Metabolic Engineering Communications最新文献

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. Subsequently engineered hypoxia sensitive CHO cell lines significantly induced protein expression (SEAP) during adverse oxygen limitation encountered during batch fermentations as well as high cell density perfusion processes (2.7 fold). We also exploited this novel cell system to establish a highly effective oxygen shift as innovative bioprocessing strategy using hypoxia induction to improve production titers. Thus, substantial improvements can be made to optimize CHO cell productivity for novel bioprocessing challenges as oxygen limitation, providing an avenue to establish better cell systems by exploiting adverse process conditions for optimized biotherapeutic production.

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. We show that P. furfuracea PFUR17_02294 produces lecanoric acid, the depside dimer of orsellinic acid, at 360 mg/L in small-scale yeast cultures. Our results unequivocally identify PFUR17_02294 as a lecanoric acid synthase and establish that a single lichen PKS synthesizes two phenolic rings and joins them by an ester linkage to form the depside product.

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. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

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. First, we constructed the resveratrol producing strain by introducing genes coding the essential enzymes for resveratrol biosynthesis [tyrosine ammonia-lyase 1 derived from Herpetosiphon aurantiacus (HaTAL1), 4-coumarate: CoA ligase 2 derived from Arabidopsis thaliana (At4CL2), and stilbene synthase 1 derived from Vitis vinifera (VvVST1)]. Subsequently, a feedback-insensitive allele of chorismate mutase was overexpressed in the constructed strain to improve resveratrol production. The constructed strain successfully produced resveratrol from a broad range of biomass-derived sugars [glucose, fructose, xylose, N-acetyl glucosamine (GlcNAc), galactose, cellobiose, maltose, and sucrose] in shake flask cultivation. Significant resveratrol titers were detected in cellobiose and sucrose fermentation (529.8 and 668.6 mg/L after 120 h fermentation, respectively), twice above the amount obtained with glucose (237.6 mg/L). Metabolomic analysis revealed an altered profile of the metabolites involved in the glycolysis and shikimate pathways, and also of cofactors and metabolites of energy metabolisms, depending on the substrate used. The levels of resveratrol precursors such as L-tyrosine increased in cellobiose and sucrose-grown cells. The results indicate that S. stipitis is an attractive microbial platform for resveratrol production from broad types of biomass-derived sugars and the selection of suitable substrates is crucial for improving resveratrol productivity of this yeast.

¹³C-based metabolic flux analysis (¹³C-MFA) is an essential tool for estimating intracellular metabolic flux levels in metabolic engineering and biology. In ¹³C-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 ¹³C-MFA. mfapy compels users to write a customized Python code by describing each step in the data analysis procedures of the isotope labeling experiments. The flexibility and extensibility provided by mfapy can support trial-and-error performance in the routine estimation of metabolic flux distributions, experimental design by computer simulations of ¹³C-MFA experiments, and development of new data analysis techniques for stable isotope labeling experiments. mfapy is available to the public from the Github repository (https://github.com/fumiomatsuda/mfapy).

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. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs. Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE. Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS, involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis. In this work, we have devised a useful platform for future engineering steps.

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. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.

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. The dramatically increased growth rate of the second population is enabled by expression of a phosphotransferase system protein, HPr, which is important for glucose transport. The potential to electronically modulate cell growth via direct genetic control will enable new opportunities in the treatment of disease and manufacture of biological therapeutics and other molecules.

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. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO–K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.

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. PCC 6803. Expression of cpcBACD in an ‘Olive’ mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g⁻¹ DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.