Metabolic Engineering Communications

Rhodopseudomonas palustris CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by R. palustris. The Phap1 phasin ( phaP1 ) from the PHB-producing model bacterium Cupriavidus necator H16 was expressed in R. palustris with the aim of overproducing PHBV from the lignin breakdown product p- coumarate by fostering smaller and more abundant granules...

Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost...

Resveratrol is a plant-derived aromatic compound with a wide range of beneficial properties including antioxidant and anti-aging effects. The resveratrol currently available on the market is predominantly extracted from certain plants such as grape and the Japanese knotweed Polygonum cuspidatum . Due to the unstable harvest of these plants and the low resveratrol purity obtained, it is necessary to develop a stable production process of high-purity resveratrol from inexpensive feedstocks. Here, we attempted to produce resveratrol from a wide range of sugars as carbon sources by a using the genetically-engineered yeast Scheffersomyces stipitis (formerly known as Pichia stipitis ), which possesses a broad sugar utilization capacity...

The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse. In this work, the potential of V. natriegens as an amino acid producer was investigated...

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5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues...

Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use l-phenylalanine and/or l-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds...

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E...

Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites...

Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene...

Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms...

Lichen-forming fungi produce a variety of secondary metabolites including bioactive polyketides. Advances in DNA and RNA sequencing have led to a growing database of new lichen gene clusters encoding polyketide synthases (PKS) and associated ancillary activities. Definitive assignment of a PKS gene to a metabolic product has been challenging in the lichen field due to a lack of established gene knockout or heterologous gene expression systems. Here, we report the reconstitution of a non-reducing PKS gene from the lichen Pseudevernia furfuracea and successful heterologous expression of the synthetic lichen PKS gene in engineered Saccharomyces cerevisiae ...

Oxygen deficiency (hypoxia) induces adverse effects during biotherapeutic protein production leading to reduced productivity and cell growth. Hypoxic conditions occur during classical batch fermentations using high cell densities or perfusion processes. Here we present an effort to create novel engineered Chinese hamster ovary (CHO) cell lines by exploiting encountered hypoxic bioprocess conditions to reinforce cellular production capacities. After verifying the conservation of the hypoxia-responsive pathway in CHO cell lines by analyzing oxygen sensing proteins HIF1a, HIF1β and VDL, hypoxia-response-elements (HREs) were functionally analyzed and used to create hypoxia-responsive expression vectors...

Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway...

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy...

13 C-based metabolic flux analysis (13 C-MFA) is an essential tool for estimating intracellular metabolic flux levels in metabolic engineering and biology. In 13 C-MFA, a metabolic flux distribution that explains the observed isotope labeling data was computationally estimated using a non-linear optimization method. Herein, we report the development of mfapy, an open-source Python package developed for more flexibility and extensibility for 13 C-MFA. mfapy compels users to write a customized Python code by describing each step in the data analysis procedures of the isotope labeling experiments...

There is much to be gained by enabling electronic interrogation and control of biological function. While the benefits of bioelectronics that rely on potential-driven ionic flows are well known (electrocardiograms, defibrillators, neural prostheses, etc) there are relatively few advances targeting nonionic molecular networks, including genetic circuits. Redox activities combine connectivity to electronics with the potential for specific genetic control in cells. Here, electrode-generated hydrogen peroxide is used to actuate an electrogenetic "relay" cell population, which interprets the redox cue and synthesizes a bacterial signaling molecule (quorum sensing autoinducer AI-1) that, in turn, signals increased growth rate in a second population...

Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 ( cpcBACD ) is functional in the mesophile Synechocystis sp...

Surfactin, a quorum sensing signal molecule, is correlated with carbon metabolism in Bacillus amyloliquefaciens . In the present work, we found that mutation of srfA (Δ srfA ) led to an obviously changed carbon metabolism in B. amyloliquefaciens . Firstly, the PTS-glucose system was significantly increased as a feedback to glucose exhaustion. Secondly, the basic carbon metabolism such as glycolysis and TCA cycle was obviously weakened in Δ srfA . Thirdly, the global regulator of CcpA (carbon catabolite protein A) and P ~ Ser46 -HPr (seryl-phosphorylated form of histidine-containing protein) to mediate the CcpA-dependent CCR (carbon catabolite repression) were not increased, but the ability to use extracellular non- and less-preferred carbon sources was down-regulated in Δ srfA ...

Docosadienoic acid (DDA, 22:2-13,16) and docosatrienoic acid (DTA, 22:3-13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds...