Biology subjects

Trinh, C. T.

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

30 records · Page 2Linked to original sources

bioRxiv2019

Understanding and eliminating the detrimental effect of endogenous thiamine auxotrophy on metabolism of the oleaginous yeast Yarrowia lipolytica

Thiamine is an essential vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this study, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing gene scTHI13, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase for the de novo thiamine synthesis in Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene, we engineered the first thiamine-prototrophic Y. lipolytica reported to date. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica.\n\nIMPORTANCEThiamine plays a crucial role as an essential cofactor for enzymes in carbon and energy metabolism of all living cells. Thiamine deficiency has detrimental consequences on cellular health. Yarrowia lipolytica, a non-conventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph, whose effects on cellular metabolism are not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host to study thiamine metabolism, especially as mammalian cells are also thiamine-auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamentals of thiamine deficiency on cellular metabolism of Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, discovery of thiamine-regulated elements enables development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

microbiology↗

bioRxiv2019

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

BackgroundEsters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows thermophilic production with advantageous downstream product separation. However, due to the limited thermal stability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for thermophilic ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.\n\nResultsIn this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperature. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated the engineered C. thermocellum could produce isobutyl acetate directly from cellulose.\n\nConclusionsThis study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

synthetic biology↗

bioRxiv2019

Harnessing natural modularity of cellular metabolism to design a modular chassis cell for a diverse class of products by using goal attainment optimization

Living cells optimize their fitness against constantly changing environments to survive. Goal attainment optimization is a mathematical framework to describe the simultaneous optimization of multiple conflicting objectives that must all reach a performance above a threshold or goal. In this study, we applied goal attainment optimization to harness natural modularity of cellular metabolism to design a modular chassis cell for optimal production of a diverse class of products, where each goal corresponds to the minimum biosynthesis requirements (e.g., yields and rates) of a target product. This modular cell design approach enables rapid generation of optimal production strains that can be assembled from a modular cell and various exchangeable production modules and hence accelerates the prohibitively slow and costly strain design process. We formulated the modular cell design problem as a blended or goal attainment mixed integer linear program, using mass-balance metabolic models as biological constraints. By applying the modular cell design framework for a genome-scale metabolic model of Escherichia coli, we demonstrated that a library of biochemically diverse products could be effectively synthesized at high yields and rates from a modular (chassis) cell with only a few genetic manipulations. Flux analysis revealed this broad modularity phenotype is supported by the natural modularity and flexible flux capacity of core metabolic pathways. Overall, we envision the developed modular cell design framework provides a powerful tool for synthetic biology and metabolic engineering applications such as industrial biocatalysis to effectively produce fuels, chemicals, and therapeutics from renewable and sustainable feedstocks, bioremediation, and biosensing.

synthetic biology↗

bioRxiv2019

Comparison of Multi-objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

A large space of chemicals with broad industrial and consumer applications could be synthesized by engineered microbial biocatalysts. However, the current strain optimization process is prohibitively laborious and costly to produce one target chemical and often requires new engineering efforts to produce new molecules. To tackle this challenge, modular cell design based on a chassis strain that can be combined with different product synthesis pathway modules has been recently proposed. This approach seeks to minimize unexpected failure and avoid task repetition, leading to a more robust and faster strain engineering process. The modular cell design problem was mathematically formulated using a multi-objective optimization framework.[1] In this study, we evaluated a library of the state-of-the-art multi-objective evolutionary algorithms (MOEAs) to identify the most effective method to solve the modular cell design problem. Using the best MOEA, we found better solutions for modular cells compatible with many product synthesis modules. Furthermore, the best performing algorithm could provide better and more diverse design options that might help increase the likelihood of successful experimental implementation. We identified key parameter configurations to overcome the difficulty associated with multi-objective optimization problems with many competing design objectives. Interestingly, we found that MOEA performance with a real application problem, e.g., the modular strain design problem, does not always correlate with artificial benchmarks. Overall, MOEAs provide powerful tools to solve the modular cell design problem for novel biocatalysis.

synthetic biology↗

bioRxiv2018

De novo Microbial Biosynthesis of a Lactate Ester Platform

BackgroundGreen organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated.\n\nResultsIn this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase (ldhA) to convert pyruvate to lactate, a propionate CoA-transferase (pct) to convert lactate to lactyl-CoA, and an alcohol acyltransferase (AAT) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into 1) the upstream module to generate the ethanol and lactate precursors and 2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate limiting step in biosynthesis of lactate esters likely due to its low activity and specificity towards the non-natural substrate lactyl-CoA and alcohols.\n\nConclusionsWe have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.

synthetic biology↗

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↗