Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 John UM (2013) Contribution of the Ethanol Industry to the ...Economy of the United States . Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii , which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol 12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.
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Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the ...tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.
Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for ...the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of these materials, however, present major ...barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium,
Pseudomonas putida
, to funnel these oxygenated compounds into a single exemplary chemical product, either β-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products.
Funneling mixed waste with microbes
Current plastic recycling methods require sorting by chemical composition, a method that is expensive and results in products that are of lower quality and value than the starting plastic. If plastic waste could instead be converted to valuable chemical intermediates, then economical use of mixed waste as a feedstock might be feasible. Sullivan
et al
. developed a two-stage oxidation and biological funneling approach that can break down and reform mixtures of common consumer plastics (see the Perspective by Yan). The end products can be adjusted by metabolic engineering of the microbes in the second step, which should enable tailored conversion into various platform or specialty chemicals. —MAF
Autoxidation and biological funneling converts mixed plastics to a single product.
Abstract
Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study,
...Pseudomonas putida
KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H
+
symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of
aroB
encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L
−1
muconate at 0.18 g L
−1
h
−1
and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.
Summary
Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways ...are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.
In this work, a series of metabolic engineering targets to improve mcl‐PHA production were combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene (phaZ) and genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted, while phaG, alkK, phaC1, and phaC2 were overexpressed to increase carbon flux from fatty acid biosynthesis to mcl‐PHA production. This strain demonstrated an increase in mcl‐PHA titer (g l−1) and yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and from lignin compared to the wild‐type strain, resulting in a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Consolidated bioprocessing (CBP) has the potential to reduce biofuel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of ...pre-manufactured cellulases. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Here we report the engineering of C. thermocellum to produce isobutanol. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichia coli. Then, these constructs were transformed and tested in C. thermocellum. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure. This strain produced 5.4g/L of isobutanol from cellulose in minimal medium at 50oC within 75h, corresponding to 41% of theoretical yield.
•Direct screening for promoter combinations to express isobutanol pathway in Clostridium thermocellum.•Isobutanol production from cellulose in minimal medium, achieving >5g/L titer and roughly 41% of theoretical yield.•Optimized conditions for isobutanol production.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further ...improve its CBP capabilities, a mutant strain of C. thermocellum was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulose Avicel, effectively eliminating formate, acetate, and lactate production and reducing H2 production by fivefold. On 5g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4mM in mutant AG553 on 20g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1mM ethanol on 10g/L Avicel. With the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.
•Eliminated genes involved in production of acetate, lactate, formate, and most H2.•Highest ethanol yield achieved to date in C. thermocellum.•More than doubled ethanol yield from pretreated switchgrass and poplar.•Increased ethanol titer at higher cellulose loadings.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Developing economically viable, scalable, and sustainable technologies for the conversion of lignocellulosic polysaccharides to liquid fuels is widely seen as a centerpiece of the global bioeconomy, ...and a key part of a multi-pronged approach to achieve carbon neutrality. Here we identify technology challenges and opportunities to achieve this promise. An overview of feedstocks, processes and products indicates that (1) biorefining at a scale sufficient to meaningfully impact climate change will likely involve fuels as the primary products, chemicals and biomaterials as co-products, and lignocellulose as the preferred feedstock; (2) microbial processing of cellulosic biomass will likely occur in the presence of solids, rather than involving solids-free sugar syrups, giving rise to challenges and constraints distinctive to lignocellulose; (3) anaerobic processing involves much lower costs than aerobic processing, making it more promising for fuel production; and (4) anaerobic production at high yields and broth titers has to date been reported only for molecules with ≤4 carbons. Some anaerobic bacteria are substantially more effective at polysaccharide deconstruction than aerobic fungi. Processes based on these microbes have great potential for cost reduction but require substantial research-driven advances. A mechanistic, functional group approach to product tolerance and inhibition is presented, separation technologies applicable to different product classes are surveyed, and perspectives are offered on opportunities to decrease product inhibition and the cost of product recovery. Pathways and research opportunities are considered for chemo-catalytic conversion of anaerobic fermentation products to larger fuel molecules. Fuel properties are considered for a broad range of biologically-derived products in relation to their suitability for various transport applications. Strategic perspectives are presented drawing on these diverse topics and insights. For multiple compounding reasons, features of small molecules make it less expensive to produce them biologically compared to large molecules, and this is particularly true for production from lignocellulose. Yet the fuels the world would most value producing from lignocellulosic biomass to address climate stabilization are large molecules compatible with heavy-duty, difficult-to-electrify transport applications. Hybrid processes wherein lignocellulose is converted biologically to small molecule intermediates and then converted chemo-catalytically to larger fuel molecules are a promising approach to reconciling this discrepancy.
Hybrid processes, featuring biological conversion of lignocellulose to small molecules followed by chemo-catalytic conversion to larger molecules suitable for difficult-to-electrify transport modes, are a promising route to biomass-derived fuels in demand for climate stabilization.
Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it ...for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida KT2440, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida KT2440 and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.
•Machine learning from 321 Pseudomonas putida RNA-seq samples yielded 84 iModulons.•We unveiled its transcriptional regulatory network by comparison with regulons.•Transcriptome changes can be comprehensively visualized by using the iModulons.•Activities of each iModulon in analyzed conditions are available on iModulonDB.
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