversity and a baseline for further experimental studies including PAM assays. We envision CASPERpam is a useful bioinformatic tool for understanding and harnessing the diversity of CRISPR systems.

bioinformatics↗

bioRxiv2018

Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 {+/-} 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% {+/-} 0.5% C11 alkene, 12.7% {+/-} 2.2% C13 alkene, 82.7% {+/-} 1.7% C15 alkene, and 3.7% {+/-} 0.8% C17 alkene, and a yield of 0.87 {+/-} 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased [~]2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

microbiology↗

bioRxiv2017

Understanding Functional Roles of Native Pentose-Specific Transporters for Activating Dormant Pentose Metabolism in Yarrowia lipolytica

Pentoses including xylose and arabinose are the second-most prevalent sugars of lignocellulosic biomass that can be harnessed for biological conversion. Although Yarrowia lipolytica has emerged as a promising industrial microorganism for production of high-value chemicals and biofuels, its native pentose metabolism is poorly understood. Our previous study demonstrated that Y. lipolytica (ATCC MYA-2613) has endogenous enzymes for D-xylose assimilation, but inefficient xylitol dehydrogenase causes Y. lipolytica to assimilate xylose poorly. In this study, we investigated the functional roles of native sugar-specific transporters for activating the dormant pentose metabolism in Y. lipolytica. By screening a comprehensive set of 16 putative pentose-specific transporters, we identified two candidates, YALI0C04730p and YALI0B00396p, that enhanced xylose assimilation. The engineered mutants YlSR207 and YlSR223, overexpressing YALI0C04730p and YALI0B00396p, respectively, improved xylose assimilation approximately 23% and 50% in comparison to YlSR102, a parent engineered strain overexpressing solely the native xylitol dehydrogenase gene. Further, we activated and elucidated a widely unknown, native L-arabinose-assimilating pathway in Y. lipolytica through transcriptomic and metabolic analyses. We discovered that Y. lipolytica can co-consume xylose and arabinose, where arabinose utilization shares transporters and metabolic enzymes of some intermediate steps of the xylose-assimilating pathway. Arabinose assimilation was synergistically enhanced in the presence of xylose while xylose assimilation was competitively inhibited by arabinose. L-arabitol dehydrogenase is the rate-limiting step responsible for poor arabinose utilization in Y. lipolytica. Overall, this study sheds light on the cryptic pentose metabolism of Y. lipolytica and further helps guide strain engineering of Y. lipolytica for enhanced assimilation of pentose sugars.\n\nIMPORTANCEThe oleaginous yeast Yarrowia lipolytica is a promising industrial platform microorganism for production of high-value chemicals and fuels. For decades since its isolation, Y. lipolytica has often been known to be incapable of assimilating pentose sugars, xylose and arabinose, that are dominantly present in lignocellulosic biomass. Through bioinformatic, transcriptomic and enzymatic studies, we have uncovered the dormant pentose metabolism of Y. lipolytica. Remarkably, unlike most yeast strains that share the same transporters for importing hexose and pentose sugars, we discovered that Y. lipolytica possess the native pentose-specific transporters. By overexpressing these transporters together with the rate-limiting D-xylitol and L-arabitol dehydrogenases, we activated the dormant pentose metabolism of Y. lipolytica. Overall, this study provides a fundamental understanding of the dormant pentose metabolism of Y. lipolytica and guides future metabolic engineering of Y. lipolytica for enhanced conversion of pentose sugars to high-value chemicals and fuels.

physiology↗

bioRxiv2017

Comprehensive Characterization of Toxicity of Fermentative Metabolites on Microbial Growth

BackgroundVolatile carboxylic acids, alcohols, and esters are natural fermentative products, typically derived from anaerobic digestion. These metabolites have important functional roles to regulate cellular metabolisms and broad use as food supplements, flavors and fragrances, solvents, and fuels. Comprehensive characterization of toxic effects of these metabolites on microbial growth under similar conditions is very limited.\n\nResultsWe characterized a comprehensive list of 32 short-chain carboxylic acids, alcohols, and esters on microbial growth of Escherichia coli MG1655 under anaerobic conditions. We analyzed toxic effects of these metabolites on E. coli health, quantified by growth rate and cell mass, as a function of metabolite types, concentrations, and physiochemical properties including carbon chain lengths and associated functional groups, chain branching features, hydrophobicity, and energy density. Strain characterization reveals these metabolites exerted distinct toxic effects on E. coli health. We find that higher concentrations and/or longer carbon lengths of metabolites cause more severe growth inhibition. For the same carbon lengths and metabolite concentrations, alcohols are most toxic followed by acids then esters. We also discover that branched chain metabolites are less toxic than linear chain metabolites for the same carbon lengths and metabolite concentrations. Remarkably, shorter alkyl esters (e.g., ethyl butyrate) are found to be less toxic than longer alkyl esters (e.g., butyl acetate) for the same carbon lengths and metabolite concentrations. Regardless of metabolite types, longer chain metabolites are less soluble and have higher energy densities but are more toxic to microbial growth.\n\nConclusionsMetabolite hydrophobicity, correlated with carbon chain length, associated functional group, chain branching feature, and energy density, is a good quantitative index to evaluate toxic effect of a metabolite on microbial health. The results provide better understanding of degrees of toxicity of fermentative metabolites on microbial growth and further help selection of desirable metabolites and hosts for industrial fermentation to overproduce them.

physiology↗

bioRxiv2017

Enhanced Guide-RNA Design And Targeting Analysis For Precise CRISPR Genome Editing Of Single And Consortia Of Industrially Relevant And Non-Model Organisms

MotivationGenetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.\n\nResultsTo accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CASPER (CRISPR Associated Software for Pathway Engineering and Research), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, p<0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA (gRNA) requirement) and multispecies population analysis (i.e. gRNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.

bioinformatics↗