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Article ; Online: Recent progress in the synthesis of advanced biofuel and bioproducts.

Pfleger, Brian F / Takors, Ralf

2023 Volume 80, Page(s) 102913

Abstract: Energy is one of the most complex fields of study and an issue that influences nearly every aspect of modern life. Over the past century, combustion of fossil fuels, particularly in the transportation sector, has been the dominant form of energy release. ...

Abstract	Energy is one of the most complex fields of study and an issue that influences nearly every aspect of modern life. Over the past century, combustion of fossil fuels, particularly in the transportation sector, has been the dominant form of energy release. Refining of petroleum and natural gas into liquid transportation fuels is also the centerpiece of the modern chemical industry used to produce materials, solvents, and other consumer goods. In the face of global climate change, the world is searching for alternative, sustainable means of producing energy carriers and chemical building blocks. The use of biofuels in engines predates modern refinery optimization and today represents a small but significant fraction of liquid transportation fuels burnt each year. Similarly, white biotechnology has been used to produce many natural products through fermentation. The evolution of recombinant DNA technology into modern synthetic biology has expanded the scope of biofuels and bioproducts that can be made by biocatalysts. This opinion examines the current trends in this research space, highlighting the substantial growth in computational tools and the growing influence of renewable electricity in the design of metabolic engineering strategies. In short, advanced biofuel and bioproduct synthesis remains a vibrant and critically important field of study whose focus is shifting away from the conversion of lignocellulosic biomass toward a broader consideration of how to reduce carbon dioxide to fuels and chemical products.
MeSH term(s)	Biofuels ; Biotechnology/methods ; Fossil Fuels ; Carbon Dioxide ; Metabolic Engineering ; Biomass
Chemical Substances	Biofuels ; Fossil Fuels ; Carbon Dioxide (142M471B3J)
Language	English
Publishing date	2023-02-26
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2023.102913
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Article ; Online: Recent progress in the synthesis of advanced biofuel and bioproducts

Pfleger, Brian F. / Takors, Ralf

Current Opinion in Biotechnology. 2023, p.102913-

2023 , Page(s) 102913–

Abstract	Energy is one of the most complex fields of study and an issue that influences nearly every aspect of modern life. Over the past century, combustion of fossil fuels, particularly in the transportation sector, has been the dominant form of energy release. Refining of petroleum and natural gas into liquid transportation fuels is also the centerpiece of the modern chemical industry used to produce materials, solvents, and other consumer goods. In the face of global climate change, the world is searching for alternative, sustainable means of producing energy carriers and chemical building blocks. The use of biofuels in engines predates modern refinery optimization and today represents a small but significant fraction of liquid transportation fuels burnt each year. Similarly, white biotechnology has been used to produce many natural products through fermentation. The evolution of recombinant DNA technology into modern synthetic biology has expanded the scope of biofuels and bioproducts that can be made by biocatalysts. This opinion examines the current trends in this research space, highlighting the substantial growth in computational tools and the growing influence of renewable electricity in the design of metabolic engineering strategies. In short, advanced biofuel and bioproduct synthesis remains a vibrant and critically important field of study whose focus is shifting away from the conversion of lignocellulosic biomass towards a broader consideration of how to reduce carbon dioxide to fuels and chemical products.
Keywords	biofuels ; biomass ; biotechnology ; carbon dioxide ; chemical industry ; climate change ; combustion ; evolution ; fermentation ; lignocellulose ; liquids ; natural gas ; petroleum ; recombinant DNA ; renewable electricity ; synthetic biology ; transportation ; transportation industry
Language	English
Publishing place	Elsevier Ltd
Document type	Article ; Online
Note	Pre-press version
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2023.102913
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Article ; Online: Overcoming barriers to medium-chain fatty alcohol production.

Su, Yun / Mangus, Anna M / Cordell, William T / Pfleger, Brian F

Current opinion in biotechnology

2024 Volume 85, Page(s) 103063

Abstract: Medium-chain fatty alcohols ( ...

Abstract	Medium-chain fatty alcohols (
MeSH term(s)	Fatty Alcohols/metabolism ; Alcohols ; Metabolic Engineering ; Fatty Acids/metabolism
Chemical Substances	Fatty Alcohols ; Alcohols ; Fatty Acids
Language	English
Publishing date	2024-01-13
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2023.103063
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Article ; Online: Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production.

Mishra, Shivangi / Perkovich, Paul M / Mitchell, Wayne P / Venkataraman, Maya / Pfleger, Brian F

Journal of industrial microbiology & biotechnology

2024 Volume 51

Abstract: The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack ... ...

Abstract	The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.
MeSH term(s)	Cupriavidus necator/genetics ; Cupriavidus necator/metabolism ; Fatty Acids/metabolism ; Synthetic Biology ; Promoter Regions, Genetic ; Codon/genetics
Chemical Substances	Fatty Acids ; Codon
Language	English
Publishing date	2024-02-17
Publishing country	Germany
Document type	Journal Article
ZDB-ID	1482484-X
ISSN	1476-5535 ; 1367-5435
ISSN (online)	1476-5535
ISSN	1367-5435
DOI	10.1093/jimb/kuae008
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Article ; Online: Milligrams to kilograms: making microbes work at scale.

Cordell, William T / Avolio, Gennaro / Takors, Ralf / Pfleger, Brian F

Trends in biotechnology

2023 Volume 41, Issue 11, Page(s) 1442–1457

Abstract: If biomanufacturing can become a sustainable route for producing chemicals, it will provide a critical step in reducing greenhouse gas emissions to fight climate change. However, efforts to industrialize microbial synthesis of chemicals have met with ... ...

