Microbial Production Of Chemicals Research Articles

Microbial Degradation and Valorization of Plastic Wastes.

A growing accumulation of plastic wastes has become a severe environmental and social issue. It is urgent to develop innovative approaches for the disposal of plastic wastes. In recent years, reports on biodegradation of synthetic plastics by microorganisms or enzymes have sprung up, and these offer a possibility to develop biological treatment technology for plastic wastes. In this review, we have comprehensively summarized the microorganisms and enzymes that are able to degrade a variety of generally used synthetic plastics, such as polyethylene (PE), polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PUR), and polyethylene terephthalate (PET). In addition, we have highlighted the microbial metabolic pathways for plastic depolymerization products and the current attempts toward utilization of such products as feedstocks for microbial production of chemicals with high value. Taken together, these findings will contribute to building a conception of bio-upcycling plastic wastes by connecting the biodegradation of plastic wastes to the biosynthesis of valuable chemicals in microorganisms. Last, but not least, we have discussed the challenges toward microbial degradation and valorization of plastic wastes.

High rate 2,3-butanediol production with Vibrio natriegens

The aim of this study was to investigate if the high substrate turnover rates of Vibrio natriegens enable 2,3-butanediol production at high productivities. Introduction of a heterologous pathway led to 2,3-butanediol production at yields comparable to Escherichia coli using glucose, sucrose and sugar beet molasses. Microaerobic fed-batch cultivations maintained at 0–1% dissolved oxygen yielded a combined diol titer of 49.9 g L−1 of 2,3-butanediol and acetoin and a production rate of 3.9 g L−1 h−1. Optimization of the oxygen supply in chemostat cultivations increased the product yield to 70% of the theoretical maximum at volumetric and specific production rates of 2 g L−1 h−1 and 0.66 g g−1 h−1, respectively. In conclusion, this study could for the first time demonstrate V. natriegens as novel host for 2,3-butanediol production with high volumetric and specific productivity, underlining its potential for microbial chemical production.

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review.

Metabolic regulation of gene expression for the microbial production of fine chemicals, such as organic acids, is an important research topic in post-genomic metabolic engineering. In particular, the ability of transcription factors (TFs) to respond precisely in time and space to various small molecules, signals and stimuli from the internal and external environment is essential for metabolic pathway engineering and strain development. As a key component, TFs are used to construct many biosensors in vivo using synthetic biology methods, which can be used to monitor the concentration of intracellular metabolites in organic acid production that would otherwise remain “invisible” within the intracellular environment. TF-based biosensors also provide a high-throughput screening method for rapid strain evolution. Furthermore, TFs are important global regulators that control the expression levels of key enzymes in organic acid biosynthesis pathways, therefore determining the outcome of metabolic networks. Here we review recent advances in TF identification, engineering, and applications for metabolic engineering, with an emphasis on metabolite monitoring and high-throughput strain evolution for the organic acid bioproduction.

The effects of kanamycin concentration on gene transcription levels in Escherichia coli.

In the area of microbial production of valuable chemicals, plasmids have been widely applied for overexpressing rate-limiting enzymes with high yields. However, the effect of antibiotic concentrations on the transcription of target genes in E. coli is less involved in previous reports. In this study, we constructed E. coli strains expressing the reporter gene and the kanamycin resistant gene in an operon, and analyzed the transcription levels of the reporter gene and the fluorescent intensity of the recombinant E. coli under differentkanamycin concentrations. We found that the growth and gene transcription of the recombinant strain were affected obviously by the concentration of kanamycin, indicating the importance of fine-tuning of antibiotic concentrations in microbial fermentation.

Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis

Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here, we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic and thus far have proven difficult to recapitulate in the common hosts Escherichia coli and Saccharomyces cerevisiae.

Microbial production of bioactive chemicals for human health

Bioactive compounds are a collection of compounds that have certain effects on humans and animals that consume them. Since many bioactive compounds are beneficial to human health, they are attracting increasing attention in modern life, and their rising consumption stimulates the development of novel production modes that are more efficient than the traditional way, which relies on chemical synthesis or extraction from natural tissues. Among the emerging production techniques, biotechnology-based generation using fermentation of genetically engineered microorganisms shows great potential as an alternative to the current manufacturing systems, and has actually been applied for the industrial supply of some bioactive compounds. In this review, we highlight the microbial production of some typical bioactive compounds, including polyphenols, polysaccharides, amino acids, and vitamins. Also, we discuss the underlying disadvantages of some microbial systems and point out the future directions for system optimization.

Engineering transport systems for microbial production.

The rapid development in the field of metabolic engineering has enabled complex modifications of metabolic pathways to generate a diverse product portfolio. Manipulating substrate uptake and product export is an important research area in metabolic engineering. Optimization of transport systems has the potential to enhance microbial production of renewable fuels and chemicals. This chapter comprehensively reviews the transport systems critical for microbial production as well as current genetic engineering strategies to improve transport functions and thus production metrics. In addition, this chapter highlights recent advancements in engineering microbial efflux systems to enhance cellular tolerance to industrially relevant chemical stress. Lastly, future directions to address current technological gaps are discussed.

Acetogenic Fermentation From Oxygen Containing Waste Gas.

The microbial production of bulk chemicals from waste gas is becoming a pertinent alternative to industrial strategies that rely on fossil fuels as substrate. Acetogens can use waste gas substrates or syngas (CO, CO2, H2) to produce chemicals, such as acetate or ethanol, but as the feed gas often contains oxygen, which inhibits acetogen growth and product formation, a cost-prohibitive chemical oxygen removal step is necessary. Here, we have developed a two-phase microbial system to facilitate acetate production using a gas mixture containing CO and O2. In the first phase the facultative anaerobic carboxydotroph Parageobacillus thermoglucosidasius was used to consume residual O2 and produce H2 and CO2, which was subsequently utilized by the acetogen Clostridium ljungdahlii for the production of acetate. From a starting amount of 3.3 mmol of CO, 0.52 mmol acetate was produced in the second phase by C. ljungdahlii. In this set-up, the yield achieved was 0.16 mol acetate/mol CO, a 63% of the theoretical maximum. This system has the potential to be developed for the production of a broad range of bulk chemicals from oxygen-containing waste gas by using P. thermoglucosidasius as an oxygen scrubbing tool.

Yarrowia lipolytica: more than an oleaginous workhorse

Microbial production of fuels and chemicals offers a means by which sustainable product manufacture can be achieved. In this regard, Yarrowia lipolytica is a unique microorganism suitable for a diverse array of biotechnological applications. As a robust oleaginous yeast, it has been well studied for production of fuels and chemicals derived from fatty acids. However, thanks in part to newfound genetic tools and metabolic understanding, Y. lipolytica has been explored for high-level production of a variety of non-lipid products. This mini-review will discuss some of the recent research surrounding the ability of Y. lipolytica to support bio-based chemical production outside the realm of fatty acid metabolism including polyketides, terpenes, carotenoids, pentose phosphate-derived products, polymers, and nanoparticles.

Synthesis and analysis of separation processes for extracellular chemicals generated from microbial conversions

Recent advances in metabolic engineering have enabled the production of chemicals via bio-conversion using microbes. However, downstream separation accounts for 60–80% of the total production cost in many cases. Previous work on microbial production of extracellular chemicals has been mainly restricted to microbiology, biochemistry, metabolomics, or techno-economic analysis for specific product examples such as succinic acid, xanthan gum, lycopene, etc. In these studies, microbial production and separation technologies were selected apriori without considering any competing alternatives. However, technology selection in downstream separation and purification processes can have a major impact on the overall costs, product recovery, and purity. To this end, we apply a superstructure optimization based framework that enables the identification of critical technologies and their associated parameters in the synthesis and analysis of separation processes for extracellular chemicals generated from microbial conversions. We divide extracellular chemicals into three categories based on their physical properties, such as water solubility, physical state, relative density, volatility, etc. We analyze three major extracellular product categories (insoluble light, insoluble heavy and soluble) in detail and provide suggestions for additional product categories through extension of our analysis framework. The proposed analysis and results provide significant insights for technology selection and enable streamlined decision making when faced with any microbial product that is released extracellularly. The parameter variability analysis for the product as well as the associated technologies and comparison with novel alternatives is a key feature which forms the basis for designing better bioseparation strategies that have potential for commercial scalability and can compete with traditional chemical production methods.

