Biomedical subjects

Jay D Keasling

Publications and source records attributed to Jay D Keasling.

At least 19 recordsLinked to original sources

PubMed2026

Nitrene generation and transfer for unnatural biosynthesis in living cells.

Synthetic biology has enabled the production of natural and unnatural products from inexpensive sustainable feedstocks. Yet, the scope of available products has been limited largely to compounds accessible from nature's chemical reactions. Evolved enzymes can catalyse reactions of unnatural substrates or reactions not found in nature but often require the addition of synthetic reagents to purified enzymes or to resting cells containing those enzymes. Here we show that chemical reactions of metabolic intermediates produced intracellularly in living cells can include intermolecular nitrene transfers. The biosynthesis of N-acetoxyanilines, in combination with the generation and transfer of N-aryl nitrene intermediates from them catalysed by a cytochrome P450, generates amino alcohols, diamines, diarylamines and aminoalkyl arenes from simple carbon feedstocks. These products-common substructures of pharmaceuticals and agrochemicals-are challenging to synthesize by standard organic chemistry and were produced from inexpensive, renewable feedstocks. Evolution of the enzymes in this pathway showed that titres can be increased by engineering and that the products can be synthesized with high enantioselectivity.

Journal Article↗

PubMed2025

Engineering Polyketide Stereocenters with Ketoreductase Domain Exchanges.

Polyketide synthases (PKSs) are versatile biosynthetic megasynthases capable of producing a diverse range of natural products with many applications, including in pharmaceuticals. The stereochemical precision of PKSs makes them a powerful tool for engineering tailored, unnatural polyketides; however, modifying the stereocenters of a PKS product while maintaining production levels remains a significant challenge. In this study, we systematically tested and evaluated strategies for ketoreductase (KR) domain exchanges, the domain responsible for setting stereocenters of polyketide products. After first optimizing the method for KR exchanges, we then performed 44 KR domain exchanges on three different PKSs to obtain high production of all four stereoisomers in vivo. By testing both one- and two-module PKS systems, we investigated how downstream modules process intermediates with altered stereochemistry and found that the configuration of the α-substituents was critical for gatekeeping by the ketosynthase (KS). To overcome this constraint, we investigated two different strategies for altering the KS domain, including introducing targeted mutations in the downstream KS, and exploring boundaries in exchanging the entire functional unit from the donor PKS. Both strategies successfully modified the KS stereocontrol with distinct trade-offs; the functional unit exchange resulted in higher titer improvements, though it was more likely to break the entire PKS. This study demonstrates a comprehensive approach to successfully engineering all four stereochemical configurations in multiple PKS systems, advancing our understanding of and ability to rationally modify polyketide stereochemistry through multiple engineering strategies.

Polyketides↗

PubMed2024

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Genetic Vectors↗

PubMed2007

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion.

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.

Acetates↗

PubMed2006

Biosynthesis and engineering of isoprenoid small molecules.

Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways. From these universal precursors, the biosynthetic pathways to specific isoprenoids diverge resulting in a staggering array of products. Taking advantage of this diversity has been the focus of much effort in metabolic engineering heterologous hosts. In addition, the engineering of the mevalonate pathway has increased levels of the universal precursors available for heterologous production. Finally, we will describe the efforts to produce to commercial terpenoids, paclitaxel and artemisinin.

Artemisinins↗

PubMed2006

Engineering the lycopene synthetic pathway in E. coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis.

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-D: -thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli.

Bacterial Proteins↗

PubMed2006

Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids.

Amorphadiene, a sesquiterpene precursor to the anti-malarial drug artemisinin, is synthesized by the cyclization of farnesyl pyrophosphate (FPP). Saccharomyces cerevisiae produces FPP through the mevalonate pathway using acetyl-CoA as a starting compound. In order to enhance the supply of acetyl-CoA to the mevalonate pathway and achieve high-level production of amorphadiene, we engineered the pyruvate dehydrogenase bypass in S. cerevisiae. Overproduction of acetaldehyde dehydrogenase and introduction of a Salmonella enterica acetyl-CoA synthetase variant increased the carbon flux into the mevalonate pathway resulting in increased amorphadiene production. This work will be generally applicable to the production of a broad range of isoprenoids in yeast.

Acetyl Coenzyme A↗

PubMed2006

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling.

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.

Acetates↗

PubMed2006

Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry.

Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.

Acetyl Coenzyme A↗

PubMed2006

Shewanella oneidensis MR-1 fluxome under various oxygen conditions.

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

Algorithms↗

PubMed2006

High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli.

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.

Antimalarials↗

PubMed2006

Evaluation of the effects of various culture conditions on Cr(VI) reduction by Shewanella oneidensis MR-1 in a novel high-throughput mini-bioreactor.

