Publications by authors named "Kristala L J Prather"

72 Publications

Dynamic Control of Metabolism.

Annu Rev Chem Biomol Eng 2021 Mar 30. Epub 2021 Mar 30.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA; email:

Metabolic engineering reprograms cells to synthesize value-added products. In doing so, endogenous genes are altered and heterologous genes can be introduced to achieve the necessary enzymatic reactions. Dynamic regulation of metabolic flux is a powerful control scheme to alleviate and overcome the competing cellular objectives that arise from the introduction of these production pathways. This review explores dynamic regulation strategies that have demonstrated significant production benefits by targeting the metabolic node corresponding to a specific challenge. We summarize the stimulus-responsive control circuits employed in these strategies that determine the criterion for actuating a dynamic response and then examine the points of control that couple the stimulus-responsive circuit to a shift in metabolic flux. Expected final online publication date for the , Volume 12 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-chembioeng-091720-125738DOI Listing
March 2021

Prospecting Biochemical Pathways to Implement Microbe-Based Production of the New-to-Nature Platform Chemical Levulinic Acid.

ACS Synth Biol 2021 Mar 25. Epub 2021 Mar 25.

Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.

Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed , where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in or . Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.
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http://dx.doi.org/10.1021/acssynbio.0c00518DOI Listing
March 2021

Natural combinatorial genetics and prolific polyamine production enable siderophore diversification in Serratia plymuthica.

BMC Biol 2021 Mar 15;19(1):46. Epub 2021 Mar 15.

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA.

Background: Iron is essential for bacterial survival. Bacterial siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe. Siderophores chelate Fe for uptake into the cell, where it is reduced to soluble Fe. Siderophores are key molecules in low soluble iron conditions. The ability of bacteria to synthesize proprietary siderophores may have increased bacterial evolutionary fitness; one way that bacteria diversify siderophore structure is by incorporating different polyamine backbones while maintaining the catechol moieties.

Results: We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin A and B with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore found in the bacterium Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact the production of serratiochelins. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness.

Conclusions: This study shows the natural occurrence of serratiochelins, photobactin, enterobactin, and aerobactin in a single bacterial species and illuminates the interplay between siderophore biosynthetic pathways and polyamine production, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 μmol/g DW) and putrescine (30.83 μmol/g DW), S. plymuthica can be exploited for the industrial production of these compounds.
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http://dx.doi.org/10.1186/s12915-021-00971-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962358PMC
March 2021

Production of D-Glyceric acid from D-Galacturonate in Escherichia coli.

J Ind Microbiol Biotechnol 2020 Dec 14;47(12):1075-1081. Epub 2020 Oct 14.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

A microbial production platform has been developed in Escherichia coli to synthesize D-glyceric acid from D-galacturonate. The expression of uronate dehydrogenase (udh) from Pseudomonas syringae and galactarolactone isomerase (gli) from Agrobacterium fabrum, along with the inactivation of garK, encoding for glycerate kinase, enables D-glyceric acid accumulation by utilizing the endogenous expression of galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3-oxopropionate reductase (garR). Optimization of carbon flux through the elimination of competing metabolic pathways led to the development of a ΔgarKΔhyiΔglxKΔuxaC mutant strain that produced 4.8 g/l of D-glyceric acid from D-galacturonate, with an 83% molar yield. Cultivation in a minimal medium produced similar yields and demonstrated that galactose or glycerol serve as possible carbon co-feeds for industrial production. This novel platform represents an alternative for the production of D-glyceric acid, an industrially relevant chemical, that addresses current challenges in using acetic acid bacteria for its synthesis: increasing yield, enantio-purity and biological stability.
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http://dx.doi.org/10.1007/s10295-020-02323-2DOI Listing
December 2020

Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae.

Enzyme Microb Technol 2020 Oct 16;140:109623. Epub 2020 Jun 16.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. Electronic address:

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ± 0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.
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http://dx.doi.org/10.1016/j.enzmictec.2020.109623DOI Listing
October 2020

The importance and future of biochemical engineering.

Biotechnol Bioeng 2020 08 29;117(8):2305-2318. Epub 2020 May 29.

