Broad Biotechnological Applications Research Articles

SPLICER: A Highly Efficient Base Editing Toolbox That Enables In Vivo Therapeutic Exon Skipping.

Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular biology, medicine, and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases, and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings, including transient effects for oligonucleotides, genotoxicity for nucleases and inconsistent exon skipping for base editors. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing with SPLICER improves exon skipping, reduces aberrant outcomes, including cryptic splicing and intron retention, and enables skipping of exons refractory to single splice-site editing. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which encodes the amino acid residues that are cleaved to form the Aβ plaques in Alzheimer's disease. SPLICER reduced the formation of Aβ42 peptides in vitro and enabled efficient exon skipping in a mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and efficient toolbox for exon skipping with broad therapeutic applications.

Engineering functional materials through bacteria-assisted living grafting

Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper's transparent peer review process is included in the supplemental information.

Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback

IntroductionTranscription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. ObjectivesDevelopment of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. MethodsWe designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. ResultsWhen this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. ConclusionMany positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

Aerobic Granular Sludge: Perspectives for Excess Sludge Management and Resource Recovery

AbstractAerobic granular sludge (AGS) technology is gaining increasing interest globally. However, several aspects are still challenging its installation, as the integration into existing municipal wastewater treatment plants. The operation of AGS units in continuous flow mode could facilitate that integration; yet has only been experienced at lab and pilot scales. Further, handling of excess granular sludge constitutes a prominent challenge. Even that, adopting effective strategies for its management holds promising contributions to the circular economy by fostering the reuse and recovery of valuable resources. The recovery of phosphorus and alginate‐like polymers holds significant commercial potential worldwide, with broad applications in agriculture, food, pharmaceuticals, and biotechnology.

CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion

BackgroundHalophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis.ResultsThree different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system.ConclusionsThis was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.

A multiphase ciliary flow of Casson fluid in a porous channel under the effects of electroosmosis and MHD: Exact solutions

The electro-osmosis effects on ciliary multiphase flow have broad applications in diverse fields, including microfluidics, biotechnology, chemical engineering, and environmental sciences. In the current analysis, the authors have elaborated the electro-osmosis effects on a ciliary two-phase channel flow of Casson fluid in the presence of solid particles and magnetic effects. The governing equations have been considered for the physical problem by applying the wave frame and dimensionless transformations. A lubrication approach has also been followed by the equations. Systems of differential equations have been solved on well-known software Mathematica by a built-in DSolve tool to acquire the exact solutions. From graphical evidences, it is observed that Helmholtz Smoluchowski velocity factor and solid particles contribution affects the electro-kinetic energy and reduces the flow speed.

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

Polish Contribution to Global Research on Somatic Embryogenesis

AbstractSomatic embryogenesis (SE) is a complex process that begins with regaining totipotency in some somatic cells, proceeds through embryo development and maturation, and ends with the formation of a whole plant. Since the first publications on SE in 1958, this regeneration process has been applied to the in vitro propagation of many plant species and has led to the development of some specific model systems. SE has been used to expand our understanding of the cytomorphological, physiological, biochemical, and genetic processes that govern the earliest developmental events in the life of plants. This paper summarizes the achievements of Polish research groups working on SE systems established for several plants (gentians, the tree fernCyathea delgadiiSternb., and conifers) and three model species (Arabidopsis thaliana,Medicagosativa, andM. truncatula). SE systems have used a broad spectrum of experimental approaches involving genomic tools (transcriptomics, proteomics, and chromatin analyses), physiological methods which focus on phytohormones, and cytological techniques. Studies on the experimental models ofA. thalianaandMedicagospp. have resulted in the identification of new genetic and epigenetic elements of the complex regulatory network controlling embryogenic induction in plant somatic cells. The protocol developed for ferns has provided a unique and simple system for cytological analysis of early SE events that occur in a single cell of initial explants. Gentian embryogenic suspension cultures have successfully been used in broad biotechnological applications, including plant transformation, protoplast isolation, culture, and fusion. Systems described for coniferous species effectively produced many vigorous somatic seedlings and cost-efficient storage of genotypes during clonal field-testing. The research undertaken by Polish scientists has resulted in developing experimental systems that have enabled significant advances in SE knowledge.

Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Chemoselective restoration of para-azido-phenylalanine at multiple sites in proteins

The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para-azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates "click" reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%-60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology.

A thermostable extracellular α-amylase from Aspergillus flavus S2-OY: Purification, characterisation and application in raw starch hydrolysis

An amylolytic fungus was isolated locally from the soil of a cassava waste dumpsite. It was identified as Aspergillus flavus S2-OY using 18S rRNA gene sequencing with gene bank accession number MZ267053.1. α-Amylase was produced from the fungus through solid substrate fermentation of potato (Solanum tuberosum L.) peel, a cheap and renewable substrate. The fungal enzyme was purified 13.41-fold by ammonium sulphate and ion exchange chromatography (CM Sephadex C-50), with final yield of 13.41%. Apparent molecular weight of 55.0 kDa was obtained for the purified enzyme, using SDS-PAGE. Optima pH (6.0) and temperature (70 °C) of α-amylase activity were determined for the enzyme which also showed thermal stability up to 70 °C, with 80% residual activity after 120 min. The enzyme exhibited a wide range of pH stability (4.0 to 7.0). Kinetic parameters Km and Vmax values obtained with soluble starch were 158 µg/ml/min and 1.51 mg/ml, respectively. The enzyme was slightly inhibited by Mg2+. Mn2+, Zn2+, Na+, K+, Hg2+ and the chelating agent, EDTA, at 5.0 mM concentration. However, it was activated by Sn2+. The enzyme hydrolysed gelatinised and ungelatinized raw yam, corn, rice and millet starches strongly but cassava starch moderately. These results revealed that cost-effective α-amylase production from Aspergillus flavus S2-OY could be realised with the use of potato peel as a cheap substrate, under solid substrate fermentation condition. Also, the fungal α-amylase is a raw starch-hydrolysing, acid to neutral and thermostable enzyme with potential for broad biotechnological applications.

Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown.

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

Molecular, morphological, and toxinological characterizations of an Argentinean strain of Halamphora coffeaeformis with potential biotechnological applications

Diatoms are a diverse group of unicellular microalgae with broad biotechnological and industrial applications. Recent studies have revealed that some species from the genus Halamphora accumulate high quantities of lipids, which make them potentially suitable for biodiesel production. However, due to their high phenotypic plasticity, molecular phylogenetic and ultrastructural analyses are usually needed for precise species identification, which in turn is crucial to define the cultivation strategies to obtain the main target compound. Moreover, the use of diatoms for nutrition, health, and aquaculture requires prior analysis of the potential toxicity of the selected species. In the present study we provide a detailed morphological, molecular, and toxinological evaluations of an Argentinian strain of Halamphora coffeaeformis with potential as feedstock of triglycerides adequate for biodiesel as well as a source of high-value metabolite production, in order to confirm its identification and to define the safety of its biomass in relation to domoic acid production. The obtained results will strengthen and diversify the use of this species in the microalgal market.

Design, synthesis and cell imaging of a simple peptide-based probe for the selective detection of RNA.

Here we present a novel peptide-based fluorescent "turn-on" molecule P1 for detecting RNA, in a double or single strand, AU-rich or CG-rich. Both computational and experimental studies indicate that the detection efficiency depends on the binding affinity of P1 and conformational changes. P1 could be applied for cell imaging without any additional transfection vectors. Selective detection of RNA in cells was determined by RNase digestion. Successful application of P1 for RNA imaging in cell mitosis reveals that it may have broad applications in research, biotechnology and medical science.

