Biology subjects

Trinh, C. T.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Establishing Design Principles for CRISPR-Cas Antifungals in Candida albicans

Drug-resistant fungal pathogens pose a growing public health threat, causing millions of infections and deaths annually. Limited antifungal drug classes and rising resistance highlight the urgent need for novel therapies. CRISPR-Cas systems offer sequence-specific antimicrobial potential, but their efficacy is influenced by organism-specific DNA repair outcomes. Here, we demonstrate that in Candida albicans, which predominantly relies on homology-directed repair (HDR), both repair template availability and DNA repair enzyme activity critically determine Cas9-induced lethality. By providing Trojan Horse donor DNA repair templates when targeting essential and DNA repair genes, we show that Cas9 lethality can be selectively tuned. Furthermore, multiplexed gRNA targeting to modulate DNA repair capacity reveals strong synergistic interactions when co-targeting HDR components, which is corroborated by enhanced killing in HDR-compromised strains. These results establish DNA repair as a programmable determinant of CRISPR-Cas antifungal activity and provide a mechanistic framework for combinatorial targeting strategies, advancing the development of CRISPR-Cas antifungals.

synthetic biology↗

bioRxiv2026

Synergistic CRISPR-Cas Antimicrobials through Essential and Defensive Gene Cotargeting in Staphylococcus aureus

Multidrug-resistant pathogens pose a major threat to One Health. Within the past decade, CRISPR-Cas systems have been explored as sequence-specific antimicrobials. While chromosomal injury has been considered the primary mechanism underlying pathogen killing by CRISPR-Cas antimicrobials, the synergistic role of gene disruption together with chromosomal injuries remains poorly understood. In this study, we characterized a new class of CRISPR-Cas antimicrobials that simultaneously cotarget essential and defensive genes to enhance potency against the clinically relevant pathogen Staphylococcus aureus. High-throughput CRISPR screening identified top-performing guide RNAs for twenty functionally diverse essential and defensive genes across the S. aureus genome. CRISPR-Cas antimicrobials were modularly formulated to target single or multiple gene loci and packaged in phage-like particles for specific delivery. By engineering an S. aureus production host with a chromosomally integrated anti-CRISPR protein, we demonstrated efficient production of CRISPR-Cas antimicrobials targeting any S. aureus chromosomal locus without self-targeting. Characterization of CRISPR-Cas antimicrobials with single guide RNA designs revealed that potency varied according to targeted gene function, achieving up to a 4-log10 reduction in viability and outperforming traditional antibiotics. Multiplexed configurations were consistently more effective than single-targeting designs, with the top-performing design demonstrating a 4.7-log10 reduction in viability. Cotargeting essential and defensive genes revealed synergies that led to improved lethality and attenuated resistance, with enhanced activity in biofilms compared to traditional antibiotics. Genes involved in signaling and stress responses were important defensive targets for developing cotargeting CRISPR-Cas antimicrobials. Overall, this study establishes design principles for synergistic CRISPR-Cas antimicrobials applicable to next-generation precision antimicrobial development. SIGNIFICANCEThe ability to effectively combat multidrug-resistant pathogens is of primary importance to One Health. This study develops a generalizable design principle for formulating potent CRISPR-Cas antimicrobials that exploit synergistic cotargeting strategies for enhanced pathogen killing. In addition to chromosomal injuries, we found that disruption of gene function plays a crucial role in determining the lethality of CRISPR-Cas antimicrobials, providing a generalizable framework for effective CRISPR-Cas antimicrobial design. The development of a CRISPR-Cas antimicrobial production host with stable, chromosomally integrated anti-CRISPR genes greatly expands the modularity, adaptability, and efficiency of formulating CRISPR-Cas antimicrobials and enables deeper insights into the molecular mechanisms involved in eliminating multidrug-resistant pathogens.

synthetic biology↗

bioRxiv2026

Cellular Stress Tolerance Governs Genetic Transformability in Recalcitrant Candida Species

Candida species are fungal pathogens whose rapidly increasing antifungal resistance poses a substantial public health challenge. High-throughput CRISPR-based screening could accelerate antifungal target discovery, yet its application in Candida has been limited by low DNA transformation efficiency. Chemical transformation exposes cells to environmental stresses to permit DNA uptake, but the physiological constraints on transformability remain poorly defined. Here, we show that genetic transformability in C. albicans is governed by the cellular capacity to withstand and recover from transformation-induced stress. Nutrient limitation markedly enhances transformation efficiency, while extracellular pH and lithium acetate chemistry strongly modulate this response. Systems-level proteomic analyses reveal that nutrient limitation and transformation chemistry prime oxidative stress tolerance, and transformation efficiency correlates with the expression of oxidative stress response proteins. Guided by these insights, we developed a generalizable fungal advanced chemical transformation (FACT) method that increases transformation efficiency across diverse Candida species and enables robust pooled CRISPR screening.

