Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae.

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

Rapid and reliable DNA assembly via ligase cycling reaction.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin.

Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940-943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.

BACKGROUND: Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. METHODOLOGY/ PRINCIPAL FINDINGS: By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. CONCLUSIONS/ SIGNIFICANCE: Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene.

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid.

BACKGROUND: Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. RESULTS: Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. CONCLUSION: The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

Identification of isopentenol biosynthetic genes from Bacillus subtilis by a screening method based on isoprenoid precursor toxicity.

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6,051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.

Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induction of a lactose-inducible (P(lac)) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli.

Engineering biosynthetic pathways in microbes for the production of complex chemicals and pharmaceuticals is an attractive alternative to chemical synthesis. However, in transferring large pathways to alternate hosts and manipulating expression levels, the native regulation of carbon flux through the pathway may be lost leading to imbalances in the pathways. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway [Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D., 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796-802]. The strain produces high levels of isoprenoids, but upon further investigation we discovered that the accumulation of pathway intermediates limited flux and that high-level expression of the mevalonate pathway enzymes inhibited cell growth. Gene titration studies and metabolite profiling using liquid chromatography-mass spectrometry linked the growth inhibition phenotype with the accumulation of the pathway intermediate 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA). Such an accumulation implies that the activity of HMG-CoA reductase was insufficient to balance flux in the engineered pathway. By modulating HMG-CoA reductase production, we eliminated the pathway bottleneck and increased mevalonate production. These results demonstrate that balancing carbon flux through the heterologous pathway is a key determinant in optimizing isoprenoid biosynthesis in microbial hosts.

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.

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%.

Catabolite repression of the propionate catabolic genes in Escherichia coli and Salmonella enterica: evidence for involvement of the cyclic AMP receptor protein.

Previous studies with Salmonella enterica serovar Typhimurium LT2 demonstrated that transcriptional activation of the prpBCDE operon requires the function of transcription factor PrpR, sigma-54, and IHF. In this study, we found that transcription from the prpBCDE and prpR promoters was down-regulated by the addition of glucose or glycerol, indicating that these genes may be regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Targeted mutagenesis of a putative CRP-binding site in the promoter region between prpR and prpBCDE suggested that these genes are under the control of CRP. Furthermore, cells with defects in cya or crp exhibited reduced transcriptional activation of prpR and prpBCDE in Escherichia coli. These results demonstrate that propionate metabolism is subject to catabolite repression by the global transcriptional regulator CRP and that this regulation is effected through control of both the regulator gene prpR and the prpBCDE operon itself. The unique properties of the regulation of these two divergent promoters may have important implications for mechanisms of CRP-dependent catabolite repression acting in conjunction with a member of the sigma-54 family of transcriptional activators.

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR.

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.

Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 microg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available.