Petroleum-based Production Research Articles

Cell-free systems: A synthetic biology tool for rapid prototyping in metabolic engineering.

Microbial cell factories provide sustainable alternatives to petroleum-based chemical production using cost-effective substrates. A deep understanding of their metabolism is essential to harness their potential along with continuous efforts to improve productivity and yield. However, the construction and evaluation of numerous genetic variants are time-consuming and labor-intensive. Cell-free systems (CFSs) serve as powerful platforms for rapid prototyping of genetic circuits, metabolic pathways, and enzyme functionality. They offer numerous advantages, including minimizing unwanted metabolic interference, precise control of reaction conditions, reduced labor, and shorter Design-Build-Test-Learn cycles. Additionally, the introduction of in vitro compartmentalization strategies in CFSs enables ultra-high-throughput screening in physically separated spaces, which significantly enhances prototyping efficiency. This review highlights the latest examples of using CFS to overcome prototyping limitations in living cells with a focus on rapid prototyping, particularly regarding gene regulation, enzymes, and multienzymatic reactions in bacteria. Finally, this review evaluates CFSs as a versatile prototyping platform and discusses its future applications, emphasizing its potential for producing high-value chemicals through microbial biosynthesis.

Mid-Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects.

Mid-to-long-chain dicarboxylic acids (DCAi, i ≥ 6) are organic compounds in which two carboxylic acid functional groups are present at the terminal position of the carbon chain. These acids find important applications as structural components and intermediates across various industrial sectors, including organic compound synthesis, food production, pharmaceutical development, and agricultural manufacturing. However, conventional petroleum-based DCA production methods cause environmental pollution, making sustainable development challenging. Hence, the demand for eco-friendly processes and renewable raw materials for DCA production is rising. Owing to advances in systems metabolic engineering, new tools from systems biology, synthetic biology, and evolutionary engineering can now be used for the sustainable production of energy-dense biofuels. Here, we explore systems metabolic engineering strategies for DCA synthesis in various chassis via the conversion of different raw materials into mid-to-long-chain DCAs. Subsequently, we discuss the future challenges in this field and propose synthetic biology approaches for the efficient production and successful commercialization of these acids.

Purple non-sulfur bacteria and the circular economy

Purple non-sulfur bacteria and the circular economy Arpita Bose, Associate Professor at Washington University in St. Louis, discusses the potential of microbial solutions in supporting sustainable and environmentally responsible alternatives to the traditional linear economy. Earth’s climate is undergoing unprecedented changes due to human activities, primarily the emission of greenhouse gases. Widespread petroleum-based production of fuels and plastics releases large amounts of pollution, contributing to rising global temperatures, extreme weather events, and ecosystem disruptions. Finding feasible solutions to the climate crisis is crucial to preserve essential resources and protect human and environmental health. Harnessing and strengthening the natural capabilities of microorganisms and microbial communities with synthetic biology will be the key to reducing and upcycling waste for a greener global economy.

Current Status and Challenges for Metal-Organic-Framework-Assisted Conversion of Biomass into Value-Added Chemicals.

Owing to the abundance of availability, low cost, and environmental-friendliness, biomass waste could serve as a prospective renewable source for value-added chemicals. Nevertheless, biomass conversion into chemicals is quite challenging due to the heterogeneous nature of biomass waste. Biomass-derived chemicals are appealing sustainable solutions that can reduce the dependency on existing petroleum-based production. Metal-organic frameworks (MOFs)-based catalysts and their composite materials have attracted considerable amounts of interest in biomass conversion applications recently because of their interesting physical and chemical characteristics. Due to their tunability, the catalytic activity and selectivity of MOF-based catalyst/composite materials can be tailored by functionalizing them with a variety of functional groups to enhance biomass conversion efficiency. This review focuses on the catalytic transformation of lignocellulosic biomass into value-added chemicals by employing MOF-based catalyst/composite materials. The main focus is given to the production of the platform chemicals HMF and Furfural from the corresponding (hemi)cellulosic biomass, due to their versatility as intermediates for the production of various biobased chemicals and fuels. The effects of different experimental parameters on the conversion of biomass by MOF-based catalysts are also included. Finally, current challenges and perspectives of biomass conversion into chemicals by MOF-based catalysts are highlighted.

