Biodegradation of oxidized low density polyethylene by Pelosinus fermentans lipase.

Polyethylene (PE) exhibits high resistance to degradation, contributing to plastic pollution. PE discarded into the environment is photo-oxidized by sunlight and oxygen. In this study, a key enzyme capable of degrading oxidized PE is reported for the first time. Twenty different enzymes from various lipase families were evaluated for hydrolytic activity using substrates mimicking oxidized PE. Among them, Pelosinus fermentans lipase 1 (PFL1) specifically cleaved the ester bonds within the oxidized carbon-carbon backbone. Moreover, PFL1 (6 µM) degraded oxidized PE film, reducing the weight average and number average molecular weights by 44.6 and 11.3 %, respectively, within five days. Finally, structural analysis and molecular docking simulations were performed to elucidate the degradation mechanism of PFL1. The oxidized PE-degrading enzyme reported here will provide the groundwork for advancing PE waste treatment technology and for engineering microbes to repurpose PE waste into valuable chemicals.

Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

Biocompatible Cu/NiMo Composite Electrocatalyst for Hydrogen Evolution Reaction in Microbial Electrosynthesis; Unveiling the Self-Detoxification Effect of Cu.

H2-driven microbial electrosynthesis (MES) is an emerging bioelectrochemical technology that enables the production of complex compounds from CO2. Although the performance of microbial fermentation in the MES system is closely related to the H2 production rate, high-performing metallic H2-evolving catalysts (HEC) generate cytotoxic H2O2 and metal cations from undesirable side reactions, severely damaging microorganisms. Herein, a novel design for self-detoxifying metallic HEC, resulting in biologically benign H2 production, is reported. Cu/NiMo composite HEC suppresses H2O2 evolution by altering the O2 reduction kinetics to a four-electron pathway and subsequently decomposes the inevitably generated H2O2 in sequential catalytic and electrochemical pathways. Furthermore, in situ generated Cu-rich layer at the surface prevents NiMo from corroding and releasing cytotoxic Ni cations. Consequently, the Cu/NiMo composite HEC in the MES system registers a 50% increase in the performance of lithoautotrophic bacterium Cupriavidus necator H16, for the conversion of CO2 to a biopolymer, poly(3-hydroxybutyrate). This work successfully demonstrates the concept of self-detoxification in designing biocompatible materials for bioelectrochemical applications as well as MES systems.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Biotechnological Plastic Degradation and Valorization Using Systems Metabolic Engineering.

Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated 3-hydroxyvalerate fraction by overexpressing acetolactate synthase in Cupriavidus necator H16.

The elastomeric properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable copolymer, strongly depend on the molar composition of 3-hydroxyvalerate (3HV). This paper reports an improved artificial pathway for enhancing the 3HV component during PHBV biosynthesis from a structurally unrelated carbon source by Cupriavidus necator H16. To increase the intracellular accumulation of propionyl-CoA, a key precursor of the 3HV monomer, we developed a recombinant strain by genetically manipulating the branched-chain amino acid (e.g., valine, isoleucine) pathways. Overexpression of the heterologous feedback-resistant acetolactate synthase (alsS), (R)-citramalate synthase (leuA), homologous 3-ketothiolase (bktB), and the deletion of 2-methylcitrate synthase (prpC) resulted in biosynthesis of 42.5 % (g PHBV/g dry cell weight) PHBV with 64.9 mol% 3HV monomer from fructose as the sole carbon source. This recombinant strain also accumulated the highest PHBV content of 54.5 % dry cell weight (DCW) with 24 mol% 3HV monomer from CO2 ever reported. The lithoautotrophic cell growth and PHBV production by the recombinant C. necator were promoted by oxygen stress. The thermal properties of PHBV showed a decreasing trend of the glass transition and melting temperatures with increasing 3HV fraction. The average molecular weights of PHBV with modulated 3HV fractions were between 20 and 26 × 104 g/mol.

Effective hexanol production from carbon monoxide using extractive fermentation with Clostridium carboxidivorans P7.