The growth and Cr(VI) reduction by Shewanella oneidensis MR-1 was examined using a mini-bioreactor system that independently monitors and controls pH, dissolved oxygen (DO), and temperature for each of its 24, 10-mL reactors. Independent monitoring and control of each reactor in the cassette allows the exploration of a matrix of environmental conditions known to influence S. oneidensis chromium reduction. S. oneidensis MR-1 grew in minimal medium without amino acid or vitamin supplementation under aerobic conditions but required serine and glycine supplementation under anaerobic conditions. Growth was inhibited by DO concentrations >80%. Lactate transformation to acetate was enhanced by low concentration of DO during the logarithmic growth phase. Between 11 and 35 degrees C, the growth rate obeyed the Arrhenius reaction rate-temperature relationship, with a maximum growth rate occurring at 35 degrees C. S. oneidensis MR-1 was able to grow over a wide range of pH (6-9). At neutral pH and temperatures ranging from 30 to 35 degrees C, S. oneidensis MR-1 reduced 100 microM Cr(VI) to Cr(III) within 20 min in the exponential growth phase, and the growth rate was not affected by the addition of chromate; it reduced chromate even faster at temperatures between 35 and 39 degrees C. At low temperatures (<25 degrees C), acidic (pH < 6.5), or alkaline (pH > 8.5) conditions, 100 microM Cr(VI) strongly inhibited growth and chromate reduction. The mini-bioreactor system enabled the rapid determination of these parameters reproducibly and easily by performing very few experiments. Besides its use for examining parameters of interest to environmental remediation, the device will also allow one to quickly assess parameters for optimal production of recombinant proteins or secondary metabolites.

Biodegradation, Environmental↗

PubMed2006

Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate.

To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD(600) value of 15 for 72 h.

Carotenoids↗

PubMed2006

Microbial sensors for small molecules: development of a mevalonate biosensor.

We describe a novel biosensor strain for detection and quantification of a small molecule, mevalonate. The biosensor strain is an Escherichia coli mevalonate auxotroph that expresses the green fluorescent protein and reports on the mevalonate concentration in the growth medium through a change in growth rate. A model describing the growth rate dependence on mevalonate was developed in order to use the biosensor strain for high-throughput screening (HTS) and quantitative measurement of mevalonate in the extracellular environment. In general, this method should be applicable to the quantification of any small molecule for which an auxotroph can be developed and will be useful for HTS of evolved metabolic pathways for which there is no readily available screen or selection.

Aldose-Ketose Isomerases↗

PubMed2006

Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes.

Many applications of synthetic biology require the balanced expression of multiple genes. Although operons facilitate coordinated expression of multiple genes in prokaryotes and eukaryotes, coordinating the many post-transcriptional processes that determine the relative levels of gene expression in operons by a priori design remains a challenge. We describe a method for tuning the expression of multiple genes within operons by generating libraries of tunable intergenic regions (TIGRs), recombining various post-transcriptional control elements and screening for the desired relative expression levels. TIGRs can vary the relative expression of two reporter genes over a 100-fold range and balance expression of three genes in an operon that encodes a heterologous mevalonate biosynthetic pathway, resulting in a sevenfold increase in mevalonate production. This technology should be useful for optimizing the expression of multiple genes in synthetic operons, both in prokaryotes and eukaryotes.

Algorithms↗

PubMed2006

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray.

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.

Asteraceae↗

PubMed2006

Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics.

The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.

Desulfovibrio vulgaris↗

PubMed2006

Coenzyme Q10 production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway.

In the present work, Escherichia coli DH5alpha was metabolically engineered for CoQ(10) production by the introduction of decaprenyl diphosphate synthase gene (ddsA) from Agrobacterium tumefaciens. Grown in 2YTG medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, and 0.5% glycerol) with an initial pH of 7, the recombinant E. coli was capable of CoQ(10) production up to 470 microg/gDCW (dry cell weight). This value could be further elevated to 900 microg/gDCW simply by increasing the initial culture pH from 7 to 9. Supplementation of 4-hydroxy benzoate did not improve the productivity any further. However, engineering of a lower mevalonate semi-pathway so as to increase the isopentenyl diphosphate (IPP) supply of the recombinant strain using exogenous mevalonate efficiently increased the CoQ(10) production. Lower mevalonate semi-pathways of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Saccharomyces cerevisiae were tested. Among these, the pathway of Streptococcus pneumoniae proved to be superior, yielding CoQ(10) production of 2,700+/-115 microg/gDCW when supplemented with exogenous mevalonate of 3 mM. In order to construct a complete mevalonate pathway, the upper semi-pathway of the same bacterium, Streptococcus pneumoniae, was recruited. In a recombinant E. coli DH5alpha harboring three plasmids encoding for upper and lower mevalonate semi-pathways as well as DdsA enzyme, the heterologous mevalonate pathway could convert endogenous acetyl-CoA to IPP, resulting in CoQ(10) production of up to 2,428+/-75 microg/gDCW, without mevalonate supplementation. In contrast, a whole mevalonate pathway constructed in a single operon was found to be less efficient. However, it provided CoQ(10) production of up to 1,706+/-86 microg/gDCW, which was roughly 1.9 times higher than that obtained by ddsA alone.

Alkyl and Aryl Transferases↗