Expression and characterization of family 40 Carbohydrate Binding Module (CBM) from Vibrio cholerae Non-O1 sialidase

Carbohydrate Binding Module (CBM) is a non-catalytic protein domain found in carbohydrate-active enzyme (glycoside hydrolase) and its role is to bring carbohydrates in close proximity to the enzyme catalytic site for complete hydrolysis. The removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. In this study, a gene encoding for family 40 CBM from Vibrio cholerae Non-O1 sialidase was cloned and successfully expressed in E. coli BL21 (DE3) strain. The CBM40 encoded 195 amino acids with 585 bp of nucleotide sequence. The protein was successfully expressed at 18°C when induced with 1 mM IPTG. Maximum expression was achieved at 20 hours after post-induction time. For purification of the protein, an anionic denaturing detergent method was used containing 1% SDS and 0.1% sarkosyl with gradient affinity elution at 50 mM imidazole concentrations. SDS-PAGE analysis of the purified CBM40 protein displayed a protein band with a molecular mass of 21 kDa. Protein characterization showed optimum stability in 100 mM citrate buffer pH 5.5, with the highest Tm value of 40 °C. The protein was stable between pH 5.5–6.2 and able to retain its activity at 27–56°C. The addition of Mn2+ and Mg2+ increased the protein melting temperature to 56°C. Meanwhile, the addition of reagents, such as 1% SDS and 1 M urea increased the protein melting temperature (Tm) to approximately 55°C. Protein stability can be influenced by many factors, including different buffers, pHs, temperatures, ionic strengths, and chemical reagents used in a study. The optimum characterization conditions established would further lead to the discovery of CBM40 protein true potential in enhancing substrate binding affinity and protein-carbohydrate recognition, which underpins its broad applications in biotechnology and protein engineering fields.

Gibbsian Surface Thermodynamics.

Gibbsian composite-system thermodynamics is the framework governing the equilibrium of composite systems, including systems that at equilibrium have more than one value of pressure because of the action of surface tension, semipermeable membranes, or fields, and thus cannot be treated as simple systems. J. W. Gibbs's paper that lays out composite-system thermodynamics, "On the Equilibrium of Heterogeneous Substances", communicated in two parts in 1876 and 1878, is widely regarded as one of the most important pieces of scientific literature of its century. Many scientists adopted and stressed the importance of Gibbsian thermodynamics. In 1960, H. B. Callen wrote a textbook that made Gibbsian composite-system thermodynamics more accessible to thermodynamicists. However, Callen's book left out Gibbs's work on curved fluid interfaces and did not treat the complicated nonideal systems of interest to today's thermodynamicists. In this Feature Article, I have attempted to convey in a comprehensive manner the framework of Gibbsian composite-system thermodynamics including in detail the treatment of systems with interface effects and with nonideal, multicomponent phases. This work lays out the relationships between important equilibrium equations including the following: the Gibbs-Duhem equation, the Gibbs adsorption equation, the Young-Laplace equation, the Young equation, the Cassie-Baxter equation, the Wenzel equation, the Kelvin equation, the Gibbs-Thompson equation, and the Ostwald-Freundlich equation, including nonideal and multicomponent forms. Equations of state that are often useful for Gibbsian composite-system thermodynamics are reviewed including adsorption isotherms and our own work on two semiempirical equations of state: the Elliott et al. form of the osmotic virial equation and the Shardt-Elliott-Connors-Wright equation for the temperature and composition dependence of surface tension. I summarize the work of our group developing Gibbisan composite-system thermodynamics including new equations for such things as the curvature-induced depression of the eutectic temperature or the removal of azeotropes by nanoscale fluid interface curvature. Gibbsian composite-system thermodynamics has broad applications in biotechnology, nanostructured materials, surface textures and coatings, microfluidics, nanoscience, atmospheric and environmental physics, among others.

Characterization and Optimization of a Deoxyribozyme with a Short Left Binding Arm.

Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have broad applications in biotechnology. In vitro selected RNA-cleaving deoxyribozymes normally contain two substrate-binding arms and a central catalytic core region. Here, we describe the systematic characterization and optimization of an RNA-cleaving deoxyribozyme with an unusually short left binding arm, and its special sequence requirement for its optimal catalytic activity.

Comparative Evaluation on Tannase Production by Lasiodiplodia plurivora ACN-10 under Submerged Fermentation (SmF) and Solid State Fermentation (SSF)

Aim: Tannase (tannin acyl hydrolase, E.C. 3.1.1.20) catalyzes the hydrolysis of ester bonds from complex hydrolysable tannins with the production of gallic acid and glucose and possess broad applications in biotechnology. This study is aimed at the production of tannase by Lasiodiplodia plurivora ACN-10 in SmF and SSF using Terminalia cattapa (almond leaves) and Magnifera indica (mango leaves) as substrates.
 Study Design: The design adopted to evaluate the production of tannase is submerged (SmF) and solid state (SSF) fermentation.
 Study Area: Federal Institute of Industrial Research Oshodi, Lagos State Nigeria.
 Methodology: Fifteen different soil samples were indiscriminately collected within Oshodi, Lagos, Nigeria and were inoculated on PDA plates at 30°C for 3-5 days. A total of 30 isolates was screened on Czapek dox minimal agar incorporated with 1% tannic acid and plates were incubated for 96 h at 30°C. Fungal isolates which were able to disintegrate tannic acid produced a clear halo zone around the colony diameter and were selected to be positive for tannase activity. The best isolate was identified based on its morphological, microscopic and molecular characteristics. Thereafter production and extraction of tannase was carried out in SmF for 0-120 h and in SSF for 0-144 h using Terminalia cattapa (Almond leaves) and Magnifera indica (Mango leaves) as substrates.
 Results: The total fungal count ranged from 1.0×104 to 3.5×105 CFU/g. A total of 30 fungal isolates produced clear halo zones (ranging from 20 to 70 mm) around the colonies during the screening with tannic acid. Isolate ACN-10, which showed the highest tannic degradation was identified based on its morphological and microscopic characteristics. On the basis of 18S rRNA gene sequence studies, the isolate was identified as Lasiodiplodia plurivora strain ACN-10 and the sequence was submitted to the Genbank with the accession number: MG250374. Results obtained in this study indicated that both substrates can be used by the isolate for tannase production in both SmF and SSF. The result of our investigation on the use of Terminalia cattapa (almond leaves) and Magnifera indica (mango leaves) as substrates for tannase production showed that optimum yield (6.064 U/ml) was obtained at 120 h in SSF while optimum production (4.623 U/ml) was observed in SmF at 96 h using Terminalia cattapa as substrate.
 Conclusion: Results obtained from this study indicated higher tannase production in solid-state fermentation compared to submerged fermentation. This is the first report to the best of our knowledge that Lasiodiplodia plurivora strain is implicated in tannase secretion. The result also demonstrated high production of extracellular tannases from low cost substrates which can be optimized and scaled up for industrial processes.

Microfluidic-assisted polymer-protein assembly to fabricate homogeneous functionalnanoparticles.

Functional polymer-protein nanoparticles (NPs) have broad applications in biotechnology and nanotechnology. In principle, controllable and vigorous mixing is required to fabricate homogeneous NPs, which remains a challenge via conventional bulk synthetic methods. In this study, an electrokinetics (EK) based microfluidic reactor with fast mixing is explored to assemble functional proteins with polymers in an ethanol/water co-solvent system. The resultant NPs show significantly improved size distribution by comparison with the ones prepared using conventional bulk method, while the NPs size can be tuned by adjusting the mass ratio of polymer to protein. The functionalities of the assembled proteins are sustained upon the EK based microfluidic mixing, indicating the application potential of our method in the controlled assembly of different functional proteins.

Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica.

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in 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 elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain.IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.