systems biology↗

bioRxiv2025

Proteome Reallocation Reveals Dynamics and Mechanisms of Phage Infection and Defense in Staphylococcus aureus

The coexistence of bacteria and phages is marked by dynamic interactions that determine infection outcomes. However, the mechanisms by which the host allocates its resources to cope with phage infection remain largely unknown. In this study, using longitudinal proteomics, we elucidated these interactions for the temperate staphylococcal phage{phi} NM1 and strains of Staphylococcus aureus either harboring its cognate prophage or lacking it. We demonstrated that infection of non-lysogenic S. aureus with{phi} NM1 induces a dramatic shutdown of host translation, reducing proteome allocation by over 20%. Quantitative analysis of the economics of{phi} NM1 infection revealed that the diversion of these cellular resources toward phage replication imposes a significant metabolic burden, thereby impairing cell growth. In contrast, lysogenic cells cope with phage infection and prevent culture collapse through a coordinated response of prophage-encoded defenses, host-encoded stress effectors, and reprogrammed cellular metabolism, thereby avoiding translation shutdown. Through coinfection with the wildtype phage and an engineered phage-like particle carrying a CRISPR-Cas phagemid, we revealed that synthetic DNA cargos evade host defenses and hijack the transcriptional machinery, altering infection outcomes. Without immunity, coinfection could collapse the non-lysogens more quickly than the native phage by overexpressing the cargo proteins, suppressing carbohydrate metabolism, and accelerating structural phage protein production through increased phage genome replication. Together, these findings provide a systems-level understanding of phage infection in S. aureus, uncovering the mechanisms for host takeover and prophage-mediated defense, with implications for next-generation phage therapy. SignificanceMultidrug-resistant bacteria pose a major threat to human health. Phage therapy offers a precise therapeutic approach by leveraging the specificity of phage infection and cargo delivery. However, as with conventional antibiotics, phage resistance can develop. Understanding the dynamic interactions between bacterial hosts and phages is therefore essential for predicting infection outcomes and designing precision phage therapies to suppress resistance. Using longitudinal proteomics, we elucidated the dynamics by which bacterial hosts reallocate cellular resources to cope with phage infection at the systems level. Coordinated defenses and reprogrammed metabolism are critical for host survival, but phage-delivered cargos can effectively bypass these barriers. These insights into the intricate interplay between the phage and host are crucial for designing next-generation precision phage therapies with greater lethality and less resistance.

systems biology↗

bioRxiv2024

Expanded Genome and Proteome Reallocation in a Novel, Robust Bacillus coagulans Capable of Utilizing Pentose and Hexose Sugars

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and TCA cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate. IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications, yet this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

systems biology↗

bioRxiv2023

CRISPR-GRIT: Guide-RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that causes severe infections in immunocompromised individuals. Overuse of antifungals coupled with climate change has led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes which require editing both alleles for elucidating gene function. Even though recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale functional genomic studies are still hindered by the necessity of co-transforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT, a repair template-integrated guide RNA design for expedited gene knockouts in C. albicans, and demonstrate its utility for multiplexed editing. We envision that this method can be employed for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

synthetic biology↗

bioRxiv2023

Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars

Short-chain esters are versatile chemicals with use as flavors, fragrances, solvents, and fuels. The de novo ester biosynthesis consists of diverging and converging pathway submodules, which is challenging to engineer to achieve optimal metabolic fluxes and selective product synthesis. Compartmentalizing the pathway submodules into specialist cells that facilitate pathway modularization and labor division can present a promising solution. Here, we engineered a synthetic Escherichia coli coculture with the compartmentalized sugar utilization and ester biosynthesis pathways to produce isobutyl butyrate from a mixture of glucose and xylose. To compartmentalize the sugar-utilizing pathway submodules, we engineered a xylose-utilizing E. coli specialist that selectively consumes xylose over glucose and bypasses the carbon catabolite repression (CCR) while leveraging the native CCR machinery to activate a glucose-utilizing E. coli specialist. Upon compartmentalizing the isobutyl butyrate pathway submodules into these sugar-utilizing specialist cells, a robust synthetic coculture could be engineered to selectively produce isobutyl butyrate at a level of 392 mg/L, about 31-fold higher than the monoculture.

synthetic biology↗

bioRxiv2023

Proteomes Reveal Metabolic Capabilities of Yarrowia lipolytica for Biological Upcycling of Polyethylene into High-Value Chemicals