Reconstruction of metabolic pathway for isobutanol production in Escherichia coli

BackgroundThe microbial production of useful fuels and chemicals has been widely studied. In several cases, glucose is used as the raw material, and almost all microbes adopt the Embden–Meyerhof (EM) pathway to degrade glucose into compounds of interest. Recently, the Entner–Doudoroff (ED) pathway has been gaining attention as an alternative strategy for microbial production.ResultsIn the present study, we attempted to apply the ED pathway for isobutanol production in Escherichia coli because of the complete redox balance involved. First, we generated ED pathway-dependent isobutanol-producing E. coli. Thereafter, the inactivation of the genes concerning organic acids as the byproducts was performed to improve the carbon flux to isobutanol from glucose. Finally, the expression of the genes concerning the ED pathway was modified.ConclusionsThe optimized isobutanol-producing E. coli produced 15.0 g/L of isobutanol as the final titer, and the yield from glucose was 0.37 g/g (g-glucose/g-isobutanol).

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.

Design and development of a “Y-shaped” microbial consortium capable of simultaneously utilizing biomass sugars for efficient production of butanol

Microbial production of chemicals from lignocellulosic biomass is usually hampered by the low efficiency of the simultaneous utilization of C5 and C6 sugars. In nature, this is not a problem because different C5- and C6-utilizing microorganisms cooperate. Nevertheless, the diverse metabolism of microorganisms in nature makes it difficult to synchronize the utilization of biomass sugars toward a specific goal. To address this problem, we sought to develop a novel microbial consortium that can mimic nature's ability of efficiently use biomass sugars, while synchronizing this capability toward a useful goal to maximize the power of nature and engineering. Starting from a completely chromosomally engineered butanol hyper-producing Escherichia coli strain that we developed previously, we developed a consortium comprising two E. coli strains with nearly identical genomic backgrounds, thus creating a “Y-shaped” consortium with two different “heads” (using xylose or glucose) but the same “body” (from glycolysis to butanol production). This “Y-shaped” chimeric consortium achieved the most efficient butanol production from mixed sugars reported to date, by equally efficient and orthogonal consumption of C5 and C6 sugars. Furthermore, we show that the consortium structure is not only adaptive to environmental perturbations, but can be arbitrarily changed to simultaneously utilize C5/C6 sugars in different ratio. The design and development of such a “Y-shaped” chimeric consortium provides a novel approach to address the need for simultaneous efficient utilization of different biomass sugars for the production of useful chemicals.

Production of Natural 2-Phenylethanol from Glucose or Glycerol with CoupledEscherichia coliStrains Expressingl-Phenylalanine Biosynthesis Pathway and Artificial Biocascades

2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high price and very limited availability. Microbial synthesis from glucose or glycerol is an attractive way to produce natural 2-PE, but suffers from low product titer. Escherichia coli NST74-Phe-Sty was engineered to coexpress the l-phenylalanine (l-Phe) biosynthesis pathway and enzyme cascade of l-Phe to 2-PE, producing only 0–0.17 g/L 2-PE from glucose at 22–37 °C due to the incompatibility of the temperature for enzyme expression and activity. Enhanced production of 2-PE (8.4–9.1 g/L) from glucose or glycerol was achieved by coupling of E. coli NST74-Phe expressing the l-Phe biosynthesis pathway for l-Phe production at the optimal temperature of 37 °C with E. coli T7-Sty expressing enzyme cascades of l-Phe to 2-PE at the optimal expression temperature of 22 °C and the optimal biotransformation temperature of 30 °C. The 2-PE titer is 4.7-fold higher than the best reported 2-PE concentration produced from glucose. The coupled strains approach could be generally applicable for enhancing microbial production of useful chemicals from sugars or glycerol.

Metabolic engineering of Corynebacterium glutamicum for hyperproduction of polymer-grade L- and D-lactic acid.

Strain development is critical for microbial production of bio-based chemicals. The stereo-complex form of polylactic acid, a complex of poly-L- and poly-D-lactic acid, is a promising polymer candidate due to its high thermotolerance. Here, we developed Corynebacterium glutamicum strains producing high amounts of L- and D-lactic acid through intensive metabolic engineering. Chromosomal overexpression of genes encoding the glycolytic enzymes, glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, triosephosphate isomerase, and enolase, increased L- and D-lactic acid concentration by 146% and 56%, respectively. Chromosomal integration of two genes involved in the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-dehydro-3-deoxyphosphogluconate aldolase), together with a gene encoding glucose-6-phosphate dehydrogenase from Zymomonas mobilis, to bypass the carbon flow from glucose, further increased L- and D-lactic acid concentration by 11% and 44%, respectively. Finally, additional chromosomal overexpression of a gene encoding NADH dehydrogenase to modulate the redox balance resulted in the production of 212g/L L-lactic acid with a 97.9% yield and 264g/L D-lactic acid with a 95.0% yield. The optical purity of both L- and D-lactic acid was 99.9%. Because the constructed metabolically engineered strains were devoid of plasmids and antibiotic resistance genes and were cultivated in mineral salts medium, these strains could contribute to the cost-effective production of the stereo-complex form of polylactic acid in practical scale.