Enhancing 3-hydroxypropionic acid production in Saccharomyces cerevisiae through enzyme localization within mitochondria

Microbial 3-hydroxypropionic acid (3-HP) production can potentially replace petroleum-based production methods for acrylic acid. Here, we constructed a yeast strain that expressed enzymes related to 3-HP biosynthesis within the mitochondria. This approach aimed to enhance the 3-HP production by utilizing the mitochondrial acetyl-CoA, an important intermediate for synthesizing 3-HP. The strain that expressed 3-HP-producing enzymes in the mitochondria (YPH-mtA3HP) showed improved production of 3-HP compared to that shown by the strain expressing 3-HP-producing enzymes in the cytosol (YPH-cyA3HP). Additionally, cMCR was overexpressed, which regulates a rate-limiting reaction in synthesizing 3-HP. In this study, we aimed to further enhance 3-HP production by expressing multiple copies of cMCR in the mitochondria using the δ-integration strategy to optimize the expression level of cMCR (YPH-mtA3HPx*). The results of flask-scale cultivation showed that 3-HP production by cMCR δ-integration was significantly higher, exhibiting a yield of 160 mg/L in YPH-mtA3HP6* strain and 257 mg/L in YPH-mtA3HP22* strain. Notably, YPH-mtA3HP22*, exhibited the highest 3-HP titer, which was 3.2-fold higher than that of YPH-cyA3HP. Our results demonstrated the potential of utilizing the mitochondrial compartment within S. cerevisiae for enhancing 3-HP production.

CO2 Emissions from Plastic Consumption Behaviors in Thailand

Plastic waste is an environmental crisis that is becoming increasingly well-documented. The rapid expansion of plastic manufacturing and consumption has led to a harmful cycle of pollution and greenhouse gas emissions due to petroleum-based production and plastic waste disposal. Plastic production and disposal depend on the consumption behavior of people. This study aimed to examine the plastic consumption behavior in Thailand and its impact on climate change at the end-of-life stage. The general information, plastic consumption, and plastic waste management were collected via questionnaires for each product lifetime, including single-use, medium-use, and long-use plastics. Based on 567 questionnaires, the results showed that people consumed single-use plastic, e.g., plastic bag, food container, cutlery, straws, and bottles, at a rate of about nine pieces/household/day or three pieces/cap/day. The medium-use and long-use plastic were 10 pieces/household/month and 50 pieces/household/year, respectively. It should be remarked that population density, education, and number of household members affected plastic consumption behavior, especially for single-use plastic. Regarding the disposal of end-of-life plastics, Thai people, on average, contribute 0.15 kg CO2eq/household/day to climate change. Many households have mismanaged waste by open dumping and open burning. Therefore, practicing proper waste management will help Thailand on the path to carbon neutrality in the future.

Succinic Acid Production from Glycerol by Actinobacillus succinogenes: Techno-economic, environmental, and exergy analyses

A significant commercial chemical namely succinic acid can be produced using glycerol from the biodiesel industry. Previously, key attributes of the upstream and the downstream processing in the synthesis of bio-based succinic acid were identified, which substantially influence the technological competitiveness compared to petroleum-based production. Accordingly, this study aims to simulate succinic acid production from glycerol with the most promising technology. The techniques of reactive extraction and direct crystallization, as well as the use of dimethyl sulfoxide (DMSO) as an electron acceptor, are investigated. Selected scenarios are evaluated based on techno-economic, energy, exergy, and greenhouse gas (GHG) emissions performances to acquire a broader perspective on the sustainability of the technology. The obtained results indicate that the addition of DMSO in the fermentation is key for the bio-based succinic acid production – the best profit of 190 million USD of the net present value, 33.3% of the internal rate of return, and 4.48 years of the payout period are estimated. Additionally, bio-based succinic acid production could compete with the petrochemical route, particularly the reduction of GHG by 26%. Therefore, the production of succinic acid from glycerol is feasible given the positive net present value and lower GHG emissions compared to the petrochemical route.

