A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Membrane transporter identification and modulation via adaptive laboratory evolution.

Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L-histidine, L-phenylalanine, L-threonine, and L-methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L-phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production.

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.

Regulatory control circuits for stabilizing long-term anabolic product formation in yeast.

Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of high-performing engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-ß-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-ß-glucoside pathway intermediates in order to stabilize vanillin-ß-glucoside production over ~55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-ß-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and almost 2-fold higher vanillin-ß-glucoside production, including 5-fold increase in total vanillin-ß-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-ß-glucoside producing cells without control circuits.

A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids.

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via ß-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.

Engineering Oleaginous Yeast as the Host for Fermentative Succinic Acid Production From Glucose.

Oleaginous yeast Yarrowia lipolytica is a prospective host for production of succinic acid. The interruption of tricarboxylic acid cycle through succinate dehydrogenase gene (SDH) deletion was reported to result in strains incapable of glucose utilization and this ability had to be restored by chemical mutation or long adaptive laboratory evolution. In this study, a succinate producing strain of Y. lipolytica was engineered by truncating the promoter of SDH1 gene, which resulted in 77% reduction in SDH activity but did not impair the ability of the strain to grow on glucose. The flux toward succinic acid was further improved by overexpressing the genes in the glyoxylate pathway and the oxidative TCA branch, and expressing phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes. A short adaptation on glucose reduced the lag phase of the strain and increased its tolerance to high glucose concentrations. The resulting strain produced 7.8 ± 0.0 g/L succinic acid with a yield of 0.105 g/g glucose in shake flasks without pH control, while mannitol (11.8 ± 0.8 g/L) was the main by-product. Further investigations showed that mannitol accumulation was caused by low pH stress and buffering the fermentation medium eliminated mannitol formation. In a fed-batch bioreactor in mineral medium at pH 5, at which point according to Ka values of succinic acid, the major fraction of product was in acidic form rather than dissociated form, the strain produced 35.3 ± 1.5 g/L succinic acid with 0.26 ± 0.00 g/g glucose yield.

Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents; here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.

Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway.

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards ß-carotene. The platform strain produced 331 ± 66 mg/L of ß-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding ß-ketolase and ß-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal ß-ketolase and ß-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

An Orthogonal and pH-Tunable Sensor-Selector for Muconic Acid Biosynthesis in Yeast.

Microbes offer enormous potential for production of industrially relevant chemicals and therapeutics, yet the rapid identification of high-producing microbes from large genetic libraries is a major bottleneck in modern cell factory development. Here, we develop and apply a synthetic selection system in Saccharomyces cerevisiae that couples the concentration of muconic acid, a plastic precursor, to cell fitness by using the prokaryotic transcriptional regulator BenM driving an antibiotic resistance gene. We show that the sensor-selector does not affect production nor fitness, and find that tuning pH of the cultivation medium limits the rise of nonproducing cheaters. We apply the sensor-selector to selectively enrich for best-producing variants out of a large library of muconic acid production strains, and identify an isolate that produces more than 2 g/L muconic acid in a bioreactor. We expect that this sensor-selector can aid the development of other synthetic selection systems based on allosteric transcription factors.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction.

The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.

BACKGROUND: Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for copper-dependent protein production, Pichia pastoris. RESULTS: At copper concentrations ranging from 0.1 mM (6.35 mg/L) to 2 mM (127 mg/L), growth rates of 0.25 h(-1) to 0.16 h(-1) were observed with copper uptake of as high as 20 mgcopper/gCDW. The intracellular copper content was estimated by subtracting the copper adsorbed on the cell wall from the total copper concentration in the biomass. Higher copper concentrations led to stronger cell growth retardation and, at 10 mM (635 mg/L) and above, to growth inhibition. To test the determined copper concentration range for optimal recombinant protein production, a laccase gene from Aspergillus clavatus [EMBL: EAW07265.1] was cloned under the control of the constitutive glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for expression in P. pastoris. Notably, in the presence of copper, laccase expression improved the specific growth rate of P. pastoris. Although copper concentrations of 0.1 mM and 0.2 mM augmented laccase expression 4 times up to 3 U/mL compared to the control (0.75 U/mL), while higher copper concentrations resulted in reduced laccase production. An intracellular copper content between 1 and 2 mgcopper/gCDW was sufficient for increased laccase activity. The physiology of the yeast could be excluded as a reason for the stop of laccase production at moderate copper concentrations as no flux redistribution could be observed by (13)C-metabolic flux analysis. CONCLUSION: Copper and its pivotal role to sustain cellular functions is noteworthy. However, knowledge on its cellular accumulation, availability and distribution for recombinant protein production is limited. This study attempts to address one such challenge, which revealed the fact that intracellular copper accumulation influenced laccase production and should be considered for high protein expression of copper-dependent enzymes when using P. pastoris. The results are discussed in the context of P. pastoris as a general host for copper -dependent enzyme production.

Dynamics of benzoate metabolism in Pseudomonas putida KT2440.

Soil microorganisms mineralize lignin-derived aromatic carbon sources using oxidative catabolic pathways, such as the ß-ketoadipate pathway. Although this aromatic pathway is one of the best-studied pathways in biochemistry, the complete pathway, including its regulation by aromatic carbon sources, has not been integrated into the metabolic network. In particular, information about the in vivo operation (e.g., kinetics and flux capacity) of the pathway is lacking. In this contribution, we use kinetic modeling and thermodynamic analysis to evaluate the in vivo operation of this key aromatic multi-step pathway. The resulting ab initio deterministic model of benzoate degradation via the ß-ketoadipate (ortho-cleavage) pathway in Pseudomonas putida KT2440 is presented. The kinetic model includes mechanistic rate expressions for the enzymes and transport processes. The design and experimental validation of the model are driven by data generated from short-term perturbation experiments in a benzoate-limited continuous culture. The results of rigorous modeling of the in vivo dynamics provide strong support for flux regulation by the benzoate transporter and the enzymes forming and cleaving catechol. Revisiting the ß-ketoadipate pathway might be valuable for applications in different fields, such as biochemistry and metabolic engineering, that use lignin monomers as a carbon source.

The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella Typhimurium LT2.

BACKGROUND: Metabolic reconstructions (MRs) are common denominators in systems biology and represent biochemical, genetic, and genomic (BiGG) knowledge-bases for target organisms by capturing currently available information in a consistent, structured manner. Salmonella enterica subspecies I serovar Typhimurium is a human pathogen, causes various diseases and its increasing antibiotic resistance poses a public health problem. RESULTS: Here, we describe a community-driven effort, in which more than 20 experts in S. Typhimurium biology and systems biology collaborated to reconcile and expand the S. Typhimurium BiGG knowledge-base. The consensus MR was obtained starting from two independently developed MRs for S. Typhimurium. Key results of this reconstruction jamboree include i) development and implementation of a community-based workflow for MR annotation and reconciliation; ii) incorporation of thermodynamic information; and iii) use of the consensus MR to identify potential multi-target drug therapy approaches. CONCLUSION: Taken together, with the growing number of parallel MRs a structured, community-driven approach will be necessary to maximize quality while increasing adoption of MRs in experimental design and interpretation.