Bioprospecting of Native Efflux Pumps To Enhance Furfural Tolerance in Ethanologenic Escherichia coli.

Efficient microbial conversion of lignocellulose into valuable products is often hindered by the presence of furfural, a dehydration product of pentoses in hemicellulose sugar syrups derived from woody biomass. For a cost-effective lignocellulose microbial conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic activities. A comprehensive plasmid-based library encoding native multidrug resistance (MDR) efflux pumps, porins, and select exporters from Escherichia coli was screened for furfural tolerance in an ethanologenic E. coli strain. Small multidrug resistance (SMR) pumps, such as SugE and MdtJI, as well as a lactate/glycolate:H+ symporter, LldP, conferred furfural tolerance in liquid culture tests. Expression of the SMR pump potentially increased furfural efflux and cellular viability upon furfural assault, suggesting novel activities for SMR pumps as furfural efflux proteins. Furthermore, induced expression of mdtJI enhanced ethanol fermentative production of LY180 in the presence of furfural or 5-hydroxymethylfurfural, further demonstrating the applications of SMR pumps. This work describes an effective approach to identify useful efflux systems with desired activities for nonnative toxic chemicals and provides a platform to further enhance furfural efflux by protein engineering and mutagenesis.IMPORTANCE Lignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction of hemicellulose by thermochemical processes, side products that inhibit cell growth and production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance by expressing multidrug resistance (MDR) pumps with putative efflux activities for furan aldehydes. The developed plasmid library and screening methods may facilitate new discoveries of MDR pumps for diverse toxic chemicals important for microbial conversion.

Engineering of phosphoenolpyruvate: carbohydrate phosphotransferase system increased acetate assimilation in Escherichia coli.

Acetate is a potential low-cost carbon source and can be used for microbial production of valuable chemicals in Escherichia coli. In this study, separate and simultaneous inactivation of the ptsG, ptsI, and ptsP genes involved in the phosphoenolpyruvate:carbohydrate phosphotransferase system was performed in E. coli and the effects on cell growth and acetate assimilation were evaluated. The mutant strain with double deletion of ptsG and ptsP exhibited faster acetate use than the other mutants. Inactivation of ptsI seriously reduced acetate consumption. This work provides a novel engineering target for improving acetate use rates.

Evolutionary engineering of Escherichia coli for improved anaerobic growth in minimal medium accelerated lactate production

Anaerobic fermentation is a favorable process for microbial production of bulk chemicals like ethanol and organic acids. Low productivity is the bottleneck of several anaerobic processes which has significant impact on the technique competitiveness of production strain. Improving growth rate of production strain can speed up the total production cycle and may finally increase productivity of anaerobic processes. In this work, evolutionary engineering of wild-type strain Escherichia coli W3110 was adopted to improve anaerobic growth in mineral medium. Significant increases in exponential growth rate and stationary cell density were achieved in evolved strain WE269, and a 96.5% increase in lactate productivity has also been observed in batch fermentation of this strain with M9 minimal medium. Then, an engineered strain for lactate production (BW100) was constructed by using WE269 as a platform and 98.3g/L lactate (with an optical purity of D-lactate above 95%) was produced in a 5-L bioreactor after 48h with a productivity of 2.05g/(L·h). Finally, preliminary investigation demonstrated that mutation in sucD (sucD M245I) (encoding succinyl-CoA synthetase); ilvG (ilvG Δ1bp) (encoding acetolactate synthase 2 catalytic subunit), and rpoB (rpoB T1037P) (encoding RNA polymerase β subunit) significantly improved anaerobic growth of E. coli. Double-gene mutation in ilvG and sucD resumed most of the growth potential of evolved strain WE269. This work suggested that improving anaerobic growth of production host can increase productivity of organic acids like lactate, and specific mutation-enabled improved growth may also be applied to metabolic engineering for production of other bulk chemicals.

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis

BackgroundEfficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production.ResultsThe aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l−1 h−1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l−1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l−1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals.ConclusionA potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5–C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l−1 of C6 and C7 ethyl ketones and several hundred mg · l−1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.