This study achieved high production of hexanol via gas fermentation using Clostridium carboxidivorans P7 by extracting hexanol from the fermentation broth. The hexanol extraction efficiency and inhibitory effects on C. carboxidivorans P7 of 2-butyl-1-octanol, hexyl hexanoate and oleyl alcohol were examined, and oleyl alcohol was selected as the extraction solvent. Oleyl alcohol was added at the beginning of fermentation and during fermentation or a small volume of oleyl alcohol was repeatedly added during fermentation. The addition of a small volume of oleyl alcohol during fermentation was the most effective for CO consumption and hexanol production (5.06 g/L), yielding the highest known hexanol titer through any type of fermentation including gas fermentation. Hexanol production was further enhanced to 8.45 g/L with the repeated addition of oleyl alcohol and ethanol during gas fermentation. The results of this study will enable sustainable and carbon-neutral hexanol production via gas fermentation.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2.

BACKGROUND: A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization. RESULTS: Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2-fixing metabolism and PHB production were investigated. CONCLUSIONS: The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Production of Hexanol as the Main Product Through Syngas Fermentation by Clostridium carboxidivorans P7.

The production of hexanol from syngas by acetogens has gained attention as a replacement for petroleum-derived hexanol, which is widely used in the chemical synthesis and plastic industries. However, acetogenic bacteria generally produce C2 compounds (e.g., acetate and ethanol) as the main products. In this study, the gas fermentation conditions favorable for hexanol production were investigated at different temperatures (30-37°C) and CO gas contents (30-70%) in batch gas fermentation. Hexanol production increased from 0.02 to 0.09 g/L when the cultivation temperature was lowered from 37 to 30°C. As the CO content increased from 30 to 70%, the CO consumption rate and hexanol production (yield, titer, and ratio of C6 compound to total products) increased with the CO content. When 70% CO gas was repeatedly provided by flushing the headspace of the bottles at 30°C, the total alcohol production increased to 4.32 g/L at the expense of acids. Notably, hexanol production (1.90 g/L) was higher than that of ethanol (1.20 g/L) and butanol (1.20 g/L); this is the highest level of hexanol produced in gas fermentation to date and the first report of hexanol as the main product. Hexanol production was further enhanced to 2.34 g/L when 2 g/L ethanol was supplemented at the beginning of 70% CO gas refeeding fermentation. Particularly, hexanol productivity was significantly enhanced to 0.18 g/L/day while the supplemented ethanol was consumed, indicating that the conversion of ethanol to acetyl-CoA and reducing equivalents positively affected hexanol production. These optimized culture conditions (gas fermentation at 30°C and refeeding with 70% CO gas) and ethanol supplementation provide an effective and sustainable approach for bio-hexanol production.

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification.

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.

Glucose/Xylose Co-Fermenting Saccharomyces cerevisiae Increases the Production of Acetyl-CoA Derived n-Butanol From Lignocellulosic Biomass.

Efficient xylose catabolism in engineered Saccharomyces cerevisiae enables more economical lignocellulosic biorefinery with improved production yields per unit of biomass. Yet, the product profile of glucose/xylose co-fermenting S. cerevisiae is mainly limited to bioethanol and a few other chemicals. Here, we introduced an n-butanol-biosynthesis pathway into a glucose/xylose co-fermenting S. cerevisiae strain (XUSEA) to evaluate its potential on the production of acetyl-CoA derived products. Higher n-butanol production of glucose/xylose co-fermenting strain was explained by the transcriptomic landscape, which revealed strongly increased acetyl-CoA and NADPH pools when compared to a glucose fermenting wild-type strain. The acetate supplementation expected to support acetyl-CoA pool further increased n-butanol production, which was also validated during the fermentation of lignocellulosic hydrolysates containing acetate. Our findings imply the feasibility of lignocellulosic biorefinery for producing fuels and chemicals derived from a key intermediate of acetyl-CoA through glucose/xylose co-fermentation.

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae.

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.

Improved 2,3-butanediol yield and productivity from lignocellulose biomass hydrolysate in metabolically engineered Enterobacter aerogenes.

We previously engineered Enterobacter aerogenesfor glucose and xylose co-utilization and 2,3-butanediol production. Here, strain EMY-22 was further engineered to improve the 2,3-butanediol titer, productivity, and yield by reducing the production of byproducts. To reduce succinate production, the budABC operon and galP gene were overexpressed, which increased 2,3-butanediol production. For further reduction of succinate and 2-ketogluconate production, maeA was selected and overexpressed in EMY-22. The optimally engineered strain produced 2,3-butanediol for a longer time and showed reduced byproduct formation from sugarcane bagasse hydrolysate under flask cultivation conditions. The engineered strain displayed 66.6, 13.4, and 16.8% improvements in titer, yield, productivity of 2,3-butanediol, respectively, compared to its parental strain under fed-batch fermentation conditions. The data demonstrate that the metabolic engineering to reduce byproduct formation is a promising strategy to improve 2,3-butanediol production from lignocellulosic biomass.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocellulosic biorefinery.