Department of Chemical and Environmental Engineering, University of California, Riverside, California.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.
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http://dx.doi.org/10.1002/bit.27364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354896PMC
August 2020

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450.

Microb Cell Fact 2020 Feb 11;19(1):26. Epub 2020 Feb 11.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, USA.

Background: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.

Results: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive.

Conclusions: The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.
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http://dx.doi.org/10.1186/s12934-020-01300-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011507PMC
February 2020

Development of a Quorum-Sensing Based Circuit for Control of Coculture Population Composition in a Naringenin Production System.

ACS Synth Biol 2020 03 21;9(3):590-597. Epub 2020 Feb 21.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

As synthetic biology and metabolic engineering tools improve, it is feasible to construct more complex microbial synthesis systems that may be limited by the machinery and resources available in an individual cell. Coculture fermentation is a promising strategy for overcoming these constraints by distributing objectives between subpopulations, but the primary method for controlling the composition of the coculture of production systems has been limited to control of the inoculum composition. We have developed a quorum sensing (QS)-based growth-regulation circuit that provides an additional parameter for regulating the composition of a coculture over the course of the fermentation. Implementation of this tool in a naringenin-producing coculture resulted in a 60% titer increase over a system that was optimized by varying inoculation ratios only. We additionally demonstrated that the growth control circuit can be implemented in combination with a communication module that couples transcription in one subpopulation to the cell-density of the other population for coordination of behavior, resulting in an additional 60% improvement in naringenin titer.
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http://dx.doi.org/10.1021/acssynbio.9b00451DOI Listing
March 2020

Development of an autonomous and bifunctional quorum-sensing circuit for metabolic flux control in engineered .

Proc Natl Acad Sci U S A 2019 12 3;116(51):25562-25568. Epub 2019 Dec 3.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Metabolic engineering seeks to reprogram microbial cells to efficiently and sustainably produce value-added compounds. Since chemical production can be at odds with the cell's natural objectives, strategies have been developed to balance conflicting goals. For example, dynamic regulation modulates gene expression to favor biomass and metabolite accumulation at low cell densities before diverting key metabolic fluxes toward product formation. To trigger changes in gene expression in a pathway-independent manner without the need for exogenous inducers, researchers have coupled gene expression to quorum-sensing (QS) circuits, which regulate transcription based on cell density. While effective, studies thus far have been limited to one control point. More challenging pathways may require layered dynamic regulation strategies, motivating the development of a generalizable tool for regulating multiple sets of genes. We have developed a QS-based regulation tool that combines components of the and QS systems to simultaneously and dynamically up- and down-regulate expression of 2 sets of genes. Characterization of the circuit revealed that varying the expression level of 2 QS components leads to predictable changes in switching dynamics and that using components from 2 QS systems allows for independent tuning capability. We applied the regulation tool to successfully address challenges in both the naringenin and salicylic acid synthesis pathways. Through these case studies, we confirmed the benefit of having multiple control points, predictable tuning capabilities, and independently tunable regulation modules.
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http://dx.doi.org/10.1073/pnas.1911144116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926038PMC
December 2019

Development of a Vanillate Biosensor for the Vanillin Biosynthesis Pathway in .

ACS Synth Biol 2019 09 3;8(9):1958-1967. Epub 2019 Sep 3.

Department of Chemical Engineering , Massachusetts Institute of Technology , 77 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.

The engineered vanillin biosynthesis pathway constructed in is industrially relevant but limited by the reaction catalyzed by catechol -methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To identify alternative -methyltransferases, we constructed a vanillate sensor based on the VanR-VanO system. Using an promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We found that this construct and an evolved variant demonstrate remarkable substrate selectivity, exhibiting no detectable response to the regioisomer byproduct isovanillate and minimal response to structurally similar pathway intermediates. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol -methyltransferases and identified three previously uncharacterized but active -methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.
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http://dx.doi.org/10.1021/acssynbio.9b00071DOI Listing
September 2019

From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.

Biotechnol Adv 2019 11 7;37(7):107397. Epub 2019 May 7.

CEB-Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Electronic address:

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value.
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http://dx.doi.org/10.1016/j.biotechadv.2019.05.003DOI Listing
November 2019

Engineered microbial biofuel production and recovery under supercritical carbon dioxide.

Nat Commun 2019 02 4;10(1):587. Epub 2019 Feb 4.

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Culture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. To solve these problems, we propose a high-pressure fermentation strategy, coupled with in situ extraction using the abundant and renewable solvent supercritical carbon dioxide (scCO), which is also known for its broad microbial lethality. Towards this goal, we report the domestication and engineering of a scCO-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO field, to produce branched alcohols that have potential use as biofuels. After establishing induced-expression under scCO, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield with co-produced isopentanol. Finally, we present a process model to compare the energy required for our process to other in situ extraction methods, such as gas stripping, finding scCO extraction to be potentially competitive, if not superior.
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http://dx.doi.org/10.1038/s41467-019-08486-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361901PMC
February 2019

Chemistry as biology by design.

Microb Biotechnol 2019 01 28;12(1):30-31. Epub 2018 Nov 28.

Department of Chemical Engineering, Microbiology Graduate Program, Synthetic Biology Center at MIT, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

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http://dx.doi.org/10.1111/1751-7915.13345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302718PMC
January 2019

Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610.

Carbohydr Polym 2019 Feb 3;205:176-183. Epub 2018 Oct 3.

CEB-Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Electronic address:

The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2%). The maximum production yield expressed as total XOS per amount of xylan (306 ± 4 mg/g) was achieved after 8 h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5 °C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield.
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http://dx.doi.org/10.1016/j.carbpol.2018.09.088DOI Listing
February 2019

Isolation, Development, and Genomic Analysis of SR7 for Growth and Metabolite Production Under Supercritical Carbon Dioxide.

Front Microbiol 2018 25;9:2152. Epub 2018 Sep 25.

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States.

Supercritical carbon dioxide (scCO) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO are mainly limited to processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO reservoir in aqueous media under scCO headspace enabled the isolation of spore-forming strain SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO headspace. Detection of endogenous fermentative compounds in cultures grown under scCO represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of SR7 represent initial advancements in the effort toward enabling exploitation of scCO as a sustainable solvent for bioprocessing.
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http://dx.doi.org/10.3389/fmicb.2018.02152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167967PMC
September 2018

Single-step production of arabino-xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewers' spent grain.

Carbohydr Polym 2018 Nov 7;199:546-554. Epub 2018 Jul 7.

CEB-Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Electronic address:

Brewers' spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG.
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http://dx.doi.org/10.1016/j.carbpol.2018.07.017DOI Listing
November 2018

Rational design of thiolase substrate specificity for metabolic engineering applications.

Biotechnol Bioeng 2018 09 29;115(9):2167-2182. Epub 2018 Jun 29.

Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse β-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.
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http://dx.doi.org/10.1002/bit.26737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131064PMC
September 2018

Layered dynamic regulation for improving metabolic pathway productivity in .

Proc Natl Acad Sci U S A 2018 03 5;115(12):2964-2969. Epub 2018 Mar 5.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139;

Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from "growth" to "production" mode. The second strategy, developed in this work, uses a biosensor for -inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date in K-12 strains.
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http://dx.doi.org/10.1073/pnas.1716920115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866568PMC
March 2018

Synthetic biology strategies for improving microbial synthesis of "green" biopolymers.

J Biol Chem 2018 04 16;293(14):5053-5061. Epub 2018 Jan 16.

From the Department of Chemical Engineering and Center for Integrative Synthetic Biology (CISB), Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Polysaccharide-based biopolymers have many material properties relevant to industrial and medical uses, including as drug delivery agents, wound-healing adhesives, and food additives and stabilizers. Traditionally, polysaccharides are obtained from natural sources. Microbial synthesis offers an attractive alternative for sustainable production of tailored biopolymers. Here, we review synthetic biology strategies for select "green" biopolymers: cellulose, alginate, chitin, chitosan, and hyaluronan. Microbial production pathways, opportunities for pathway yield improvements, and advances in microbial engineering of biopolymers in various hosts are discussed. Taken together, microbial engineering has expanded the repertoire of green biological chemistry by increasing the diversity of biobased materials.
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http://dx.doi.org/10.1074/jbc.TM117.000368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892568PMC
April 2018

A Robust CRISPR Interference Gene Repression System in Pseudomonas.

J Bacteriol 2018 04 12;200(7). Epub 2018 Mar 12.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

spp. are widely used model organisms in different areas of research. Despite the relevance of in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in spp. using a nuclease-null Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in and 300-fold repression in pyoverdine production in This inducible system enables the study of essential genes, as shown by depletions in , , and that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first characterization of protospacer adjacent motif (PAM) site preferences of dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of dCas9, especially in GC-rich organisms. spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Although spp. are widely used as model organisms in different areas of research, existing tools to deplete a protein of interest in these organisms remain limited. We have developed a robust and inducible gene repression tool in , , and using the dCas9. This method of protein depletion is superior to existing methods, such as promoter replacements and addition of degradation tags, because it does not involve genomic modifications of the target protein, is titratable, and is capable of repressing multiple genes simultaneously. This gene repression system now enables easy depletion of specific proteins in , accelerating the study and engineering of this widely used model organism.
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http://dx.doi.org/10.1128/JB.00575-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847647PMC
April 2018

Pathway towards renewable chemicals.

Nat Microbiol 2017 12;2(12):1580-1581

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41564-017-0071-9DOI Listing
December 2017

Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

Nat Biotechnol 2017 03 13;35(3):273-279. Epub 2017 Feb 13.

Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.
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http://dx.doi.org/10.1038/nbt.3796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340623PMC
March 2017

Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.

Curr Protoc Mol Biol 2017 01 5;117:31.8.1-31.8.20. Epub 2017 Jan 5.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.

The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.29DOI Listing
January 2017

Porting the synthetic D-glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae.

Biotechnol J 2016 Sep 29;11(9):1201-8. Epub 2016 Jun 29.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.

D-Glucaric acid can be produced as a value-added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH-mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo-inositol oxygenase (MIOX) enzyme to be rate-limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid-tolerant and evaluate a codon-optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene - either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon-optimized for expression in S. cerevisiae - in order to identify the rate-limiting steps for D-glucaric acid production both from a fermentative and non-fermentative carbon source. myo-Inositol availability was found to be rate-limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate-limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed-batch mode, and 1.6 g/L from glucose supplemented with myo-inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.
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http://dx.doi.org/10.1002/biot.201500563DOI Listing
September 2016

Towards effective non-viral gene delivery vector.

Biotechnol Genet Eng Rev 2015 Apr-Oct;31(1-2):82-107

a MIT-Portugal Program.

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.
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http://dx.doi.org/10.1080/02648725.2016.1178011DOI Listing
January 2017

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

Appl Microbiol Biotechnol 2016 Aug 5;100(15):6725-6737. Epub 2016 May 5.

iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.
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http://dx.doi.org/10.1007/s00253-016-7565-xDOI Listing
August 2016

Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway.

Microb Cell Fact 2016 Apr 11;15:61. Epub 2016 Apr 11.

Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room E17-504G, Cambridge, MA, 02139, USA.

Background: Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin.

Results: Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and L-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-L-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33% (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25% increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability.

Conclusions: Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or methionine biosynthesis, we pursued two orthogonal strategies that are each aimed at deregulating multiple reactions. Our results increase the working knowledge of SAM biosynthesis engineering and provide a framework for improving titers of metabolic products dependent upon methylation reactions.
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http://dx.doi.org/10.1186/s12934-016-0459-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828866PMC
April 2016

Modular and selective biosynthesis of gasoline-range alkanes.

Metab Eng 2016 Jan 10;33:28-40. Epub 2015 Nov 10.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement.
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http://dx.doi.org/10.1016/j.ymben.2015.10.010DOI Listing
January 2016

Biological synthesis unbounded?

Nat Biotechnol 2015 Nov;33(11):1148-9

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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http://dx.doi.org/10.1038/nbt.3399DOI Listing
November 2015

Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae.

ACS Synth Biol 2016 Feb 18;5(2):116-24. Epub 2015 Nov 18.

Department of Chemical Engineering, ‡MIT Center for Integrative Synthetic Biology, §Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae. In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.
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http://dx.doi.org/10.1021/acssynbio.5b00164DOI Listing
February 2016