Construction of novel metabolic pathways with artificial enzymes

Non-fossil raw materials can be utilized for the production of useful compounds by way of microbial fermentation . Sugars are obtained from carbon fixations of plants or photosynthetic microorganisms, and are used as a carbon source for the biosynthesis of useful target compounds by genetically modified microorganisms. In order for a microorganism to produce enough target compound, techniques for optimal metabolic design must include balance of energy production/consumption, redox pathways, and intracellular carbon flow. With recent innovations in genome analysis technology and information processing technology, computational design tools that can describe more than 1000 genome-scale metabolic reactions to efficiently produce target compounds have been developed worldwide. However, the established tools are not designed to search and create biosynthetic pathways for production of non-natural compounds from fossil resources. We developed BioProV and M-path, new simulation tools that enable metabolic design for the biosynthesis of unnatural compounds. By combining these tools with enzyme engineering technology, we succeeded in expanding the scope of bioproduction targets. The first example is construction of an artificial metabolic pathway to biosynthesize isoprene. Isoprene the raw material for production of synthetic rubber that can be used in automobile tires. Currently, isoprene is industrially produced as a by-product of naphtha pyrolysis. Therefore, by establishing green isoprene production technology, dependence upon petroleum can be reduced. Isoprene is a substance that can exist within cells of many organisms as a monomer of polyisoprene rubber, and also as a structural unit of secondary metabolites. It is difficult to optimize its synthentic pathway due to shortages of intracellular ATP supply, and challenges in the introduction of improved biosynthetic pathways. In nature, isoprene is produced from mevalonic acid through a five-step reaction, but the newly constructed artificial metabolic pathway consists of just two steps from mevalonic acid to isoprene. This results in a three-fold reduction in cellular energy consumption. Furthermore, we succeeded in constructing a highly active enzyme that exhibits 10,000-fold higher isoprene-producing activity relative to natural enzymes. By introducing these artificial metabolic reactions into Escherichia coli, efficient artificial isoprene production was achieved. In addition, we have developed a microbial production system for 1,3-butadiene, another alternative source for synthetic rubber. Moreover, rationally engineered enzymes from insects and plants enzymes have resulted in the construction of an artificial pathway to benzylisoquinoline alkaloids and downstream opioid analgesics

Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy

BACKGROUND: Biodiesel production from marine microalgae has received much attention as microalgae can be cultivated on non-arable land without the use of potable water, and with the additional benefits of mitigating CO(2) emissions and yielding biomass. However, there is still a lack of effective operational strategies to promote lipid accumulation in marine microalgae, which are suitable for making biodiesel since they are mainly composed of saturated and monounsaturated fatty acids. Moreover, the regulatory mechanisms involved in lipid biosynthesis in microalgae under environmental stress are not well understood. RESULTS: In this work, the combined effects of salinity and nitrogen depletion stresses on lipid accumulation of a newly isolated marine microalga, Chlamydomonas sp. JSC4, were explored. Metabolic intermediates were profiled over time to observe transient changes during the lipid accumulation triggered by the combination of the two stresses. An innovative cultivation strategy (denoted salinity-gradient operation) was also employed to markedly improve the lipid accumulation and lipid quality of the microalga, which attained an optimal lipid productivity of 223.2 mg L(-1) d(-1) and a lipid content of 59.4% per dry cell weight. This performance is significantly higher than reported in most related studies. CONCLUSIONS: This work demonstrated the synergistic integration of biological and engineering technologies to develop a simple and effective strategy for the enhancement of oil production in marine microalgae

Cell-surface display engineering of industrial Saccharomyces cerevisiae for hemicellulosic-to-ethanol consolidated bioprocesses

The utilization of lignocellulosic biomass to produce biofuels and commodity chemicals has appeared as a solution to alleviate the envisioned depletion of fossil resources. Nevertheless, the attainment of economically viable lignocellulosic-based processes requires an effective utilization of the hemicellulosic fraction, which may comprise up to 40% of the total biomass[1]. This represents a major bottleneck, mainly due to the requirement of chemical/enzymatic treatments for the hydrolysis of hemicellulose into fermentable sugars, and the fact that hemicellulose is mainly composed of xylose, a sugar that is not readily consumed by Saccharomyces cerevisiaethe most used organism in industrial biotechnology. In this context, consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism/consortium, appears as a solution to decrease environmental and economic costs in lignocellulosic biorefineries. Therefore, in this work, hemicellulolytic enzymes were displayed on the cell surface of robust industrial S. cerevisiae strains with advantageous traits (e.g. thermotolerance and inhibitor tolerance). These strains were also engineered for xylose consumption with both the isomerase and the oxidoreductase pathways, which was previously optimized for fermentation of inhibitor-containing hydrolysates[2]. The combination of these modifications allowed the direct production of 11.1 g/L of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, representing an ethanol yield of 0.327 g/g of potential xylose/glucose. To the extent of our knowledge, this is the highest ethanol concentration reported from direct conversion of a lignocellulosic-derived hemicellulose by S. cerevisiae without the addition of external hydrolytic catalysts. Additionally, the cell-surface display of hemicellulases presented a fermentative advantage in simultaneous saccharification and fermentation of corn cob hemicellulosic fraction, greatly decreasing the necessity for commercial enzymes. These results prove the value of industrial S. cerevisiae strains as hosts for the construction of whole-cell biocatalysts for hemicellulosic-based processes, without the need for expensive exogenous enzymes, chemical catalysts or laborious detoxification steps.Portuguese Foundation for Science and Technology (FCT, Portugal) strategic funding of UID/BIO/04469/2019 unit and COMPETE2020(POCI-01-0145-FEDER-006684). MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha). BioTecNorte operation (NORTE-01-0145- FEDER000004) funded by European Regional Development Fund under the scope of Norte2020— Programa Operacional Regional do Norte and MultiBiorefinery project (POCI-01–0145-FEDER-016403).info:eu-repo/semantics/publishedVersio

Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

AbstractRre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation

A Systematic Approach to Timeseries Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture

Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture

Synthesis of Sulfo-Sialic Acid Analogues: Potent Neuraminidase Inhibitors in Regards to Anomeric Functionality

The design, synthesis and application of N-acetylneuraminic acid-derived compounds bearing anomeric sulfo functional groups are described. These novel compounds, which we refer to as sulfo-sialic acid analogues, include 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid and its 4-deoxy-3,4-dehydrogenated pseudoglycal. While 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid contains no further modifications of the 2-deoxy-pyranose ring, it is still a more potent inhibitor of avian-origin H5N1 neuraminidase (NA) and drug-resistant His275Tyr NA as compared to the oxocarbenium ion transition state analogue 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The sulfo-sialic acid analogues described in this report are also more potent inhibitors of influenza NA (up to 40-fold) and bacterial NA (up to 8.5-fold) relative to the corresponding anomeric phosphonic acids. These results confirm that this novel anomeric sulfo modification offers great potential to improve the potency of next-generation NA inhibitors including covalent inhibitors