Polyolefins derived from plastic wastes are recalcitrant for biological upcycling. However, chemical depolymerization of polyolefins can generate depolymerized plastic (DP) oil comprising of a complex mixture of saturated, unsaturated, even and odd hydrocarbons suitable for biological conversion. While DP oil contains a rich carbon and energy source, it is inhibitory to cells. Understanding and harnessing robust metabolic capabilities of microorganisms to upcycle the hydrocarbons in DP oil, both naturally and unnaturally occurring, into high-value chemicals are limited. Here, we discovered that an oleaginous yeast Yarrowia lipolytica undergoing short-term adaptation to DP oil robustly utilized a wide range of hydrocarbons for cell growth and production of citric acid and neutral lipids. When growing on hydrocarbons, Y. lipolytica partitioned into planktonic and oil-bound cells with each exhibiting distinct proteomes and amino acid distributions invested into establishing these proteomes. Significant proteome reallocation towards energy and lipid metabolism, belonging to two of the 23 KOG (Eukaryotic Orthologous Groups) classes C and I, enabled robust growth of Y. lipolytica on hydrocarbons, with n-hexadecane as the preferential substrate. This investment was even higher for growth on DP oil where both the KOG classes C and I were the top two, and many associated proteins and pathways were expressed and upregulated including the hydrocarbon degradation pathway, Krebs cycle, glyoxylate shunt and, unexpectedly, propionate metabolism. However, a reduction in proteome allocation for protein biosynthesis, at the expense of the observed increase towards energy and lipid metabolisms, might have caused the inhibitory effect of DP oil on cell growth. MPORTANCESustainable processes for biological upcycling plastic wastes in a circular bioeconomy are needed to promote decarbonization and reduce environmental pollution due to increased plastic consumption, incineration, and landfill storage. Strain characterization and proteomic analysis revealed the robust metabolic capabilities of Y. lipolytica to upcycle polyethylene into high-value chemicals. Significant proteome reallocation towards energy and lipid metabolisms was required for robust growth on hydrocarbons with n-hexadecane as the preferential substrate. However, an apparent over-investment in these same categories to utilize complex DP oil came at the expense of protein biosynthesis, limiting cell growth. Taken together, this study elucidates how Y. lipolytica activates its metabolism to utilize DP oil and establishes Y. lipolytica as a promising host for the upcycling of plastic wastes.

systems biology↗

bioRxiv2023

Consolidated Bioprocessing of Lignocellulosic Biomass Poplar to Produce Short-Chain Esters by Clostridium thermocellum

Consolidated bioprocessing (CBP) of lignocellulosic biomass using cellulolytic microorganisms presents a promising sustainable and economical biomanufacturing platform where enzyme production, biomass hydrolysis, and fermentation to produce biofuels, biochemicals, and biomaterials occur in a single step. However, understanding and redirecting metabolism of microorganisms to be compatible with CBP to produce non-native metabolites are limited. In this study, we metabolically engineered a cellulolytic thermophile Clostridium thermocellum and demonstrated its compatibility with CBP integrated with a mild Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters (i.e., ethyl acetate, ethyl isobutyrate, isobutyl acetate, isobutyl isobutyrate) with broad use as solvents, flavors, fragrances, and biofuels. A recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of a thermostable alcohol acetyltransferase improved the target esters production without compromised deacetylation activities. We discovered these esterases exhibited promiscuous thioesterase activities and their deletion improved ester production by increasing isobutanol flux and rerouting the native electron and carbon fermentative metabolism besides their known major function of ester degradation. The total ester production could be further enhanced up to 80-fold and the composition of short-chain esters could be modified by deleting lactate biosynthesis and/or CELF-pretreated poplar under different pretreatment conditions.

bioengineering↗

bioRxiv2022

Gene Co-expression Connectivity Predicts Gene Targets Underlying High Ionic Liquid Tolerance in Yarrowia lipolytica

Microbial tolerance to organic solvents such as ionic liquids (ILs) is a robust phenotype beneficial for novel biotransformation. While most microbes become inhibited in 1%-5% (v/v) IL (e.g., 1-ethyl-3-methylimidazolium acetate), we engineered a robust Yarrowia lipolytica (YlCW001) that tolerates a record high of 18% (v/v) IL via adaptive laboratory evolution. Yet, genotypes conferring high IL tolerance in YlCW001 remain to be discovered. Using dynamic RNA-Seq data, we shed light on the underlying cellular processes that Y. lipolytica responds to IL. We introduced Gene Co-expression Connectivity (GeCCo) to discover genotypes conferring desirable phenotypes that might not be found by the conventional differential expression (DE) approaches. GeCCo selects genes based on their number of co-expressed genes in a sub-network of upregulated genes by the target phenotype. We experimentally validated GeCCo by reversely engineering high IL-tolerant phenotype in wildtype Y. lipolytica. We found that gene targets selected by both DE and GeCCo exhibited the best statistical chance at increasing IL tolerance when individually overexpressed. Remarkably, the best combination of dual-overexpressed genes were genes selected by GeCCo alone. This non-intuitive combination of genes, BRN1 and OYE2, are involved in guiding/regulating mitotic cell division, chromatin segregation/condensation, microtubule and cytoskeletal organization, and Golgi vesicle transport.

systems biology↗

bioRxiv2022

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including keto-isovalerate submodule for converting pyruvate to keto-isovalerate, ketoacid elongation submodule for producing longer carbon-chain keto-acids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. The engineered strains produced isoamyl acetate at a titer of 8.8 g/L (> 0.25 g/L toxicity limit), a yield of 0.17 g/g (47% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

synthetic biology↗

bioRxiv2021

Controlling Selectivity of Modular Microbial Biosynthesis of Butyryl-CoA-Derived Designer Esters

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. Here, we present a generalizable framework to enable the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

synthetic biology↗

bioRxiv2021

Probing Specificities of Alcohol Acyltransferases for Designer Ester Biosynthesis with a High-Throughput Microbial Screening Platform

Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high-throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate-based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CATSa) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate-based culturing technique with a previously established colorimetric assay, we developed a high-throughput microbial screening platform for AATs. We demonstrated that this platform could not only confirm CATSa F97W with enhanced isobutyl acetate synthesis but also identify three ATF1Sc (P348M, P348A, and P348S) variants, derived from Saccharomyces cerevisiaes AAT and engineered by model-guided protein design, for enhanced butyl acetate production. We anticipate the high-throughput microbial screening platform is a useful tool to identify novel AATs that have important roles in nature and industrial biocatalysis for designer bioester production.

bioengineering↗

bioRxiv2021

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, {beta}-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates. IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

systems biology↗

bioRxiv2021

Computational Design and Analysis of Modular Cells for Large Libraries of Exchangeable Product Synthesis Modules

Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method, named ModCell-HPC, capable of designing modular cells for large libraries with hundredths of products with a highly-parallel and multi-objective evolutionary algorithm. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner under different carbons sources. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells. Furthermore, we used ModCell-HPC to identify design features that allow an existing modular cell to be re-purposed towards production of new molecules. Overall, ModCell-HPC is a useful tool towards more efficient and generalizable design of modular cells to help reduce research and development cost in biocatalysis.

synthetic biology↗

bioRxiv2020

Repurposing chloramphenicol acetyltransferase for a robust and efficient designer ester biosynthesis platform

Robust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as an interchangeable metabolic module for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and repurposed chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacterium Escherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinant E. coli was also capable of simulating the ester profile of roses with high conversion (> 97%) and titer (> 1 g/L) from fermentable sugars at 37{degrees}C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacterium Clostridium thermocellum harboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50{degrees}C) due to the engineered enzymes remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.

synthetic biology↗

bioRxiv2020

Development of a genome-scale metabolic model of Clostridium thermocellum and its applications for integration of multi-omics datasets and strain design

Solving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as catalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade untreated lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

systems biology↗

bioRxiv2019

Endogenous esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Medium chain esters are potential drop-in biofuels and versatile chemicals. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either esterases/lipases or alcohol acyltransferase (AAT), but the AAT-dependent pathway is more thermodynamically favorable in aqueous fermentation environment. Even though cellulolytic thermophiles such as Clostridium thermocellum harboring the engineered AAT-dependent pathway can directly convert lignocellulosic biomass into esters, the production is currently not efficient and requires optimization. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. In this study, we developed a simple, high-throughput colorimetric assay to screen the endogenous esterases of C. thermocellum responsible for ester hydrolysis. We identified, characterized, and disrupted two critical endogenous esterases that significantly contributes to isobutyl acetate degradation in C. thermocellum. We demonstrated that not only did the engineered esterase-deficient strain alleviate ester hydrolysis but also helped improve isobutyl acetate production while not affecting its robust metabolism for effective cellulose assimilation.\n\nIMPORTANCECarbohydrate esterases (CEs) are important enzymes in the deconstruction of lignocellulosic biomass by the cellulolytic thermophile C. thermocellum, yet some are potential ester degraders in a microbial ester production system. Currently, the functional roles of CEs for hydrolyzing medium chain esters and negatively affecting the ester microbial biosynthesis are not well understood. This study discovered novel CEs responsible for isobutyl acetate degradation in C. thermocellum and hence identified one of the critical bottlenecks for direct conversion of lignocellulosic biomass into esters.

microbiology↗