Phenol-rich bio-oil from pyrolysis of palm kernel shell and its isolated lignin

Phenol and phenolic compounds are the important chemicals for several industries. To reduce the dependence on the petroleum-based production, lignocellulosic biomass becomes the promising resource. Palm kernel shell and its real isolated lignin were comparatively studied for the potential on the production of phenol and phenolic compounds via the pyrolysis process. Both samples were subjected to various chemical and thermal analyses to compare their chemical structures and thermal behavior. The pyrolysis process of palm kernel shell and its lignin was performed with a fixed-bed reactor to generate bio-oil with a high fraction of phenol and phenolic compounds. The extraction process could affect the structure of lignin and the pyrolysis behavior of the lignin. Pyrolysis experiments showed that the isolated lignin gave a higher yield of bio-oil than the palm kernel shell and about twice the total fraction of phenol and phenolic compounds. Phenol production from palm kernel shell was highest at a pyrolysis temperature of 500 °C, while yields of other phenolic compounds were highest at 400 °C. Including phenol, H-type phenolic compounds were the main product from both palm kernel shell and its isolated lignin. C-type phenolic compounds were found in significant quantities in the bio-oil from lignin. The total fraction of phenol and other phenolic compounds from the lignin bio-oil was up to 78 % and higher than that from the palm kernel shell bio-oil. • Isolated lignin of PKS was studied for phenol and phenolic production. • Highest production of phenol was from the pyrolysis of PKS at 500 °C. • Lignin generated more total phenol and phenolic compounds than PKS. • Lignin gave 78 % of phenol and phenolic compounds in bio-oil.

PHB Processability and Property Improvement with Linear-Chain Polyester Oligomers Used as Plasticizers.

In 2021, global petroleum-based plastic production reached over 400 million metric tons (Mt), and the accumulation of these non-biodegradable plastics in the environment is a worldwide concern. Polyhydroxybutyrate (PHB) offers many advantages over traditional petroleum-based plastics, being biobased, completely biodegradable, and non-toxic. However, its production and use are still challenging due to its low deformation capacity and narrow processing window. In this work, two linear-chain polyester oligomers were used as plasticizers to improve the processability and properties of PHB. Thermal analyses, XRD, and polarized optical microscopy were performed to evaluate the plasticizing effect on the PHB and the reflection on the mechanical behavior. Both oligomers acted as PHB plasticizers, with a reduction in Tg and Tm as a function of the plasticizer concentration, which can make it easier to handle the material in thermal processing and reduce the probability of thermal degradation. Plasticizer 2 proved to be the most promising between the two with an optimized condition of 20%, in which there was a decrease in elastic modulus of up to 72% and an increase in the maximum elongation of 467%.

Regulation of carbon flux and NADH/NAD+ supply to enhance 2,3-butanediol production in Enterobacter aerogenes

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD+ was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD+ by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD+ by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications.

Opportunities in the microbial valorization of sugar industrial organic waste to biodegradable smart food packaging materials

Many petroleum-derived plastics, including food packaging materials are non-biodegradable and designed for single-use applications. Annually, around 175 Mt. of plastic enters the land and ocean ecosystems due to mismanagement and lack of techno economically feasible plastic waste recycling technologies. Renewable sourced, biodegradable polymer-based food packaging materials can reduce this environmental pollution. Sugar production from sugarcane or sugar beet generates organic waste streams that contain fermentable substrates, including sugars, acids, and aromatics. Microbial metabolism can be leveraged to funnel those molecules to platform chemicals or biopolymers to generate biodegradable food packaging materials that have active or sensing molecules embedded in biopolymer matrices. The smart package can real-time monitor food quality, assure health safety, and provide economic and environmental benefits. Active packaging materials display functional properties such as antimicrobial, antioxidant, and light or gas barrier. This article provides an overview of potential biodegradable smart/active polymer packages for food applications by valorizing sugar industry-generated organic waste. We highlight the potential microbial pathways and metabolic engineering strategies to biofunnel the waste carbon efficiently into the targeted platform chemicals such as lactic, succinate, muconate, and biopolymers, including polyhydroxyalkanoates, and bacterial cellulose. The obtained platform chemicals can be used to produce biodegradable polymers such as poly (butylene adipate-co-terephthalate) (PBAT) that could replace incumbent polyethylene and polypropylene food packaging materials. When nanomaterials are added, these polymers can be active/smart. The process can remarkably lower the greenhouse gas emission and energy used to produce food-packaging material via sugar industrial waste carbon relative to the petroleum-based production. The proposed green routes enable the valorization of sugar processing organic waste into biodegradable materials and enable the circular economy.

The Effect of Alkali Treatment on Pineapple Leaf Fibers (PALF) on the Performance of PALF Reinforced Rice Starch Biocomposites

ABSTRACT In recent years, petroleum-based plastic production has increased exponentially, but its negative impact on the environment, wildlife, and human life is not getting much attention. It is high time now to search for possible alternative materials to replace these plastics. The present study investigates the change in surface morphology, mechanical, and thermal properties of rice starch bioplastic films reinforced with untreated and treated pineapple leaf fibers (PALF). Three types of films were synthesized: Type A (Pure rice starch film), Type B (Rice starch film reinforced with untreated PALF), Type C (Rice starch film reinforced with alkaline-treated PALF) using starch and PALF in the 10:1 weight ratio. It was observed that after alkaline treatment of PALF, interfacial bonding between PALF and rice starch matrix was improved, which further helped to increase the tensile strength of the films. Dynamic mechanical analysis of films showed an increase in storage and loss modulus values after incorporating PALF into the rice starch matrix. The thermal stability of films was also improved after incorporating treated PALF into it because of the removal of low molecular weight components from the surface of PALF. Synthesized bioplastic-composite films were also biodegradable.