BACKGROUND: Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. RESULTS: In this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L-1 glucose and 23.1 g L-1 xylose, within 24 h producing 30.1 g L-1 ethanol with a yield of 0.48 g g-1. CONCLUSIONS: Owing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.

Genomic and phenotypic characterization of a refactored xylose-utilizing Saccharomyces cerevisiae strain for lignocellulosic biofuel production.

BACKGROUND: Engineered strains of Saccharomyces cerevisiae have significantly improved the prospects of biorefinery by improving the bioconversion yields in lignocellulosic bioethanol production and expanding the product profiles to include advanced biofuels and chemicals. However, the lignocellulosic biorefinery concept has not been fully applied using engineered strains in which either xylose utilization or advanced biofuel/chemical production pathways have been upgraded separately. Specifically, high-performance xylose-fermenting strains have rarely been employed as advanced biofuel and chemical production platforms and require further engineering to expand their product profiles. RESULTS: In this study, we refactored a high-performance xylose-fermenting S. cerevisiae that could potentially serve as a platform strain for advanced biofuels and biochemical production. Through combinatorial CRISPR-Cas9-mediated rational and evolutionary engineering, we obtained a newly refactored isomerase-based xylose-fermenting strain, XUSE, that demonstrated efficient conversion of xylose into ethanol with a high yield of 0.43 g/g. In addition, XUSE exhibited the simultaneous fermentation of glucose and xylose with negligible glucose inhibition, indicating the potential of this isomerase-based xylose-utilizing strain for lignocellulosic biorefinery. The genomic and transcriptomic analysis of XUSE revealed beneficial mutations and changes in gene expression that are responsible for the enhanced xylose fermentation performance of XUSE. CONCLUSIONS: In this study, we developed a high-performance xylose-fermenting S. cerevisiae strain, XUSE, with high ethanol yield and negligible glucose inhibition. Understanding the genomic and transcriptomic characteristics of XUSE revealed isomerase-based engineering strategies for improved xylose fermentation in S. cerevisiae. With high xylose fermentation performance and room for further engineering, XUSE could serve as a promising platform strain for lignocellulosic biorefinery.

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production.

Advances in cellulosic conversion to fuels: engineering yeasts for cellulosic bioethanol and biodiesel production.

Cellulosic fuels are expected to have great potential industrial applications in the near future, but they still face technical challenges to become cost-competitive fuels, thus presenting many opportunities for improvement. The economical production of viable biofuels requires metabolic engineering of microbial platforms to convert cellulosic biomass into biofuels with high titers and yields. Fortunately, integrating traditional and novel engineering strategies with advanced engineering toolboxes has allowed the development of more robust microbial platforms, thus expanding substrate ranges. This review highlights recent trends in the metabolic engineering of microbial platforms, such as the industrial yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, for the production of renewable fuels.

Effect of manganese ions on ethanol fermentation by xylose isomerase expressing Saccharomyces cerevisiae under acetic acid stress.

The efficient fermentation of lignocellulosic hydrolysates in the presence of inhibitors is highly desirable for bioethanol production. Among the inhibitors, acetic acid released during the pretreatment of lignocellulose negatively affects the fermentation performance of biofuel producing organisms. In this study, we evaluated the inhibitory effects of acetic acid on glucose and xylose fermentation by a high performance engineered strain of xylose utilizing Saccharomyces cerevisiae, SXA-R2P-E, harboring a xylose isomerase based pathway. The presence of acetic acid severely decreased the xylose fermentation performance of this strain. However, the acetic acid stress was alleviated by metal ion supplementation resulting in a 52% increased ethanol production rate under 2g/L of acetic acid stress. This study shows the inhibitory effect of acetic acid on an engineered isomerase-based xylose utilizing strain and suggests a simple but effective method to improve the co-fermentation performance under acetic acid stress for efficient bioethanol production.

Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates.