Next generation biofuel engineering in prokaryotes

Gronenberg, Luisa S.; Marcheschi, Ryan J.; Liao, James C.

doi:10.1016/j.cbpa.2013.03.037

Cited by 139 publications

(88 citation statements)

References 90 publications

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“…A yield of 36.7 Mg·ha −1 was achieved in field trials in Oklahoma (9), and switchgrass has the potential to produce 500% or more energy than is used for its cultivation (10). The use of abundant lignocellulosic plant biomass as feedstock is environmentally desirable and economically essential for enabling a viable biofuels industry (11). Current strategies for bioethanol production from lignocellulosic feedstocks require three major operational steps: physicochemical pretreatment, enzymatic saccharification, and fermentation ( Fig.…”

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confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

198

171

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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. metabolic engineering | bioenergy and thermophiles

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mentioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

198

171

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“…Since these organisms do not require fixed carbon feedstocks, they have great potential for synthetic biology and metabolic engineering applications (1)(2)(3)(4). Several strains of cyanobacteria have been engineered to act as microbial cellular factories for the production of fuels and chemicals, such as isobutanol, 2,3-butanediol, free fatty acids, and D-lactate (5)(6)(7). Despite these advances, none of these engineered strains has been able to achieve industrially relevant levels of productivity.…”

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confidence: 99%

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Berla

Pakrasi

2015

Appl Environ Microbiol

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Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO 2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the P cpc560 or the P trc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.C yanobacteria are oxygenic photosynthetic prokaryotes that use solar energy to fix CO 2 , converting it into biomass and valuable products. Since these organisms do not require fixed carbon feedstocks, they have great potential for synthetic biology and metabolic engineering applications (1-4). Several strains of cyanobacteria have been engineered to act as microbial cellular factories for the production of fuels and chemicals, such as isobutanol, 2,3-butanediol, free fatty acids, and D-lactate (5-7). Despite these advances, none of these engineered strains has been able to achieve industrially relevant levels of productivity. A lack of effective and well-characterized tools for synthetic biology in cyanobacteria has limited progress relative to that with other established microbial chassis, such as Escherichia coli or Saccharomyces cerevisiae (1). Synechocystis sp. strain PCC 6803 is a naturally transformable cyanobacterial chassis for synthetic biology. This strain carries several endogenous plasmids in addition to its single chromosome (8-11). Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13). Additionally, these plasmids increase their copy numbers during the transition from the exponential-growth phase to the stationary phase (14). Thus, we have hypothesized that the expression of heterologous genes from sites in these plasmids could be autoinduced by such a growth transition. This induction could be advantageous in a production system that first yields a dense culture of light-harvesting cyanobacteria and later uses those microbial cell factories to churn out a product of interest.One strat...

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“…While many thioesterases have been ex-plored for long-chain fatty acid production (14)(15)(16)(17), few studies have focused on those that prefer short-chain acyl-CoAs. Several broad-specificity acyl-CoA thioesterases, including E. coli TesB and Saccharomyces cerevisiae Pte1p, can be used for SCFA production but lack the specificity necessary for optimizing biosynthetic pathways (11,18).…”

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confidence: 99%

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

McMahon

Prather

2014

Appl Environ Microbiol

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b Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.

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Next generation biofuel engineering in prokaryotes

Cited by 139 publications

References 90 publications

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

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