Surfactant-Induced Reconfiguration of Urea-Formaldehyde Resins Enables Improved Surface Properties and Gluability of Bamboo.

The high-efficiency development and utilization of bamboo resources can greatly alleviate the current shortage of wood and promote the neutralization of CO2. However, the wide application of bamboo-derived products is largely limited by their unideal surface properties with adhesive as well as poor gluability. Herein, a facile strategy using the surfactant-induced reconfiguration of urea-formaldehyde (UF) resins was proposed to enhance the interface with bamboo and significantly improve its gluability. Specifically, through the coupling of a variety of surfactants, the viscosity and surface tension of the UF resins were properly regulated. Therefore, the resultant surfactant reconfigured UF resin showed much-improved wettability and spreading performance to the surface of both bamboo green and bamboo yellow. Specifically, the contact angle (CA) values of the bamboo green and bamboo yellow decreased from 79.6° to 30.5° and from 57.5° to 28.2°, respectively, with the corresponding resin spreading area increasing from 0.2 mm2 to 7.6 mm2 and from 0.1 mm2 to 5.6 mm2. Moreover, our reconfigured UF resin can reduce the amount of glue spread applied to bond the laminated commercial bamboo veneer products to 60 g m−2, while the products prepared by the initial UF resin are unable to meet the requirements of the test standard, suggesting that this facile method is an effective way to decrease the application of petroleum-based resins and production costs. More broadly, this surfactant reconfigured strategy can also be performed to regulate the wettability between UF resin and other materials (such as polypropylene board and tinplate), expanding the application fields of UF resin.

Bioaugmented Mixed Culture by Clostridium aceticum to Manipulate Volatile Fatty Acids Composition From the Fermentation of Cheese Production Wastewater.

Production of targeted volatile fatty acid (VFA) composition by fermentation is a promising approach for upstream and post-stream VFA applications. In the current study, the bioaugmented mixed microbial culture by Clostridium aceticum was used to produce an acetic acid dominant VFA mixture. For this purpose, anaerobic sequencing batch reactors (bioaugmented and control) were operated under pH 10 and fed by cheese processing wastewater. The efficiency and stability of the bioaugmentation strategy were monitored using the production and composition of VFA, the quantity of C. aceticum (by qPCR), and bacterial community profile (16S rRNA Illumina Sequencing). The bioaugmented mixed culture significantly increased acetic acid concentration in the VFA mixture (from 1170 ± 18 to 122 ± 9 mgCOD/L) compared to the control reactor. Furthermore, the total VFA production (from 1254 ± 11 to 5493 ± 36 mgCOD/L) was also enhanced. Nevertheless, the bioaugmentation could not shift the propionic acid dominancy in the VFA mixture. The most significant effect of bioaugmentation on the bacterial community profile was seen in the relative abundance of the Thermoanaerobacterales Family III. Incertae sedis, its relative abundance increased simultaneously with the gene copy number of C. aceticum during bioaugmentation. These results suggest that there might be a syntropy between species of Thermoanaerobacterales Family III. Incertae sedis and C. aceticum. The cycle analysis showed that 6 h (instead of 24 h) was adequate retention time to achieve the same acetic acid and total VFA production efficiency. Biobased acetic acid production is widely applicable and economically competitive with petroleum-based production, and this study has the potential to enable a new approach as produced acetic acid dominant VFA can replace external carbon sources for different processes (such as denitrification) in WWTPs. In this way, the higher treatment efficiency for WWTPs can be obtained by recovered substrate from the waste streams that promote a circular economy approach.