Abstract	If biomanufacturing can become a sustainable route for producing chemicals, it will provide a critical step in reducing greenhouse gas emissions to fight climate change. However, efforts to industrialize microbial synthesis of chemicals have met with varied success, due, in part, to challenges in translating laboratory successes to industrial scale. With a particular focus on Escherichia coli, this review examines the lessons learned when studying microbial physiology and metabolism under conditions that simulate large-scale bioreactors and methods to minimize cellular waste through reduction of maintenance energy, optimizing the stress response and minimizing culture heterogeneity. With general strategies to overcome these challenges, biomanufacturing process scale-up could be de-risked and the time and cost of bringing promising syntheses to market could be reduced.
MeSH term(s)	Bioreactors ; Industrial Microbiology
Language	English
Publishing date	2023-06-03
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	47474-5
ISSN	1879-3096 ; 0167-7799
ISSN (online)	1879-3096
ISSN	0167-7799
DOI	10.1016/j.tibtech.2023.05.002
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Article ; Online: Microbes paired for biological gas-to-liquids (Bio-GTL) process.

Pfleger, Brian F

Proceedings of the National Academy of Sciences of the United States of America

2016 Volume 113, Issue 14, Page(s) 3717–3719

MeSH term(s)	Greenhouse Effect ; Humans
Language	English
Publishing date	2016-04-05
Publishing country	United States
Document type	Journal Article ; Editorial ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't ; Comment
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1601926113
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Article ; Online: MALDI-MS screening of microbial colonies with isomer resolution to select fatty acid desaturase variants

Choe, Kisurb / Jindra, Michael A. / Hubbard, Susan C. / Pfleger, Brian F. / Sweedler, Jonathan V.

Analytical Biochemistry. 2023 July, v. 672 p.115169-

2023

Abstract: Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput ... ...

Abstract	Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid MS assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.
Keywords	Escherichia coli ; Thunbergia alata ; Umbellularia californica ; fatty acid desaturase ; fatty acids ; genes ; isomers ; ozone ; ozonolysis ; regioselectivity ; Double bonds ; Enzyme engineering ; Membrane lipids ; ACP ; CoA ; MALDI ; MS ; ToF ; E. coli ; IPTG ; PCR ; TaDes ; WT ; DMDS ; GC ; FID ; FFA ; KO ; OD ; T. alata ; BTE ; UVPD ; PB ; LB
Language	English
Dates of publication	2023-07
Publishing place	Elsevier Inc.
Document type	Article ; Online
ZDB-ID	1110-1
ISSN	1096-0309 ; 0003-2697
ISSN (online)	1096-0309
ISSN	0003-2697
DOI	10.1016/j.ab.2023.115169
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Article ; Online: Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol.

Purdy, Hugh M / Pfleger, Brian F / Reed, Jennifer L

Metabolic engineering

2021 Volume 69, Page(s) 87–97

Abstract: Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic ... ...

Abstract	Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.
MeSH term(s)	1-Butanol/metabolism ; Butanols ; NAD/genetics ; NAD/metabolism ; Nitrates/metabolism ; Synechococcus/genetics ; Synechococcus/metabolism
Chemical Substances	Butanols ; Nitrates ; NAD (0U46U6E8UK) ; isobutyl alcohol (56F9Z98TEM) ; 1-Butanol (8PJ61P6TS3)
Language	English
Publishing date	2021-11-10
Publishing country	Belgium
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2021.11.003
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Article ; Online: Revisiting metabolic engineering strategies for microbial synthesis of oleochemicals.

Yan, Qiang / Pfleger, Brian F

Metabolic engineering

2019 Volume 58, Page(s) 35–46

Abstract: Microbial production of oleochemicals from renewable feedstocks remains an attractive route to produce high-energy density, liquid transportation fuels and high-value chemical products. Metabolic engineering strategies have been applied to demonstrate ... ...

Abstract	Microbial production of oleochemicals from renewable feedstocks remains an attractive route to produce high-energy density, liquid transportation fuels and high-value chemical products. Metabolic engineering strategies have been applied to demonstrate production of a wide range of oleochemicals, including free fatty acids, fatty alcohols, esters, olefins, alkanes, ketones, and polyesters in both bacteria and yeast. The majority of these demonstrations synthesized products containing long-chain fatty acids. These successes motivated additional effort to produce analogous molecules comprised of medium-chain fatty acids, molecules that are less common in natural oils and therefore of higher commercial value. Substantial progress has been made towards producing a subset of these chemicals, but significant work remains for most. The other primary challenge to producing oleochemicals in microbes is improving the performance, in terms of yield, rate, and titer, of biocatalysts such that economic large-scale processes are feasible. Common metabolic engineering strategies include blocking pathways that compete with synthesis of oleochemical building blocks and/or consume products, pulling flux through pathways by removing regulatory signals, pushing flux into biosynthesis by overexpressing rate-limiting enzymes, and engineering cells to tolerate the presence of oleochemical products. In this review, we describe the basic fundamentals of oleochemical synthesis and summarize advances since 2013 towards improving performance of heterotrophic microbial cell factories.
MeSH term(s)	Biofuels ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Metabolic Engineering ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Yarrowia/genetics ; Yarrowia/metabolism
Chemical Substances	Biofuels
Language	English
Publishing date	2019-04-22
Publishing country	Belgium
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2019.04.009
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Article ; Online: Leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous hosts.

Cook, Taylor B / Pfleger, Brian F

MedChemComm

2019 Volume 10, Issue 5, Page(s) 668–681

Abstract: Bacteria have historically been a rich source of natural products ( ...

Abstract	Bacteria have historically been a rich source of natural products (
Language	English
Publishing date	2019-04-25
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	2545949-1
ISSN	2040-2511 ; 2040-2503
ISSN (online)	2040-2511
ISSN	2040-2503
DOI	10.1039/c9md00055k
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