Techno-economic and life cycle analysis of biofuel production via hydrothermal liquefaction of microalgae in a methanol-water system and catalytic hydrotreatment using hydrochar as a catalyst support

The hydrochar, a by-product of hydrothermal liquefaction (HTL) of algal biomass, was utilized through two methods; combustion and activation, for its usage as a source of heat and a catalyst support for hydrodeoxygenation (HDO) process in the production of algal biofuels. In this study, techno-economic analysis (TEA) and life cycle assessment (LCA) of algal biofuels production in a two-stage process (HTL and HDO) were investigated. Aspen plus simulation and SimaPro software were used to analyze process economics and greenhouse gas (GHG) emissions. Microalgae at 200 dry metric tonnes day−1 was the basis for its conversion to biocrude oil through HTL in the methanol-water system followed by catalytic upgrading to produce biofuels. According to HTL experimental results, maximum biocrude oil yield of 57.8 wt% was obtained using microalgae-solvent mass ratio and methanol-water mass ratio of 1:5 and 3:1, respectively. Produced biocrude oil contained 14.5 wt% of oxygen and HHV of 33.4 MJ kgbiocrude oil−1 which required upgrading to be utilized as a transportation fuel. HDO was employed to enhance the quality of biocrude oil with decrease in oxygen content (3.1 wt%) and increase in HHV (42 MJ kgbiofuel−1). The minimum fuel selling price (MFSP) for using method #2 (activation) was 2.2 $ L−1 to breakeven the cost of operation, which was about 10% lower than that from method #1 (combustion). The GHG emissions performance was estimated at −1.13 gCO2-eq MJ−1 indicating the significant GHG emissions reduction compared to petroleum-based fuels production.

Co-production of acetoin and succinic acid by metabolically engineered Enterobacter cloacae

BackgroundRenewable chemicals have attracted attention due to increasing interest in environmental concerns and resource utilization. Biobased production of industrial compounds from nonfood biomass has become increasingly important as a sustainable replacement for traditional petroleum-based production processes depending on fossil resources. Therefore, we engineered an Enterobacter cloacae budC and ldhA double-deletion strain (namely, EC∆budC∆ldhA) to redirect carbon fluxes and optimized the culture conditions to co-produce succinic acid and acetoin.ResultsIn this work, E. cloacae was metabolically engineered to enhance its combined succinic acid and acetoin production during fermentation. Strain EC∆budC∆ldhA was constructed by deleting 2,3-butanediol dehydrogenase (budC), which is involved in 2,3-butanediol production, and lactate dehydrogenase (ldhA), which is involved in lactic acid production, from the E. cloacae genome. After redirecting and fine-tuning the E. cloacae metabolic flux, succinic acid and acetoin production was enhanced, and the combined production titers of acetoin and succinic acid from glucose were 17.75 and 2.75 g L−1, respectively. Moreover, to further improve acetoin and succinic acid production, glucose and NaHCO3 modes and times of feeding were optimized during fermentation of the EC∆budC∆ldhA strain. The maximum titers of acetoin and succinic acid were 39.5 and 20.3 g L−1 at 72 h, respectively.ConclusionsThe engineered strain EC∆budC∆ldhA is useful for the co-production of acetoin and succinic acid and for reducing microbial fermentation costs by combining processes into a single step.

Engineering of Saccharomyces cerevisiae for efficient fermentation of cellulose.

Conversion of lignocellulosic biomass to biofuels using microbial fermentation is an attractive option to substitute petroleum-based production economically and sustainably. The substantial efforts to design yeast strains for biomass hydrolysis have led to industrially applicable biological routes. Saccharomyces cerevisiae is a robust microbial platform widely used in biofuel production, based on its amenability to systems and synthetic biology tools. The critical challenges for the efficient microbial conversion of lignocellulosic biomass by engineered S. cerevisiae include heterologous expression of cellulolytic enzymes, co-fermentation of hexose and pentose sugars, and robustness against various stresses. Scientists developed many engineering strategies for cellulolytic S. cerevisiae strains, bringing the application of consolidated bioprocess at an industrial scale. Recent advances in the development and implementation of engineered yeast strains capable of assimilating lignocellulose will be reviewed.

Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase

Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well.

The current strategies and parameters for the enhanced microbial production of 2,3-butanediol

2,3-Butanediol (2,3-BD) is a propitious compound with many industrial uses ranging from rubber, fuels, and cosmetics to food additives. Its microbial production has especially attracted as an alternative way to the petroleum-based production. However, 2,3-BD production has always been hampered by low yields and high production costs. The enhanced production of 2,3-butanediol requires screening of the best strains and a systematic optimization of fermentation conditions. Moreover, the metabolic pathway engineering is essential to achieve the best results and minimize the production costs by rendering the strains to use efficiently low cost substrates. This review is to provide up-to-date information on the current strategies and parameters for the enhanced microbial production of 2,3-BD.

Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.